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ViroLIEgy

ViroLIEgy
26 May 2022 | 5:14 pm

Monkeypox Party With Patrick Timpone


Yesterday, I had the privilege once again of discussing all things virology with Patrick Timpone. This time, we focused our attention on moneypox…for some reason. 😉 We also spoke about the misuse of PCR, the problems with antibodies, the well-orchestrated fear-campaign as a tool to gain control, the dangers of vaccination, the lack of proof for sexually transmitted disease, and many other topics. I hope that the information here is useful!

ViroLIEgy
24 May 2022 | 1:58 pm

Monkey Business


If you have been paying attention to the (fake) news recently, you may have heard a little something about a crop of monkeypox cases popping up around the world. Normally, most people wouldn't bat an eye at any headlines concerning the monkeypox yet the mainstream media is making a strong push to ensure that these headlines catch your attention. As can be seen, the usual suspects such as President Biden, the CDC, and the WHO are all involved in sounding the alarm over what is considered to be a mild and relatively hard to transmit "viral" disease.

Cue the FEAR propaganda.

It makes one wonder what (or who) could possibly be behind such a push to strike fear into the minds of a populace already on high alert over the emerging "6th wave of Covid-19." It's not like they have these things planned out well in advance now, do they?

Monkeypox outbreak exercise. Reminds me of something…

In March of 2021, the Nuclear Threat Initiative (NTI), a nonprofit group created in 2001 by Senator Sam Nunn and philanthropist Ted Turner, hosted a monkeypox pandemic exercise that just so happened to be forecasted to take place in May 2022. What an interesting coincidence, right? If you are unfamiliar with NTI, according to the organization's homepage, it is a "nonprofit, nonpartisan global security organization focused on reducing nuclear and biological threats imperiling humanity." From the Wikipedia page, it states that NTI "serves as the Secretariat for the 'Nuclear Security Project', in cooperation with the Hoover Institution at Stanford. It is guided by former Secretary of State George P. Shultz, former Secretary of Defense William J. Perry, former Secretary of State Henry A. Kissinger and Nunn," who are collectively known as the "four horsemen of the nuclear apocalypse." Nothing suspicious about that.

If we look at the summary of the monkeypox pandemic exercise, we can gather some additional insight:

Strengthening Global Systems to Prevent and Respond to High-Consequence Biological Threats

"In March 2021, NTI partnered with the Munich Security Conference to conduct a tabletop exercise on reducing high-consequence biological threats. The exercise examined gaps in national and international biosecurity and pandemic preparedness architectures—exploring opportunities to improve prevention and response capabilities for high-consequence biological events. Participants included 19 senior leaders and experts from across Africa, the Americas, Asia, and Europe with decades of combined experience in public health, biotechnology industry, international security, and philanthropy."

Exercise Summary

"Developed in consultation with technical and policy experts, the fictional exercise scenario portrayed a deadly, global pandemic involving an unusual strain of monkeypox virus that first emerged in the fictional nation of Brinia and spread globally over 18 months. Ultimately, the exercise scenario revealed that the initial outbreak was caused by a terrorist attack using a pathogen engineered in a laboratory with inadequate biosafety and biosecurity provisions and weak oversight. By the end of the exercise, the fictional pandemic resulted in more than three billion cases and 270 million fatalities worldwide.

Discussions throughout the tabletop exercise generated a range of valuable insights and key findings. Most significantly, exercise participants agreed that, notwithstanding improvements following the global response to COVID-19, the international system of pandemic prevention, detection, analysis, warning, and response is woefully inadequate to address current and anticipated future challenges. Gaps in the international biosecurity and pandemic preparedness architecture are extensive and fundamental, undermining the ability of the international community to prevent and mount effective responses to future biological events—including those that could match the impacts of COVID-19 or cause damage that is significantly more severe."

Strengthening Global Systems to Prevent and Respond to High-Consequence Biological Threats
https://www.nti.org/wp-content/uploads/2021/11/NTI_Paper_BIO-TTX_Final.pdf

Upon opening the report of the event, we find some interesting representatives listed among the 19 senior leaders and experts who attended the drill including:

  1. Dr. Ruxandra Draghia-Akli
    • Global Head Johnson & Johnson Global Public Health R&D 
    • Janssen Research & Development
  2. Dr. Chris Elias President, Global
    • Development Division Bill & Melinda Gates Foundation
  3. Sir Jeremy Farrar
    • Director Wellcome Trust
  4. Dr. George Gao
    • Director-General, Chinese Center for Disease Control and Prevention (China CDC)
    • Vice President, the National Natural Science Foundation of China (NSFC)
    • Director and Professor, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences
      Dean, Medical School, University of Chinese Academy of Sciences
  5. Dr. John Nkengasong
    • Director Africa Centres for Disease Control and Prevention
  6. Dr. Michael Ryan
    • Executive Director WHO Health Emergencies Programme
  7. Dr. Petra Wicklandt
    • Head of Corporate Affairs Merck KGaA

We have representatives from pharmaceutical companies (J&J, Merck, GlaxoSmithKline), the Bill and Melinda Gates Foundation, the African and Chinese CDC, and the WHO. As is always seemingly the case, the very people who control the response and who profit off of these "pandemics" are the very ones executing the drills that "predict" the pandemics. Don't believe me? Let's take a trip down memory lane back to October 2019 with Event 201:

Event 201

"The Johns Hopkins Center for Health Security in partnership with the World Economic Forum and the Bill and Melinda Gates Foundation hosted Event 201, a high-level pandemic exercise on October 18, 2019, in New York, NY. The exercise illustrated areas where public/private partnerships will be necessary during the response to a severe pandemic in order to diminish large-scale economic and societal consequences."

The Event 201 scenario

"Event 201 simulates an outbreak of a novel zoonotic coronavirus transmitted from bats to pigs to people that eventually becomes efficiently transmissible from person to person, leading to a severe pandemic. The pathogen and the disease it causes are modeled largely on SARS, but it is more transmissible in the community setting by people with mild symptoms."

https://www.centerforhealthsecurity.org/event201/about

As can be seen, the Bill and Melinda Gates Foundation was once again heavily involved with this "pandemic" exercise which accurately predicted a "coronavirus" outbreak resulting in a pandemic 6 weeks before the "real pandemic" emerged. Among the participants in Event 201 that also participated in the NTI monkeypox drill were:

  1. Dr. Chris Elias President,
    • Global Development Division Bill & Melinda Gates Foundation
  2. Dr. George Gao
    • Director-General, Chinese Center for Disease Control and Prevention (China CDC)

We can also find representatives from Johnson & Johnson (again), the UN, and the CDC.

Why do we regularly see such drills and exercises taking place before the exact same events play out? Are these pandemic drills, which are sponsored by the same players who eerily predict the very "virus" within months, merely coincidences or something else entirely?

While the timing of these pandemic drills should raise red flags in regards to showing that this media push is a carefully controlled narrative, the threat of the "virus" behind the media stories is just as fictional as that presented in the drills. If you are here reading this article, you are either questioning the official narrative or you understand that whatever "virus" is chosen, they have never been scientifically proven to exist. This holds true for the monkeypox just as it does for any other "virus."

Previously, I wrote about how the monkeypox was just the rebranding and relabelling of smallpox. In 1958, the WHO decided to make a push to eradicate smallpox through the use of what is considered the deadliest vaccine known to man, even though smallpox had steadily declined throughout the proceeding decades. Coincidently (or not), the monkeypox "virus" was "discovered" in two colonies of test monkeys in a Danish lab that same year. In my previous article, I did not highlight the papers related to the isolation of this "virus" in monkeys and humans so it seems to be the perfect time to do so now. As the initial 1958 paper itself is rather long, I am only focusing on the methods used to "isolate" the monkeypox "virus" originally in monkeys. You will be able to see that, as always, it is just another unpurified tissue/cell cultured creation that was compared to other unpurified cultured concoctions with indirect immunological testimg. At no point were the particles assumed to be monkeypox ever purified and isolated directly from the fluids of the monkeys. Upon separation from the monkeys, the samples were immediately subjected to numerous chemical additives such as McIlvain's buffer saline and Bacto-tryptose "Difco" containing Penicillin and Streptomycin. The samples were then inoculated onto the chorio-allantoic membrane of 11-12 day old Leghorn embryos and into tissue cultures of monkey kidney, human amnion and HeLa cells:

A POX-LIKE DISEASE IN CYNOMOLGUS MONKEYS

"During the summer and fall of 1958 two outbreaks of a non-fatal pox-like disease in cynomolgus monkeys have been observed in the
monkey colony in this institute. Both outbreaks occurred rather late after the monkeys had been received, i.e. 51 and 62 days after arrival and only a small percentage of the exposed animals showed signs of illness.

This paper presents observations on the epidemiological and clinical 
manifestations of the disease. The isolation of a virus from the diseased animals will also he described as well as some studies on the properties of the agent which in this paper will be referred to as monkey pox virus. 

MATERIAL AND METHODS 

Virus isolation: Material for egg inoculation was collected from the pustular lesions and diluted approximately 1:10 in 0.004 hl. McIlvain's buffer saline pH 7.2. Material for tissue culture experiments was collected from the pustules by means of a cotton swab, which was immediately immersed into 2 ml of Bacto-tryptose "Difco". Both diluents contained Penicillin (100 mg per ml) and Streptomycin (0.1 mg per ml). After clarification by low speed centrifugation the supernatants were used for inoculation onto the chorio-allantoic membrane of 11-12 day old Leghorn embryos and into tissue cultures of monkey kidney, human amnion and HeLa cells. A total of three virus strains was isolated, one during the first and two during the second outbreak. Most of the studies to be reported were carried out using the strain which was isolated first. 

Vaccinia strain: Lot NO. 1/50 111 received from the smallpox vaccine department in this institute was used in some experiments in order to compare it with the monkey virus. This strain had heen maintained for several years by serial cutaneous passages in rabbits and calves. Either the glycerinated third calf passage or a subsequent first passage of this material on the chorio-allantois was used. 

Egg passages: The technique descrihed by Westwood et al. was employed. After inoculation the eggs were incubated for 48 hours at 37° C when vaccinia virus was used for infection and for 72 hours when the monkey virus was used. 

Neutralization tests on the chorio-allantoic membrane: Undiluted serum was mixed with equal volumes of increasing dilutions of virus. After incubation for 2-3 hours at room temperature 0.1 ml amounts of the serum-virus mixtures were inoculated onto the chorio-allantoic membranes of groups of five 11-12 day old embryos. The membranes were harvested after 48-72 hours continued incubation and the reduction effected in the number of typical pocks was determined.

Tissue cultures: The techniques employed for the preparation of tissue cultures for infectivity titrations and for neutralization tests have been described in earlier publications. An inoculum of 0.2 ml was used for tissue culture passages and the tubes were kept stationary at 35° C. During the first experiments they were examined daily for cytopathic changes but in later ones microscopic examination was carried out only every fourth or fifth day. Serial passages were usually made with 0.2 ml amounts of undiluted nutrient fluid harvested 5 days after inoculation.

Hemagglutination: The pattern method of Salk was employed. The technique has been described in detail in previous papers.

Complement-fixation tests were carried out according to the method of Fulton & Dumbell as modified by Svedmyr et al. Antigens were prepared from nutrient fluid of tissue cultures as well as from chorio-allantoic membranes infected with monkey-pox or vaccinia virus.

Rabbit immune sera: Hyperimmune sera against monkey pox and vaccinia viruses were prepared in rabbits by repeated intravenous injections of 0.5 to 2.0 ml amounts of 10 percent suspensions of infected chorio-allantoic membranes. The animals received a total of 7 intravenous injections (on day 1, 9, 11, 16, 18, 23 and 25). They were bled 10 days after the last injection.

Another hyperimmune serum prepared by immunizing rabbits with calf-lymph vaccinia antigen which had been inactivated by heating at 70° C for 60 minutes was also used. In addition, an hyperimmune vaccinia rabbit serum kindly supplied by
dr. F. 0. MacCallum, England was used in some experiments.

A human hyperimmune serum was finally used in some egg-neutralization tests.

Diffusion-precipitation test was performed according to the method described by Gispen.

Electron microscopy: A prototype of the Phillips electron microscope type EM 100 B was used. Exposures were made at a magnification of 3000 X and photographically enlarged as desired.

The pus-like material from the pustular lesions of a monkey was removed by means of a capillary pipette, placed on formvar coated grids and allowed to dry. Following the method of Van Royen & Scoff the grids were treated with 0.25 percent crystalline trypsin solution for 4 hours at 37° C after which they had 2 brief washings in distilled water. Fixation was carried out in the vapours of a 1 percent osmiumtetroxide solution for 15 minutes, and in order to minimize the risk of infection the grids were heated to 70″ C for one hour (1) prior to shadowcasting with platinum. The egg adapted virus grown on chorio-allantoic membranes was purified for electron microscopy by means of differential centrifugation and precipitation with citric acid according to the method of Henderson & McClean. The final suspension was placed directly on formvar coated grids, dried and further treated as stated above with the exception that no digestion with trypsin was carried out."

Virus Isolation

"Virus isolation was attempted only from one monkey during outbreak No. 1 and from two monkeys during outbreak No. 2. Virus was recovered from all three animals. The strain isolated first has been most extensively studied but there is no indication of any difference between this strain and those isolated during outbreak No. 2. 

Virus isolation in eggs: Scrapings from several papules which were diluted lO^-3, 10^-5, and 10^-7 produced greyish oedematous changes in the membranes. In addition small discrete lesions showing a tendency to spread along the blood vessels could be seen in the eggs inoculated with the highest dilution of virus (Fig. 4). 

On continued passage, using dilute membrane suspensions as seed, the oedematous reaction disappeared and the small opaque dome-shaped pocks became predominant ( Fig. 5). In membranes harvested after incubation for 3 days these pocks resembled closely those described for variola virus. The lesions developed later and were much smaller than those seen with vaccinia virus (Fig. 6). The titer of the original pustule material was 7 x 10^8 and no significant increase in titer was observed on continued passage in eggs. 

Virus isolation in tissue cultures: Pustular scrapings were emulsified in tryptose-broth, clarified by low speed centrifugation, and the supernatant was inoculated into tissue cultures of monkey kidney, human amnion, and HeLa cells. Cytopathic lesions developed in all cell types after continued incubation for 2-3 days and destruction was almost complete after 5 days.

The cytopathogenic effect was characterized by a rounding of the affected cells which subsequently became granulated and condensed.
The cells retained their rounded shape for several days, eventually slipping off the side of the tube leaving macroscopically visible holes in the cell sheet. In the monkey kidney and human amnion cell cultures the affected cells were interconnected by threadlike syncytial elongations (Fig. 7). In the HeLa cell cultures these formations were not observed but otherwise the cytopathogenic effect was similar to that observed in the other cell types used.

On continued tissue culture passage the cytopathogenic changes developed somewhat more slowly, suggesting that the passage fluid contained less virus than the pustular material. This assumption was supported by egg titrations. Tissue culture passage fluid contained only 10^6 to 10^7 pock forming units while the pustular material had a titer of about 10^8. On titration in tissue cultures the titer of passage fluid varied between 10^-4 and 10^-6."

https://doi.org/10.1111/j.1699-0463.1959.tb00328.x

"Partially purified."

It is rather interesting that this new "virus," which just so happens to share the exact same symptoms of smallpox, was conveniently discovered at the same time that the WHO declared that they would eradicate smallpox through a mass vaccination campaign.  It is also interesting to note that many of the symptoms associated with both smallpox and monkeypox can be side-effects from the very vaccine given to protect against the diseases. This could lead one to believe that the "discovery" of the monkeypox in monkeys and subsequently in humans a decade later was the perfect scapegoat to blame for any cases of the same symptoms of disease that occurred after the WHO officially declared smallpox eradicated.

In any case, the monkeypox itself was said not to be able to infect humans, only monkeys. That all changed in 1970 when a case of smallpox was identified in a 9-month-old unvaccinated child from the village of Bokenda in Basankusu Province, Democratic Republic of the Congo. The whole village (and presumably the mother and father of the child) had been mass vaccinated for smallpox the year before. No one was said to have become infected with the "virus" from the child. Upon clinical examination and "isolation" of the "virus," it was originally said to be smallpox (i.e. variola) that the child was suffering from. However, after further incubating the sample in an egg, which was subjected to toxic culturing conditions for a longer period of time, the membrane broke down some more and it was decided by the researchers that the pock formations noticed were now different than those observed in smallpox.

As in the previous paper from 1958, you will see that the "virus" is once again an unpurified mixture of host material (skin lesions) along with other materials such as chicken eggs, chick embryo fibroblast, monkey kidney cells, and four continuous cell lines (VERO, A-1, PEK, HEP-2). Sadly, the actual ingredients added beyond the cell type were left to methods utilized in previous unreferenced research but it was stated by the researchers that they used the "usual" isolation techniques which means that antibiotics/antifungals, fetal bovine serum, and other additives were most likely applied in the creation of this "virus."

The remainder of the paper involves the indirect comparison of the size and shape of pock formations in egg embryos and antibody testing between the cultured concoctions. Differences in pock formation/morphology could easily be explained due to differences in the culture ingredients/conditions and not because of any imaginary "virus." In fact, a change in the length of incubation periods in the eggs between smallpox (vaccinia as a stand-in) at 48 hours and monkeypox "virus" at 72 hours was noted in both the 1958 and 1972 papers. It can be seen that with 48 hours of incubation, the pocks resembled smallpox. At 72 hours, the pock formations did not match smallpox and resembled monkeypox. It was not explained why it was decided that it was necessary to incubate the monkeypox samples an extra 24 hours in either study. Thus, it can be seen that the researchers are creating the effect that they want to see by manipulating the time of incubation.

1958:

"After inoculation the eggs were incubated for 48 hours at 37° C when vaccinia virus was used for infection and for 72 hours when the monkey virus was used."

1972:

"After incubation for 48 hours, as already stated the lesions on chick-embryo CAM differed little from those caused by ordinary smallpox virus. However, after 72 hours they had already become flatter and haemorrhages appeared in the centre of most of the pocks."

This discrepancy was even pointed out in Chapter 29 of Smallpox and its Eradication by Frank Fenner where it is revealed that the WHO experts agreed in 1969 that the appearance of the pocks after 3 days of incubation was the first indication of monkeypox over smallpox:

"At the first meeting of the WHO Informal Group on Monkeypox and Related Viruses, in Moscow in March 1969, the experts agreed that the first indication that virus recovered from a skin lesion might be monkeypox virus would be the haemorrhagic appearance of the pocks produced on the chorioallantoic membrane after 3 days' incubation at 35 ° C.

On 23 September 1970 Dr S. S. Marennikova, Dr E. M. Shelukhina and Dr N. N. Maltseva, of the WHO collaborating centre in Moscow, recovered a virus on the chorioallantoic membrane from material sent from a patient in Zaire. When examined after incubation for 2 days, the pocks were "perfectly typical" of variola virus. However, after another day's incubation at 35 ° C, there was some haemorrhage around the pocks, a feature never seen with variola virus and characteristic of monkeypox virus."

https://www.google.com/url?sa=t&source=web&rct=j&url=https://biotech.law.lsu.edu/blaw/bt/smallpox/who/red-book/Chp%252029.pdf&ved=2ahUKEwiJi_G355X1AhWKZM0KHd9IAA8QFnoECDEQAQ&usg=AOvVaw0o1ZKfIZFos-R50YesS-Wp

As for the antibody results, since antibodies have never been purified  and isolated nor scientifically proven to exist, it should be obvious that using one fictional entity (antibodies) to identify another fictional entity ("virus") is not proof that a "virus" exists nor that it is either the same or different from other fictional "viruses" also assumed to exist. Sadly, this simple bit of logic never seems to enter into the minds of these researchers for some reason.

The entirety of the 1972 paper identifying the first case of human monkeypox infection and the "isolation" of the "virus" is presented below:

Isolation and properties of the causal agent of a new variola-like disease (monkeypox) in man

The causal agent of a case of disease in man occurring in the Democratic Republic of the Congo with a similar clinical picture to smallpox was isolated and studied. The agent was identified as monkeypox virus. A comparative study of the isolated strain (Congo-8) and of viruses isolated from similar cases of illness in Liberia (Liberia-i and Liberia-2 strains) and Sierra Leone (V-70 1 266 strain) showed that they were identical. A number of local species of monkeys and apes were examined serologically in the Congo region to determine the probability of human infection with monkeypox virus. It was confirmed that the animals had had contact with an agent of the poxvirus group. In 2 of the 7 sera examined, antibodies of the variola-vaccinia group of poxviruses were discovered (virus-neutralizing
antibodies, precipitins, and antihaemagglutinins). In a chimpanzee, antihaemagglutinins
were found in a titre of 1:1,280, and in the same animal a variola-like virus was isolated from the kidneys. In the course of the investigation, it was shown conclusively that monkeypox virus and the strains under investigation could be distinguished from ordinary variola and vaccinia viruses on the basis of their behaviour in pig embryo kidney continuous cell line culture.

Until fairly recently it was believed that only two poxviruses other than variola and alastrim viruses could cause generalized infection in man accompanied by skin lesions. These were the true cowpox agent and the vaccinia virus. As a rule, both these
viruses cause lesions at the point of infection, and the infection becomes generalized only in occasional cases. In 1958 a new representative of the poxvirus group, the monkeypox virus, was discovered (Magnus et al., 1959). Since then a number of outbreaks of monkeypox among trapped animals kept in nature reserves and zoological gardens have been recorded and studied (Prier et al., 1960; McConnell et al., 1962; Peters, 1966; Gispen, Verlinde & Zwart, 1967).

It has been established that the infection caused by the virus can be transmitted to some other animals (anteaters) and to various species of monkey and ape, including the anthropoid apes. The severity of the disease in monkeys varies and depends to a considerable extent on the species. The disease follows its most severe course in anthropoid apes. Nevertheless, no illness in man caused by monkey-pox virus has been noted in any of these outbreaks. The data quoted in this paper, however, indicate that monkeypox virus can cause a disease similar to ordinary smallpox in man. The virus was isolated from material obtained from a 9-month-old unvaccinated child (A. I.) from the village of Bokenda in Basankusu Province, Democratic Republic of the Congo, who was suffering from a disease suspected to be smallpox. The village concerned is situated in a remote locality in the depths of a tropical rain forest. No cases of smallpox had occurred during the 2 previous years either in the village or in the surrounding area. No cases of infection with the variola-like disease spreading from the child A. I. were recorded (Ladnyj, Ziegler & Kima, 1971). Practically the whole population of Bokenda village had been vaccinated against smallpox a year before this case occurred during a mass smallpox vaccination campaign throughout the country.

In addition to reporting the results of the isolation and identification of the Congo-8 strain of virus from the child A. I. this paper gives the results of a serological examination of a group of monkeys and apes from the focus, and also the results of a study of Liberia-i, Liberia-2, and V-70 1 266 viruses isolated in the Center for Disease Control, Atlanta, Ga., USA, from patients suffering from similar variola-like illnesses in Liberia and Sierra Leone.

MATERIAL AND METHODS

Material from the patients in the Democratic Republic of the Congo was obtained through the World Health Organization by air in a special packing, and consisted of the contents of skin lesions (scabs and smears on slides). The Liberia-I, Liberia-2, and V-70 1 266 strains were obtained through WHO on 11 December 1970, 18 December 1970, and 30 April 1971, respectively, in the form of suspensions of chorioallantoic membrane (CAM), infected CAM, and scabs from a patient (Liberia-1). These strains had been passaged once or twice in the laboratory and were used in the experiments in the form of first-or second-passage suspensions of CAM (disregarding passages undergone before they were received in the laboratory).

Strains Congo-8, Liberia-1, Liberia-2, and V-70 1 266 were compared with viruses of variola (strains MT-60 and MK-60-Harvey), cowpox (Brighton strain), monkeypox (Copenhagen strain), and vaccinia (strains Tanzania-3 and France). The viruses were used in the form of a suspension of infected CAM after one or two further passages in chick embryos.

The sera and organs of monkeys caught in the area of Bokenda village were received in the laboratory in a frozen state in a container of liquid nitrogen.

The usual methods were used to isolate the virus from materials taken from patients and from the organs of monkeys and to investigate them by means of the agar gel microprecipitation test (World Health Organization, 1969).

In studying the genetic features of the viruses (the type of pock on CAM and the plaques in cell cultures, haemagglutinating activity, pathogenicity for rabbits following infection by intradermal inoculation or scarification and other properties), standard methods were used (Marennikova & gafikova, 1969; Marennikova, Gurvic & geluhina, 1970). The animals used in the experiments were white chinchilla rabbits weighing 2.5 kg, and randomly bred white mice weighing 10-11 g.

The type of cytopathic effect was determined in the following cell cultures: two primary (chick embryo fibroblast and monkey kidney cells) and four continuous (VERO, A-1, PEK, HEP-2). The morphology of the plaques was studied both with and without agar overlay (Porterfield & Allison, 1960; Gendon & ternos, 1964).

In the comparative studies equal doses of the viruses were used. The doses given to rabbits intradermally and by scarification were 10^6 pock-forming units per 0.1 ml. The doses used to study pathogenicity in chick embryos ranged from 10 to 10^7 pock-forming units per 0.1 ml, the results being read after 72 hours. In studying the ceiling temperature for the development of specific lesions, 100 and 1,000 pock-forming units were used. Except in special tests, the morphology of pocks was determined on CAM after
72 hours, when there were 10-100 pocks on the membrane. The type of cytopathic effect was studied by infection with doses of virus ranging from 10 to 10^7 TCD50, the results being read from 24 hours to 7 days after infection.

Before examination the monkey sera were thawed and heated at 56°C for 30 minutes. The haemagglutination-inhibition test was carried out with 2 agglutinating units (AU) of the test virus and a 1% suspension of chick red cells. The neutralization test was carried out by the usual method (Boulter, 1957),
while the Ouchterlony method (1949) was used in the agar gel precipitation test.

RESULTS

Research on the laboratory diagnosis of smallpox carried out in this institute in 1970, with material taken from patients during the period 18 December 1969 to 22 October 1970, enabled us to confirm the presence of smallpox in a number of provinces of the Democratic Republic of the Congo (Dendale Kinshasa, Kalomy, Longo Ngaba Kinshasa, and Kabulo Kalukivu). The strains of variola virus isolated did not differ from the typical virus of natural smallpox.

During the same period a virus was isolated from material taken on 1 September 1970 in the village of Bokenda in the province of Basankusu from the child A. I. as already stated. On the basis of an examination of infected CAM after 48 hours and serological identification by means of the agar gel microprecipitation test, this virus was initially identified as variola virus (23 September 1970). However, the character of the pocks on CAM had changed sharply after 72 hours and this observation, together with results of haemagglutinating activity tests, suggested that the agent isolated was of a different nature. A detailed study was then undertaken and the virus was also re-isolated from material taken from the patient.

Table I shows the results of isolating the virus from material taken from the patient and testing the basic properties of the strain. Investigations showed that the isolated virus (strain Congo-8) produced a peculiar type of pock on CAM, distinguishing the strain from both vaccinia virus and ordinary variola virus. This virus also showed a high degree of haemagglutinating activity and a marked tendency to cause lesions on scarified rabbit skin. On the third to fourth day after infection of the rabbit, the scarified area stood out above the healthy skin surrounding it as a result of the development of confluent papular eruptions without any marked induration of the underlying tissues. The confluent elements took the form of moist ridges along the lines of scarification (see Fig. 1).

When the rabbits were infected intradermally, necrosis developed in the centre of the dense infiltrated area that formed at the site of inoculation. The area of infiltration itself was slightly haemorrhagic. In rabbits infected by scarification and intradermally, the infection assumed a generalized character.

After incubation for 48 hours, as already stated the lesions on chick-embryo CAM differed little from those caused by ordinary smallpox virus. However, after 72 hours they had already become flatter and haemorrhages appeared in the centre of most of the pocks (Fig. 2). By that time the non-uniformity of the pocks in respect of morphology and size had become obvious; side by side with small pocks containing haemorrhages, a small number of large white pocks without haemorrhages could be observed. This feature was seen at an incubation temperature of 35°C. An increase in the incubation temperature by as little as 1 deg C led to changes in the nature of the pocks. Pocks that developed on CAM showed no haemorrhage.

The unusual combination of properties and the close similarity of a number of the features of the Congo-8 strain to those of other viruses in the pox group made it necessary to carry out a detailed comparison of the virus in experiments with cowpox, monkeypox, variola, and vaccinia viruses. The strains of Liberia-1, Liberia-2, and V-70 1 266 from Liberia and Sierra Leone, which had been isolated from cases of variola-like illness in man, similar to that from which A. I. was suffering, were included in the same tests. The results of the comparative study showed that Congo-8, Liberia-I, Liberia-2, and V-70 1 266 did not differ substantially one from another according to the tests used, nor did they differ from the classical type of monkeypox virus of the Copenhagen strain (Tables 2 and 3).

Not only was these a close affinity between these four strains according to the tests already described (type of pock on CAM, skin lesions in rabbits, and haemagglutinating activity) but they all produced plaques of identical morphology and size, whether agar overlay was used (in chick embryo fibroblast cells) or not (Vero continuous cell line). Unlike ordinary variola virus, which under agar overlay produces small plaques less than 1 mm in diameter, monkeypox virus and the Congo-8, Liberia-1, and Liberia-2 isolates formed larger plaques with a visible internal structure and an uneven margin. In experiments without agar overlay, variola virus gave scarcely visible plaques with an intensely stained rim, whereas the isolates formed large plaques with a transparent centre and two peripheral zones: an inner reticular zone with an uneven edge and an outer intensely stained zone (Fig. 3). The differences in the type of cytopathic effect between these viruses and the variola virus were less marked than the differences in plaque formation. The isolates produced a cytopathic effect of focal type in the initial stages with breaks in the cell layer and a zone of cell proliferation on the periphery. The cells bounding the focus were rounded and highly refractive. In the centre of the focus were cells or groups of cells that had lost the capacity for process formation. At higher doses of the virus (over 10^5 TCD5O) there was a "diffuse" cytopathic effect throughout the cell layer (Fig. 4).

The study of a number of cell cultures infected with Congo-8, Liberia-2, and Copenhagen viruses showed that the most sensitive were VERO cells (titres of 107-5 and 106.2 TCD5O) compared with chick embryo fibroblast cells (titres of 105-5-106 5 TCD,O) and the A-1 cells (titres of 105.5-106 5 TCDS,O). These viruses showed marked haemadsorption phenomena in chick embryo fibroblasts, monkey kidney cells, Vero cells, A-1 cells, and HEP-2 cells.

It has been shown previously (Marennikova, Gurvic & Seluhina, 1970) that a feature of monkeypox virus that distinguishes it from variola and vaccinia viruses is its incapacity for active replication and the absence of the haemadsorption phenomenon in a culture of the continuous pig embryo kidney (PEK) cell line. Study of the behaviour of Congo-8, Liberia-1, and Liberia-2 strains showed that they behave similarly: their titre in PEK cells did not exceed 10^2-6 TCD50/ml and the haemadsorption phenomenon did not occur even when cytopathic changes had taken place (Table 2).

Determination of the ceiling temperature for the development of pocks on chick embryo CAM showed, that, unlike vaccinia and cowpox viruses, strains Congo-8, Liberia-1, and Liberia-2, as well as monkeypox virus, did not cause lesions at a temperature of 39.6°C following the administration of 100 and 1,000 pock-forming units. However, this group of strains and the monkeypox virus caused the development of pocks on CAM at a temperature at which no lesions developed following infection with the MT-60 strain of variola virus (38.6.39°C). The test strains differed from variola virus and cowpox virus in their higher haemagglutinating activity (see Table 3). The Congo-8 strain and the three other isolates tested proved highly pathogenic for chick embryos. Following intracerebral infection of white mice, these strains, like monkeypox virus, were considerably more pathogenic than variola virus for adult mice (Table 3).

In the serological studies use was made of hyper-immune rabbit sera against vaccinia and variola viruses. In the haemagglutination-inhibition test with both antisera, the test viruses produced titres identical with those produced by monkeypox virus (Table 4). In the agar gel precipitation test, all the viruses except cowpox virus were found to have an identical antigenic structure (Fig. 5).

In view of the fact that the experimental material obtained enabled the Congo-8 virus to be identified as monkeypox virus, it seemed useful to investigate monkeys and apes living in the area of the infective focus. In January 1971 Dr I. D. Ladnyj and Dr P. Ziegler trapped a group of monkeys of different species whose blood, serum, and organs (liver, spleen, and kidneys) were studied. The results of serological examinations of the animals are given in Table 5. Of 7 sera tested, 2 showed antihaemagglutinins in titres of 1:16 and 1:1,280. In these same sera (Nos 4 and 9), virus-neutralizing antibodies, in a titre of more than 1:40, and precipitins were discovered. The detection of antibodies to vaccinia virus shows that an agent of the poxvirus group is circulating among certain local species of monkey. Furthermore, the presence of precipitins and the high level of antihaemagglutinins found in a chimpanzee suggested that it had been infected. This view is supported by the isolation from the kidneys of the same chimpanzee of a variola-like virus. The results of this study are given by Marennikova et al. (1971).

DISCUSSION

Experience gained in recent years in studying variola virus has shown that it is distinguished, like a number of other viruses, although to a lesser degree, by intraspecific variability or natural variation (Bedson, Dunbell & Thomas, 1963; Sarkar & Mitra, 1967; Marennikova & gafikova, 1969). The differences found were related to the degree of pathogenicity of the strains for chick embryos and white mice, the ceiling temperature for pock development, and some other indices. However, not one of  the strains of variola and alastrim viruses investigated proved capable of causing the development of lesions on scarified skin in rabbits. The Congo-8 strain isolated from the patient A. I. with an illness resembling smallpox caused a marked specific reaction when it was placed on scarified rabbit skin. This feature of the Congo-8 virus, combined with other characteristics (the haemorrhagic type of pocks on CAM, types of plaque in tissue culture, high degree of haemagglutinating activity, ceiling temperature for the development of lesions, etc.) makes it impossible for it to be considered as a variant of variola virus. On the other hand, the properties of Congo-8 proved to be indistinguishable from those of the classic representative of monkeypox virus (the Copenhagen strain), and provided good grounds for identifying it as a monkeypox virus itself. The evidence thus indicates that the monkeypox virus can cause disease in man.

The fact that examinations of numerous samples over a number of years of material from patients from different countries with suspected smallpox have not previously revealed an agent identical with, or close to, the Congo-8 virus in its properties, and that no such cases have been reported by other authors, may indicate that this infection has a low degree of contagiousness and a comparatively limited area of distribution. This view is supported particularly by the results of examinations of other material taken from patients in the Democratic Republic of the Congo. Except for Congo-8, the virus isolated in each case did not differ from the variola virus. However, the results of examining Liberia-1 and Liberia-2 strains, which according to the results we obtained did not differ substantially in their properties from the Congo-8 strain, suggest that monkeypox in man is not strictly limited in distribution since the Democratic Republic of the Congo and Liberia have no common frontier and are fairly far apart. It may be assumed that cases of monkeypox in man are caused by the presence of a reservoir of the virus in certain species of monkeys and apes in the areas concerned. This view is supported by the results of serological examinations of animals trapped in the focus of infection. The low degree of contagiousness of monkeypox to man or the complete absence of infectivity for other persons in the patient's household may be the reason why the disease was not discovered earlier. There is no doubt that the discovery of this infection was aided by the establishment of an epidemiological surveillance system sufficiently sensitive to discover single cases of illness.

https://apps.who.int/iris/bitstream/handle/10665/263482/PMC2480798.pdf?sequence=1&isAllowed=y

In Summary:
  • In March 2021, NTI partnered with the Munich Security Conference to conduct a tabletop exercise on reducing high-consequence biological threats
  • Participants included 19 senior leaders and experts from across Africa, the Americas, Asia, and Europe with decades of combined experience in public health, biotechnology industry, international security, and philanthropy
  • Developed in consultation with technical and policy experts, the fictional exercise scenario portrayed a deadly, global pandemic involving an unusual strain of monkeypox "virus" that first emerged in the fictional nation of Brinia and spread globally over 18 months
  • Ultimately, the exercise scenario revealed that the initial outbreak was caused by a terrorist attack using a pathogen engineered in a laboratory with inadequate biosafety and biosecurity provisions and weak oversight
  • By the end of the exercise, the fictional pandemic resulted in more than three billion cases and 270 million fatalities worldwide
  • Most significantly, exercise participants agreed that, notwithstanding improvements following the global response to "COVID-19," the international system of pandemic prevention, detection, analysis, warning, and response is woefully inadequate to address current and anticipated future challenges
  • The Johns Hopkins Center for Health Security in partnership with the World Economic Forum and the Bill and Melinda Gates Foundation hosted Event 201, a high-level pandemic exercise on October 18, 2019, in New York, NY
  • Event 201 simulates an outbreak of a novel zoonotic "coronavirus" transmitted from bats to pigs to people that eventually becomes efficiently transmissible from person to person, leading to a severe pandemic
  • The pathogen and the disease it causes are modeled largely on "SARS," but it is more transmissible in the community setting by people with mild symptoms
  • Two outbreaks of smallpox-like disease occurred in lab monkeys in 1958 rather late after the monkeys had been received, i.e. 51 and 62 days after arrival and only a small percentage of the exposed animals showed signs of illness
  • Material for egg inoculation was collected from the pustular lesions and diluted approximately 1:10 in 0.004 hl. McIlvain's buffer saline pH 7.2
  • Material for tissue culture experiments was collected from the pustules by means of a cotton swab, which was immediately immersed into 2 ml of Bacto-tryptose "Difco"
  • Bacto™ Tryptone is a pancreatic digest of casein. It was developed by Difco Laboratories while investigating a peptone particularly suitable for the elaboration of indole by bacteria. It is also notable for the absence of detectable levels of carbohydrates. https://www.americanpharmaceuticalreview.com/25246-Cell-Culture-Media-Supplements/5821993-Bacto-Tryptone/
  • Both diluents contained Penicillin (100 mg per ml) and Streptomycin (0.1 mg per ml)
  • After clarification by low speed centrifugation the supernatants were used for inoculation onto the chorio-allantoic membrane of 11-12 day old Leghorn embryos and into tissue cultures of monkey kidney, human amnion and HeLa cells
  • Vaccinia strain (i.e. not variola) lot NO. 1/50 111 received from the smallpox vaccine department in this institute was used in some experiments in order to compare it with the monkey "virus"
  • This strain had heen maintained for several years by serial cutaneous passages in rabbits and calves
  • Either the glycerinated third calf passage or a subsequent first passage of this material on the chorio-allantois was used
  • After inoculation the eggs were incubated for 48 hours at 37° C when vaccinia "virus" was used for infection and for 72 hours when the monkey "virus" was used (i.e. the samples being compared were not treated the same)
  • For neutralization tests, undiluted serum was mixed with equal volumes of increasing dilutions of "virus"
  • After incubation for 2-3 hours at room temperature 0.1 ml amounts of the serum-"virus" mixtures were inoculated onto the chorio-allantoic membranes of groups of five 11-12 day old embryos
  • Tissue culture methods were not described in this paper yet it was stated that during the first experiments they were examined daily for cytopathic changes but in later ones microscopic examination was carried out only every fourth or fifth day
  • Serial passages were usually made with 0.2 ml amounts of undiluted nutrient fluid harvested 5 days after inoculation
  • For complement fixation tests, antigens were prepared from nutrient fluid of tissue cultures as well as from chorio-allantoic membranes infected with monkey-pox or vaccinia "virus"
  • Hyperimmune sera against monkey pox and vaccinia "viruses" were prepared in rabbits by repeated intravenous injections of 0.5 to 2.0 ml amounts of 10 percent suspensions of infected chorio-allantoic membranes
  • The animals received a total of 7 intravenous injections (on day 1, 9, 11, 16, 18, 23 and 25)
  • They were bled 10 days after the last injection
  • Another hyperimmune serum prepared by immunizing rabbits with calf-lymph vaccinia antigen which had been inactivated by heating at 70° C for 60 minutes was also used
  • In addition, an hyperimmune vaccinia rabbit serum kindly supplied by dr. F. 0. MacCallum, England was used in some experiments
  • In other words, the sera used for antibody experiments came from many sources with different preparation methods
  • Electron microscopy followed the method of Van Royen & Scoff:
    1. The grids were treated with 0.25 percent crystalline trypsin solution for 4 hours at 37° C after which they had 2 brief washings in distilled water
    2. Fixation was carried out in the vapours of a 1 percent osmiumtetroxide solution for 15 minutes
    3. In order to minimize the risk of infection the grids were heated to 70° C for one hour (1) prior to shadowcasting with platinum
    4. The egg adapted "virus" grown on chorio-allantoic membranes was "purified" for electron microscopy by means of differential centrifugation and precipitation with citric acid according to the method of Henderson & McClean
    5. The final suspension was placed directly on formvar coated grids, dried and further treated as stated above with the exception that no digestion with trypsin was carried out
  • In other words, the "purified virus" from egg membranes was heavily treated and coated with platinum before imaging
  • "Virus" isolation was attempted only from one monkey during outbreak  No. 1 and from two monkeys during outbreak No. 2
  • Most of the studies were done from the "isolate" of the first monkey
  • For "virus isolation" in eggs, scrapings from several papules which were diluted lO^-3, 10^-5, and 10^-7 produced greyish oedematous changes in the  membranes
  • In addition small discrete lesions showing a tendency to spread along the blood vessels could be seen in the eggs inoculated with the highest dilution of "virus"
  • On continued passage, using dilute membrane suspensions as seed, the oedernatous reaction disappeared and the small opaque dome-shaped pocks became predominant
  • In membranes harvested after incubation for 3 days these pocks resembled closely those described for variola "virus"
  • For "virus isolation" in tissue cultures, pustular scrapings were emulsified in tryptose-broth, clarified by low speed centrifugation, and the supernatant was inoculated into tissue cultures of monkey kidney, human amnion, and HeLa cells.
  • Cytopathic lesions developed in all cell types after continued incubation for 2-3 days and destruction was almost complete after 5 days
  • In other words, the longer they incubated their toxic cell soup, the more the cells died
  • In the HeLa cell cultures, formations were not observed as in the other cell lines but otherwise the cytopathogenic effect was similar to that observed in the other cell types used
  • On continued tissue culture passage the cytopathogenic changes developed somewhat more slowly, suggesting that the passage fluid contained less "virus" than the pustular material
  • This assumption was supported by egg titrations
  • The causal agent of a case of disease in man occurring in the Democratic Republic of the Congo with a similar clinical picture to smallpox was said to be isolated and studied
  • The agent was identified as monkeypox "virus"
  • A number of local species of monkeys and apes were examined serologically in the Congo region to determine the probability of human infection with monkeypox "virus"
  • In the course of the investigation, it was shown "conclusively" that monkeypox "virus" and the strains under investigation could be distinguished from ordinary variola and vaccinia "viruses" on the basis of their behaviour in pig embryo kidney continuous cell line culture
  • In other words, they looked at the INDIRECT chemical reactions and determined different "viruses" based on this rather than DIRECT evidence of purified/isolated particles directly from the fluids
  • It was believed that only two "poxviruses" other than variola and alastrim "viruses" could cause generalized infection in man accompanied by skin lesions: the cowpox and vaccinia "virus"
  • In 1958 a new representative of the "poxvirus" group, the monkeypox "virus," was discovered
  • Since then a number of outbreaks of monkeypox among trapped animals kept in nature reserves and zoological gardens have been recorded and studied (no outbreaks in animals in the wild?)
  • The severity of the disease in monkeys varies and depends to a considerable extent on the species
  • The disease follows its most severe course in anthropoid apes
  • No illness in man caused by monkeypox "virus" had been noted in any of these outbreaks
  • The data quoted in this paper, however, was said to indicate that monkeypox "virus" can cause a disease similar to ordinary smallpox in man
  • The "virus" was "isolated" from material obtained from a 9-month-old unvaccinated child (A. I.) from the village of Bokenda in
    Basankusu Province, Democratic Republic of the Congo, who was suffering from a disease suspected to be smallpox
  • No cases of infection with the variola-like disease spreading from the child A. I. were recorded
  • Practically the whole population of Bokenda village had been vaccinated against smallpox a year before this case occurred during a mass smallpox vaccination campaign throughout the country
  • Material from the patients in the Democratic Republic of the Congo was obtained through the World Health Organization by air in a special packing (no mention of what this consisted of), and consisted of the contents of skin lesions (scabs and smears on slides)
  • The Liberia-I, Liberia-2, and V-70 1 266 strains were obtained through WHO on 11 December 1970, 18 December 1970, and 30 April 1971, respectively, in the form of suspensions of chorioallantoic membrane (CAM) (highly vascularized membrane found in the eggs of certain amniotes like birds and reptiles), infected CAM, and scabs from a patient (Liberia-1)
  • These strains had been passaged once or twice in the laboratory and were used in the experiments in the form of first-or second-passage suspensions of CAM (disregarding passages undergone before they were received in the laboratory)
  • Strains Congo-8, Liberia-1, Liberia-2, and V-70 1 266 were compared with "viruses" of variola (strains MT-60 and MK-60-Harvey), cowpox (Brighton strain), monkeypox (Copenhagen strain), and vaccinia (strains Tanzania-3 and France)
  • The "viruses" were used in the form of a suspension of infected CAM after one or two further passages in chick embryos
  • The sera and organs of monkeys caught in the area of Bokenda village were received in the laboratory in a frozen state in a container of liquid nitrogen
  • The usual methods (i.e. tissue and cell culture) were used to "isolate" the "virus" from materials taken from patients and from the organs of monkeys and to investigate them by means of the agar gel microprecipitation test
  • Standard methods used for studying the genetic features of the "viruses" included:
    1. The type of pock on CAM and the plaques in cell cultures
    2. Haemagglutinating activity
    3. Pathogenicity for rabbits following infection by intradermal inoculation or scarification and other properties
  • The type of cytopathic effect was determined in the following cell cultures:
    1. Two primary (chick embryo fibroblast and monkey kidney cells)
    2. Four continuous (VERO, A-1, PEK, HEP-2)
  • The doses given to rabbits intradermally and by scarification were 10^6 pock-forming units per 0.1 ml.
  • The doses used to study pathogenicity in chick embryos ranged from 10 to 10^7 pock-forming units per 0.1 ml, the results being read after 72 hours
  • The type of cytopathic effect was studied by infection with doses of "virus" ranging from 10 to 10^7 TCD50, the results being read from 24 hours to 7 days after infection
  • In other words, the doses used and the timing to determine results varied
  • Before examination the monkey sera were thawed and heated at 56°C for 30 minutes
  • On the basis of an examination of infected CAM after 48 hours and serological identification by means of the agar gel microprecipitation test, this "virus" was initially identified as variola "virus" (23 September 1970)
  • However, the character of the pocks on CAM had changed sharply after 72 hours and this observation, together with results of haemagglutinating activity tests, suggested that the agent isolated was of a different nature
  • In other words, the initial examination and serological investigation told them that the boy had smallpox but after 72 hours of further culturing, the pocks in the chorioallantoic membrane changed and they decided it was no longer smallpox
  • Investigations showed that the isolated "virus" (strain Congo-8) produced a peculiar type of pock on CAM, distinguishing the strain from both vaccinia "virus" and ordinary variola "virus" (i.e. they could not see the "virus" particles but assumed a difference in the type of pock in CAM meant it was different)
  • This "virus" also showed a high degree of haemagglutinating activity and a marked tendency to cause lesions on scarified rabbit skin (how shocking that lesions occurred where rabbits were scraped and cut)
  • When the rabbits were infected intradermally, necrosis (the death of most or all of the cells in an organ or tissue due to disease, injury, or failure of the blood supply) developed in the centre of the dense infiltrated area that formed at the site of inoculation
  • In rabbits infected by scarification and intradermally, the infection assumed a generalized character
  • After incubation for 48 hours, as already stated the lesions on chick-embryo CAM differed little from those caused by ordinary smallpox "virus"
  • However, after 72 hours they had already become flatter and haemorrhages appeared in the centre of most of the pocks
  • The unusual combination of properties and the close similarity of a number of the features of the Congo-8 strain to those of other "viruses" in the pox group made it necessary to carry out a detailed comparison of the "virus" in experiments with cowpox, monkeypox, variola, and vaccinia "viruses"
  • The results of the comparative study showed that Congo-8, Liberia-I, Liberia-2, and V-70 1 266 did not differ substantially one from another according to the tests used, nor did they differ from the classical type of monkeypox "virus" of the Copenhagen strain
  • The 4 strains all produced plaques of identical morphology and size, whether agar overlay was used (in chick embryo fibroblast cells) or not (Vero continuous cell line)
  • Unlike ordinary variola "virus," which under agar overlay produces small plaques less than 1 mm in diameter, monkeypox "virus" and the Congo-8, Liberia-1, and Liberia-2 isolates formed larger plaques with a visible internal structure and an uneven margin
  • In other words, they determined similarity of the invisible "viruses" by plaques produced and not by showing purified/isolated particles in EM
  • The differences in the type of cytopathic effect between these "viruses" and the variola "virus" were less marked than the differences in plaque formation (i.e. the CPE was not distinct)
  • The study of a number of cell cultures infected with Congo-8, Liberia-2, and Copenhagen viruses showed that the most sensitive were VERO cells (titres of 10^7-5 and 10^6.2 TCD5O) compared with chick embryo fibroblast cells (titres of 10^5-5-10^6 5 TCD5O) and the A-1 cells (titres of 10^5.5-10^6 5 TCD5O)
  • These "viruses" showed marked haemadsorption phenomena in chick embryo fibroblasts, monkey kidney cells,Vero cells, A-1 cells, and HEP-2 cells
  • It had been shown previously (Marennikova, Gurvic & Seluhina, 1970) that a feature of monkeypox "virus" that distinguishes it from variola and vaccinia "viruses" is its incapacity for active replication and the absence of the haemadsorption phenomenon in a culture of the continuous pig embryo kidney (PEK) cell line (more indirect non-specific chemical reactions)
  • As the behaviour of Congo-8, Liberia-1, and Liberia-2 strains showed that they behave similarly, they were determined to be monkeypox rather than smallpox
  • Following intracerebral infection of white mice, these strains, like monkeypox "virus," were considerably more pathogenic than variola "virus" for adult mice
  • Upon investigating monkeys and apes, the detection of antibodies to vaccinia "virus" showed that an agent of the "poxvirus" group was circulating among certain local species of monkey
  • The presence of precipitins and the high level of antihaemagglutinins found in a chimpanzee suggested that it had been infected
  • This view was supported by the "isolation" from the kidneys of the same chimpanzee of a variola-like "virus"
  • The lesions upon scarification of rabbits from the Congo-8 "virus," combined with other (indirect) characteristics (the haemorrhagic type of pocks on CAM, types of plaque in tissue culture, high degree of haemagglutinating activity, ceiling temperature for the development of lesions, etc.) made it impossible for it to be considered as a variant of variola "virus" (even though they originally claimed that it was)
  • The evidence indicated that the monkeypox "virus" can cause disease in man (even though this was never shown)
  • It may be assumed that cases of monkeypox in man are caused by the presence of a reservoir of the "virus" in certain species of monkeys and apes in the areas concerned
  • This view is supported by the results of serological examinations of animals trapped in the focus of infection
  • The low degree of contagiousness of monkeypox to man or the complete absence of infectivity for other persons in the patient's household may be the reason why the disease was not discovered earlier

Should everybody be concerned about monkeypox as Joe Biden has warned? If you understand that the original evidence for the monkeypox "virus" in both animal and man lacks the physical evidence of purified and isolated particles directly from the host fluids which were then proven pathogenic in a natural way, you will be able to easily brush aside the latest attempt at fear propaganda. Taking samples from monkeys and subjecting them to tissue, egg, and cell culturing with the addition of various other chemicals and looking for patterns formed after incubation does not prove a "virus" is present. Taking this toxic soup and injecting it in numerous ways and/or scarifying it onto the skin of animals and seeing lesions form is not a recreation of the same disease. It is an unnatural experimental reaction to various forms of torture and abuse the animals are subjected to and does not prove a "virus" as the cause. Performing various tests mixing the blood of animals and humans and claiming the results mean that there is a specific antibody reacting to a specific "virus" when neither have been properly purified and isolated does not prove a "virus" either. All of these examples are of the indirect evidence used to state the existence of an invisible "virus" which are the result of unnatural lab-created chemical reactions and concoctions. They are performed and evaluated without physically having direct evidence of the assumed "virus" particles in question in order to infer its presence due to the resulting reactions. These researchers are trying to sell you on the idea of Santa Claus by way of half-eaten cookies, presents under the tree, and a colorful note that looks suspiciously like your mother's handwriting.

A similar conclusion about the lack of evidence regarding the existence of the monkeypox "virus" was made by Dr.'s Sam and Mark Bailey in their recent excellent article on this topic:

"In any case, a review of the scientific evidence revealed that with regards to monkeypox: (a) there is no evidence of a physical particle that meets the definition of a virus, (b) there is no evidence of anything transmitting between humans, and (c) there is no way to confirm a diagnosis of monkeypox unless you believe in clinically-unvalidated tests such as the PCR kits that have been produced. In other words, if we see a monkeypox "pandemic" that is used as an excuse to role out more globalist terrorism, it will be on the back of another PCR pandemic, not one that has any basis in nature."

Monkeypox Mythology

They go into great detail breaking down the CDC's deceptive practices in a supposed monkeypox outbreak in the US in 2003 and the (surprise surprise) misuse of PCR to diagnose cases. I highly recommend reading the whole article.

The bottom line is that there is no reason to fear monkeypox today any more than there was before the drill and the ensuing fear-mongering headlines. Monkeypox is nothing but the reclassification of the same symptoms of disease that belong to a select group including smallpox, chickenpox, shingles, measles, etc. They are all varying levels of the same detoxification process. Could there be an increase in the amount of people suffering these sets of symptoms? Sure, as a vast amount of people have repeatedly subjected themselves to experimental injections over and over again during the last two years. Eventually, the body must rid itself of the toxic overload which may result in a similar set of symptoms as seen in cases of so-called monkeypox. However, as always, there is no need to fear any "virus" nor any infection. There is nothing to this monkeypox business other than a well-orchestrated fear-campaign. The only thing to fear regarding this media blitz in need of a poster child is this:

ViroLIEgy
21 May 2022 | 3:55 pm

The Clonal Selection Antibody Theory (1957)


Immunology does not suffer from a lack of experimental data, but still some of the most elementary questions are undecided, and it is not yet possible to choose between instructive and elective theories."

Joshua Ledergerg, Genes and Antibodies https://doi.org/10.1126/science.129.3364.1649

Every scientific theory relies on the scientific method. A scientist may make an observation and devise a hypothesis to explain that observation, then design an experiment to test that hypothesis. If the hypothesis is shown to be incorrect, the scientist will develop a new hypothesis and begin the process again. If the hypothesis is supported by the results of the experiment, it will go on to be tested again. If the hypothesis isn't disproven or surpassed by a better explanation, the scientist may incorporate it into a larger theory that helps to explain the observed phenomenon and relates it to other phenomena, according to the Field Museum

A scientific theory is not the end result of the scientific method; theories can be proven or rejected, just like hypotheses. And theories are continually improved or modified as more information is gathered, so that the accuracy of the prediction becomes greater over time.

https://www.livescience.com/21491-what-is-a-scientific-theory-definition-of-theory.html

At the time that Frank MacFarlane Burnet proposed the Clonal Selection theory of antibody formation in 1957, there were a few different explanations floating about, including Burnet's own Indirect Template theory which was suggested almost a decade earlier. They included:

  1. Side Chain Theory
  2. Direct Template Theory
  3. Indirect Template Theory
  4. Natural Selection Theory

Each of these theories proposed different ways antibodies formed based on the "scientific" evidence of the time. Naturally, Burnet saw the burning need to discredit his previous theory in order to throw a new one into the mix. To do so, he utilized Niels Jerne's Natural Selection theory and reworked it so that he could cover-up the holes and lack of evidence in his previous attempt. Thus, the Clonal Selection theory was born and it has gone on to become the most widely accepted explanation for the formation of the unseen particles assumed to be within the blood. The acceptance of this "theory" is clearly pointed out by these first three sources, which use some interesting wording describing Burnet's last contribution to the antibody narrative:

"Clonal selection theory is a scientific theory in immunology that explains the functions of cells of the immune system (lymphocytes) in response to specific antigens invading the body. The concept was introduced by Australian doctor Frank Macfarlane Burnet in 1957, in an attempt to explain the great diversity of antibodies formed during initiation of the immune response.[1][2] The theory has become the widely accepted model for how the human immune system responds to infection and how certain types of B and T lymphocytes are selected for destruction of specific antigens.[3]"

https://en.m.wikipedia.org/wiki/Clonal_selection

"The clonal selection hypothesis is a widely accepted model for the immune system's response to infection.

"clonal selection: An hypothesis which states that an individual lymphocyte (specifically, a B cell) expresses receptors specific to the distinct antigen, determined before the antibody ever encounters the antigen. Binding of Ag to a cell activates the cell, causing a proliferation of clone daughter cells.

The clonal selection hypothesis has become a widely accepted model for how the immune system responds to infection and how certain types of B and T lymphocytes are selected for destruction of specific antigens invading the body."

https://bio.libretexts.org/Bookshelves/Microbiology/Book%3A_Microbiology_(Boundless)/11%3A_Immunology/11.07%3A_Antibodies/11.7C%3A_Clonal_Selection_of_Antibody-Producing_Cells

"In 1957, Burnet proposed that central tolerance to self-antigens occurred by clonal deletion of self-reactive lymphocytes [7]. However, as we will discuss, this model has been 'updated' on several occasions."

https://www.cell.com/trends/immunology/fulltext/S1471-4906(04)00311-4

Notice the words hypothesis and model thrown about in these descriptions of what is considered to be the most widely accepted scientific theory for antibody production? What could they possibly be referring to? Let's see if we can clear this up by looking at the definitions:

scientific hypothesis, an idea that proposes a tentative explanation about a phenomenon or a narrow set of phenomena observed in the natural world. 

https://www.britannica.com/science/scientific-hypothesis

scientific modeling, the generation of a physical, conceptual, or mathematical representation of a real phenomenon that is difficult to observe directly. 

https://www.britannica.com/science/scientific-modeling

scientific theory, systematic ideational structure of broad scope, conceived by the human imagination, that encompasses a family of empirical (experiential) laws regarding regularities existing in objects and events, both observed and posited. A scientific theory is a structure suggested by these laws and is devised to explain them in a scientifically rational manner.

https://www.britannica.com/science/scientific-theory

Putting it all together, Burnet's Clonal Selection theory conceived of by the human imagination, which can be rejected just like any other tentative explanation otherwise known as a hypothesis, is the current model, which is a conceptual representation of a real phenomenon that is "difficult to observe directly." This explanation obviously creates a bit of a problem. In order for this hypothesis/model/theory to be considered scientific, it must adhere to the scientific method:

"A theory is a carefully thought-out explanation for observations of the natural world that has been constructed using the scientific method, and which brings together many facts and hypotheses."

https://www.fieldmuseum.org/blog/what-do-we-mean-theory-science

The scientific method requires the observation of a real phenomena in nature. In fact, this is the very first step:

  1. Observe a natural phenomenon
  2. Alternate hypothesis
    • Independent variable (the presumed cause)
    • Dependent variable (the observed effect)
    • Control variables
  3. Null hypothesis
  4. Test/experiment
  5. Analyze the observation/data
  6. Validate/invalidate hypothesis

It requires the use of a valid independent variable, i.e. purified/isolated particles assumed to be antibodies, to vary and manipulate in order to determine a cause and effect relationship. In other words, the independent variable must be something that has been shown to physically exist in reality. As antibodies have never been purified and isolated directly from humans in order to be used as a valid independent variable and these particles can not be observed directly in nature, none of the indirect experimental data generated and utilized to formulate these theories was scientific. None of the antibody "theories" adhered to the scientific method in order to develop the hypotheses which were used to craft the experiments that were needed to produce the data which was utilized to create the theory. They all started by presupposing the existence of the very entity they were trying to conjure up evidence for in order to formulate a theory to explain the effects seen as well as the shape and function of the presupposed entity for which they could not observe directly.

Basically, this is a long way of saying that we can exclude scientific from the definition of the word "theory" used to describe Burnet's Clonal Selection as it is obvious that it did not adhere to the scientific method and is thus not scientific at all. Therefore, we can replace the scientific definition with the common definition:

theory, an idea that is suggested or presented as possibly true but that is not known or proven to be true

https://www.britannica.com/dictionary/theory

This can be applied to all antibody theories which amount to nothing more than dreamt up speculative fantasies crafted from non-scientific experimental data which combined human and animal blood with various chemicals to create a non-specific reaction in order to blame on invisible entities. These mad science experiments are obviously the exact opposite of a natural phenoma observed in nature. All antibody theories therefore start from a fraudulent foundation.

In any case, as the previous theories/models of this unseen entity were unscientific and could not hold up to scrutiny, they were eventually unceremoniously thrown out in favor of Burnet's Clonal theory. For decades it was considered as the "truth" even though it was nothing but speculation masquerading as science. However, even the unscientific Clonal theory has being challenged recently. In 1992, Irun Cohen, an immunologist at the Weizmann Institute of Science in Israel who developed the theory of the immunological homunculus in 1989, wrote a paper challenging the Clonal Selection theory. Unfortunately, I was unable to aquire the whole paper. However, the abstract gives us an idea as to the grounds Cohen challenged the theory on:

The cognitive principle challenges clonal selection

"Here, Irun Cohen argues that the clonal selection paradigm is no longer a convenient paradigm for organizing thinking about the immune system. He contends that most immunologists now investigate questions for which the clonal selection paradigm makes no provision and that one of its major tenets is contradicted by the prevalence of natural autoimmunity. Instead, he proposes a cognitive paradigm."

https://pubmed.ncbi.nlm.nih.gov/1476598/

In a page referencing the work of Cohen, further light can be shed on his criticisms as he claimed that the tenets of classic Clonal Selection are not compatible with the findings reported in a paper on the success of a therapeutic peptide vaccination. Cohen felt that the success of the clinical trial was compatible instead with the homunculus concept of autoimmunity:

DiaPep277 Peptide and Type 1 Diabetes

Raz I., Elias D., Avron A., Tamir M., Metzger M., Cohen I.R.
-cell function in new-onset type 1 diabetes and immunomodulation with a heat-shock protein peptide (DiaPep277): A randomized, double-blind, phase II trial. The Lancet, 2001; 358: 1749-53.

"At the practical level, this paper shows that autoimmune destruction of beta cells in patients with newly diagnosed type 1 diabetes can be arrested by the administration of DiaPep277, a peptide fragment of the 60kDa heat shock protein molecule. Three subcutaneous injection of the peptide, 1 mg each in an oil vehicle, were effective. The optimal dose of peptide and the most effective schedule of injections remain to be worked out. Nevertheless, the confirmation of effectiveness and safety in continuing trials would indicate that immunologically specific treatment of autoimmune disease is feasible.

At the theoretical level, these results serve as a proof-of-concept in the controversy between contending theories regarding the basic organization of the immune system. The classical clonal selection theory holds that:

  1. Self-non-self discrimination is the aim of the immune system;
  2. Autoimmunity is deleted from the healthy repertoire of antigen receptors;
  3. Autoimmunity arises by accident;
  4. Autoimmune disease therapy requires blocking self-recognition, or removal of self-recognizing lymphocytes.

These tenets of classic clonal selection are not compatible with the findings reported in the paper. On the contrary, the success of the clinical trial of therapeutic peptide vaccination is compatible with the homunculus concept of autoimmunity. This theory includes the following ideas:

  1. The aim of the immune system is not to discriminate self from non-self, but to organize inflammation in a way that maintains, heals and, where possible, regenerates damaged cells and tissues; the healthy system is continuously responding to self.
  2. The healthy repertoire includes a high frequency of lymphocytes that recognize key self-antigens: the immunological homunculus.
  3. Autoimmune diseases arise from a failure of normal regulation, and the resulting diseases are predictably associated with collectives of particular self-antigens.
  4. The natural cure to autoimmune disease is to activate and reinstate the regulatory mechanisms built into the homunculus.

The paper demonstrates that a single peptide can indeed activate regulation; the immune system is best controlled by supplying the immune information the system is organized to seek. The immune system is a cognitive system."

https://www.weizmann.ac.il/immunology/sci/CohenPage2.html

Irun Cohen's visual representation of natural autoimmunity–the immunological
homunculus https://doi.org/10.1016/j.jaut.2007.07.016

In 2002, Arthur M. Silverstein, an American immunologist and science historian who has written extensively on the history of immunology, wrote a paper attempting to defend the Clonal Selection theory. However, in the paper, he noted the criticism of the Clonal Selection theory by Cohen and others as an obsolete and outdated theory and pointed to some very important questions that CST needed to answer which were largely unaddressed:

The Clonal Selection Theory: what it really is and why modern challenges are misplaced

"The Clonal Selection Theory (CST)2 of Macfarlane Burnet and David Talmage seems to be under fire currently from several directions. Irun Cohen, in considering the role of autoimmunity in the economy of the body, suggested that, "Progress in immunology appears to have rendered the clonal selection paradigm incomplete, if not
obsolete; true it accounts for the importance of clonal activation, but it fails to encompass, require, or explain most of the subjects being studied by immunologists today …". Polly Matzinger has gone even further: based upon her view that the "danger theory" has negated Burnet's classical view of self-nonself discrimination, she extends this to suggest that the entire clonal selection paradigm that has ruled immunology for some 35 years has been overthrown."

"Even philosophers of science have occasionally blurred the important distinction between the core hypotheses of CST and the ancillary
hypotheses that may stem from it. In his book The Immune Self: Theory or Metaphor?, A. Tauber calls Burnet's view of tolerance "…a cornerstone of his later immune theory [CST]", and throughout appears to accept the various challenges to Burnet's ideas on tolerance and self-nonself as challenges to the central meaning of CST. Again, in their book The Generation of Diversity, S. Podolsky and A. Tauber discuss the several challenges to Burnet's idea of self-nonself and conclude that, "Specifically, we must ponder whether CST, as constructed by Burnet, Talmage, and Lederberg [sic!8] … is now being seriously challenged". Kenneth Schaffner, in his elegant discussion of the philosophical bases of CST, Discovery and Explanation in Biology and Medicine, formally defines three levels of hypothesis in CST and actually assigns Burnet's tolerance hypothesis to a secondary level. But even he sometimes seems to suggest that tolerance experiments may serve as tests of CST.

From instruction to selection

Between 1930 and the early 1960s, the accepted explanation for the large repertoire of antibody specificities was that antigen somehow acts as a template to transfer information to the globulin-producing mechanism. This was termed an "instruction theory" and was advanced in different forms. At a time when immunology was preoccupied with chemical approaches, and when little was known about how proteins are formed, these instruction theories seemed plausible. No matter that they could not explain such observations as the persistence of antibody formation, the accelerated and enhanced secondary response or what would later be termed affinity maturation.

Then in 1955, in the context of increasing interest in tissue transplantation, immunodeficiency diseases, autoimmunity and tolerance, Niels Jerne suggested that the information for all antibody specificities is inherent in the host and expressed as "natural" antibodies. Now the sole function of antigen was to select the matching antibody and transport it to the appropriate cells where, somehow, they would be stimulated to produce more of the same. Jerne's idea attracted little support at the time; its surviving claim-to-fame lies in the fact that David Talmage and Macfarlane Burnet independently saw in its selectionist approach the seed of an interesting idea."

Silverstein offered questions CST must answer that were largely unaddressed. I'm paraphrasing them here for length considerations:

  1. If a "Landsteiner-size" repertoire arises spontaneously, what is the mechanism for its generation?
  2. If a repertoire is generated randomly and somatically, why are destructive autoantibodies not formed against native antigens to engender immediate autoimmune disease?
  3. Is there more than one type of receptor on a single cell?
  4. If somatic mutation persists after clonal expansion, how can clonal specificity be maintained?
  5. How can interaction of antigen with a surface antibody receptor induce cell proliferation and differentiation?
  6. What determines and regulates which clonal daughters differentiate to form antibody and which survive as memory cells?

"Any valid theory of antibody formation (and that is what CST is) must satisfactorily explain these and other questions. However, the central theory need not fall just because its promulgator was wrong in proposing a mechanism to answer one of its subsidiary questions. Some of these questions address the "selection" component of CST, whereas others deal with the "clonal" component; the former might survive the disproof of the latter. It is curious that CST has been challenged based upon Burnet's error in explaining self-nonself discrimination, but no one has suggested that CST might be challenged because Burnet was wrong about the mechanism for the generation of diversity, although these are hierarchically equal hypotheses. Yet self-nonself is chosen among all the other possibilities, for reasons that we will explore below."

"Burnet became so involved with the question of self and with his explanation of the mechanism of tolerance that even he began to view it as an integral part and even a test of CST, rather than as merely a subsidiary question to be approached by trial and error. In discussing the foundations of CST, Burnet admitted that if immunologists are correct in doubting that "tolerance is wholly a matter of the absence of the immunocyte … an extensive reorientation [of CST] will become necessary". No wonder that others might feel the same! (We might note also that Burnet came close to giving up on CST in 1962, when reports came in of two and even four different antibody specificities produced by a single cell; when Szenberg et al. found too many pocks on the chorioallantoic membrane of chick embryos injected with small numbers of lymphocytes (that is, the proportion of cells specific for MHC alloantigens appeared to be much too high)40; and when Trentin and Fahlberg found that a single clone of cells used to reconstitute a lethally irradiated mouse seemed able to form antibodies of different specificities.)"

"Given this wide-open theoretical terrain, it is no wonder that debate continues on such questions as a "big bang" versus the continuous generation of diversity, the relative roles of central versus peripheral mechanisms of tolerance, the number and type of signals required for one or the other response, whether autoimmunity is dangerous or beneficial (Cohen) and whether the immune apparatus evolved to recognize infectious pathogens (Cohn and Janeway), "danger" (Matzinger) or, following Jerne, "self" (Coutinho and Cohen)."

"In the end, however, we must not lose sight of the fact that the CST is only a theory of how antibodies are formed, not a theory of why they are formed."

DOI:10.1038/ni0902-793

As can be seen, Burnet's theory, while presented as the truth regarding antibody production and formation, is not without its detractors. Even Burnet himself waffled on the correctness of his own theory in 1962, stated in 1967 that it was incomplete and the terms were obscure and meaningless, and considered his work speculative in his own paper. The entire 1957 paper detailing his ideas is presented below:

A Modification of Jerne's Theory of Antibody Production using the
Concept of Clonal Selection

There are three current theoretical interpretations of antibody production which, following Talmage (1957), may be referred to as the direct template theory in which the antigen serves as a template against which the specific pattern of the antibody is synthesized, the indirect template theory which postulates a secondary template incorporated into the genetic-synthetic processes of the antibody producing cells (Burnet, 1956), and the natural selection theory in which the antigen acts essentially by selection for excess production of natural antibody molecules of corresponding type (Jerne, 1955).

The two latter theories were devised primarily to account for two sets of phenomena for which the direct template theory seems quite irrelevant. The first is the absence of immunological response to "self" constituents and the related phenomena of immunological tolerance; the second is the evidence that antibody production can continue in the absence of antigen. Some means for the recognition and differentiation of potentially antigenic components of the body from foreign organic material must be provided in any acceptable formulation. In Burnet and Fenner's (1949) account, a positive recognition of "self" material was ascribed to the presence of "self
markers" in all potentially antigenic
macromolecules, and corresponding recognition units in the scavenger cells of the body. At the time it was regarded as inconceivable that a mechanism could exist which would recognise in positive fashion all foreign material and no attempt was made to devise one, despite the fact that we have always recognised the clumsy character of the self-marker, self-recognition scheme.

It is the great virtue of Jerne's hypothesis that it provides an approach to this alternative method of recognising self from not self. There is no doubt about the presence in all mammalian or avian sera of a wide range of reactive globulins which can legitimately be called "natural antibodies." Jerne assumed that amongst these globulin molecules were all the possible patterns needed for specific immunological type reaction with any antigen, except for those patterns corresponding to body antigens which would be eliminated by in vivo absorption. When a foreign antigen enters the blood it unites, according to Jerne's scheme, with one of the corresponding natural antibody molecules. The complex is taken up by a phagocytic cell in which the antigen plays no further part, but the antibody globulin provokes the production by the cell of a fresh crop of similar molecules which are liberated as antibody. If this basis is accepted, most immunological phenomena can be well described in terms of the theory. Its major objection is the absence of any precedent for, and the intrinsic unlikelihood of, the suggestion that a molecule of partially denatured antibody could stimulate a cell, into which it had been taken, to produce a series of replicas of the molecule.

Talmage (1957) has suggested that Jerne's view is basically an extension of Ehrlich'side chain theory of antitoxin production and that it would be more satisfactory if the replicating elements essential to any such theory were cellular in character ab initio rather than extracellular protein which can replicate only when taken into an appropriate cell. Talmage does not elaborate this point of view but clearly accepts it as the best basis for the future development of antibody theory. He stresses the multiplicity of the globulin types that can be present in the blood and is profoundly sceptical of any approach which attempts to "unitarian" an interpretation of antibody. In his view properdin has as much right to be called an antibody as any other globulin.

Before receiving Talmage's review we had adopted virtually the same approach but had developed it from what might be called a "clonal" point of view. This is simply a recognition that the expendable cells of the body can be regarded as belonging to clones which have arisen as a result of somatic mutation or conceivably other inheritable changes. Each such clone will have some individual characteristic and in a special sense will be subject to an evolutionary process of selective survival within the internal environment of the body.

It is believed that the advantages of Jerne'stheory can be retained and its difficulties overcome if the recognition of foreign pattern is ascribed to clones of lymphocytic cells and not to circulating natural antibody. The resulting formulation may be stated as follows:

The plasma-globulins comprise a wide variety of individually patterned molecules and probably several types of physically distinct structure. Amongst them are molecules with reactive sites which can correspond probably with varying degrees of precision to all, or virtually all, the antigenic determinants that occur in biological material other than that characteristic of the body itself. Each type of pattern is a specific product of a clone of mesenchymal cells and it is the essence of the hypothesis that each cell automatically has available on its surface representative reactive sites equivalent to those of the globulin they produce. For the sake of ease of exposition these cells will be referred to as lymphocytes, it being understood that other mesenchymal types may also be involved. Under appropriate conditions, cells of most clones can either liberate soluble antibody or give rise to descendant cells which can.

It is assumed that when an antigen enters the blood or tissue fluids it will attach to the surface of any lymphocyte carrying reactive sites which correspond to one of its antigenic determinants. The capacity of a circulating lymphocyte to pass to tissue sites and there to initiate
proliferation is now relatively well established (cf. Gowens, 1957; Simonsen, 1957). It is postulated that when antigen-natural antibody contact takes place on the surface of a lymphocyte the cell is activated to settle in an appropriate tissue, spleen, lymph node or local inflammatory accumulation, and there undergo proliferation to produce a variety of descendants. In this way preferential proliferation will be initiated of all those clones whose reactive sites correspond to the antigenic determinants on the antigen used. The descendants will include plasmacytoid forms capable of active liberation of soluble antibody and lymphocytes which can fulfill the same functions as the parental forms. The net result will be a change in the composition of the globulin molecule population to give an excess of molecules capable of reacting with the antigen, in other words the serum will now take on the qualities of specific antibody. The increase in the number of circulating lymphocytes of the clones concerned will also ensure that the response to a subsequent entry of the same antigen will be extensive and rapid, i.e. a secondary type immunological response will occur.

Such a point of view is basically an attempt to apply the concept of population genetics to the clones of mesenchymal cells within the body. It is clear that the internal environment involved is an exceedingly complex one and in all probability many factors will impinge on clones of antibody-producing cells from that environment. It is equally certain that inheritable changes (at the clonal level) will occur as a result of somatic mutation or of the still obscure processes responsible for differentiation during development of regeneration and repair.

It would be inappropriate to elaborate this view much further in a preliminary communication, but it should be immediately evident that it has highly relevant implications for the general function of the lymphocyte, for the fact that sensitization and homograft immunity reactions seem to be mediated by lymphocytes or other mesenchymal cells without liberation of classical antibody, and for recent findings of extremely rapid liberation of antibody from normal cells. A preliminary survey of a variety of pathological conditions which involve anomalous immune reactions also suggests that this cellular approach has greater relevance to the problems than any of the other hypotheses. These aspects will be elaborated in a more extensive contribution now in preparation.

One aspect, however, should be mentioned. The theory requires at some stage in early embryonic development a genetic process for which there is no available precedent. In some way we have to picture a "randomization" of the coding responsible for part of the specification of gamma globulin molecules, so that after several cell generations in early mesenchymal cells there are specifications in the genomes for virtually every variant that can exist as a gamma globulin molecule. This must then be followed by a phase in which the randomly developed specification is stabilized and transferred as such to descendant cells. At this stage, again following Jerne, any clones of cells which carry reactive sites corresponding to body determinants will be eliminated. The necrotic effect of the tuberculin on sensitived fibroblasts might be taken as a crude analogue of the process by which clones with unwanted reactivity can be eliminated in the late embryonic period with the concomitant development of immune tolerance.

The hypothesis has many of the same implications as the Burnet-Fenner and the Jerne theories. Its chief advantage
over the former is its relevance to the nature of normal antibodies including the red cell isoagglutinins and the simpler interpretation of tolerance to potential antigens experienced in embryonic life. Its advantages over Jerne's theory are its capacity to cover homograft and related types of immunity as well as the production of classical antibody, and to eliminate the very unlikely assumption that entry of a globulin molecule into a cell will stimulate the cell to produce exact replicas of that globulin.

Despite the speculative character of much of the detail of this modification of Jerne's theory–which might be referred to as the "clonal selection hypothesis"–it has so many implications calling for experimental inquiry that it has been thought justifiable to submit this preliminary account for publication.

DOI:10.3322/canjclin.26.2.119

In Summary:
  • Irun Cohen, an immunologist at the Weizmann Institute of Science, Israel, argued that the clonal selection paradigm is no longer a convenient paradigm for organizing thinking about the immune system
  • He contended that most immunologists now investigate questions for which the clonal selection paradigm makes no provision and that one of its major tenets is contradicted by the prevalence of natural autoimmunity
  • Cohen stated that the results of a diabetes drug trial served as a proof-of-concept in the controversy between contending theories regarding the basic organization of the immune system
  • Cohen argued that the tenets of classic clonal selection are not compatible with the findings reported in the paper
  • On the contrary, the success of the clinical trial of therapeutic peptide vaccination was compatible with the homunculus concept of autoimmunity, which states:
    1. The aim of the immune system is not to discriminate self from non-self, but to organize inflammation in a way that maintains, heals and, where possible, regenerates damaged cells and tissues; the healthy system is continuously responding to self.
    2. The healthy repertoire includes a high frequency of lymphocytes that recognize key self-antigens: the immunological homunculus.
    3. Autoimmune diseases arise from a failure of normal regulation, and the resulting diseases are predictably associated with collectives of particular self-antigens.
    4. The natural cure to autoimmune disease is to activate and reinstate the regulatory mechanisms built into the homunculus.
  • Cohen argued that the immune system is a cognitive system
  • In considering the role of autoimmunity in the economy of the body, he suggested that, "Progress in immunology appears to have rendered the clonal selection paradigm incomplete, if not obsolete; true it accounts for the importance of clonal activation, but it fails to encompass, require, or explain most of the subjects being studied by immunologists today …".
  • Polly Matzinger has gone even further: based upon her view that the "danger theory" has negated Burnet's classical view of self-nonself discrimination, she extends this to suggest that the entire clonal selection paradigm that has ruled immunology for some 35 years has been overthrown
  • In his book The Immune Self: Theory or Metaphor?, A. Tauber calls Burnet's view of tolerance "…a cornerstone of his later immune theory [CST]", and throughout appears to accept the various challenges to Burnet's ideas on tolerance and self-nonself as challenges to the central meaning of CST
  • Again, in their book The Generation of Diversity, S. Podolsky and A. Tauber discuss the several challenges to Burnet's idea of self-nonself and conclude that, "Specifically, we must ponder whether CST, as constructed by Burnet, Talmage, and Lederberg [sic!8] … is now being seriously challenged".
  • Between 1930 and the early 1960s, the accepted explanation for the large repertoire of antibody specificities was that antigen somehow acts as a template to transfer information to the globulin-producing mechanism.
  • This was termed an "instruction theory" and was advanced in different forms
  • At a time when immunology was preoccupied with chemical approaches, and when little was known about how proteins are formed, these instruction theories seemed plausible
  • No matter that they could not explain such observations as the persistence of antibody formation
  • Niels Jerne suggested that the information for all antibody specificities is inherent in the host and expressed as "natural" antibodies
  • Jerne's idea attracted little support at the time; its surviving claim-to-fame lies in the fact that David Talmage and Macfarlane Burnet independently saw in its selectionist approach the seed of an interesting idea
  • Arthur M. Silverstein, immunologist and historian, submitted questions CST must answer:
    1. If a "Landsteiner-size" repertoire arises spontaneously, what is the mechanism for its generation?
    2. If a repertoire is generated randomly and somatically, why are destructive autoantibodies not formed against native antigens to engender immediate autoimmune disease?
    3. Is there more than one type of receptor on a single cell?
    4. If somatic mutation persists after clonal expansion, how can clonal specificity be maintained?
    5. How can interaction of antigen with a surface antibody receptor induce cell proliferation and differentiation?
    6. What determines and regulates which clonal daughters differentiate to form antibody and which survive as memory cells?
  • Any valid theory of antibody formation must satisfactorily explain these and other questions
  • However, Silverstein suggested that the central theory need not fall just because its promulgator was wrong in proposing a mechanism to answer one of its subsidiary questions
  • Some of these questions address the "selection" component of CST, whereas others deal with the "clonal" component; the former might survive the disproof of the latter
  • CST has been challenged based upon Burnet's error in explaining self-nonself discrimination
  • In discussing the foundations of CST, Burnet admitted that if immunologists are correct in doubting that "tolerance is wholly a matter of the absence of the immunocyte … an extensive reorientation [of CST] will become necessary".
  • Burnet came close to giving up on CST in 1962, when reports came in of two and even four different antibody specificities produced by a single cell
  • Given this wide-open theoretical terrain, it is no wonder that debate continues on such questions such as:
    1. A "big bang" versus the continuous generation of diversity
    2. The relative roles of central versus peripheral mechanisms of tolerance
    3. The number and type of signals required for one or the other response
    4. Whether autoimmunity is dangerous or beneficial (Cohen)
    5. Whether the immune apparatus evolved to recognize infectious pathogens
  • In the end it is stated that we must not lose sight of the fact that the CST is only a theory of how antibodies are formed, not a theory of why they are formed
  • According to Burnet, there were three (actually four yet Burnet excluded Ehrlich's Side-chain theory for some reason) theoretical interpretations of antibody production:
    1. The direct template theory
      • The antigen serves as a template against which the specific pattern of the antibody is synthesized
    2. The indirect template theory
      • Postulates a secondary template incorporated into the genetic-synthetic processes of the antibody producing cells
    3. The natural selection theory
      • The antigen acts essentially by selection for excess production of natural antibody molecules of corresponding type
  • The two latter theories were devised primarily to account for two sets of phenomena for which the direct template theory seems quite irrelevant:
    1. The first is the absence of immunological response to "self" constituents and the related phenomena of immunological tolerance
    2. The second is the evidence that antibody production can continue in the absence of antigen
  • In Burnet's own Indirect Template theory, a positive recognition of "self" material was ascribed to the presence of "self markers" in all potentially antigenic macromolecules, and corresponding recognition units in the scavenger cells of the body
  • At the time it was regarded as inconceivable that a mechanism could exist which would recognise in positive fashion all foreign material and no attempt was made to devise one
  • Burnet admitted that they always recognised the clumsy character of the self-marker, self-recognition scheme
  • Burnet applauded Jerne's Natural Selection hypothesis (odd he called it a hypothesis rather than a theory) as it provided an approach to this alternative method of recognising self from not self
  • Jerne assumed that amongst the globulin molecules considered "natural antibodies," there were all the possible patterns needed for specific immunological type reaction with any antigen, except for those patterns corresponding to body antigens which would be eliminated by in vivo absorption
  • When a foreign antigen enters the blood it unites, according to Jerne's scheme, with one of the corresponding natural antibody molecules
  • Burnet pondered that if this basis is accepted, most immunological phenomena can be well described in terms of the theory
  • However, its major objection is the absence of any precedent for, and the intrinsic unlikelihood of, the suggestion that a molecule of partially denatured antibody could stimulate a cell, into which it had been taken, to produce a series of replicas of the molecule
  • Talmage suggested that Jerne's view is basically an extension of Ehrlich's side chain theory of antitoxin production and that it would be more satisfactory if the replicating elements essential to any such theory were cellular in character ab initio rather than extracellular protein which can replicate only when taken into an appropriate cell
  • Talmage did not elaborate this point of view but clearly accepted it as the best basis for the future development of antibody theory
  • He stressed the multiplicity of the globulin types that can be present in the blood and was profoundly sceptical of any approach which attempts to "unitarian" an interpretation of antibody
  • In his view properdin had as much right to be called an antibody as any other globulin
  • In other words, Talmage didn't want the unseen antibody to be locked into a definite descriptive box and felt there were many globulin types which could be called antibodies
  • Before receiving Talmage's review, Burnet claimed he had adopted virtually the same approach but had developed it from what might be called a "clonal" point of view
  • This was simply a recognition that the expendable cells of the body can be regarded as belonging to clones which have arisen as a result of somatic mutation or conceivably other inheritable changes
  • Burnet believed that the advantages of Jerne's theory (wait…is it a theory or a hypothesis…) could be retained and its difficulties overcome if the recognition of foreign pattern is ascribed to clones of lymphocytic cells and not to circulating natural antibody
  • The plasma-globulins comprise a wide variety of individually patterned molecules and probably several types of physically distinct structure
  • Amongst them are molecules with reactive sites which can correspond probably with varying degrees of precision to all, or virtually all, the antigenic determinants that occur in biological material other than that characteristic of the body itself
  • Each type of pattern is a specific product of a clone of mesenchymal cells and it is the essence of the hypothesis that each cell automatically has available on its surface representative reactive sites equivalent to those of the globulin they produce
  • It was assumed that when an antigen enters the blood or tissue fluids it will attach to the surface of any lymphocyte carrying reactive sites which correspond to one of its antigenic determinants
  • It was postulated (i.e. suggest or assume the existence, fact, or truth of something as a basis for reasoning, discussion, or belief) that when antigen-natural antibody contact takes place on the surface of a lymphocyte the cell is activated to settle in an appropriate tissue, spleen, lymph node or local inflammatory accumulation, and there undergo proliferation to produce a variety of descendants
  • Such a point of view was basically an attempt to apply the concept of population genetics to the clones of mesenchymal cells within the body
  • It was clear that the internal environment involved is an exceedingly complex one and in all probability many factors will impinge on clones of antibody-producing cells from that environment
  • Burnet felt that it would be inappropriate to elaborate this view much further in a preliminary communication
  • A preliminary survey of a variety of pathological conditions which involve anomalous immune reactions suggested that this cellular approach has greater relevance to the problems than any of the other hypotheses (back to hypotheses instead of theories again)
  • Burnet stated that his theory required at some stage in early embryonic development a genetic process for which there is no available precedent
  • He claimed that they have to, in some way, picture a "randomization" of the coding responsible for part of the specification of gamma globulin molecules, so that after several cell generations in early mesenchymal cells there are specifications in the genomes for virtually every variant that can exist as a gamma globulin molecule
  • In other words, one must use ones imagination in order for this "theory" to work
  • Burnet claimed that the hypothesis had many of the same implications as the Burnet-Fenner and the Jerne theories (it seems one can switch between hypothesis and theory interchangeably)
  • Burnet stated that its chief advantage over his former theory is its relevance to the nature of normal antibodies including the red cell isoagglutinins and the simpler interpretation of tolerance to potential antigens experienced in embryonic life
  • Its advantages over Jerne's theory are its capacity to cover homograft and related types of immunity as well as the production of classical antibody, and to eliminate the very unlikely assumption that entry of a globulin molecule into a cell will stimulate the cell to produce exact replicas of that globulin
  • Due to the speculative character of much of the detail of his modification of Jerne's theory– Burnet referred to his latest as the "clonal selection hypothesis"

In 2007, Arthur M. Silverstein participated with a few other esteemed immunologists in reflecting back on the 50 year impact of the Clonal Selection "theory." In it, he explained some of the opposition to CST and offered an ominous warning for what had to occur in order for the speculative hypothesis masquerading as a theory to be universally accepted by the immunology community:

Reflections on the clonal-selection theory

"There were two types of opposition to the CST, one early and continued, and the other of more recent vintage. The first was the resistance to this biological theory on the part of the chemically oriented immunologists who had introduced and believed in the instructionist theories of antibody formation that the CST replaced. Many of these immunologists chose to disregard the CST, continuing their physico-chemical studies of antibody and antigen (such as David Pressman, a student of Linus Pauling). Others sought to disprove it (such as Dan Campbell, also a Pauling student, who searched for persisting antigen, and Alain Bussard, who wrote a highly entertaining philosophical challenge to the CST). Some (like Elvin Kabat and Fred Karush) made peace with it. The rest (like Haurowitz and Bussard) never gave in. It was ever thus, as Thomas Kuhn pointed out that the full acceptance of a radically new paradigm occurs only when believers in the old one die out!"

doi: 10.1038/nri2177

According to Silverstein, in order for Frank MacFarlane Burnet's 1957 Clonal Selection to be universally accepted, the old guard opposing it needs to die out. For whatever reason, Burnet's storytelling happened to be the last one standing in a long line of theories about the formation and function of antibodies, even though it is still being challenged today. However, calling any of the proposed explanations which came before or after a scientific theory is completely unjustified as not a single one ever used evidence built from the scientific method. In no instance was any natural phenoma ever observed. Combining human and animal blood with various substances in a lab is not a natural phenoma and claiming the resulting effect has significance as to what occurs inside the human body is beyond ridiculous. At no point was a valid independent variable of purified and isolated particles assumed to be antibodies ever observed in order to determine cause and effect nor the form and function of the theorized entities. The idea of antibodies existed first in order to attempt to explain the lab-created effects. Without the direct evidence of these particles in the blood of humans, all of the hypotheses and all of the experimental data used to devise the competing theories amount to nothing but unscientific pseudoscience propping up the imagination of the interpretor. The resulting theories are fictional narratives explaining fictional entities.

If these theories are identified for what they truly are, which is an accumulation of guesswork, hunches, assumptions, and suppositions cobbled together into a fantastical narrative, there would be no problem. If people need to believe in invisible entities belonging to the realm of the imagination in order to have an explanation to justify their belief in a perceived immunity from invading boogeymen, so be it. The problems arise when these exercises in creative writing are presented as the truth rather than the science fiction that they most certainly are. When these stories are used to promote dangerous medical treatments and to determine one either free of or infected with a "deadly pathogen," that is where the line needs to be drawn. We can not allow the wool to be pulled over our eyes any longer while pseudoscience is being presented as science and fiction is presented as truth. It is far past time to sound the alarm and shine the spotlight on these fraudulent practices before the narrative solidifies further as the truth in the minds of our youth.

ViroLIEgy
18 May 2022 | 3:03 pm

The Natural Selection Theory of Antibody Formation (1955)


Such a scientist does not allow himself to be blocked by inconvenient facts or by the confusing life of the laboratory. Such a scientist can attain the sublime through science by abstracting from concrete details, thereby reaching out to universal connections, and by putting his personal imprint on the world" -Niels Jerne

https://brunolemaitre.ch/narcissism-science/testp-page/

Jerne never took satisfaction from small discoveries — in this respect he resembled an artist more than a biomedical researcher — and he struggled to formulate theories of great breadth, wishing to impose his own worldview and his personality on nature." Later in life, Jerne complained that immunology was becoming too complicated and technical. 

https://www.nature.com/articles/424253a

Six years after Frank MacFarlane Burnet proposed his Indirect Template theory for antibody production and with three major competing theories already available to choose from, it was time once again to throw yet another explanation into the mix to describe the formation of the still unseen, still unobserved, and still assumed to exist fictional entities. This new proposal came from Niels Jerne, a Danish immunologist who became known for supplying three "significant" ideas into the antibody story:

  1. Instead of the body creating antibodies from the antigens, the immune system already had all of the antibodies available in order to fight off any pathogen (Jerne's Natural Selection theory proposed in 1955).
  2. The immune system can identify and distinguish what is from the "self" (or host) and what is not (proposed by Jerne in 1971).
  3. The idea that not only do antibodies attach to an antigen, but that they can also become attached to other antibodies (which became known as the Network theory proposed by Jerne in 1974)

This post will look specifically at Jerne's Natural Selection paper and the idea that the body naturally has everything needed to fight off invading pathogens. In other words, it is not the antigen that is either directly or indirectly responsible for creating the antibodies as proposed by other theories. Jerne obviously had his own ideas for how these invisible entities formed, functioned, and existed in the body. Before delving into his paper, let's see what we can uncover about some of his work and how he came about his ideas.

This first source is from Jerne's obituary published by the New York Times. It gives us an idea as to what Jerne is recognized for:

"He was cited especially for three important theories of immunology that together offered a coherent general image of the immune defenses. They were built on findings from research that Dr. Jerne himself carried out, and also incorporated the fruits of work by many other researchers.

The first theory provided an explanation, which was generally accepted by 1984, of the fashion in which antibodies are generated to match any possible bacterium, virus or other intruder into the body. The second theory presented an inclusive picture of how the immune defense system develops and matures.

The third and newest of the theories was first propounded in the mid- 1970's and became known as the network theory. This was a logical and elaborate explanation of the interactive processes by which the body's immune system goes into action, when needed to fight disease, and later lapses into inactivity when it is no longer needed."

Jerne was recognized for creating a "coherent general image" of how these unseen entities formed and functioned by using his research and the work of others to create his "coherent" narrative. This "general image" relates to the three main ideas mentioned briefly above which are further fleshed out in this next source. We see that, based on his own interpretation of the indirect experimental evidence, Jerne challenged beliefs that were held by most immunologists at the time. Jerne, who was known to take the opposing viewpoint regularly in conversation, essentially took the opposing position of the main theories of the time and was given a Nobel Prize in 1984 for bringing together his ideas and the (presumably) incoherent work of others into a "coherent" narrative, thus providing evidence that the Nobel Prize is awarded to the best storytellers:

THEORIES OF ANTIBODIES PRODUCTION

"Niels K. Jerne, M.D., was awarded the 1984 Nobel Prize in Physiology or Medicine for developing three theories that form the basis of modern cellular immunology. He shared the Nobel jointly with César Milstein and Georges J.F. Köhler (immunologists honored for developing the hybridoma technique for producing monoclonal antibodies), for his theories concerning "the specificity in development and control of the immune system."

Jerne's three main theories challenged widely held views concerning the development of antibodies and laid new foundations for contemporary immunology. Jerne published his first theory, the "natural-selection theory" of antibody formation, in 1955. At the time, immunologists believed that specific antibodies were nonexistent until their corresponding antigens entered the system and served as templates upon which the antibodies were created. Another leading theory at the time held that antigens introduced into cells were modified by enzymes and that repeated antigen exposure caused replication of antibodies that were partial replicas of these enzyme-modified antigens. Jerne challenged both of these notions, hypothesizing that all antibodies are formed during fetal development and are present in the body from birth. He suggested that when an antigen enters the body, it binds to a pre-existing complementary antibody and stimulates the rapid production of identical antibodies.

With his second theory, first set forth in 1971, Jerne sought to explain how the immune system learns to distinguish self from non-self. Immunologists at the time thought that the body's self-tolerance could not be inherited as a standard pattern but must be learned. Jerne theorized that this "learning" takes place in the thymus gland in the upper chest, where different populations of lymphocytes are exposed to histocompatibility antigens. Lymphocytes that recognize self-antigens are suppressed, whereas non-self lymphocytes, which have accumulated spontaneous mutations, develop and multiply into lymphocytes that can detect foreign antigens.

In 1974, Jerne published his third and most significant theory, his "network theory," which revolutionized the way immunologists thought about adaptive immunity and immune regulation. Jerne posited that an antibody can be produced and bind to the antigen-specific variable region of another antibody, being called as anti-antibodies, a process, which, in turn, triggers a successive cascade of anti-anti-antibody production. This cascade broadens the diversity of the antibody population, and the network attains a state of balance under normal conditions, which can be perturbed and restored during additional antigen exposures."

https://www.google.com/url?sa=t&source=web&rct=j&url=https://www.ndvsu.org/images/StudyMaterials/Micro/Theories-of-Antibody-Production.doc.pdf&ved=2ahUKEwjv9pv8nuT3AhUfjIkEHYaDB3gQFnoECAYQAQ&usg=AOvVaw1SEkGBAviylhNpfgoUJmnw

If we needed any extra evidence that Jerne's contributions to immunology relate more to great science fiction writers than to scientific discoveries, this brief passage from Jerne's friend and biographer Thomas Söderqvist may help to sell the portrait of a man who used his imagination over experimental evidence:

The Life and Work of Niels Kaj Jerne as a Source of Ethical Reflection

"There was no crucial experimental evidence to suggest either of these two theories. Of course, they had both been preceded by several years of experimental work, but in the final outcome they were both a matter of creative thinking – or to be more precise, speculation. The short space does not allow me to go into detail here. Suffice it to say that there is very good evidence in Jerne's documents – now deposited in the Royal Library in Copenhagen – to suggest that the decisive factor that set him on the speculative track to the selection and the network theories was his view of his own personal self. To put it shortly, in both cases Jerne seems to have used his understanding of himself as a cognitive resource, as a metaphor, for his construction of the theories. From early on in life, he had thought of himself as a person who would never accept being moulded by the outer circumstances."

https://onlinelibrary.wiley.com/doi/10.1046/j.1365-3083.2002.01082.x

While it should be clear that Niels Jerne was more speculatist than scientist, was his Natural Selection theory, which amounted to conjecture created without firm evidence, actually accurate? Fortunately, we have hindsight to guide us rather than the proclamations of the Nobel Prize. We can see how Jerne's theories played out by looking at the views of his contemporaries. Regarding his Natural Selection paper, this next source is from immunologist Melvin Cohn who basically claimed that Jerne's paper was laughed at by himself and his colleagues for proposing the idea of self-replicating proteins:

THE WISDOM OF HINDSIGHT!

"I immediately presented this paper at our department journal club. The response was like mine; it seemed inconceivable that one could propose a self-replicating protein, given what we knew about DNA structure and genetics in 1955. Neither was there any need to convince me that template theories were ruled out. What was needed was a plausible selectionist theory, and it had to begin with a receptor on cells not in solution. Lennox and I were already at that point in our thinking. I was rather surprised that Delbruck submitted Jerne's paper to Publications of the National Academy of Science, but immunology was only an eccentricity and could be allowed to run counter to the rest of biology."

This final source is a 2012 article from molecular biologist and immunologist Donald Forsdyke who detailed how Jerne's theory "somehow" explained the experimental observations of others in a better way than all of the other theories. Forsdyke proclaimed this even though he admitted that Jerne's ideas are considered primitive to Paul Ehrlich's own theoretical observations from over 55 years prior and that, due to the rapid rise of molecular biology, Jerne's ideas were essentially "proven" wrong at the time of publication:

Immunology (1955-1975): The Natural Selection Theory, the Two Signal Hypothesis and Positive Repertoire Selection

"For both Jerne and myself, ideas were triggered by experimental observations relating to 'natural antibody' – antibody present in an organism prior to that organism's exposure to the specific antigen to which the antibody could bind. When studying the reactivity of viruses with blood serum in the spring of 1954, Jerne unexpectedly discovered an activity – dubbed 'P-star' – which he interpreted as due to natural antibody. This set off a train of thought leading to a theory of antibody formation that seemed to unify disparate observations better than some alternatives (Jerne 1955). He postulated a process by which an organism would, somehow, randomly generate a repertoire of antibodies covering a wide range of specificities. An incoming antigen (e.g. virus) would select the antibody that best complemented it, and would then conduct it to a cell where the antibody would, somehow, be replicated and secreted in large quantities (thus increasing protection against the virus). At the time of their first 'natural' generation, antibodies that reacted with 'self' antigens would have been absorbed by 'structures in the body of the animal itself' so that auto-immune reactivity would not develop."

"From a 'presentist' perspective (Harrison 1987), Jerne's first theoretical immunology paper (the natural selection theory; Jerne, 1955) later appeared primitive relative to the sophisticated cellular hypothesis advanced by Ehrlich a half century earlier (Forsdyke 1995a). Indeed, so rapid were developments in molecular biology that, by the time of publication Jerne's proposed mechanisms were already obsolete (Cohn 1994). Protein directly begetting protein (more antibody) was inconsistent with much of genetics and molecular biology. If a cell could somehow 'read' a protein and turn that information into nucleic acid, Jerne's idea might have worked, because nucleic acid can beget nucleic acid, and that nucleic acid can then beget more of the protein. But no mechanism for directly 'reading' a protein into nucleic acid was known then, or has since emerged."

https://www.queensu.ca/academia/forsdyke/theorimm8.htm

As we can see that Jerne's Natural Selection ideas were considered obsolete by the time he published them, we really don't need to waste too much more of our time on his speculation disguised as a theory. Presented below is the entire 9-page paper from 1955 by Niels Jerne:

THE NATURAL-SELECTION THEORY OF ANTIBODY FORMATION

An immense amount of experimental data related to the problem of antibody formation has accumulated. Theories offering a basic interpretation of these observations have, in contrast, been few. The theory formulated in the present paper, though highly speculative, attempts to provide a framework for the interpretation of the main features of antibody appearance in response to the injection of antigen into an animal.

Two views concerning the mechanism of antibody formation are at present most widely favored. One is the "antigen-template" theory, developed by Breinl, Haurowitz, Mudd, Alexander, and Pauling. This theory assumes that antibodies can be produced only by cells in which the antigen is present. The specific affinity of an antibody molecule toward the antigen is due to a complementarity ln structure derived from the folding of part of the polypeptide chain of a globulin molecule in direct contact with a determinant or haptenic region of the antigen. The antigen thus serves as a template in the final stage of formation of a globulin molecule.

The other view tries to establish a similarity between antibody formation and adaptive enzyme formation and allows for the continued production of antibody
after the antigen has disappeared from the body. This is the "modified-enzyme" theory, formulated by Burnet and Fenner. They propose that the introduction of an antigen into cells, containing enzymes directed toward the disposal of effete cells and cellular debris from the organism itself, induces the formation of "enzymic units" adapted toward the destruction of the antigen. A renewed contact with the antigen stimulates the replication of these enzymic units. Circulating antibody molecules are partial replicas of the modified enzymic units, carrying specificity but lacking enzymic action.

The "natural-selection" theory, proposed in the present paper, may be stated as follows: The role of the antigen is neither that of a template nor that of an enzyme modifier. The antigen is solely a selective carrier of spontaneously circulating antibody to a system of cells which can reproduce this antibody. Globulin molecules are continuously being synthesized in an enormous variety of different configurations. Among the population of circulating globulin molecules there will, spontaneously, be fractions possessing affinity toward any antigen to which the animal can respond. These are the so-called "natural" antibodies. The introduction of an antigen into the blood or into the lymph leads to the selective attachment to the antigen surface of those globulin molecules which happen to have a complementary configuration. The antigen carrying these molecules may then be engulfed by a phagocytic cell. When the globulin molecules thus brought into a cell have been dissociated from., the surface of the antigen, the antigen has accomplished its role and can be eliminated.

The introduction of the selected globulin molecules into a cell or the transfer of these molecules into another cell is the signal for the synthesis or reproduction of molecules identical to those introduced, i.e., of specific antibodies. The release of these antibody molecules into the circulation will shift the composition of the population of circulating globulin molecules. Antigen, secondarily introduced into the circulation, now meets a larger concentration of specific molecules and carries a larger quantity of these, selected for the better-fitting ones, to the antibody-producing apparatus, which already contains many cells engaged in the synthesis of molecules of these types of specificity. This leads to the more rapid reproduction of an
improved assortment of antibody molecules, followed by a further directional shift in the circulating globulin population. The reproduction need not be highly faithful; copying mistakes will be harmless, and may occasionally produce an improved fit. When, in the absence of further antigen stimuli, no more pressure is exerted, the population will slowly revert toward a normal condition of equilibrium. The normal equilibrium may be upheld by a continuous reproduction of samples of the population of molecules circulating at any given moment. Somewhere, however, either in the beginning of the life of an animal or continuously, a spontaneous production of random specificities must take place. The spontaneously produced globulin molecules may be formed only in small numbers. Those among them that will attach themselves to structures in the body of the animal itself will be removed and will therefore not be available for reproduction. The absence of globulin molecules carrying these specificities will prevent a response to antigens of these specific types.

Discussion.-Burnet and Fenner list a number of essential immunological observations which are not satisfactorily accounted for by the antigen-template theory.

  1. The booster effect. A secondary stimulus with the same antigen provokes a more active production of antibody than does a primary stimulus. According to the natural-selection theory, this would be due to the fact that antigen injected secondarily encounters a larger concentration of specific antibodies in the circulation
    than were present at the time for the primary injection. More antibody molecules are therefore brought to the globulin-reproducing cells.
  2. The change in character of the antibody produced in response to repeated inoculations of the same antigen. The main changes observed are from "low-grade" antibody of low combining capacity, produced in the beginning of an immunization course, toward "more avid" antibody of high combining power, produced later. Besides this improvement in quality, an increase has been observed in the range of cross-reactions with related antigens. The present theory explains this development by natural selection. At the time of the primary stimulus the antigen injected finds only few globulin molecules in the circulation, showing various degrees of affinity toward the antigen surface patterns. At the time of a later stimulus in the course of immunization, when these molecules have been replicated in large numbers, the antigen will find a larger concentration of globulin molecules fitting all its surface patterns and will preferentially carry those which show the highest combining capacity to the globulin-reproducing cells.
  3. The apparent exponential rise in circulating antibody during the first period of production. On the present theory this may be due to an autocatalytic replication of the specific globulin molecules and to a multiplication of the cells.
  4. The continued production of antibody for long periods. The antigen-template theory could deal satisfactorily with this point if it could be shown that sufficient antigen remains present during the entire period of antibody production. On the other hand, this theory would have to be abandoned if it could be shown that antibody production continues after the antigen has been eliminated from the body. The latter view is held by Burnet and Fenner, for the following reasons: Since circulating antibody has a rapid turnover, it must be continuously synthesized for long periods, and not only by the cells originally stimulated. The reticuloendothelial cells and the plasma cells which are believed to be engaged in antibody production are short-lived cells. The repeated transfer of the original antigen from a disintegrating cell to another appropriate cell seems unlikely. According to the natural-selection theory, the continued antibody production after elimination of the antigen is natural, because the antigen will have completed its role when it has carried the antibody molecules to the reproducing apparatus.
  5. To this list we would add the observation that the surface of particulate antigens seems to play a dominant part in determining the specificity of the antibody molecules produced. Avery and Neil found that the antibody produced against suspensions of pneumococci is mainly directed against the surface presented by these bacteria. White who studied the antibody produced as a response to injections of Salmonella cells, concluded that "it would seem that the antiserum of the bacillus is overwhelmingly the antiserum of its surface rather than of its substance." Henle et al. studying spermatozoa as antigens, and Morgan who investigated the antigens of Shigella, arrived at similar conclusions. This predominance of the antigen surface is in harmony with the natural-selection theory of antibody formation because the globulin molecules that are eligible for preferential reproduction are those that can attach themselves to the surface presented by the antigen before it is removed from the circulation. It would seem, however, to involve the antigen-template theory in difficulties. From the well-known experiments of Topley it can be calculated that a rabbit, after two intravenous injections of formalin-killed Salmonella cells, synthesized about 100,000 antibody molecules per second per bacterium that had been injected, and continued this rate of production for a period of many weeks. If we imagine that the bacteria serving as antigen templates were not broken down but resided in toto each in an antibody producing cell, this would entail impossible quantitative implications. If, however, the bacteria were broken down into many fragments, how would a cell in which one of these fragments served as antigen template be able to distinguish what part of the fragment had originally been at the surface of the intact bacterium?

It seems worthwhile to consider more closely a theory which seems to provide simple explanations of these important immunological phenomena. Except for specificity toward an antigen, no known properties distinguish normal serum globulin from antibody. In the absence of antigen no directional pressure is imposed upon globulin synthesis, and it seems reasonable to assume that a great variety of configurations, due, perhaps, to various amino acid sequences at the specific sites of the globulin molecules, may develop at random.

An immense body of experimental data testifies to the fact that normal sera from one animal species may contain antibodies against an enormous number of different
bacteria, some of which are not known to be natural parasites to this species. Though these natural antibodies are often supposed to have evolved as a result of previous exposure to antigen, this attitude seems merely to reflect the definition of an antibody as a substance produced in response to an antigen, which, however, does not imply that antibodies are not produced in the absence of, and prior to, exposure to an antigen. Doerr, summing up an extensive review of experimental data in this field, states: "We must accept that it has been definitely demonstrated that natural antibodies can develop without an antigenic stimulus, and that this spontaneous formation is by far the most frequent origin of natural antibodies against bacteria erythrocytes, toxins, and virus particles." The slight phagocytosis promoting activity found in normal sera has been ascribed to the presence of normal specific opsonins. These substances may be equated with natural antibodies. Their activity can be specifically inhibited by hapten. It has been observed that the ability of a rabbit to produce antibodies against bovine serum albumin or against an artificially conjugated antigen is related to the rate at which the injected antigen is removed from the blood. In poorly responding animals the antigen remains in circulation much longer than in good responders. We interpret this in terms of the spontaneous presence in the circulation of less or more specificially fitting globulin, which determines both the rate of phagocytic removal and the amount of specific globulin that will be carried to the globulin-repioducing system.

The methods available for demonstrating the presence of specific antibody in serum rarely permit the detection of concentrations lower than 10^12 or 10^11 antibody molecules per milliliter. Even diphtheria antitoxin, by the extremely sensitive rabbit skin test, cannot be detected in dilutions containing less that 5 X 10^10 antitoxin molecules per milliliter. Most sensitive, probably, is the demonstration of antibodies against bacteriophage, which can be measured quantitatively in sera from normal animals.

Among the comparatively small number, perhaps a few thousand, of antigen-antibody systems investigated, cross-reactions are by no means rare, suggesting that the number of specific configurations which a globulin molecule can exhibit is large but limited. Since normal mammalian serum contains more than 10^17 globulin molecules per milliliter, these may include a million 10^11 fractions of different specificity. This would seem an amply sufficient number.

In what type of cells does the postulated replication of globulin molecules take place? The lymphoid tissue contains cells which are capable of producing globulin and antibody. This tissue is scattered throughout the body but is mainly concentrated in the bone marrow, the thymus, the spleen, the appendix, and various lymph nodes, totaling about 0.5 percent of the body weight. The mesenchymal reticulum cells of the lymphoid tissue develop through a matuiation series to one of three types of cells: phagocytic endothelial cells, lymphocytes, and plasma cells. The developmental and functional relationship between these closely associated ceils is not clear. The phagocytic cells take up foreign particles from the blood and the lymph, and, since the blood is filtered through bone marrow and spleen, and the lymph through the lymph nodes, circulating phagocytes also come into close contact with the lymphoid tissue. Lymphocytes have a life-span of not more than a few days. It can be calculated that the daily output in the rabbit is of the order of 10^10 of these cells. They circulate in the lymph and blood, and many seem to return to the germinal centers of the lymphoid tissue. The lymphocytes seem to pioduce some gamma globulin; but whether they take part in the production of antibody after an antigen stimulus is undecided. The plasma cells, on the other hand, are definitely involved. In conditions of hyperglobulinemia and of hyperimmunization there is a large increase in plasma cells in the bone marrow and elsewhere. The injection of an antigen into the tissues of an animal leads to the production of antibody in the lymph node draining the injection site. During the days following the injection this lymph node exhibits marked mitotic activity. There is an increase in weight and a parallel increase in desoxyribonucleic acid (DNA). This is followed by a large increase in ribonucleic acid (RNA), which reaches a peak around the fifth day, coinciding with the period of greatest antibody increase. The RNA is mainly concentrated in the freely multiplying plasma cell precursors. The antibody content of the lymph leaving the lymph node may be a hundred times greater than that of the lymph entering the node, and most of this antibody leaving the node is inclosed in cells suspended in the lymph plasma, about 5 percent of which may be plasma cells, the rest lymphocytes. After intravenous injections antibody-producing plasma cells can be recognized in the red pulp of the spleen.

The thymus, though rich in nucleic acid and lymphocytes, develops no plasma cells and does not show any immunological activity. Burnet and Fenner, in spite of this, suggest that the lymphocytes may be responsible for maintenance of low levels of antibody long after the plasma cells have initiated the formation during the peak phase of the antibody response. Though they infer that, if this suggestion is true, the immunological function of the lymphocytes can hardly be their major one, it would be a different matter if the function included the maintenance of the circulating gamma globulin. In a rabbit the half-life of circulating globulin is about 5 days. This means that the rabbit must daily synthesize about 10^18 globulin molecules. For 10^10 lymphocytes to accomplish this task, each would have to synthesize about a thousand globulin molecules per second.

If it were true that antibody production after an antigen stimulus is the preferential replication of selected globulin molecules, the production of normal globulin might be an unselective reproduction of the circulating globulin. This could be accomplished either by a mechanism by which a small fraction of circulating globulin could enter into lymphoid cells or by the transfer of synthesizing units from worn-out lymphocytes and plasma cells, returning to the lymphoid tissue, to young cells. In this way the animal would tend to preserve the spectrum of globulin specificities already present, and a drastic change could be accomplished only by a mechanism of preferential replication of a selected fraction of the circulating globulin. This would essentially reduce the production of both normal gamma globulin and antibody to one mechanism: selective and unselective reproduction of circulating globulin molecules.

Somewhere in the beginning, however, we have to postulate a spontaneous production of globulin molecules of a great variety of random specificities in order to start the process. Possibly a specialized lymphoid tissue, such as that of the thymus which is most active in embryonic and early independent life and decays soon after, is engaged in this function. If this small spontaneous production of globulin took place mainly in embryonic and early life, before the much larger body of cells later engaged in maintaining the composition of the circulating globulin by reproduction had started to function, the early removal of a specific fraction of molecules might lead to the permanent disappearance of this type of specificity. Such a mechanism could explain the absence in the blood of specific globulin against antigens of the organism itself, since such globulin molecules, if spontaneously produced, would be removed by attachment to the auto-antigens and would no longer be available for reproduction. The absent specificities would include, besides auto-antibodies, natural antibody against antigens implanted in the animal during embryonic life. The absence from the circulation of such antibodies would, in turn, prevent response to a later antigenic stimulus of this type.

This might occur also if a specific type of globulin molecules could be removed from an adult animal, and might be the explanation of the "immunological paralysis" described by Felton: the blocking of subsequent response by the administration of a large initial dose of pneumococcal polysaccharide. This hapten will induce the formation of antibody in mice only if the dose injected does not exceed a certain very small amount. Felton reported that only doses below 0.001 mg. of a certain preparation of such a polysaccharide were effective in engendering antibody formation in mice. Thus the injection of more than 10^14 molecules of polysaccharide of molecular weight 4,000 inhibited the response. Perhaps a certain fraction of the haptenic particles, by association with a protein, are capable of antigenic stimulation but cannot act when the larger, purely haptenic fraction eliminates all the spontaneously present antibody molecules.

The crucial point of the natural-selection theory is the postulate that the introduction of antibody molecules into appropriate cells can be the signal for the production of more of their kind. This notion is unfamiliar. However, as nothing is known about the mechanism of antibody synthesis in a cell, it would seem a priori more reasonable to assume that an animal can translate a stimulus, introduced by protein molecules which it has itself at one time produced, into an increased synthesis of this same type of molecules than to suppose that an animal can utilize all sorts of foreign substances and can build them functionally and semipermanently into the most intimate parts of its globulin-synthesizing cells.

Any attempt to elaborate this notion into a picture of what may be the mechanism of a replication of specific protein must, at present, be highly speculative. It would seem profitable if, as the modified-enzyme theory tries to do, an analogy could be established on the cellular level between the induction of antibody synthesis in animals and the induction of adaptive enzyme synthesis in microorganisms, since both entail the increased formation of a specific protein. Recent discussions of the nature of adaptive enzyme formation are, however, also still in a speculative stage.

Prior to induction, a small amount of the enzyme appears already to be spontaneously present in the adaptable cells. Induction may involve the replication either of the enzyme molecules or of the elements which form the enzyme. Since RNA seems to play a part in the organization of protein synthesis, it has been suggested that RNA acts as a template on which amino acids are assembled. RNA may thus determine the order of amino acid residues in the protein chain, and. reciprocally, as suggested by Caldwell and Hinshelwood, a protein molecule may determine the order of the nucleotides in the synthesis of RNA. A reversible situation of this kind, as Gale has pointed out, might account for his finding that labeled amino acids are incorporated into proteins of cells that have been prevented from synthesizing new protein by the lack of other essential amino acids. RNA does not seem to be synthesized unless amino acids are present, and optimal RNA synthesis occurs under conditions of optimal protein synthesis. If these syntheses were coupled, it would seem feasible that the introduction of a protein molecule into a cell could initiate a replication of its specific structure.

A mechanism of this sort would be needed for the present theory of antibody formation. If RNA is the template on which protein molecules are assembled, an antibody molecule introduced into the appropriate cell must be able either to initiate the synthesis of specific RNA or to combine with pre-existing RNA. The latter possibility would imply either that the cells already contain a large variety of RNA structures of various specificities-i.e., that a cell is already potentially capable of synthesizing a large variety of globulin molecules of different specificity, the desired type being favored by the introduction of a globulin molecule of this type-or that there are only a smaller variety of different RNA structures present in the cell, upon a related one of which the inducing antibody molecule can impose its specificity.

The present theory predicts that the amount of antibody produced in response to an antigen stimulus depends on the concentration of a type of circulating globulin which can attach itself specifically to the surface of this antigen. This could be tested in experiments of the following types.

The rate of antibody production after a primary stimulus should depend on the concentration of spontaneous antibody already present in the circulation. It is a common observation that the response to an antigen of an animal whose serum already contains a low level of specific antibody is greater than that of animals in which no antibody can be demonstrated. It should be possible, therefore, to increase the rate of antibody production by increasing the concentration of antibody circulating at the time of the primary antigenic stimulus. By introducing antibody from an immunized donor animal into the blood of a recipient animal, prior to the injection of the antigen into the latter, enhanced antibody response to this antigen stimulus should be expected. The common finding in such an experiment is a depression of the response. However, experiments designed to test this point should take into account the fact that even when an animal of the same species is used as the donor of antibody, individual immunological differences residing in the globulin molecules may prevent the recipient animal from utilizing the globulin molecules supplied by the donor. Experiments would therefore have to be carried out between identical twins. Also, when serum from a donor animal is used, the possibility should be considered that the antibody molecules may have been altered, during clotting, separation, and storage of the serum, from the form in which they existed in the circulating plasma.

Also, it should be possible to decrease the rate of antibody production by decreasing the concentration of antibody circulating at the time of the antigenic stimulus. Thus the injection of the corresponding hapten into the blood, prior to the antigen, should depress the antibody response. This may be the explanation of Felton's experiments mentioned above. The booster effect, i.e., the greater response to a secondary antigenic stimulus than to a primary one, should depend on the presence, at the time of the secondary stimulus, of antibody due to the primary stimulus. It has been observed that a booster response to diphtheria toxin does not occur in an animal after the effects, produced by the primary stimulus, have worn off and antitoxin is no longer demonstrable.

In cases in which cross-reactivity between two pairs of antigen-antibody systems is due to related surface patterns, a secondary stimulus with the second antigen following a primary stimulus with the first antigen may lead to the increased production of antibody of the first type. The second antigen would carry many molecules, formed in response to the first antigen, to the reproducing cells. This is what is usually called the "anamnestic reaction."

Summary.-A theory of antibody formation is proposed which postulates the spontaneous presence, in the blood of an animal, of small numbers of antibody molecules against all antigens to which the animal can respond, and delegates to the antigen the sole role of carrying such specific globulin molecules from the circulation into cells in which these molecules can induce the production of more of their kind.

The theory offers an explanation for the presence in blood of a large pool of normal globulins, for the presence of natural antibodies, for the dominant part played by the surface of antigen particles in antibody induction, for the change in character of antibody during the course of immunization (by natural selection), for the exponential increase during part of antibody production, for a continued production of antibody in the absence of the antigen, for the booster phenomenon, for the absence of auto-antibodies, for immunological paralysis and haptenic inhibition, and for the anamnestic reaction.

doi: 10.1073/pnas.41.11.849

In Summary:
  • Niels Jerne is known for three main ideas that he added to the antibody narrative:
    1. Instead of the body creating antibodies from the antigens, the immune system already had all of the antibodies available in order to fight off any pathogen (Jerne's Natural Selection theory proposed in 1955).
    2. The immune system can identify and distinguish what is from the "self" (or host) and what is not (proposed by Jerne in 1971).
    3. The idea that not only do antibodies attach to an antigen, but that they can also become attached to other antibodies (which became known as the Network theory proposed by Jerne in 1974)
  • He was recognized for these three "important" theories (i.e. ideas) of immunology that together offered a coherent general image of the immune defenses
  • They were built on findings from research that Dr. Jerne himself carried out, and also incorporated the fruits of work by many other researchers (in other words, Jerne took from his own and the many unrelated experimental observations/data of others to create his own story on how it all worked)
  • The first theory provided an explanation, which was generally accepted by 1984, of the fashion in which antibodies are generated to match any possible bacterium, "virus" or other intruder into the body
  • The second theory presented an inclusive picture of how the immune defense system develops and matures
  • The third theory was a logical and elaborate explanation of the interactive processes by which the body's immune system goes into action, when needed to fight disease, and later lapses into inactivity when it is no longer needed
  • Jerne shared the 1984 Nobel Prize jointly with César Milstein and Georges J.F. Köhler (immunologists honored for developing the hybridoma technique for producing monoclonal antibodies), for his theories concerning "the specificity in development and control of the immune system."
  • Jerne's three main theories (i.e. ideas) challenged widely held views concerning the development of antibodies and laid new foundations for contemporary immunology
  • At the time, immunologists believed that specific antibodies were nonexistent until their corresponding antigens entered the system and served as templates upon which the antibodies were created (Direct Template theory)
  • Another leading theory at the time held that antigens introduced into cells were modified by enzymes and that repeated antigen exposure caused replication of antibodies that were partial replicas of these enzyme-modified antigens (Indirect Template theory)
  • Jerne challenged both of these notions, hypothesizing that all antibodies are formed during fetal development and are present in the body from birth
  • He suggested that when an antigen enters the body, it binds to a pre-existing complementary antibody and stimulates the rapid production of identical antibodies
  • Immunologists at the time thought that the body's self-tolerance could not be inherited as a standard pattern but must be learned
  • Jerne theorized in 1971 that this "learning" takes place in the thymus gland in the upper chest, where different populations of lymphocytes are exposed to histocompatibility antigens
  • Jerne posited (i.e. assumed as a fact) in 1974 that an antibody can be produced and bind to the antigen-specific variable region of another antibody, being called as anti-antibodies, a process, which, in turn, triggers a successive cascade of anti-anti-antibody production
  • There was no crucial experimental evidence to suggest either of Jerne's two theories
  • Of course, they had both been preceded by several years of experimental work, but in the final outcome they were both a matter of creative thinking – or to be more precise, speculation
  • There is very good evidence in Jerne's documents – now deposited in the Royal Library in Copenhagen – to suggest that the decisive factor that set him on the speculative track to the selection and the network theories was his view of his own personal self
  • To put it shortly, in both cases Jerne seemed to have used his understanding of himself as a cognitive resource, as a metaphor, for his construction of the theories
  • When immunologist Melvin Cohn submitted Jerne's 1955 paper to his colleagues, the response was unanimous in that it seemed inconceivable that one could propose a self-replicating protein, given what they knew about DNA structure and genetics in 1955
  • Cohn stated that what was needed was a plausible selectionist theory, and it had to begin with a receptor on cells not in solution
  • Cohn also stated that immunology was only an eccentricity and could be allowed to run counter to the rest of biology
  • According to microbilogist/immunologist Donald Forsdyke, Jerne's ideas were triggered by experimental observations relating to 'natural antibody' – antibody present in an organism prior to that organism's exposure to the specific antigen to which the antibody could bind
  • When studying the reactivity of "viruses" with blood serum in the spring of 1954, Jerne unexpectedly discovered an activity – dubbed 'P-star' – which he interpreted as due to natural antibody
  • This set off a train of thought leading to a theory of antibody formation that seemed to unify disparate observations better than some alternatives
  • He postulated a process by which an organism would somehow randomly generate a repertoire of antibodies covering a wide range of specificities
  • An incoming antigen (e.g. "virus") would select the antibody that best complemented it, and would then conduct it to a cell where the antibody would somehow be replicated and secreted in large quantities (thus increasing protection against the "virus")
  • Jerne's first theoretical immunology paper (the natural selection theory; Jerne, 1955) later appeared primitive relative to the sophisticated cellular hypothesis advanced by Ehrlich a half century earlier in 1900
  • Rapid developments in molecular biology made Jerne's proposed mechanisms already obsolete by the time of publication
  • Jerne's idea of protein directly begetting protein (more antibody) was inconsistent with much of genetics and molecular biology
  • No mechanism for directly 'reading' a protein into nucleic acid was known then, or has since emerged
  • An immense amount of experimental data related to the problem of antibody formation has accumulated
  • Theories offering a basic interpretation of these observations have, in contrast, been few
  • Jerne admitted that his theory was highly speculative and attempted to provide a framework for the interpretation of the main features of antibody appearance in response to the injection of antigen into an animal
  • Two views concerning the mechanism of antibody formation at that time were most widely favored
    1. Direct Template
      • The "antigen-template" theory, developed by Breinl, Haurowitz,  Mudd, Alexander, and Pauling
      • This theory assumes that antibodies can be produced only by cells in which the antigen is present
    2. Indirect Template
      • The "modified-enzyme" theory, formulated by Burnet and Fenner
      • They proposed that the introduction of an antigen into cells, containing enzymes directed toward the disposal of effete cells and cellular debris from the organism itself, induces the formation of "enzymic units" adapted toward the destruction of the antigen
      • They tried to establish a similarity between antibody formation and adaptive enzyme formation and allowed for the continued production of antibody after the antigen has disappeared from the body
  • In Natural Selection, the role of the antigen is neither that of a template nor that of an enzyme modifier (i.e. in opposition to both Direct and Indirect theory)
  • The antigen is solely a selective carrier of spontaneously circulating antibody to a system of cells which can reproduce this antibody
  • The introduction of the selected globulin molecules into a cell or the transfer of these molecules into another cell is the signal for the synthesis or reproduction of molecules identical to those introduced, i.e., of specific antibodies
  • Somewhere, however, either in the beginning of the life of an animal or continuously, a spontaneous production of random specificities must take place
  • Burnet and Fenner list a number of essential immunological observations which are not satisfactorily accounted for by the antigen-template theory
  • According to the natural-selection theory, the booster effect is because the antigen injected secondarily encounters a larger concentration of specific antibodies in the circulation than were present at the time for the primary injection
  • There is a change in character of the antibody produced in response to repeated inoculations of the same antigen
  • The main changes observed are from "low-grade" antibody of low combining capacity, produced in the beginning of an immunization course, toward "more avid" antibody of high combining power, produced later
  • Besides this improvement in quality, an increase had been observed in the range of cross-reactions with related antigens
  • According to Natural Selection, the apparent exponential rise in circulating antibody during the first period of production may be due to an autocatalytic replication of the specific globulin molecules and to a multiplication of the cells
  • The antigen-template theory could deal satisfactorily with antibodies persisting after the antigen is gone if it could be shown that sufficient antigen remains present during the entire period of antibody production
  • On the other hand, this theory would have to be abandoned if it could be shown that antibody production continues after the antigen has been eliminated from the body
  • The reticuloendothelial cells and the plasma cells are believed to be engaged in antibody production are short-lived cells
  • According to the natural-selection theory, the continued antibody production after elimination of the antigen is natural, because the antigen will have completed its role when it has carried the antibody molecules to the reproducing apparatus
  • The observation that the surface of particulate antigens seems to play a dominant part in determining the specificity of the antibody molecules produced
  • According to Jerne, this predominance of the antigen surface is in harmony with the natural-selection theory of antibody formation because the globulin molecules that are eligible for preferential reproduction are those that can attach themselves to the surface presented by the antigen before it is removed from the circulation
  • Except for specificity toward an antigen, no known properties distinguish normal serum globulin from antibody
  • In the absence of antigen no directional pressure is imposed upon globulin synthesis, and it seemed reasonable to assume that a great variety of configurations, due, perhaps, to various amino acid sequences at the specific sites of the globulin molecules, may develop at random
  • Doerr, summing up an extensive review of experimental data in this field, stated: "We must accept that it has been definitely demonstrated that natural antibodies can develop without an antigenic stimulus, and that this spontaneous formation is by far the most frequent origin of natural antibodies against bacteria erythrocytes, toxins, and virus particles."
  • The slight phagocytosis promoting activity found in normal sera has been ascribed to the presence of normal specific opsonins and these substances may be equated with natural antibodies
  • In poorly responding animals the antigen remains in circulation much longer than in good responders which was interpreted by Jerne to mean the spontaneous presence in the circulation of less or more specificially fitting globulin, which determines both the rate of phagocytic removal and the amount of specific globulin, will be carried to the globulin-repioducing system
  • The methods available for demonstrating the presence of specific antibody in serum (i.e. indirectly) rarely permit the detection of concentrations lower than 10^12 or 10^11 antibody molecules per milliliter
  • Among the comparatively small number, perhaps a few thousand, of antigen-antibody systems investigated, cross-reactions are by no means rare, suggesting that the number of specific configurations which a globulin molecule can exhibit is large but limited
  • The developmental and functional relationship between phagocytic endothelial cells, lymphocytes, and plasma cells is not clear
  • The lymphocytes seem to pioduce some gamma globulin; but whether they take part in the production of antibody after an antigen stimulus is undecided
  • Jerne believed that the plasma cells are definitely involved in antibody production
  • In conditions of hyperglobulinemia and of hyperimmunization there is a large increase in plasma cells in the bone marrow and elsewhere
  • After intravenous injections antibody-producing plasma cells can be recognized in the red pulp of the spleen
  • In other word, Jerne equated seeing increased plasma cells as signs of antibodies without directly observing antibodies
  • The thymus, though rich in nucleic acid and lymphocytes, develops no plasma cells and does not show any immunological activity
  • Burnet and Fenner, in spite of this, suggest that the lymphocytes may be responsible for maintenance of low levels of antibody long after the plasma cells have initiated the formation during the peak phase of the antibody response
  • Though they infer that, if this suggestion is true, the immunological function of the lymphocytes can hardly be a major one
  • If it were true that antibody production after an antigen stimulus is the preferential replication of selected globulin molecules, the production of normal globulin might be an unselective reproduction of the circulating globulin
  • This could be accomplished either by a mechanism by which a small fraction of circulating globulin could enter into lymphoid cells or by the transfer of synthesizing units from worn-out lymphocytes and plasma cells, returning to the lymphoid tissue, to young cells (i.e. Jerne is throwing out random possibilities from his imagination for how this could work)
  • Jerne felt it must be postulated that, somewhere in the beginning, a spontaneous production of globulin molecules of a great variety of random specificities in order to start the process
  • Possibly a specialized lymphoid tissue, such as that of the thymus which is most active in embryonic and early independent life and decays soon after, is engaged in this function
  • He claimed that if this small spontaneous production of globulin took place mainly in embryonic and early life, before the much larger body of cells later engaged in maintaining the composition of the circulating globulin by reproduction had started to function, the early removal of a specific fraction of molecules might lead to the permanent disappearance of this type of specificity
  • He felt such a mechanism could explain the absence in the blood of specific globulin against antigens of the organism itself
  • This might occur also if a specific type of globulin molecules could be removed from an adult animal, and might be the explanation of the "immunological paralysis" described by Felton
  • He felt that perhaps a certain fraction of the haptenic particles, by association with a protein, are capable of antigenic stimulation but cannot act when the larger, purely haptenic fraction eliminates all the spontaneously present antibody molecules
  • The crucial point of the natural-selection theory is the postulate that the introduction of antibody molecules into appropriate cells can be the signal for the production of more of their kind
  • Jerne admitted that this notion was unfamiliar, however, as nothing is known about the mechanism of antibody synthesis in a cell, he felt it would seem a priori more reasonable to assume that an animal can translate a stimulus, introduced by protein molecules which it has itself at one time produced, into an increased synthesis of this same type of molecules than to suppose that an animal can utilize all sorts of foreign substances and can build them functionally and semipermanently into the most intimate parts of its globulin-synthesizing cells
  • He stated that any attempt to elaborate on this notion into a picture of what may be the mechanism of a replication of specific protein must be highly speculative
  • Discussions of the nature of adaptive enzyme formation (as championed by Burnet) were also in a speculative stage
  • A mechanism of this sort would be needed for the present theory of antibody formation: if RNA is the template on which protein molecules are assembled, an antibody molecule introduced into the appropriate cell must be able either to initiate the synthesis of specific RNA or to combine with pre-existing RNA
  • The present theory predicted that the amount of antibody produced in response to an antigen stimulus depends on the concentration of a type of circulating globulin which can attach itself specifically to the surface of this antigen
  • By introducing antibody from an immunized donor animal into the blood of a recipient animal, prior to the injection of the antigen into the latter, enhanced antibody response to this antigen stimulus should be expected, yet the common finding in such an experiment is a depression of the response
  • When serum from a donor animal is used, the possibility should be considered that the antibody molecules may have been altered, during clotting, separation, and storage of the serum, from the form in which they existed in the circulating plasma
  • It had been observed that a booster response to diphtheria toxin does not occur in an animal after the effects, produced by the primary stimulus, have worn off and antitoxin is no longer demonstrable
  • In cases in which cross-reactivity between two pairs of antigen-antibody systems is due to related surface patterns, a secondary stimulus with the second antigen following a primary stimulus with the first antigen may lead to the increased production of antibody of the first type
  • Jerne summarized his theory of antibody formation as a proposal which postulates the spontaneous presence, in the blood of an animal, of small numbers of antibody molecules against all antigens to which the animal can respond, and delegates to the antigen the sole role of carrying such specific globulin molecules from the circulation into cells in which these molecules can induce the production of more of their kind
  • In other words, Jerne proposed a theory that antibodies exist naturally in the blood of animals as these entities still remained unseen in a purified/isolated state
  • Jerne felt his theory offered an explanation for:
    1. The presence in blood of a large pool of normal globulins
    2. The presence of natural antibodies
    3. The dominant part played by the surface of antigen particles in antibody induction
    4. The change in character of antibody during the course of immunization (by natural selection)
    5. The exponential increase during part of antibody production
    6. The continued production of antibody in the absence of the antigen
    7. The booster phenomenon
    8. The absence of auto-antibodies
    9. The immunological paralysis and haptenic inhibition
    10. The anamnestic reaction

And they had deep discussions on many nights, leaving Bussard [another immunologist] with the conclusion that Jerne was a person who primarily "plays with ideas….he plays in his own mind"'

https://brunolemaitre.ch/narcissism-science/testp-page/

Niels Jerne created theories with no crucial experimental evidence to back them up. His ideas belonged to the realm of the artist. He used creative thinking to conjure up fantasies that amounted to nothing but pure speculation, as admitted in his biography as well as by Jerne himself in the first paragraph of his 1955 paper. Jerne used his own view of his personal self to construct his ideas into theories. As the best storytellers often do, he weaved together disparate elements from unrelated experiments into a "cohesive general image" of how antibodies form and function within the body, even though the entities themselves had never been seen and this entire process could never be observed. Jerne was awarded a Nobel Prize in 1984 "for theories concerning the specificity in development and control of the immune system." In other words, he was awarded for his fictional storytelling skills.

Because of Jerne's contributions, there were now four competing narratives in 1955 explaining the unobservable. While these are all given the name "theories," they really should be called hypotheses as nowhere in the creation of these differing tentative explanations was the scientific method ever applied. The entities being discussed were still unpurified and never isolated. Antibodies had never been observed. They were not available to be used as a physically existing independent variable in order to determine cause and effect. They were assumed to be within the blood and causing certain chemical reactions during experimentation. Thus, the unrelated experimental data could be interpreted many different ways and depended upon the eye and imagination of the beholder in order to try and explain the contradictory findings. The data needed to be continually massaged into a "cohesive general image" that would be accepted by the majority as the truth rather than the pure speculation that it actually was. In essence, these antibody "theories" amount to nothing more than ink blots waiting to be reinterpreted by the next observer.

ViroLIEgy
15 May 2022 | 4:05 pm

The Indirect Template Theory of Antibody Production (1949)


From 1900 to 1949, there were two main competing theories attempting to explain antibody production and formation as they had never been purified nor isolated directly from the fluids and proven to exist. Antibodies were still unseen fictional creations assumed to be within the blood acting as defenders against invading pathogens, thus researchers needed to take unrelated experimental results produced by different researchers over the years and cram them together in order to fit into a cohesive narrative. The first of these was the Side-Chain theory formulated by Paul Ehrlich in 1900. It is considered part of the Selection theories of antibody production which believes that antigens react to antibodies already existing within the body. It was the main theory explaining what antibodies are and how they form and function for the better part of three decades. However, in 1930 the Direct Template theory was proposed by Fritz Breinl and Felix Haurowitz and later revised by Linus Pauling in 1940. This was considered an Instruction theory as antigens were thought to play a prominent role by serving as a genetic template used to create specific antibodies. These two theories created a bit of a divide between biologically-trained and chemically-trained immunologists as they attempted to explain, according to their backgrounds, how antibodies and the immune system worked.

Fortunately or unfortunately, depending on how you look at it,  Austrailian virologist Frank MacFarlane Burnet determined in 1949 that a third interpretation was warranted in order to rectify some of the loopholes in the earlier theories. His view of the sea of indirect evidence, hypotheses, and theories produced over the decades led him to be in lockstep with Breinl, Haurowitz, and Pauling's instructionist view. However, there were some key differences which led MacFarlane to propose what became known as the Indirect Template theory in the second edition of his book fittingly titled "The Production of Antibodies." The book itself is 142 pages long so, needless to say, I will not be posting the entirety of it here. However, we can find a few sources which paraphrase the theory directly from the book to give us an idea of what MacFarlane proposed.

From Immounopedia.org:

The Indirect Template Theory

"(i) To account for the non-antigenicity of body components these were assumed to carry 'self-markers;" at some point in the antibody-producing sequence a 'recognition unit' was postulated to act as a means of detecting material carrying self-markers and deflecting it from the possibility of immune response;

(ii) To account for the persistence of antibody-producing capacity it was postulated that a 'genocopy' of the antigenic determinant was incorporated in the genome of the Stem cell concerned, so allowing the indefinite production of descendant antibody-producing cells;

(iii) This incorporation of pattern determinants into the genetic structure of antibody-producing cells provided some basis for the changes in antibody character that may result from secondary antigenic stimuli or simple lapse of time."

Burnet, F.M & Fenner, F. The Production of Antibodies. Monograph of the Walter and Eliza Hall Institute, Melbourne, 2nd Edition, 1953

https://www.immunopaedia.org.za/timeline/the-indirect-template-theory/

From this short excerpt, we can see that MacFarlane was attempting to explain how antibodies persist in the blood to provide immunity after the antigen has been cleared out of the body. As he believed that antibodies continued to circulate in the body long after the antigen was gone, this meant that the antigen could not be the template continuing to produce the antibodies one by one by direct contact. This was in opposition to Breinl, Haurowitz, and Pauling's Direct Template theory which proposed that the antigen goes into the cell and stamps its code onto each antibody after it is produced. Thus, MacFarlane alternately proposed the Indirect Template theory which stated that the antigen incorporates itself into the genetics of the cell in order to produce an endless supply of antibodies.

In 1950, Nature wrote an article which provides us with further insight into Burnet's Indirect Template theoty. There are some rather interesting admissions made regarding his work:

PRODUCTION OF ANTIBODIES

"The first edition of this essay, published in 1941, has long been rather hard to come by, and the appearance of a second is therefore proportionately more welcome. Since the publication of the first edition, and to a large extent because of it, antibodies have been interesting biologists more and more, in spite of the inroads of antibiotics into their therapeutic uses.

Dr. F. M. Burnet has not changed his ground. Antibody formation is thought to be the consequence of an inherited change in the pattern of synthesis of serum globulin in mesenchymal cells; that is, a change which endures through repeated fissions long after the physical disappearance of the agent which in the first place brought it about. (It makes no important difference to the theory what sort of cell actually makes antibodies, and the authors now give due weight to recent evidence incriminating the plasma cell and the lymphocyte, or its precursor.) According to Burnet's theory, therefore, antibody-formation is the outcome of an inherited cellular transformation. The 'chemical' theory, which in one form or another the authors couple with the names of Mudd, Haurowitz and Pauling, requires the continued presence of antigen during the entire term of antibody formation, the antigen being (crudely) a mould or template for distorting normal into antibody globulin. It is clearly a matter of the utmost technical difficulty to decide whether antibody formation outlives its stimulus or not, and the evidence one way or the other is at present quite inconclusive.

The incompatibility between the chemical and biological' theories turns solely on the question of whether or not a cellular transformation is entailed. As the authors point out, the mechanism envisaged by the chemical theory may well prove to be correct in principle, though it may not take place at such a late stage in the assembly line of globulin synthesis as is commonly thought. It is essential for Burnet's theory that the formation of antibodies is subsidized by a cytoplasmic self-reproducing system. Extracellular proteins like serum globulins are the reproductively inert by-products of the synthetic activity of an enzyme system which is undergoing continual replication in the cytoplasm. Antibody is serum globulin formed as the by-product of an enzyme system which, under the impress of antigen, has submitted to a slight inherited change that is in some important ways analogous to that responsible or the formation of adaptive enzymes in bacteria an analogy supported since the publication of the first edition by Hinshelwood's interpretation for the adaptive process. The sort of molecular distortion envisaged by the chemical theory might be supposed to occur in the Burnet enzyme system itself rather than in the texture of its finished product-an idea which fits easily into the pattern of modern speculation about the nature of self-reproducing systems.

However, the authors would be the first to admit that much of the evidence they bring forward in support of their views is no more than suggestive. The existence of a short phase of exponential expansion of circulating antibody is poor evidence for the existence of a multiplicative antibody-forming system, though it is certainly consistent with such a possibility; and as evidence that antibody formation outlives its antigenic stimulus, the very long-drawn-out immunity that follows infection by some viruses is admittedly clouded by the possibility that a trace infection by virus does indeed persist. But to say this is to say the worst: this is a book which will stimulate or goad every immunologist into thinking afresh about old problems and coming to terms with new ones. Among the matter new to this edition is an ingenious and most stimulating commentary on the important and disturbing fact that embryos do not form antibodies, worked in with a theory of how antibody-forming cells come to distinguish native from foreign organic molecules. Another is the chapter on transplantation immunity, containing the suggestion that the response to the grafting of foreign homologous cells has something in common with the type of sensitivity provoked by tuberculin.

In summary, the immunologist needs no special inducement to read this essay, for he will do so anyway; but it is important that every biologist interested in the problems of cellular heredity and transformation should be aware of the rather direct bearing of antibody formation upon them."

https://doi.org/10.1038/166204a0

As can be seen from the Nature article, Burnet believed that antibody formation was "thought to be the consequence of an inherited change in the pattern of synthesis of serum globulin in mesenchymal cells." Oddly enough, it didn't really matter to his theory which cells actually produced antibodies as it seemed any old cell would do. It was also admitted that the evidence presented for his theory was only suggestive. In other words, as with the theories before it, there was no conclusive evidence proving Burnet's theory as the correct one. Perhaps this had to do with the continued lack of direct evidence in the form of purified and isolated particles assumed to be antibodies for which the researchers could study and learn from rather than throwing out guesses as to how these unseen entities look and function based on indirect chemistry experiments?

In the abstract for the book by The Journal of Immunology, it is stated that Burnet's theory is based on the assumption of a self-producing enzyme. The assumption itself comes from logarithmic curves showing an increase in antibody titer. Because of this, it was assumed that "there must be something, somewhere proliferating" which is just another way of saying "we can not observe the antibodies directly so we must assume their presence indirectly:"

"In this second edition of Burnet's well-known book on the production of antibodies the authors have clarified and extended the views advanced previously. They believe that the first injection of an antigen causes the modification of intracellular enzymes, so that these become adapted to the antigen. Subsequent antigen injections stimulate the replication of the adapted enzyme units formed. The circulating antibodies are considered as partial replicas of the enzyme units, devoid of enzymic activity.

The assumption of self-producing enzyme units is based chiefly on the initial logarithmic increase of the antibody titer. The logarithmic shape of the curve leads the authors to believe that "there must be something, somewhere proliferating". They assume that the enzymes which are normally involved in the disposal of expendable body cells, become adapted to antigens which are similar to the normal substrates of the same enzymes."

https://www.jimmunol.org/content/66/4/485

While it should be a red flag that there needed to be not one but various theories for how antibodies supposedly look, form, and function, there were a few issues related to Burnet's theory that eventually led to it being disregarded. In a 1994 article by American immunologist Melvin Cohn, it is stated that Burnet's rejection of the Direct Template theory was weak and that his evidence supporting his argument for antibody production long after the antigen was cleared was controversial and unconvincing. Cohn also pointed out that the argument rested on whether one could measure residual antibody secretion by end cells (plasmacytes) after induction had ceased which was beyond the experimental capabilities:

THE WISDOM OF HINDSIGHT

"As the vast majority of immunologists were "laissez-faire" instructionists, Burnet & Fenner's rejection of Pauling's direct template theory might have been important, but their argument was weak. They argued that if antigen were a template, then antibody synthesis would cease when antigen was ridded. They then cited evidence that antibody synthesis continued long after any antigen could possibly be present in the animal, but this remained controversial and unconvincing for a simple reason. All theories, on a priori grounds, require that induction of antibody cease when antigen is eliminated. Consequently, their argument rested on whether one could measure residual antibody secretion by end cells (plasmacytes) after induction had ceased, and this had to be beyond the experimental methodology of the time, thereby leaving in its wake a useless polemic."

doi: 10.1146/annurev.iy.12.040194.000245.

Another strike against Burnet's theory was that it was later determined that there were antibodies of different specificities with different amino acid sequences:

"This explained specificity and the secondary response but it was abandoned when it was known that antibodies of different specificities had different amino acid sequence in their combining sites."

https://ecoursesonline.iasri.res.in/mod/page/view.php?id=61813

It was also stated that in nature, there was no need for the antigen to enter the B cell in order to produce antibodies so this apparently also disproved his theory:

"Burnet and Fenner proposed this instructive theory to explain the synthesis of antibody as an adaptive protein. According to this theory, antigen enters into B cell and it binds to its DNA and modifies it and forms this modified DNA, antibodies are produced against antigen and it also specific for it.  But in nature, there is no need for an antigen to enter into B cell and modify DNA for antibody production because soluble antigen can activate B cell by binding to its cell surface receptor BCR.  Because of this reason, this theory also disproved."

https://biosiva.50webs.org/plasmacell.htm

In the end, after championing it for nearly a decade, Burnet abandoned his Indirect Template theory and eventually proposed what became the "definitive" antibody explanation known as the Clonal Selection theory in 1957.

In Summary:
  • At some point in the antibody-producing sequence, a 'recognition unit' was postulated to act as a means of detecting material carrying self-markers and deflecting it from the possibility of immune response
  • It was postulated that a 'genocopy' of the antigenic determinant was incorporated in the genome of the Stem cell concerned, so allowing the indefinite production of descendant antibody-producing cells
  • This incorporation of pattern determinants into the genetic structure of antibody-producing cells provided some basis for the changes in antibody character that may result from secondary antigenic stimuli or simple lapse of time
  • In other words, this theory was used to explain how antibodies could remain in the blood to provide "immunity" after the antigen was dealt with
  • Antibody formation was thought to be the consequence of an inherited change in the pattern of synthesis of serum globulin in mesenchymal cells; that is, a change which endures through repeated fissions long after the physical disappearance of the agent which in the first place brought it about (i.e. antibodies exist long after the antigen disappears)
  • According to Burnet's theory, therefore, antibody-formation is the outcome of an inherited cellular transformation
  • It makes no important difference to the theory what sort of cell actually makes antibodies
  • It was a matter of the utmost technical difficulty to decide whether antibody formation outlives its stimulus or not, and the evidence one way or the other was quite inconclusive
  • The incompatibility between the chemical and biological' theories turns solely on the question of whether or not a cellular transformation is entailed
  • It is essential for Burnet's theory that the formation of antibodies is subsidized by a cytoplasmic self-reproducing system
  • The sort of molecular distortion envisaged by the chemical theory might be supposed to occur in the Burnet enzyme system itself rather than in the texture of its finished product-an idea which fits easily into the pattern of modern speculation about the nature of self-reproducing systems
  • The authors would be the first to admit that much of the evidence they bring forward in support of their views is no more than suggestive
  • The existence of a short phase of exponential expansion of circulating antibody is poor evidence for the existence of a multiplicative antibody-forming system, though it is certainly consistent with such a possibility
  • Burnet also worked in a theory of how antibody-forming cells come to distinguish native from foreign organic molecules
  • The assumption of self-producing enzyme units is based chiefly on the initial logarithmic increase of the antibody titer
  • The logarithmic shape of the curve led Burnet to believe that "there must be something, somewhere proliferating"
  • Burnet assumed that the enzymes which are normally involved in the disposal of expendable body cells, become adapted to antigens which are similar to the normal substrates of the same enzymes
  • Burnet & Fenner's rejection of Pauling's direct template theory might have been important, but their argument was weak
  • They argued that if antigen were a template, then antibody synthesis would cease when antigen was ridded
  • They then cited evidence that antibody synthesis continued long after any antigen could possibly be present in the animal, but this remained controversial and unconvincing for a simple reason
  • All theories, on a priori grounds, require that induction of antibody cease when antigen is eliminated
  • Consequently, their argument rested on whether one could measure residual antibody secretion by end cells (plasmacytes) after induction had ceased, and this had to be beyond the experimental methodology of the time, thereby leaving in its wake a useless polemic

Frank MacFarlane Burnet's Indirect Template theory was a short-lived explanation for the production and formation of antibodies that never really got off the ground. It joined two other attempts at taking unrelated indirect experimental findings and forcing them together into a cohesive description for unobserved phenomena. As with the Side-Chain and Direct Template theories before it, Burnet's Indirect Template theory had its own view and interpretation of the experimental evidence accumulated over the decades. He was able to furnish his own findings as well as those of others in order to support his overall hypothesis. Much like Linus Pauling's work with the Direct Template theory, Burnet's proposal was considered a legitimate theory initially. Other researchers built off of his theory with their own work and experimentations. However, Burnet's original theory eventually fell out of favor which means much time and effort was wasted on a false theory which produced research which could only lead to results which would be considered false and erroneous. The theory led researchers in the wrong direction.

Burnet's Indirect Template theory is a shining example as to why definitive conclusions should not be made about the assumed existence, form, and function of invisible entities. It is only a matter of time before someone else comes along and interprets the indirect experimental evidence in a different light and proposes another theory which may be accepted by the consensus majority instead. This leads to a revolving door of theories for something which can not be seen nor explained as antibodies started off as an idea rather than an observed phenomena in nature. A theory is supposed to be born out of the observation of natural phenoma, not built from an idea of something invisible assumed to exist within the blood. A theory is supposed to be assembled from facts and results aquired from the implementation of the scientific method. The scientific method requires a valid independent variable in order to determine cause and effect. As antibodies have never been purified and isolated directly from the fluids, there is no independent variable in order to complete the scientific method. Thus the antibody theories are not scientific.

What this means is that there was a massive amount of wasted time and literature that was spent on unscientific and disproven theories such as the Direct and Indirect Template theories. In many cases, this would be fine as this is how science is supposed to work. Theories are made to be disproven and replaced. However, this becomes a problem when unproven theories are used as a means of implementing health measures. Antibodies are presented as if they exist and that the current theory is considered to be the stone cold truth. Medical decisions, treatments, vaccinations, therapies, etc. are made based on the latest favorable theory which may some day soon fall out of favor for something "better." Decisions are determined during supposed pandemics based on whether or not antibodies exist and function in the way that the theory proposes that they do. However, the evidence is contradictory and the theory remains unproven.

What happens when the antibody theory is ultimately disproven? Where does that leave the person who was vaccinated and believed a theoretical rise in antibodies was a good thing yet ended up injured due to the toxic side effects? How does this help the person suffering serum sickness after taking experimental monoclonal antibody therapy as a cure for a "virus?" What does this mean for the research promising medical marvels and insights which is ultimately unreproducible and irreplicable due to the non-specific reactions of the theoretical antibodies? Where does it leave us when antibodies are used as a means to determine one is "protected" or not in order to give or take away our rights?

Unproven theories are being presented as the unbridled truth and are used to impact our health and our freedoms. Antibodies are the perfect scapegoat to keep us blind to the "virus" lie and believing in the power of vaccination. They are the opposite side of the viroLIEgy coin and the lack of evidence for these theoretical entities must be scrutinized to the same extent.

ViroLIEgy
12 May 2022 | 3:13 pm

Antibodies, Plasma, and the Power of Correlation


There is a real problem in the sciences that goes far beyond the methods and experiments. It is a deeper issue that has more to do with the very ideology used by the researchers themselves which permeates throughout much of the scientific literature. It is this idea that finding indirect evidence of assumed particles or even genomic sequences in a (heavily altered) tissue, cell culture, and/or blood sample can legitimately point to a cause and effect relationship. However, this is obviously not the case and those who promote this idea are engaging in what is known as the false-cause logical fallacy. This is better known either by the phrase "with this, therefore because of this" or "correlation does not equal causation:"

Correlation vs. Causation

"The place where this fallacy shows up the most often is in (the media), which unfortunately is where most people get their science information and news. Imagine you're looking to buy a magazine. Which headline best grabs your attention:

  1. "One study on a limited population shows that when people do X, Y happens a certain percentage of the time."
  2. "Link found between doing X and Y happening!"
  3. "New research shows that X causes Y."

If you ever read a scientific paper, you'll find that almost all scientists make statements like that first one. However by the time this research hits the popular media, it's often transformed to look a lot more like that last one."

https://www.google.com/amp/s/www.quickanddirtytips.com/education/science/correlation-vs-causation%3famp

We see this all the time in science. It is very common to find correlations in relationships yet know nothing about the cause. Sadly, more often than not, these supposed relationships are assumed to be one of cause-and-effect without providing any direct evidence that this is the case.

Take, for instance, the work of Astrid Fagraeus. It was said that a "breakthrough" in Immunology occurred in 1947-48 when Astrid Fagraeus published her doctoral dissertation "Antibody Production in relation to the Development of Plasma Cells." She was given the credit for showing that the plasma cells produce antibodies. Before her paper, the function of plasma cells was unknown. But did Astrd Fagraeus really prove the correlation between plasma cells and antibodies definitively?

According to the book "Advances in Immunology" by Frederick W. Alt, she did not:

"In 1902, the Russian scientist
Alexander Maximow summed up the findings on plasma cells in a comprehensive pathological work. Based on the studies of Marschalko, Almkvist and others, Maximow described plasma cells as "mononuclear leukocytes that have emigrated from blood vessels and are particularly altered, mostly lymphocytes, which are present in normal lymphoid organs and pathological tissues and may possibly provide stable elements" (Maximow, 1902). At that time the nature of "stable elements" remained unclear, but from today's perspective, these stable elements are certainly secreted antibodies. In 1938, Fred Kolouch Jr. (Minnesota) directly correlated the formation of plasma cells with injection of the antigen. Moreover, he made the essential finding that increase of serum antibodies accompanied the accumulation of plasma cells in the bone marrow (BM) and postulated for the first time that plasma cells produce antibodies, even though he had no direct experimental evidence for his hypothesis (Kolouch, 1938). This breakthrough in immunology was achieved in 1948 by the Swedish immunologist Astrid Fagraeus (Fagraeus, 1947, 1948). During her studies on multiple myeloma (MM), she observed an increase of plasma cell numbers in hypergammaglobulinemia, a finding which had already been described earlier by Bing (Bing, 1940). Fragraeus performed in vitro studies with plasma cell-enriched spleen biopsies from horse serum-injected rabbits and found a direct correlation between plasma cell numbers in the tissue and the amount of secreted antibodies in the culture medium. However, she was unable to provide clear evidence that plasma cells were indeed the cells that produced antibodies. Finally, in 1955, Leduc and colleagues succeeded in concluding that plasma cells produce antibodies by using immunofluorescence to detect antigen-binding proteins (i.e., antibodies) in morphologically defined plasma cells in lymph nodes of diphtheria-toxin-immunized rabbits (Coons, Leduc, & Connolly, 1955; Leduc, Coons, & Connolly, 1955)."

https://doi.org/10.1016/bs.ai.2020.01.002

As can be seen by the above source, many scientists, including Fagraeus, found correlations between plasma serum and antibodies. However, Fagraeus could not provide clear evidence of the "direct correlation" that she observed. Did this lack of proof stop the accolades by the scientific institutions announcing Fagraeus's breakthrough? No, the lack of clear evidence did not stop the scientific community from recognizing Fagraeus and her findings as proof that plasma cells produce the still unseen antibodies:

"Fagraeus' doctoral dissertation, 'Antibody Production in relation to the Development of Plasma Cells', attracted international attention and was considered a milestone in modern immunology. In this work, she was the first to show that plasma cells produce antibodies (IgG). Until then their function was unknown."

https://en.m.wikipedia.org/wiki/Astrid_Fagraeus

"When Behring and Kitasato demonstrated the specific protection of rabbits by serum from infected animals in 1890 [4], it became clear that serum antibodies could provide "immunity", i.e., protection against a recurrent infectious challenge. It took another 60 years until Astrid Fagreus discovered the cells secreting those antibodies, the "plasma cells", describing them as "terminal stages of B cell differentiation" [5]. "Protective memory", as provided by serum antibodies, was later conceptually complemented by a "reactive memory" formed by expanded and long-lived populations of antigen-experienced T and B lymphocytes."

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3337994/

"In 1943, Mogens Bjørneboe and Harald Gormsen were the first to experimentally show that repeated immunization of rabbits with polyvalent vaccines leads to massive proliferation of plasma cells in most organs and that this proliferation correlates with antibody concentration.

That finding was supported a few years later by Astrid Fagraeus, who reported that plasma cells produce antibodies in vitro. Tissue cultures of spleens from rabbits immunized with live bacteria showed abundant formation of plasma cells. Fagraeus concluded that plasma cells appear in connection with strong antigen stimulation."

https://www.nature.com/articles/ni.3602

While the actual dissertation paper was difficult to find, I did manage to obtain and take an image of the Nature article in which she details her work. While I was unable to copy/paste from the article itself, there was a summary provided by the Journal of Immunology:

doi: 10.1038/159499a0

Before the summary, let's look a little closer at what Fagraeus did to achieve her results:

Pieces of rabbit spleen were cultured in normal rabbit serum, tyrode solution (an acidic solution made up of various substances), and isotonic sodium bicarbonate (generated by adding 150 mEq of sodium bicarbonate to a liter of 5% dextrose). A gas mixture of 80% oxygen, 4.5% carbon dioxide, and nitrogen was then passed into the tubes which were incubated at 37° C for 5-12 hours. From this tissue cultured mixture, Fagraeus assumed that the accumulation of plasma cells meant that they produced antibodies. Listed below is a summary of the results:

The Plasma Cellular Reaction and Its Relation to the Formation of Antibodies in Vitro

The Journal of Immunology 58 (1), 1-13, 1948

Summary

1. During secondary response, elicited in sensitized rabbits by means of intravenous injections of antigen, a great increase in the number of plasma cells (pl.c.) in the spleen was recorded simultaneously with the increase of circulating antibodies.

2. Pl.c. were confined almost exclusively to the red pulp, especially after the injection of living S. typhi. They originated apparently from reticulum cells, passing through a chain of development: transitional cell → immature pl.c. → mature pl.c.

Pieces of spleen were excised at different times during the period of antibody formation in rabbits, differential cell counts were made and the capacity of excised splenic tissue to form antibodies in vitro was investigated. The following observations were made:

a. The amount of antibody liberated in plain tissue extracts, under conditions preventing growth or metabolism of cells, was very low, whereas significant yields were obtained in tissue cultures.

b. The capacity of the red pulp abundant in pl.c. to produce antibodies in vitro was considerably superior to that of lymph follicles, rich in lymphocytes but devoid of pl.c.

c. Antibody production was comparatively poor in tissue containing only transitional cells, reached a maximum when numerous immature pl.c. were present and receded when predominantly mature pl.c. were found.

4. After intravenous injections the antigen accumulated in those places where pl.c. subsequently developed.

The conclusion is drawn that antibodies under the conditions of the experiments, are formed by cells of the R.E.S., passing through a chain of development, the final link of which is the mature pl.c.

https://www.jimmunol.org/content/58/1/1.short

doi: 10.1038/159499a0.

While the methods used to determine antibody content is not overly clear beyond staining with methyl green pyronine and the steps used for tissue culturing, it is clear that Fagraeus relied on the power of correlation to suggest that plasma cells create antibodies. This process of plasma cells producing antibodies could not be observed as antibodies themselves have never been observed in a purified/isolated state nor can such a production process be seen inside of a living organism. Fagraeus relied on the proliferation of plasma cells being a correlation of antibody content and assumed that staining patterns were able to tell her that antibodies were present in the dead tissue culture sections. Obviously, staining tissue cultures would be a form of indirect evidence which is probably why Frederick Alt concluded that even though Fagraeus found a "direct correlation" between plasma cell numbers in the tissue and the amount of secreted antibodies in the culture medium, she was unable to provide clear evidence that plasma cells were the cells that produced antibodies. Granted, Alt then immediately stated that in 1955, Leduc and colleagues were successful in concluding that plasma cells produced antibodies by using immunofluorescence to detect antibodies in morphologically defined plasma cells in lymph nodes of diphtheria-toxin-immunized rabbits. However, was this truly the case? How effective is immunofluorescence at detecting antibodies? From the first sentence in the discussion section of the 1955 paper listed as a reference, Leduc and colleagues state this:

"The method employed for the histological demonstration of antibody is clearly specific, despite the confusion caused at times by the occurrence of non-specific reactions."

doi: 10.1084/jem.102.1.49.

In other words, the method they used to determine antibody content was not specific even though they claimed that it was. This is a nice case of scientific double-speak attempting to confuse the audience into accepting the idea that the methods used are in fact valid when the evidence shows otherwise. In a follow-up paper by Coons in 1956, he admitted to some other interesting revelations:

"The use of labeled antibody requires a certain familiarity with the methods and tradition of immunology, as well as with the more strictly practicaI procedures used in the purification of antigens and the production of antisera. There are of course many antigenic substances which can
theoretically be studied under a variety of circumstances by such means. The specificity of every reaction must be established by appropriate controls, the character of which will vary with the substance and the circumstance. Labeled antibodies so employed identify objects and localize them; they are most effective when used to answer a morphological question."

"The nonspecific reactions referred to above are due to at least three elements: side reactions producing derivatives of fluorescein not readily removed by dialysis (these "stain" elastic tissue, blood vessel endothelium, and perhaps other elements), probably proteins in serum which react with tissue components (cf. Kidd and Friedewald, 1942), and finally antibodies against tissue components occurring as a result of immunization (e.g., Watson and Coons, 1954)."

"The antibody content in these cells increased, as judged by observation of the intensity of fluorescence, during this sequence."

https://doi.org/10.1016/S0074-7696(08)62565-6

Antibodies can bind non-specifically you say?

According to Coons, the purification of the antigen and the production of antisera are of critical importance to obtaining results. Oddly, he says nothing about the importance of purifying and isolating the antbodies. Theoretically, there are a variety of antigens that can be studied under a variety of circumstances using their method. However, appropriate controls are a necessity to determine specificity as specificity will vary with the substance and the circumstance. Non-specific reactions do occur and it was stated that there were at least three reasons for this:

  1. The stain reacts to other tissues
  2. Other proteins in the blood react to the tissue
  3. Antibodies are present from immunization
  4. ???

Thus, Coons believed that it was not a failure of the immunofluorescence methods they utilized to be specific in identifying antibodies. Instead, inconsistent results were explained away by his three postulated excuses, along with the possibility that other unknown excuses remained for the apparent lack of specificity. Beyond these reasons, the method itself was apparently "clearly specific."

The reason this matters is shown in the final highlight where Coons stated that the antibody content in these cells increased, as judged by observation of the intensity of fluorescence. In other words, the only way they could determine antibodies were present in the tissue culture samples was due to the fluorescence stain which they used as a guide to tell them the amount of antibody by the intensity of the fluorescent signal. Beyond the fact that this is an indirect way to detect and measure unseen entities, if the test is not specific and regularly cross-reacts with other substances for various reasons, the results can not be specific to detecting antibodies and are essentially meaningless. The use of fluorescence to determine antibody is just another example of a correlation being established as a fact when direct evidence does not establish a causal relationship. As Coons pointed out, the above 3 excuses, as well as any other unknown variable he did not think up to list, may have been the cause for any of the results rather than the assumed antibodies. Thus, the correlation between the fluorescence signal and the presence of antibodies remains unproven.

What must be understood about any of the evidence relating to antibodies is that there is nothing there but dreamt up hypothetical fantasies and unrelated chemistry experiments attempting to claim an invisible entity was the cause of the observed lab-created effects. It is the same exact trick used in virology which is why these two (pseudo)sciences go hand-in-hand. If the entity in question has never been observed in a purified and isolated state and was conjured up without ever being found in nature, none of these reactions matter as the results are being applied to a fictitious entity. A story is created in order to try and explain the unrelated experimental results in order to fit the latest and greatest revision of the agreed-upon unproven theory.

See that band-like deposit? That band represents "antibodies."

Finding plasma cells near fluorescent dyes in stained tissue cultures does not mean that antibodies are present nor that plasma cells produce these imaginary entities, especially when the method used is known to be non-specific and prone to cross-reactions. If various scientists get the same or similar results using a similar method, this still does not prove the correlation correct. All that does is strengthen the correlation. In order to prove that plasma cells produce antibodies, the antibodies themselves must be shown to exist in a purified and isolated state first. Only then would any immunofluorescence test be able to be calibrated and validated to the intended target so that the results have some semblance of any meaning. Even then, as the process of plasma cells producing antibodies is unobservable, this connection would still remain nothing more than a strong correlation.

However, none of this matters as it is very apparent that the scientific community is of the mindset that correlation does in fact equal causation. They want you to believe that the fluorescent dye equals antibodies. They want you to believe that the cultured cells dying equals "virus." Yet these researchers would be wise to remember:

"Correlation tests for a relationship between two variables. However, seeing two variables moving together does not necessarily mean we know whether one variable causes the other to occur. This is why we commonly say "correlation does not imply causation."

A strong correlation might indicate causality, but there could easily be other explanations:

  • It may be the result of random chance, where the variables appear to be related, but there is no true underlying relationship.
  • There may be a third, lurking variable that that makes the relationship appear stronger (or weaker) than it actually is.

https://www.jmp.com/en_us/statistics-knowledge-portal/what-is-correlation/correlation-vs-causation.html

ViroLIEgy
10 May 2022 | 4:02 pm

Direct Template and the Theoretical Structure of Antibodies (1930-1940)


I found that Landsteiner and I had a much different approach to science: Landsteiner would ask, 'What do these experimental observations force us to believe about the nature of the world?' and I would ask, 'What is the most simple, general and intellectually satisfying picture of the world that encompasses these observations and is not incompatible with them?'"

Linus Pauling
1970

His student Max Perutz, who shared the 1962 Nobel Prize in chemistry for his studies of the structure of hemoglobin, believes that Pauling's preoccupation with this vitamin, which "spoilt his great reputation as a chemist," might be related to "his greatest failing, his vanity." According to Perutz, Pauling "would never admit that he might have been wrong."

https://online.ucpress.edu/hsns/article/51/4/427/118595/Template-Theories-the-Rule-of-Parsimony-and

After decades of being nothing more than dreamt up fantastical theoretical creations sprawled onto the notepads of Paul Ehrlich, it was still unknown how antibodies were formed, how they looked, and how they interacted with antigens once introduced into the body. Thus in 1930, virologists Fritz Breinl and Felix Haurowitz took their stab at answering the various unknowns by coming up with their own hypothesis. The dynamic duo stated that it was the antigen itself which entered into the blood cells and acted as a template for creating the antibodies required for immunity. These antibodies were then said to be specific to the antigen which had produced them. This was the second major theoretical explanation for the creation and formation of antibodies after Paul Ehrlich's side-chain theory. It eventually became known as the direct template theory. Like Ehrlich before them, Breinl and Haurowitz also relied on assumptions to create their own hypothesis as antibodies were still unseen invisible entities believed to be floating around within the end result of certain chemical reactions. Sadly, I could not find their original paper as it was unavailable and in German. However, a few sources give us some insight into this collaboration and how their theory came to be. The first source is from the biography of Felix Haurowitz:

FELIX HAUROWITZ

"A new and lifelong research interest began in 1930 and stimulated by a phone call from a colleague, Fritz Breinl, who had just returned from a year at the Rockefeller Institute in New York. Breinl, a virologist, was excited by the experiments of Karl Landsteiner with synthetic haptens. He asked Haurowitz to read the papers and discuss with him what could be done to solve the mystery of antibody production. Thus began an exciting but short-lived collaboration that led to what was later called the template theory of antibody formation. Equally important, it committed Haurowitz to an experimental study of the role of antigen in antibody production for the rest of his research career. As might be expected, they used horse hemoglobin as an antigen in the work for their first paper. Unlike Landsteiner, who used only qualitative indices for the amount of antigen-antibody precipitate (- or +, ++, +++), Haurowitz and Breinl used quantitative methods to determine the amount of hemoglobin in the precipitate, and they also indirectly determined the amino acid content of the nonhemoglobin portion of the precipitate. In his autobiography for the National Academy, Haurowitz underlined the following statement for emphasis: "I concluded that the antibody must be serum globulin and suggested therefore that the antigen interferes with the process of globulin biosynthesis in such a way that globulins complementarily adjusted to the antigen are formed." Thus began the template theory, as it was later called, to which he adhered with some modifications for the rest of his life."

https://nap.nationalacademies.org/read/4547/chapter/7

According to this short section from Haurowitz's biography, their work with antibodies used horse blood as an antigen. They quantitatively determined the amount of hemoglobin in the blood and used indirect methods to determine the amino acid content of the nonhemoglobin part of the precipitate. Somehow, from this separation of their precipitate, Haurowitz was able to conclude (i.e. guess) that the antibody was serum globulin. He then determined that the antigen interefered with the creation of the globulins making them adjust to the antigen in order to clear the antigens out of the system.

While it is an interesting theory on how these invisible entities function and interact, there sadly wasn't much information in the above source to go off of in regards to the methods used to determine this explanation. Fortunately in 1967, Haurowitz wrote a paper discussing his work further. While it doesn't provide much insight into the exact methods used by the two researchers, it does provide us with information on the work of another man who built off of their research 10 years later:

The Evolution of Selective and Instructive Theories of Antibody Formation

THE TEMPLATE THEORY OF ANTIBODY FORMATION

"Ehrlich's view of the selective role of the antigen was accepted for a long time. However, Landsteincr's finding that antibodies are also formed against synthetic haptens which never occur in nature, made it difficult to believe that the organism could have cells precommitted to the formation of antibodies against azophenylarsonate, azophenylsulfonate, and other synthetic products of the chemical laboratories. For this reason, Breinl and Haurowitz (1930) proposed that the administered antigen might interfere directly with the mechanism of y-globulin biosynthesis and modify this process in such a manner that a complementary combining site in the newly formed y-globulin molecule is produced. Similar ideas were advanced later by Alexander (1931) and by Mudd (1932). With Breinl, I suggested that complementariness 
is accomplished by suitable orientation of the amino acid residues in the newly formed globulin molecule. I attributed this action to intermolecular forces, particularly electrostatic interaction, dipole induction, and the short-range van der Waals forces, as shown in Fig. 2 (Haurowitz, 1939). At first sight, this may seem related to the lock and key picture used for the enzyme-substrate system. However, a key cannot produce a lock. One might rather compare the antigen with an electrode and the antibody with a galvanoplastic, formed at this electrode. 

Both the selective theory and the template theory are based on the idea of mutual complementariness of the groups which are responsible for the combination of antibody with antigen. This complementariness allows the reacting groups to approach each other very closely, so that the short-range intermolecular forces become operative and bring about specific combination of antigen with antibody (Haurowitz, 1939). Abundant evidence for this view has been provided by the work of Pauling, Pressman, and their co-workers on the inhibitory action of haptens related to the antigenic determinants (Pauling et al., 1944), and by equilibrium dialyses (Carsten and Eisen, 1955; Karush, 1962) which make it possible to determine the affinity between antigenic determinants and the specific combining sites of antibody molecules.

Our view of a template role for the antigen was taken up ten years later by Linus Pauling (1940) who introduced an important modification by claiming that all antibodies formed in an organism have the same peptide chain with identical amino acid sequences, and that they differ from each other only in their conformation. This assumption was later strongly supported by the finding that the N-terminal pentapeptide of different rabbit antibodies was found to be the same (Porter 1959) and that their amino acid composition was identical (Smith et el., 1955; Fleischer et al., 1961). We now know that the typical 7 S antibodies of rabbits consist of two heavy and two light chains (Fleischman et el., 1963; Edelman and Gally, 1964) and that there are small differences in the amino acid composition of different allotypes. Moreover, it has been shown by work in the laboratories of Tanford (Whitney and Tanford, 1965) and Haber (1964) that denatured antibodies refold spontaneously if the denaturing agent is carefully removed. This rules out the presence of identical amino acid sequences in two antibodies of different specificity, but it does not allow one to choose between a selective and an instructive action of the antigen since the latter also might result in changes in the amino acid composition and sequence."

https://www.google.com/url?sa=t&source=web&rct=j&url=https://citeseerx.ist.psu.edu/viewdoc/download%3Fdoi%3D10.1.1.856.5258%26rep%3Drep1%26type%3Dpdf&ved=2ahUKEwiwz6XAltD3AhV1RDABHUx5C-AQFnoECAMQAQ&usg=AOvVaw1xA888tO37GEWbXdFG_HWI

Linus Pauling: Model Man

As we can see from Haurowitz's paper, it was Linus Pauling, an American chemist who published a vast library of work with well over 1200 papers and books, who carried on fleshing out their theory 10 years later. He is the only person to win two unshared Nobel Prizes and is considered one of the 20 greatest scientists of all time. Besides carrying on the role of the antigen as a template, Pauling also received the distinction of "proving" correct Paul Ehrlich's lock and key mechanism for how antibodies/antigens work:

"The word 'Antikörper', the German for antibody, was first used in a paper by Paul Ehrlich in 1891. Ehrlich proposed a 'lock and key' mechanism of antibody-antigen interaction but this was not confirmed until the 1940s by Linus Pauling."

https://www.google.com/url?sa=t&source=web&rct=j&url=https://www.rcpath.org/uploads/assets/077f9015-91d1-41a4-ba3fd408b884967b/17-Structure-of-an-Antibody.pdf&ved=2ahUKEwjywKCqg5fwAhW5CTQIHcpkAfgQFjAOegQICxAC&usg=AOvVaw2Qlne0rOhuwP0FF24AhlTg

Linus Pauling was able to achieve this amazing feat by providing his own theoretical explanations and drawings on the formation of the structure of antibodies. However, even though Pauling is given credit for continuing and expanding upon the direct template theory and for confirming aspects of Ehrlich's "lively imagination" with his own pretty pictures, his theoretical fantasies were shown to be completely wrong:

Thinking about the Creation of Antibodies

"In the Spring of 1936, Pauling began another collaboration, this time with Karl Landsteiner, an Austrian scientist who won a Nobel prize for discovering and developing the field of blood typing. Landsteiner invented the ABO system, and uncovered methods for making blood transfusions safe. In this research Landsteiner observed that, in instances where the wrong blood type is used in a transfusion, antibodies attacked the transfused blood. Pauling was intrigued by Landsteiner's work, and began reading about antibodies; he was interested and puzzled by what he found. While the scientific community knew that antibodies worked, how exactly they worked and how exactly they were formed were still unknown.

At the time, there were four main schools of thought regarding the creation of antibodies: the Antigen-Incorporation theory, the Side-Chain theory, the Instruction theory, and the Selection theory.

The Antigen-Incorporation theory, originally proposed by Hans Buchner in 1893, proposed that antibodies were actually the byproduct of antigens "splintering" in the human body and becoming incorporated into it. Despite the fact that this theory had been largely disproven at the time, it was proposed again by E. Hertzfeld and R. Klinger in 1918, by W.H. Manwaring in 1926, by Locke, Main, and Hirsch also in 1926, and finally once more by Gustave Ramon in 1930.

The Side-Chain theory was posited by the famous Paul Ehrlich in 1897, who argued that the body's immunological reaction to antigens was "only a repetition of the processes of normal metabolism." Ehrlich thought that cells would digest certain antigens in the same way that they digested nutrients. After repeated assimilations, or too large of an assimilation, the cells would overcompensate and release antibodies. His theory included a number of issues that the scientific community could not solve at the time, and it took over sixty years for the model to be improved upon.

The Instruction theory states that the body uses antigens as a template, then manufactures antibodies to specifically combat the antigen that the antibody is based off of. Pauling eventually belonged to this school of thought, as did Landsteiner, Michael Heidelberger, Felix Haurowitz, and Jerome Alexander. This group was far from unified however; the only point on which adherents to this school agreed was that antigens acted as templates. How antibodies worked, and how they were produced, was still a highly contentious question.

The final theory was the Selection theory, which was in concept almost identical to Ehrlich's Side-Chain theory, except that its explanations were based on more modern mechanisms. Instead of general metabolic processes, quantum mechanical forces were proposed to be the cause of the attraction between antigens and antibodies. This school of thought became more popular near the end of World War II and in the post-war era.

Pauling described Antibodies as "fantastically precise little weapons," and found it fascinating that they could identify and attack invading molecules that were different from safe molecules by only a few atoms. Antibodies are made of pure protein, are remarkably similar to one another, are relatively enormous, and also attack vastly different types of molecules.

Pauling and Landsteiner were especially vexed by how antibodies could target varied molecules so precisely when they were so similar. Pauling proceeded to read Landsteiner's book on antibodies, and began to wonder if shape affected antibodies as much as it affected regular proteins. Landsteiner had arrived at a similar conclusion, and in 1939 published a note in Science suggesting that shape was what determined the effect of antibodies.

Pauling expanded upon this idea, and in 1940 published a paper in which he hypothesized that antibodies were built as chains of non-specific proteins which collided with antigens, then compressed and shaped themselves around the antigen, "like wet clay pressed against a coin." The paper created quite a stir, and generated a lot of support for the notion of using chemistry to solve biological questions. Unfortunately for Pauling, it later turned out that his hypothesis was deeply flawed.

Another argument developed in the 1940 paper was that antibodies are bivalent – that is, they have two sites which can bind to antigens. In addition to being bivalent, Pauling hypothesized that each of the "arms" of an antibody could latch onto different kinds of antigens. While Pauling was incorrect on the latter part – antibodies can only grab onto one type of antigen – he was correct that they are bivalent.

Pauling had gotten off to a strong and noticeable start in the field of immunology. Whether correct or incorrect, he was making progress towards a greater understanding of how the body protects itself."

https://www.google.com/amp/s/paulingblog.wordpress.com/2013/08/01/thinking-about-the-creation-of-antibodies/amp/

Models galore!

As can be seen by the above source, while Pauling carried on the work and theories of Breinl and Haurowitz, much of his proposed theories were later determined to be wrong, including the idea that antibodies had "arms" which could latch on to different kinds of antigens. Instead, these "arms" can only grab ahold of only one type of antigen. While his incorrectness was excused in this source as progress towards creating a greater understanding of how these fictional creations work, we see that the inaccuracy of Pauling's theory is repeated again in these excerpts from "Linus Pauling and the Structure of Proteins:"

Antibodies

"Pauling took on the problem using antibodies as his tools. In July 1939, Landsteiner published a note in Science linking Pauling's and Mirsky's theory of protein structure – with its emphasis on long chains held in specific shapes by hydrogen bonds – with his own ideas about antibody formation. Perhaps, he wrote, antibodies were all the same basic molecule, simply folded in different ways to make them specific for certain targets.

Pauling had been thinking along the same lines. The chemical evidence still pointed, ever more strongly, toward the central importance of long chains. The physical evidence pointed toward a relatively dense, tight-knit structure for globular proteins, including antibodies. What if, Pauling reasoned, antibodies were secreted from antibody-producing cells as long filaments, chains of amino acids, that came into contact with a target substance, a virus or the wall of a bacterium or some other unwanted invader in the body. What if the loose end of the antibody formed itself in some complementary way to the structure of some part of the invader, like wet clay pushed against a coin? The two complementary structures might then stick to one another, held by a variety of weak forces that could come into play when atoms got very close to one another. Once that happened, the middle part of the antibody molecule might then fold on itself, like a stack of pancakes, creating the characteristic density of a globular protein. The back end of the antibody molecule, in this model, would be free to shape itself to another invader, making antibodies two-armed, and explaining how they were able to attack and clump target substances into a mass.

The model fit a great deal of data. It was simple and clean. Pauling wrote it up and sent what he hoped would be a landmark paper on antibody formation to the Journal of the American Chemical Society in the summer of 1940. "What is the simplest structure which can be suggested . . . for a molecule with the properties observed for antibodies, and what is the simplest reasonable process of formation for such a molecule?" he asked at the beginning. Then he elegantly and persuasively put forward his ideas.

His paper made a great stir. It was another demonstration of the power of applying chemical ideas to biology. It was a new vindication of Weaver's approach, which he was now calling "molecular biology."

It was only later that it was proven completely wrong."

https://scarc.library.oregonstate.edu/coll/pauling/proteins/narrative/page17.html

Dreaming up more models!

Pauling built upon the work of others and proposed his own theories and ideas of how antibodies look, how they form, and how they function. He is credited with providing the first model for the structure of these invisible entities. Much like Breinl and Haurowitz's direct template theory, Pauling's ideas were mostly determined to be wrong and cast aside. He even attempted to claim the ability to create artificial antibodies based on his theory which turned out to be wildly unreproducible. Pauling was called out by many of his own peers for the inability to reproduce his results and even by his own financiers, the Rockefeller Foundation. However, even though Pauling's ideas were considered inaccurate and incorrect, his work was still built upon in the following decades by other researchers, leading to scientific papers perpetuating unreproducible results:

Template theories, the rule of parsimony, and disregard for irreproducibility——The example of Linus pauling's research on antibody formation

"In 1940, Linus Pauling proposed his template theory of antibody formation, one of many such theories that rejected Paul Ehrlich's selective theory of preformed "receptors" (antibodies), assuming instead a direct molding of antibody shapes onto that of the antigen. Pauling believed that protein shapes—independently of amino acid sequences—determined antibody specificity and biological specificity in general. His theory was informed by his pioneering work on protein structure, and it was inspired by the intuitive "rule of parsimony" and simplicity. In 1942, Pauling published his alleged success in producing specific artificial antibodies through experiments based on his 1940 theory. However, his experiments could not be reproduced by prominent immunochemists at the time, and, later, it became generally accepted that antibody specificity was not generated according to Pauling's and others' "instruction" template theories. A citation analysis shows that Pauling's papers on antibody generation continue to be cited as, among other things, pioneering studies of a chemical technology called "molecular imprinting." The examples of Pauling and other protein chemists are used in this paper to demonstrate that scientific belief, philosophical concepts, and subjective theory preferences facilitated the occurrence of irreproducibility in immunochemistry and beyond. The article points to long-term consequences for the scientific community if irreproducible results are not acknowledged. It concludes by arguing that despite the risks, e.g., for the occurrence and perpetuation of irreproducible results that they entail, subjectivity and a commitment to scientific convictions have often been prerequisites for the generation, and holding on to, scientific innovation in the face of doubt and rejection from the scientific community."

"Despite the warm reception in both the popular press and some of the professional journals, the immunological community remained skeptical. Pauling and Campbell's experiments increasingly met with criticism by colleagues, including those who otherwise strongly appreciated Pauling's work, such as Michael Heidelberger, Karl Landsteiner, and Oswald Avery, as well as by the Rockefeller Foundation, which funded Pauling's protein research. Attempts to reproduce Pauling's results, such as by Landsteiner, remained unsuccessful. Campbell seemed to be the only scientist who could make specific artificial antibodies.34

In most cases, colleagues expressed their criticism in private letters as well as expert opinions sent to funding agencies.35 To mention just a few: Henry B. Bull from Northwestern University wrote in June 1943, "You have my good wishes in your endeavor to prepare artificial antibodies, but I must confess a feeling of pessimism.…Frankly, I am not impressed by experimental procedures which work sometimes but which do not at other times, and no cause can be assigned for the failure." Despite being an early promoter of template models, Felix Haurowitz from the University of Istanbul wrote in September 1943, "I tried to repeat your experiments.…In such experiments with methyl blue I did not find any trace of antibody. Experiments with resorcinol coupled to diazotized arsanilic acid failed, too." In an expert opinion for the Rockefeller Foundation, microbiologist René Dubos stated that Pauling's views "have received wide notoriety because of his great prestige," adding that many of his colleagues feel "that his claims are based on very insufficient evidence."

In contrast, Elvin A. Kabat, a co-worker of Heidelberger, not only criticized Pauling's method during the latter's visit to the Rockefeller Institute, but also published his criticism: "[the studies] of Pauling and Campbell lack the full details of control experiments necessary for a proper evaluation of their data [such] that the identity of their materials and antibody is far from established." In other words, the studies did not pay attention to unspecific precipitation. For example, dyes similar to the ones they used gave precipitates with normal horse serum. Moreover, they did not exclude the presence of natural antibodies in the starting materials. Kabat also pointed to the fact that Pauling and Campbell's experiment "contains observations which are quite in conflict with the known behavior and properties of antibodies." Therefore he considered Pauling and Campbell's claim that they had been in the "region of antibody-excess…extremely unlikely" and "even more unlikely that the precipitate…could have been antibody in the usual sense of the term."

Likewise, Jack L. Morrison from the University of Alberta, who had been a postdoctoral fellow with Pauling in 1948 and '49, believed that Pauling and Campbell's method did not demonstrate the production of specific antibodies, but that any specific precipitates of dyes (as antigens) and antibodies would be masked by nonspecific precipitation of other blood proteins by the same dye at the same pH values. Pauling's colleague at Caltech, James Bonner, later commented: "Linus's bad ideas are better than most people's good ones. But it took him a long time to let go of this one though;…Dan Campbell came specifically [to Caltech] to work on this matter. They got nowhere."

2.4. The End of the Era of Direct Template Theories in Immunology

"The idea of antibody creation on antigens as direct templates began to be increasingly challenged starting in the early 1940s. Biologists and medical immunologists realized that the immunochemical models were not able to explain basic features of the immunological response, in particular the continuous production of antibodies long after the antigen had disappeared from the organism. According to Thomas Soderqvist, a widespread but unarticulated discontent with template theories was prevalent among immunologists, including some immunochemists such as Colin MacLeod in the early 1950s. Indeed, one immunologist in New York proffered that "they knew the instruction theory isn't going to work."44 But, as Michel Morange made clear, the transition of template models to genome-based molecular selective models "was not linear"; different models of antibody synthesis coexisted after the demise of the direct template theories, and it took many years until "a full molecular description of the mechanism of antibody production" was available and became generally accepted."

4.1. Overview of the Reception

"A citation analysis, with the help of the Web of Knowledge engine in all databases of the BIOSIS Citation Index, shows that Pauling's 1940 paper on antibody formation was cited altogether a total of 776 times between 1940 and 2019. Interestingly, the paper did not receive the majority of citations in the first years after publication, but was actually cited far more often starting in the 1960s, and then especially after 2000. Between 2008 and 2018, it received up to 30 citations per year; see Figure 2.

"Interestingly, it was the prospect of the artificial synthesis of antibodies or enzyme-like polymers that seemed to have been a major impetus for this research. Praising the contributions by Pauling and Dickey, Anderson and Nicholls claim that "mankind has for decades benefited from the in vitro use of antibodies." They considered the validity of Pauling's and Campbell's experimental papers on artificial antibody synthesis irrelevant ("It appears pointless to discuss the validity of these findings today.") and highlighted the similarity of Pauling's work to today's molecular imprinting: "It is noteworthy that the procedure was in essence similar to what today is called bio-imprinting." In both cases the procedures were based on "instructive" models and used direct template methods.

This statement gives rise to the question of how it was possible that Pauling's paper, which proposed a mistaken hypothesis and an experimental method that did not work, could be cited as a paper that stimulated a methodology along similar lines, without prior analysis of the failure and suggestions for improvements."

"This reasoning can also be applied to Pauling's 1940 theory. It contained parts that were fruitful, in particular his highlighting of the (already known) immensely important principle of complementarity and of the role of weak forces in macromolecular interaction. However, his claim that the existence of direct "instruction" templates predicted by the theory could be successfully applied experimentally to produce artificial antibodies, did not result in successful research: the experimental method ended up failing. Two subsequent publications purporting its success claimed results that were irreproducible, and the attempt by Pauling's postdoctoral fellow Dickey to use Pauling's method for molecularly imprinting non-protein molecules had questionable results. Therefore, the claim of researchers of molecularly imprinted antibodies, other proteins, and non-protein molecules that their procedures, which they assert to be essentially similar to Pauling's, had been successful (a contention that is supported by the strong rise of papers in this field since the 1990s), deserves further examination."

https://online.ucpress.edu/hsns/article/51/4/427/118595/Template-Theories-the-Rule-of-Parsimony-and

I imagine his model car collection must be impressive.

It should hopefully be clear that the direct template theory of antibody formation created by Breinl and Haurowitz and supported by Pauling (amongst others) was flawed, inaccurate, and eventually abandoned. However, like the theories which came before and eventually those that came afterwards, it was a theory that was created without any direct observation of the theoretical entities it was supposed to be explaining. Linus Pauling tried to make the results and conclusions of his own chemistry experiments fit together into a simple explanation that coalesced well with often incompatible and contradictory results from the work of others. While it has been shown that his theories were largely wrong, Pauling is still credited with coming up with the first "accurate" model for the formation these unseen entities. I wanted to provide some highlights from his 15-page report from 1940 in order to gain some insight into his assumption-filled extravaganza:

A Theory of the Structure and Process of Formation of Antibodies

I. Introduction

During the past four years I have been making an effort to understand and interpret serological phenomena in terms of molecular structure and molecular interactions. The field of immunology is so extensive and the experimental observations are so complex (and occasionally contradictory) that no one has found it possible to induce a theory of the structure of antibodies from the observational material. As an alternative method of attack we may propound and attempt to answer the following questions: What is the simplest structure which can be suggested, on the basis of the extensive information now available about intramolecular and intermolecular forces, for a molecule with the properties observed for antibodies, and what is the simplest reasonable process of formation of such a molecule? Proceeding in this way, I have developed a detailed theory of the structure and process of formation of antibodies and the nature of serological reactions which is more definite and more widely applicable than earlier theories, and which is compatible with our present knowledge of the structure and properties of simple molecules as well as with most of the direct empirical information about antibodies. This theory is described and discussed below.

II. The Proposed Theory of the Structure and Process of Formation of Antibodies

When an antigen is injected into an animal some of its molecules are captured and held in the region of antibody production. An antibody to this antigen is a molecule with a configuration which is complementary to that of a portion of the antigen molecule. This complementariness gives rise to specific forces of appreciable strength between the antibody molecule and the antigen molecule; we may describe this as a bond between the two molecules. I assume, with Marrack, Heidelberger, and other investigators, that the precipitate obtained in the precipitin reaction is a framework, and that to be effective in forming the framework an antibody molecule must have two or more distinct regions with surface configuration complementary to that of the antigen. The rule of parsimony (the use of the minimum effort to achieve the result) suggests that there are only two such regions, that is, that the antibody molecules are at the most bivalent. The proposed theory is based on this reasonable assumption. It would, of course, be possible to expand the theory in such a way as to provide a mechanism for the formation of antibody molecules with valence higher than two ; but this would make the theory considerably more complex, and it is likely that antibodies with valence higher than two occur only rarely, if at all.

Antibodies are similar in amino-acid composition to one or another of the fractions of serum globulin of the animal producing the serum. It is known that there exist antibodies of different classes, with different molecular weights-the molecular weights of rabbit antibody and of monkey antibody (to pneumococcus polysaccharide) are about 157,000, whereas those of pig, cow, and horse antibodies are about 930,000. The following discussion is for antibodies with molecular weight about 160,000, and similar in constitution to the y fraction of serum globulin; the changes to be made to cause it to apply to antibodies of other classes are obvious.

The effect of an antigen in determining the structure of an antibody molecule might involve the ordering of the amino-acid residues in the polypeptide chains in a way different from that in the normal globulin, as suggested by Breinl and Haurowitz and Mudd. I assume, however, that this is not so, but that all antibody molecules contain the same polypeptide chains as normal
globulin, and difer from normal globulin only in the configuration of the chain; that is, in the way that the chain is coiled in the molecule. There is at present no direct evidence supporting this assumption. The assumption is made because, although I have found it impossible to formulate in detail a reasonable mechanism whereby the order of amino-acid residues in the chain would be determined by the antigen, a simple and reasonable mechanism, described below, can be advanced whereby the antigen causes the polypeptide chain to assume a configuration complementary to the antigen. The number of configurations accessible to the polypeptide chain is so great as to provide an explanation of the ability of an animal to form antibodies with considerable specificity for an apparently unlimited number of different antigens, without
the necessity of invoking also a variation in the amino-acid composition or amino-acid order.

The Postulated Process of Formation of Antibodies. Let us assume that the globulin molecule consists of a single polypeptide chain, containing several hundred amino-acid residues, and that the order of amino-acid residues is such that for the center of the chain one of the accessible configurations is much more stable than any other, whereas the two end parts of the chain are of such a nature that there exist for them many configurations with nearly the same energy. (This point is discussed in detail in Section IV.) Four steps in our postulated process of formation of a normal globulin molecule are illustrated on the left side of Fig. 1."

III. Some Points of Comparison with Experiment

a. The Heterogeneity Of Immune Sera

The theory requires that the serum homologous to a given antigen be not homogeneous, but heterogeneous, containing antibody molecules of greatly varied configurations. Many of the antibody molecules will be bivalent, with two active ends with configuration complementary to portions of the surface of an antigen molecule. Great variety in this complementary configuration would be expected to result from the accidental approximation to one or another surface region, and further variety from variation in position of the antigen molecule relative to the point of liberation of the globulin chain end and from accidental coiling and linking of the chain end before it comes under the influence of the antigen. Some of the antibody molecules would be univalent, one of the chain ends having, because of its too great distance from the antigen, folded into a normal globulin configuration."

b. The Bivalence of Antibodies and the Multivalence of Antigens.

"Our theory is based on the idea that the precipitate formed in the precipitin reaction is a network of antibody and antigen molecules in which many or all of the antibody molecules grasp two antigen molecules apiece and the antigen molecules are grasped by several antibody molecules. The direct experimental evidence for this picture of the precipitate has been ably discussed by its propounders and supporters, Marrack and Heidelberger and Kendall, and need not be reviewed here. To the structural chemist it is clear that this picture of the precipitate must be correct. The great specificity of antibody-antigen interactions requires that a definite bond be formed between an antibody molecule and an antigen molecule. If antibodies or antigens were univalent, this would lead to complexes of one antigen molecule and one or more antibody molecules (or of one antibody molecule and one or more antigen molecules), and we know from experience with proteins that these aggregates would in general remain in solution. If both antibody and antigen are multivalent, however, the complex will grow to an aggregate of indefinite
size, which is the precipitate."

"It seems probable that all antibodies have this structure-that they are bivalent, with their two active regions oppositely directed. Heidelberger and his collaborators and Marrack have emphasized the multivalence of antibodies and antigens, but limitation of the valence of antibodies to the maximum value two (ignoring the exceptional case of the attachment of two or more antigens or haptens to the same end region of an antibody) has not previously been made."

c. The Antibody-Antigen Molecular Ratio in Precipitates.

"Our theory provides an immediate simple explanation of the observed antibody-antigen molecular ratios in precipitates. Under optimum conditions a precipitate will be formed
in which all the valences of the antibody and antigen molecules are satisfied. An idealized representation of a portion of such a precipitate is given in Fig. 3. The figure shows a part of a layer with each antigen molecule bonded to six surrounding antibody molecules ; this structure represents the value N = 12 for the valence of the antigen, each antigen molecule being attached also to three antibody molecules above the layer represented and to three below. Each antibody molecule is bonded to two antigen molecules, one at each end. An ideal structure of the antibody-antigen precipitate for N = 12 may be described as having antigen molecules at the positions corresponding to closest packing, with the twelve antibody molecules which surround each antigen molecule lying along the lines connecting it with the twelve nearest antigen neighbors.

Similar ideal structures can be suggested for other values of the antigen valence. The antigen molecules might be arranged for N = 8 at the points of a body-centered cubic lattice, and for N = 6 at the points of a simple cubic lattice, with antibody molecules along the connecting lines. For N = 4 the antigen molecules, connected by antibody molecules, might lie at the points occupied by carbon atoms in diamond; or two such frameworks might interpenetrate, as in the cuprous oxide arrangement (copper and oxygen atoms being replaced by antibody and antigen molecules, respectively).

It is not to be inferred that the actual precipitates have the regularity of structure of these ideal arrangements. The nature of the process of antibody formation, involving the use of a portion of the antigen surface selected at random as the template for the molding of an active end of an antibody molecule, introduces so much irregularity in the framework that a regular structure analogous to that of a crystal is probably never formed. The precipitate is to be compared rather with a glass such as silica glass, in which each silicon atom is surrounded tetrahedrally by four oxygen atoms and each oxygen atom is bonded to two silicon atoms, but which lacks further orderliness of arrangement. Additional disorder is introduced in the precipitate by variation in the effective valence of the antigen molecules and by the inclusion of antibody molecules with only one active end."

"It is seen that our theory provides a simple explanation of the fact that for antigens of molecular weight equal to or less than that of the antibody the precipitate contains considerably more antibody than antigen. The values given in Table I are not to be considered as having rigorous quantitative significance. The calculated maximum molecular ratio would be larger for elongated antibody molecules than for spherical antibody molecules, and larger for non-spherical than for spherical antigen molecules, and, moreover, in many sera the antibodies might be complementary in the main only to certain surface regions of the antigen, the number of these determining the valence of the antigen. That this is so is indicated by the observation that after long immunization of a rabbit with egg albumin serum was obtained giving a precipitate with a considerably larger molecular ratio than that for earlier bleedings."

d. The Use of a Single Antigen Molecule as the Template for an Antibody Molecule.

There are two ways in which an antibody molecule with two opposed active regions complementary to the antigen might be produced. One is the way described in Section 11. The other would involve the which (with an occasional exception) both antigen manufacture of the antibody molecule in its and antibody are bivalent, the molecular ratio final configuration between two antigen molecules, one of which would serve as the pattern for one antibody end and the other for the second. No attempts to decide between these alternatives seems to have been made before; evidence, however, some of which is mentioned below, to indicate that the first method of antibody production, involving only one antigen molecule, occurs predominantly. It is for this reason that I have developed the rather complicated theory described above, with the two end portions of the antibody forming first, one (or both) then separating from the antigen, and the central part of the antibody then assuming its shape and holding the active ends in position for attachment to two antigen molecules.

This theory requires that the formation of antibody be a reaction of the first order with respect to the antigen, whereas the other alternative would require it to be of the second order. There exists very little evidence as to whether on immunization with small amounts of antigen the antibody production is proportional to the amount of antigen injected or to its square. Some support for the one-antigen-molecule theory is provided by the experiments dealing with the injection of a mixture of antigens."

e. Criteria for Antigenic Power.

There has been extensive discussion of the question of what makes a substance an antigen, but no generally accepted conclusions have been reached. Our theory permits the formulation of the following reasonable criteria for antigenic activity:

  1. The antigen molecule must contain active groups, capable of sufficiently strong interaction with the globulin chain to influence its configuration.
  2. The configuration of the antigen molecule must be well-defined over surface regions large enough to give rise to an integrated antibody- antigen force sufficient to hold the molecules together.
  3. The antigen molecule must be large enough to have two or more such surface regions, and in case that the antigenic activity depends upon a particular group the molecule must contain at least two of these groups. (This criterion applies to antibodies effective in the precipitin and agglutinin reactions and in anaphylaxis.

These criteria are satisfied by substances known to have antigenic action. Many proteins, some carbohydrates with high molecular weight (bacterial polysaccharides, invertebrate glycogen), and some lipids and carbohydrate-lipid complexes are antigenic. The simple chemical substances so far studied have been found to be inactive, except those which are capable of combining with proteins in the body. Non-antigenic substances have been reported to become antigenic when adsorbed on particles (Forssman antigen on kaolinz); in this case the particle with adsorbed hapten is to be considered the antigen "molecule" of our theory. I predict that relatively simple molecules containing two or more haptens will be found to be antigenic; experiments to test this prediction are now under way.

IV. A More Detailed Discussion of the Structure of Antibodies and Other Proteins

"There has been gathered so far very little direct evidence regarding the detailed structure of protein molecules. Chemical information is compatible with the polypeptide-chain theory of protein structure, and this theory is also supported by the rather small amount of pertinent X-ray evidence."

"Layer structures other than this one might also be assumed, in which the chains are not extended. Some fibrous proteins, such as a-keratin, are known to have structures of this general type, but the nature of the folding of the chains has not yet been determined.

We have postulated the existence of an extremely large number of accessible configurations with nearly the same energy for the end parts of the globulin polypeptide chain. A layer structure, with variety in the type of folding in the layer, would not, it seems to me, give enough configurational possibilities to explain the great observed versatility of the antibody precursor in adjusting itself to the antigen, and I think that skew configurations must be invoked. But simple considerations show that it would be difficult for the chain to assume a skew configuration in which most of the peptide carbonyl and imino groups take part in forming hydrogen bonds, as they do in the layer structures; and in consequence the skew configurations would be much less stable than the layer configurations. The way out of this difficulty is provided by the postulate that the end parts of the globulin polypeptide chains contain a very large proportion (perhaps one-third or one-half) of proline and hydroxyproline residues and other residues which prevent the assumption of a stable layer configuration."

Serological experiments with artificial conjugated antigens, especially azoproteins, have provided results of great significance to the theory of antibody structure. Many of the arguments based on these results are presented in the books of Landsteiner and Marrack. The data obtained regarding cross-reactions of azoprotein sera with related azoproteins show that electrically charged groups (carboxyl, sulfonate, arsenate) interact strongly with homologous antibodies, and that somewhat weaker interactions are produced by hydrogen-bond-forming groups and groups with large electric dipole moments (hydroxy, nitro). The principal action of a weak group such as alkyl, phenyl or halogen is steric; this is shown clearly by the strong cross-reactions between similar chloro and methyl haptens. The data on specificity of antibodies with respect to haptens indicate strongly that the hapten group fits into a pocket in the antibody, and that the fit is a close one. It should not be concluded that all antibody-antigen bonds are of just this type; for example, the fitting of an antibody group into a pocket in the antigen may also be often of importance. Extensive work will be needed to determine the detailed nature of the antibody structures complementary to particular haptens and antigens.

V. Further Comparison of the Theory with Experiment. Possible Experimental Tests of Predictions

a. Methods of Determining the Valence of Antibodies.

The following methods may be proposed to determine the valence of antibodies. First, let a serum be produced by injection of an azoprotein of the following type: its hapten is to be sufficiently strong (that is, to interact sufficiently strongly with the homologous antibody) that one hapten group forms a satisfactory antibody-antigen bond, and the number of hapten groups per molecule is to be small enough so that in the main only one group will be present in the area serving as a pattern for an antibody end. The same hapten is then attached to another protein, and this azoprotein is precipitated with the serum. If it be assumed that the precipitate is valence-saturated, the ratio of hapten groups to antibody molecules in the precipitate gives the average valence of the antibody."

"The bivalence of antibodies and our postulate that only one antigen molecule is involved in the formation of an antibody molecule require that a precipitin-effective antihapten be produced only if the injected antigen contain at least two hapten groups. Pertinent data have been obtained on this point by Haurowitz and his collaborators, who found that effective antihapten precipitin serum was produced by an azoprotein, made from arsanilic acid and horse globulin, containing 0.24% arsenic (4.8 haptens per average molecule of molecular weight 157,000), and a trace by one containing 0.13% arsenic (2.6 haptens per molecule).

FROM THE FOOTNOTES:

"From the data discussed above, which in our opinion indicate that two hapten groups combine with a bivalent antibody molecule, Haurowitz drew the different conclusion that one arsenic-containing group in the antigen combines with one antibody molecule. He reached this result by assuming 100,000 (rather than 157,000) for the molecular weight of the antibody and by assuming that the active group in the antigen consists of two haptens attached to a tyrosine or histidine residue. It is, of course, likely that this occurs in antigens with high arsenic content, but it seems probable that the haptens are mainly attached to separate residues in the antigens containing only a few haptens per molecule."

"If we assume that the conditions of each precipitation were such that only suitable bivalent antibody molecules were incorporated in the precipitate, the equations above would require the third precipitate to weigh about 24 mg; the experiment accordingly provides some support for the theory. The quantitative discrepancy may possibly be due to the incorporation of some effectively univalent antibody molecules in the first two precipitates.

The qualitative experimental results which have been reported are in part compatible and in part incompatible with the theory."

A second deduction, relating to specificity, can also be made. To achieve a sufficiently strong
antibody-antigen bond with an antigen containing only weak groups a large surface region of the antigen must come into play, whereas with an antigen containing strong groups only a smal region (in the limit one group) is needed. Hence antibodies to antigens containing strong groups show low specificity, and those to antigens containing weak groups show high specificity. This prediction is substantiated by many observations. Egg albumin, hemoglobin, and similar proteins give highly specific sera, whereas azoproteins produce sera which are less specific, strong cross-reactions being observed among various proteins with the same hapten attached. This shows, indeed, that a single hapten group gives a sufficiently strong bond to hold antibody and antigen together. In such a case the approximation of the antibody to a strong hapten is very close, and great specificity is shown with regard to the hapten itself, this specificity being the greater the stronger the hapten. Many examples of these effects are to be found in Landsteiner's work."

VI. Processes Auxiliary to Antibody Formation

"It seems not unlikely that certain processes auxiliary to antibody formation occur. The reported increase in globulin (aside from the antibody fraction) after immunization suggests the operation of a mechanism whereby the presence of antigen molecules accelerates the synthesis of the globulin polypeptide chains. There is little basis for suggesting possible mechanisms for this process at present."

Summary

"It is assumed that antibodies differ from normal serum globulin only in the way in which the two end parts of the globulin polypeptide chain are coiled, these parts, as a result of their amino-acid composition and order, having accessible a very great many configurations with nearly the same stability; under the influence of an antigen molecule they assume configurations complementary to surface regions of the antigen, thus forming two active ends. After the freeing of one end and the liberation of the central part of the chain this part of the chain folds up to form the central part of the antibody molecule, with two oppositely-directed ends able to attach themselves to two antigen molecules.

Among the points of comparison of the theory and experiment are the following: the heterogeneity of sera, the bivalence of antibodies and
multivalence of antigens, the framework structure and molecular ratio of antibody-antigen precipitates, the use of a single antigen molecule as
template for an antibody molecule, criteria for antigenic activity, the behavior of antigens containing two different haptens, the antigenic activity of antibodies, factors affecting the rate of antibody production and the specificity of antibodies, and the effect of denaturing agents. It is shown that most of the reported experimental results are compatible with the theory. Some new experiments suggested by the theory are mentioned.

https://doi.org/10.1021/ja01867a018

In Summary:
  • Fritz Breinl asked Felix Haurowitz to read the antibody papers and discuss with him what could be done to solve the mystery of antibody production
  • This began an exciting but short-lived collaboration that led to what was later called the template theory of antibody formation
  • They used horse hemoglobin as an antigen in the work for their first paper
  • Unlike Landsteiner, who used only qualitative indices for the amount of antigen-antibody precipitate (- or +, ++, +++), Haurowitz and Breinl used quantitative methods to determine the amount of hemoglobin in the precipitate, and they also indirectly determined the amino acid content of the nonhemoglobin portion of the precipitate
  • According to Haurowitz: "I concluded that the antibody must be serum globulin and suggested therefore that the antigen interferes with the process of globulin biosynthesis in such a way that globulins complementarily adjusted to the antigen are formed."
  • Ehrlich's view of the selective role of the antigen was accepted for a long time
  • However, Landsteincr's finding that antibodies are also formed against synthetic haptens (a substance said to combine with a specific antibody but lacks antigenicity of its own) which never occur in nature, made it difficult to believe that the organism could have cells precommitted to the formation  synthetic products of the chemical laboratories
  • For this reason, Breinl and Haurowitz (1930) proposed that the administered antigen might interfere directly with the mechanism of y-globulin biosynthesis and modify this process in such a manner that a complementary combining site in the newly formed y-globulin molecule is produced
  • The two researchers suggested that complementariness is accomplished by suitable orientation of the amino acid residues in the newly formed globulin molecule
  • Haurowitz attributed this action to intermolecular forces, particularly electrostatic interaction, dipole induction, and the short-range van der Waals forces (i.e. he claimed invisible forces acted upon the invisible entities in order to create their complementary form)
  • Both the selective theory and the template theory are based on the idea of mutual complementariness of the groups which are responsible for the combination of antibody with antigen
  • Haurowitz stated that his view of a template role for the antigen was taken up ten years later by Linus Pauling (1940) who introduced an important modification by claiming that all antibodies formed in an organism have the same peptide chain with identical amino acid sequences, and that they differ from each other only in their conformation
  • He claimed Pauling's assumption was later strongly supported (i.e. not proven) by the findings from two unrelated studies
  • Ehrlich proposed a 'lock and key' mechanism of antibody-antigen interaction but this was not confirmed until the 1940s by Linus Pauling
  • Karl Landsteiner observed that, in instances where the wrong blood type is used in a transfusion, antibodies attacked the transfused blood
  • Pauling was intrigued by Landsteiner's work, and began reading about antibodies; he was interested and puzzled by what he found as while the scientific community "knew" that antibodies worked, how exactly they worked and how exactly they were formed were still unknown
  • At the time, there were four main schools of thought regarding the creation of antibodies:
    1. The Antigen-Incorporation theory
      • Proposed that antibodies were actually the byproduct of antigens "splintering" in the human body and becoming incorporated into it
      • Despite the fact that this theory had been largely disproven at the time, it was proposed again by E. Hertzfeld and R. Klinger in 1918, by W.H. Manwaring in 1926, by Locke, Main, and Hirsch also in 1926, and finally once more by Gustave Ramon in 1930
    2. The Side-Chain theory
      • Paul Ehrlich's theory that the body's immunological reaction to antigens was "only a repetition of the processes of normal metabolism"
      • Ehrlich thought that cells would digest certain antigens in the same way that they digested nutrients
      • After repeated assimilations, or too large of an assimilation, the cells would overcompensate and release antibodies
      • His theory included a number of issues that the scientific community could not solve at the time, and it took over sixty years for the model to be improved upon
    3. The Instruction theory
      • States that the body uses antigens as a template, then manufactures antibodies to specifically combat the antigen that the antibody is based off of
      • Pauling eventually belonged to this school of thought, as did Landsteiner, Michael Heidelberger, Felix Haurowitz, and Jerome Alexander
      • This group was far from unified however; the only point on which adherents to this school agreed was that antigens acted as templates
      • How antibodies worked, and how they were produced, was still a highly contentious question
    4. The Selection theory
      • In concept almost identical to Ehrlich's Side-Chain theory
      • Instead of general metabolic processes, quantum mechanical forces were proposed to be the cause of the attraction between antigens and antibodies
  • Pauling described antibodies as "fantastically precise little weapons"
  • Both Pauling and Landsteiner arrived at a similar conclusion suggesting that shape was what determined the effect of antibodies
  • Pauling expanded upon this idea, and in 1940 published a paper in which he hypothesized that antibodies were built as chains of non-specific proteins which collided with antigens, then compressed and shaped themselves around the antigen, "like wet clay pressed against a coin"
  • Unfortunately for Pauling, it later turned out that his hypothesis was deeply flawed
  • Another argument developed in the 1940 paper was that antibodies are bivalent – that is, they have two sites which can bind to antigens
  • In addition to being bivalent, Pauling hypothesized that each of the "arms" of an antibody could latch onto different kinds of antigens
  • While Pauling was incorrect on the latter part – antibodies can only grab onto one type of antigen – he was "correct" that they are bivalent
  • Whether correct or incorrect, it was said Pauling was making progress towards a greater understanding of how the body protects itself
  • Landsteiner linked his theory with Pauling's and Mirsky's theory of protein structure – with its emphasis on long chains held in specific shapes by hydrogen bonds
  • Linus Pauling's "What if's?"
    1. What if antibodies were secreted from antibody-producing cells as long filaments, chains of amino acids, that came into contact with a target substance, a "virus" or the wall of a bacterium or some other unwanted invader in the body?
    2. What if the loose end of the antibody formed itself in some complementary way to the structure of some part of the invader?
      • The two complementary structures might then stick to one another, held by a variety of weak forces that could come into play when atoms got very close to one another
      • Once that happened, the middle part of the antibody molecule might then fold on itself, like a stack of pancakes, creating the characteristic density of a globular protein
      • The back end of the antibody molecule, in this model, would be free to shape itself to another invader, making antibodies two-armed, and explaining how they were able to attack and clump target substances into a mass
  • Pauling wrote up his ideas into a paper which made a great stir
  • It was another demonstration of the power of applying chemical ideas to biology
  • It was only later that it was proven completely wrong
  • Pauling's theory assumed a direct molding of antibody shapes onto that of the antigen
  • In 1942, Pauling published his alleged success in producing specific artificial antibodies through experiments based on his 1940 theory
  • However, his experiments could not be reproduced by prominent immunochemists at the time, and, later, it became generally accepted that antibody specificity was not generated according to Pauling's and others' "instruction" template theories
  • The examples of Pauling and other protein chemists demonstrate that scientific belief, philosophical concepts, and subjective theory preferences facilitated the occurrence of irreproducibility in immunochemistry and beyond
  • Pauling and Campbell's experiments increasingly met with criticism by colleagues, including those who otherwise strongly appreciated Pauling's work, such as Michael Heidelberger, Karl Landsteiner, and Oswald Avery, as well as by the Rockefeller Foundation, which funded Pauling's protein research
  • Attempts to reproduce Pauling's results, such as by Landsteiner, remained unsuccessful
  • Henry B. Bull from Northwestern University wrote in June 1943, "You have my good wishes in your endeavor to prepare artificial antibodies, but I must confess a feeling of pessimism.…Frankly, I am not impressed by experimental procedures which work sometimes but which do not at other times, and no cause can be assigned for the failure."
  • Felix Haurowitz from the University of Istanbul wrote in September 1943, "I tried to repeat your experiments.…In such experiments with methyl blue I did not find any trace of antibody. Experiments with resorcinol coupled to diazotized arsanilic acid failed, too."
  • Microbiologist René Dubos stated that Pauling's views "have received wide notoriety because of his great prestige," adding that many of his colleagues feel "that his claims are based on very insufficient evidence."
  • In contrast, Elvin A. Kabat, a co-worker of Heidelberger, not only criticized Pauling's method during the latter's visit to the Rockefeller Institute, but also published his criticism: "[the studies] of Pauling and Campbell lack the full details of control experiments necessary for a proper evaluation of their data [such] that the identity of their materials and antibody is far from established."
  • In other words, the studies did not pay attention to unspecific precipitation such as dyes similar to the ones they used gave precipitates with normal horse serum
  • Jack L. Morrison from the University of Alberta, who had been a postdoctoral fellow with Pauling in 1948 and '49, believed that Pauling and Campbell's method did not demonstrate the production of specific antibodies, but that any specific precipitates of dyes (as antigens) and antibodies would be masked by nonspecific precipitation of other blood proteins by the same dye at the same pH values
  • The idea of antibody creation on antigens as direct templates began to be increasingly challenged starting in the early 1940s
  • Biologists and medical immunologists realized that the immunochemical models were not able to explain basic features of the immunological response, in particular the continuous production of antibodies long after the antigen had disappeared from the organism
  • According to Michel Morange, the transition of template models to genome-based molecular selective models "was not linear"; different models of antibody synthesis coexisted after the demise of the direct template theories, and it took many years until "a full molecular description of the mechanism of antibody production" was available and became generally accepted."
  • Pauling's 1940 paper on antibody formation was cited altogether a total of 776 times between 1940 and 2019
  • The paper did not receive the majority of citations in the first years after publication, but was actually cited far more often starting in the 1960s, and then especially after 2000
  • Between 2008 and 2018, it received up to 30 citations per year
  • Praising the contributions by Pauling and Dickey, Anderson and Nicholls claimed that "mankind has for decades benefited from the in vitro use of antibodies."
  • They considered the validity of Pauling's and Campbell's experimental papers on artificial antibody synthesis irrelevant ("It appears pointless to discuss the validity of these findings today.") and highlighted the similarity of Pauling's work to today's molecular imprinting: "It is noteworthy that the procedure was in essence similar to what today is called bio-imprinting."
  • In both cases the procedures were based on "instructive" models and used direct template methods
  • This statement gives rise to the question of how it was possible that Pauling's paper, which proposed a mistaken hypothesis and an experimental method that did not work, could be cited as a paper that stimulated a methodology along similar lines, without prior analysis of the failure and suggestions for improvements
  • Pauling's claim that the existence of direct "instruction" templates predicted by the theory could be successfully applied experimentally to produce artificial antibodies, did not result in successful research: the experimental method ended up failing
  • Two subsequent publications purporting its success claimed results that were irreproducible, and the attempt by Pauling's postdoctoral fellow Dickey to use Pauling's method for molecularly imprinting non-protein molecules had questionable results
  • Therefore, the claim of researchers of molecularly imprinted antibodies, other proteins, and non-protein molecules that their procedures, which they assert to be essentially similar to Pauling's, had been successful deserves further examination
  • The field of immunology is so extensive and the experimental observations are so complex (and occasionally contradictory) that no one had found it possible to induce a theory of the structure of antibodies from the observational material
  • Pauling wanted to know what is the simplest structure which can be suggested, on the basis of the extensive information available about intramolecular and intermolecular forces, for a molecule with the properties observed for antibodies, and what is the simplest reasonable process of formation of such a molecule?
  • In other words, he tried to adhere to Occams Razor, aka the law of parsimony, in that the simplest answer is the preferred one
  • He therefore developed a detailed theory of the structure and process of formation of antibodies and the nature of serological reactions
  • Pauling assumed that the precipitate in the precipitin reactions was the framework for an antibody and that there must be 2 or more regions complementary to an antigen
  • The proposed theory was based on this reasonable assumption according to the law of parsimony
  • It was "known" that there exist antibodies of different classes, with different molecular weights
  • Pauling assumed that all antibodies are similar to normal globulins and differ only in the configuration of their chains
  • However. he admitted that there is no direct evidence supporting his assumptions
  • Pauling based his assumption on the fact that he could not formulate a reasonable mechanism where the amino-acid chains were determined by the antigen
  • Pauling assumed that the globulin molecule consisted of a single polypeptide chain, containing several hundred amino-acid residues, and that the order of amino-acid residues is such that for the center of the chain one of the accessible configurations is much more stable than any other, whereas the two end parts of the chain are of such a nature that there exist for them many configurations with nearly the same energy
  • The theory required that the serum homologous to a given antigen be not homogeneous, but heterogeneous, containing antibody molecules of greatly varied configurations
  • Pauling's theory was based on the idea that the precipitate formed in the precipitin reaction is a network of antibody and antigen molecules in which many or all of the antibody molecules grasp two antigen molecules apiece and the antigen molecules are grasped by several antibody molecules
  • It seemed probable that all antibodies are bivalent, with their two active regions oppositely directed
  • Pauling felt his theory provided an immediate simple explanation of the observed antibody-antigen molecular ratios in precipitates under optimum conditions
  • Similar ideal structures were suggested for other values of the antigen valence
  • Pauling provided drawings of idealized representations of antibodies and antigens
  • However, he stated it should not be inferred that the precipitates have the regularity of structure as presented in the idealized versions
  • He also stated the nature of antibody formation, using a portion of the antigen surface selected at random as the template for the molding of an active end of an antibody molecule, introduces so much irregularity in the framework that it probably never forms the perfect crystalline structure represented
  • There are two ways in which an antibody molecule with two opposed active regions complementary to the antigen might be produced
  • No attempts to decide between these alternatives seems to have been made before
  • It is for this reason that Pauling developed the rather complicated theory, with the two end portions of the antibody forming first, one (or both) then separating from the antigen, and the central part of the antibody then assuming its shape and holding the active ends in position for attachment to two antigen molecules
  • This theory requires that the formation of antibody be a reaction of the first order with respect to the antigen, whereas the other alternative would require it to be of the second order
  • There exists very little evidence as to whether on immunization with small amounts of antigen the antibody production is proportional to the amount of antigen injected or to its square
  • There had been extensive discussion of the question of what makes a substance an antigen, but no generally accepted conclusions had been reached
  • Non-antigenic substances have been reported to become antigenic when adsorbed on particles and Pauling considered the particle with adsorbed hapten to be the antigen "molecule" of his theory
  • He predicted that relatively simple molecules containing two or more haptens will be found to be antigenic
  • Pauling admitted that there had been gathered very little direct evidence regarding the detailed structure of protein molecules
  • There were potential layer structures which could be assumed but no determination had been made on the folding of the chains
  • Pauling postulated the existence of an extremely large number of accessible configurations with nearly the same energy for the end parts of the globulin polypeptide chain
  • It seemed to Pauling that a layer structure, with variety in the type of folding in the layer, would not give enough configurational possibilities to explain the great observed versatility of the antibody precursor in adjusting itself to the antigen, and he thought that skew configurations must be invoked
  • Serological experiments with artificial conjugated antigens, especially azoproteins, provided results of great significance to the theory of antibody structure
  • The data on specificity of antibodies with respect to haptens indicated strongly that the hapten group fits into a pocket in the antibody, and that the fit is a close one
  • Pauling felt it should not be concluded that all antibody-antigen bonds are of just this type; for example, the fitting of an antibody group into a pocket in the antigen may also be often of importance
  • He admitted more extensive work was needed to determine the detailed nature of the antibody structures complementary to particular haptens and antigens
  • In order to determine valence (combining power of an element), it was assumed that if the precipitate is valence-saturated, the ratio of hapten groups to antibody molecules in the precipitate gives the average valence of the antibody
  • Pauling stated the bivalence of antibodies and his postulate that only one antigen molecule is involved in the formation of an antibody molecule require that a precipitin-effective antihapten be produced only if the injected antigen contain at least two hapten groups
  • He felt pertinent data had been obtained on this point by Haurowitz and his collaborators but in the footnotes it is admitted that Haurowitz drew the different conclusion that one arsenic-containing group in the antigen combines with one antibody molecule
  • Pauling assumed that the conditions in the precipitation were such that only suitable bivalent antibody molecules were present in the precipitate
  • He felt that the quantitative discrepancy may possibly be due to the incorporation of some effectively univalent antibody molecules in the first two precipitates
  • The qualitative experimental results which had been reported were in part compatible and in part incompatible with the theory
  • Pauling deduced that antibodies to antigens containing strong groups show low specificity, and those to antigens containing weak groups show high specificity
  • Strong cross-reactions were observed among various proteins with the same hapten attached
  • While he stated that it seemed likely that certain processes auxiliary to antibody formation occur, there was little basis for suggesting possible mechanisms for this process
  • Pauling admitted that it is assumed in his theory that antibodies differ from normal serum globulin only in the way in which the two end parts of the globulin polypeptide chain are coiled, these parts, as a result of their amino-acid composition and order, having accessible a very great many configurations with nearly the same stability; under the influence of an antigen molecule they assume configurations complementary to surface regions of the antigen, thus forming two active ends
  • After the freeing of one end and the liberation of the central part of the chain this part of the chain folds up to form the central part of the antibody molecule, with two oppositely-directed ends able to attach themselves to two antigen molecules
  • He claimed that most of the reported experimental results are compatible with the theory

The direct template theory was proposed by Fritz Breinl and Felix Haurowitz 30 years after Paul Ehrlich's side-chain theory once it became apparent that researchers still had no idea how the invisible entities known as antibodies looked, formed, or functioned. It became the second major theoretical explanation attempting to combine many disparate elements and experimental research into a cohesive narrative. It stood as a valid explanation championed by immunochemists for nearly two decades. Acclaimed chemist Linus Pauling, two-time Nobel Prize winner who is considered among the 20 greatest scientists to ever live, refined and expanded upon the theory, lending it great credence in the 1940's until it was finally decided and agreed upon that the theory was not an accurate representation at all. The antibody fiasco is one of Pauling's greatest failings and was an otherwise ugly stain upon his "illustrious" career.

The problem with direct template and any of the remaining 5 antibody theories is that they are nothing but fictional explanations for unseen entities carrying out unobservable processes. Researchers use the results of unrelated chemistry experiments in a lab in order to try and explain and validate an idea and concept created without direct evidence to the physical existence of these entities. There is absolutely no way any of the theories can be considered accurate as the reactions are all studied outside of a living organism using chemicals and body fluids that would never be combined together within a living organism.

However, if you enjoy your "proof" in the form of assumptions and guesswork based on cherry-picked experimental research, papers such as Pauling's 1940 theoretical explanation of the antibody structure will be right up your alley. The amount of times he admitted to making assumptions is astonishing. Granted, it is also admitted to be a theory on the structure and formation of antibodies. However, it is lauded by some as proof of the form of antibodies as well as confirmation of Ehrlich's own theory regarding the lock-and-key mechanism of action. It should be obvious that one unproven theoretical explanation does not get to confirm another one, yet this is how science has worked for the better part of the last century. Theories are taken as the truth and are built upon by various other researchers until a consensus agreement is established. It does not matter if the original research ends up being lambasted for being unreproducible. Future researchers will base their work on the old inaccurate research which will propagate and perpetuate into a cycle of fraudulent data. With antibody research making up a bulk of the fraudulent data during the current reproducibility crisis in the sciences, the lessons from the past stand out as stark warnings as to why theories should not be held up as truth even in the face of a majority consensus.

ViroLIEgy
7 May 2022 | 3:41 pm

The Antibody Equation (1929)


It is very apparent to anyone looking into the origins of antibodies that the idea of what these entities are in terms of how they look and how they function came well before any attempts to actually purify, isolate, and characterize the assumed particles. Antibodies were (and still are) nothing more than unseen theoretical constructs used to explain chemical reactions created in a lab. These fictional creations reside in the "domain of the invisible spectrum" conjured up by the "lively imagination" of a man named Paul Ehrlich. While there was no direct proof for the existence of these entities, the antibody concept was far too important to the immunological narratives forming around the growing practice of vaccination and the increased acceptance of other unseen entities known as "viruses" to just give it up. As the purification and isolation of antibodies in order to see and study them was an impossible task, researchers sought other methods to attempt to provide indirect evidence for the existence of these theoretical creations.

One man who is credited with providing such evidece is Michael Heidelberger, considered the "Founder of Immunochemistry." He was the first to apply mathematics to the reaction of antibodies and their antigens. He is also known for "proving" that antibodies are proteins by showing that the antigens of pneumococcus bacteria are polysaccharides (or carbohydrates). Here is a brief overview of his work:

How Heidelberger and Avery sweetened immunology All about nitrogen

"Avery and Dochez's initial characterization of this pneumococcal substance showed that it was resistant to both heat and trypsin—features unbefitting most proteins—but that it did contain nitrogen, a component of proteins. But its true nature was not revealed until 1923, when Michael Heidelberger—then in the chemistry department synthesizing drugs against poliomyelitis and African sleeping sickness—teamed up with Avery.

The more they purified the reactive substance the less nitrogen it contained. When it was virtually nitrogen-free, recalled Heidelberger in a 1979 article, Avery ventured a guess: "Could it be a carbohydrate?" (2). Chemical analysis confirmed its sugary character, and subsequent studies of other pneumococcal serotypes revealed that each bacterial capsule had a distinct polysaccharide signature. It was this signature that dictated the serological specificity of the organism. The duo published these findings in two articles in the Journal of Experimental Medicine (34).

Their results were met with considerable skepticism, as it was then thought that only proteins could incite a specific immune response. "Nobody believed it," says Emil Gotschlich (Rockefeller University), whose later work on polysaccharide-based vaccines stemmed in large part from Heidelberger and Avery's discoveries. "It took them a lot of effort to convince people that the polysaccharide was the immunoreactive component."

Antibodies solidified

Heidelberger and Avery's discovery came at a time when antibodies were regarded—by those who believed they existed at all—as mysterious substances that floated around in serum. "It appeared to me that there was a crying need to determine the true nature of antibodies," wrote Heidelberger in 1979, "and that until this was done there could be no end to the polemics and uncertainties that were plaguing immunology" (2). Heidelberger later purified the antibodies from his precipitin reactions and showed that they themselves were proteins. As a result, says friend and colleague Victor Nussenzweig (New York University), "there were no more mystical ideas about what antibodies were."

Heidelberger and his postdoctoral fellow Forrest Kendall later quantitated the precipitin reaction (5), bringing much-needed mathematics to the study of antibody–antigen interactions and lifting antibodies even further out of the realm of the mysterious (see the next "From the Archive")."

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2212983/#!po=46.8750

Heidelberger hard at work using his microscopic vision.

Two of Heidelberger's papers are most often cited as the proof that antibodies are proteins. The first is a paper he did with Oswald Avery in 1923. It is used as proof that the pneumococcus antigens are carbohydrates. However, was this paper successful in drawing this conclusion? Presented here are some highlights from their collaboration:

THE SOLUBLE SPECIFIC SUBSTANCE OF PNEUMOCOCCUS.

"In 1917 Dochez and Avery (1) showed that whenever pneumococci are grown in fluid media, there is present in the cultural fluid a substance which precipitates specifically in antipneumococcus serum of the homologous type. This soluble substance is demonstrable in culture filtrates during the initial growth phase of the organisms; that is, during the period of their maximum rate of multiplication when little or no cell death or disintegration is occurring. The formation of this soluble specific material by pneumococci on growth in vitro suggested the probability of an analogous substance being formed on growth of the organism in the animal body. Examination of the blood and urine of experimentally infected animals gave proof of the presence of this substance in considerable quantities in the body fluids following intraperitoneal infection with pneumococcus. In other words, this soluble material elaborated at the focus of the disease readily diffuses throughout the body, is taken up in the blood, passes the kidney, and appears in the urine unchanged in specificity. Similarly, a study of the serum of patients suffering from lobar pneumonia has revealed a substance of like nature in the circulating blood during the course of the disease in man. Furthermore, examination of the urine of patients having pneumonia due to pneumococci of Types I, II, and III has shown the presence of this substance in some stage of the disease in approximately two-thirds of the cases. Recently from filtered alkaline extracts of pulverized bacteria of several varieties, including pneumococci, Zinsser and Parker have prepared substances which appear free from coagulable protein. These substances, called "residue antigens," are specifically predpitable by homologous antisera. These observers consider these acid- and heat-resistant antigenic materials analogous to the soluble specific substance of pneumococcus described
by Dochez and Avery. In spite of the fact that these "residue antigens" are precipitable by homologous sera produced by immunization with the whole bacteria, Zinsser and Parker have so far failed to produce antibodies in animals by injecting the residues.

In the earlier studies by Dochez and Avery certain facts were ascertained concerning the chemical characteristics of this substance. It was found that the specific substance is not destroyed by boiling; that it is readily soluble in water, and precipitable by acetone, alcohol, and ether; that it is precipitated by colloidal iron, and does not dialyze through parchment; and that the serological reactions of the substance are not affected by proteolytic digestion by trypsin. Since the substance is easily soluble, thermostable, and type-specific in the highest degree, it seemed an ideal basis for the beginning of a study of the relation between bacterial specificity and chemical constitution. The present report deals with the work done in this direction.

EXPERIMENTAL

The organism used in the present work was Pneumococcus Type II. The most abundant source of the soluble specific substance appeared to be an 8 day autolyzed broth culture; hence this material was used as the principal source of supply. For comparison dissolved pneumococci and lots of urine containing the specific substance were also worked up, with essentially the same results, as will be seen from Table I.

The process for the isolation of the soluble specific substance consisted in concentration of the broth, precipitation with alcohol,
repeated re-solution and reprecipitation, followed by a careful series of fractional precipitations with alcohol or acetone after acidification
of the solution with acetic acid, and, finally, repeated fractional precipitation with ammonium sulfate and dialysis of the aqueous solution of the active fractions.

Five lots of 15 liters each of 8 day cultures of Pneumococcus Type II in meat infusion phosphate broth are each concentrated on the water bath in large evaporating dishes to 1,000 to 1,200 cc. and precipitated in a separatory funnel by the gradual addition, with vigorous rotation, of 1.2 volumes of 95 per cent alcohol.The mixture separates into two layers, and is allowed to stand over night, or for several hours. The upper layer, which is almost black and comprises the largest part of the mixture, contains only traces of the soluble specific substance, and is siphoned off and discarded. The lower, more viscous layer is run into a 250 cc. centrifuge bottle (occasionally a second will be required), capped, and rotated at high speed for ½ hour. Three layers are formed, of which the uppermost is merely a further amount of the liquid previously discarded. The middle layer consists of a compact, greenish cake of insoluble matter and gummy material, and contains most of the soluble specific substance. The bottom layer, from which salts often separate, is a brownish syrup rich in salts and nitrogenous matter and relatively poor in specific substance, and can, by careful manipulation, be poured off
to a large extent. Although a small proportion of the specific substance is lost if this syrup is discarded, its elimination represents so considerable a purification as to warrant the sacrifice of the active material contained. The gummy cake remaining in the centrifuge bottle, together with adhering salts and syrup, is now rinsed out and ultimately combined with similar material from the other lots, All of this is then dissolved as completely as possible in water, care being taken to break up the many lumps of gummy material, diluted to 1 liter, and again precipitated with alcohol. In this case about 1.3 liters are required to precipitate all but the last traces of active material from the upper layer. This is again discarded and the lower layer treated as before. At this stage there is relatively less of the bottom layer, and it is more difficult to separate it from the cake containing the specific substance, but as much as possible is removed. The remaining material is smoothed out with water, diluted to about 500 cc., and centrifuged. The precipitate is washed twice with water, and the washings are combined with the main solution. The still turbid liquid, the volume of which should be about 750 cc., is put through the alcohol purification process a third time, about 1.1 liters of alcohol being required. After having been centrifuged, the active material is again dissolved in water, made definitely acid to litmus with acetic acid, and again centrifuged. The precipitate is washed three times with water acidulated with acetic acid, and the filtrate and washings are combined in a separatory funnel and diluted again if necessary to 750 cc. Acetone (redistilled) is now added until a permanent precipitate forms, about 250 c¢. being necessary. The precipitate is allowed to settle, whereupon the lower part of the mixture containing the precipitate is drawn off and centrifuged. The clear superuatant fluid is restored to the main solution, while the precipitate, which consists largely of insoluble material and gives an aqueous solution almost devoid of activity, is discarded. Fractional precipitation is continued, and even when the specific substance appears in quantity in the precipitate, it is occasionally possible to separate a lower, inactive, syrupy layer, as in the previous purifications by alcohol. Addition of acetone is continued until a test portion, heated on the water bath to remove acetone, diluted with saline, and neutralized, no longer gives a precipitate with immune serum, after which the upper layer may be discarded. The active precipitates are then redissolved in water, centrifuged again, and the supernatant liquid is diluted to 375 cc., reacidified with acetic acid, and again fractionated with acetone. If inactive fractions are obtained, the process is again repeated until no further purification results. Alcohol may be used for these fracfionations instead of acetone, the only difference being that a somewhat larger proportion is required. The active material is then dissolved in about 150 cc. of water and again made definitely acid with acetic acid. The solution is treated with solid ammonium sulfate until the first slight precipitate forms. This is generally inactive, and if so, may be discarded. Finally, ammonium sulfate is added to saturation, completely precipitating the specific substance if the volume of the solution is not too great. The mixture is allowed to stand for several hours and is then centrifuged and the precipitate washed with a little saturated ammonium sulfate solution. It is redissolved in about 75 cc. of water acidified with acetic acid, centrifuged if necessary, and again precipitated by saturation with ammonium sulfate. Finally, the specific substance so obtained is dissolved in water and dialyzed first against running tap water in the presence of chloroform and toluene, and finally against distilled water until tests for sulfate and phosphate ion are negative. Addition of acetic acid during the early stages of the dialysis assists in the removal of calcium, which otherwise forms a large part of the ash.

The dialyzed solution is concentrated to dryness on the water bath and the residue redissolved in hot water. If the solution is not perfectly clear, it is centrifuged again before being evaporated to dryness, and the whole process is repeated as long as insoluble material separates. Toward the end of the final concentration absolute alcohol may be added to assist in the precipitation of the substance.

Variations in the exact volumes given are often necessary with different lots of broth, but this will occasion little difficulty if all fractionations are controlled by the specific precipitin test.

As so obtained the soluble specific substance forms an almost colorless varnish-like mass which may be broken up and dried to constant weight at 100°C. in vacuo. The yield from 75 liters of broth averages about 1 gin., although it varies within rather wide limits in individual lots.

By the method outlined above all substances precipitable with
phosphotungstic acid or capable of giving the biuret reaction were eliminated. The residual material (Preparation 17, in Table I), for which no claim of purity is made, as efforts at its further purification are still under way, contained, on the ash-free basis, 1.2 percent of nitrogen. It was essentially a polysaccharide, as shown by the formation of 79 percent of reducing sugars on hydrolysis, and by the isolation and identification of glucosazone from the products of hydrolysis."

"Table I represents a summary of the reactions of some of the earlier preparations worked with, as well as the later ones. Preparation 4 was obtained from the urine of a patient with a Type II pneumococcus infection, while No. 8 was obtained from an antiforrain solution of the pnemnococci. In both of these cases, as well as in Nos. 9, 11, and 15, the method of purification given above had not been fully worked out.

Attempts to stimulate antibody production by the immunization of animals with the purified substance yielded negative results.

DISCUSSION.

While it has long been known that the capsular material of many microorganisms consists, at least in part, of carbohydrates, any connection between this carbohydrate material and the specificity relationships of bacteria appears to have remained unsuspected. While it cannot be said that the present work establishes this relationship, it certainly points in this direction. Evidence in favor of the probable carbohydrate nature of the soluble specific substance is the increase in specific activity with reduction of the nitrogen content, the increase in optical rotation with increase in specific activity, the parallelism between the Molisch reaction and specific activity, the high yield of reducing sugars on hydrolysis, and the actual isolation of glucosazone from a small quantity of the material. The small amounts of substance available up to the present have hindered the solution of the problem, and it is hoped that efforts at further purification of the soluble specific substance, now in progress with larger amounts of material, will definitely settle the question.

SUMMARY.

  1. A method is given for the concentration and purification of the soluble specific substance of the pneumococcus.
  2. The material obtained by this method is shown to consist mainly of a carbohydrate which appears to be a polysaccharide built up of glucose molecules.
  3. Whether the soluble specific substance is actually the polysaccharide, or occurs merely associated with it, is still undecided, although the evidence points in the direction of the former possibility."

https://doi.org/10.1084/jem.38.1.73

A beautiful mind?

Heidelberger's original 1923 paper can hardly be claimed to be the slam-dunk proof that bacteria antigens are carbohydrates. For starters, Heidelberger admitted that he was unsure if the presumed "antigen" substance was a carbohydrate or if it was merely associated with it. Even more importantly, he could not produce any antibody response upon injecting his presumed antigen into animals. This would indicate that the substance was not an antigen whatsoever as antigens are specifically defined as "a toxin or other foreign substance which induces an immune response in the body, especially the production of antibodies." Thus, it seems rather odd to assume antibodies are proteins based off of this work, but assume they did:

MICHAEL HEIDELBERGER
1888–1991

"Since the pneumococcal capsular antigen was a polysaccharide, and antibodies were thought to be proteins, Heidelberger realized that by measuring the amount of protein in specific precipitates made with the capsular antigen he could determine their antibody content. Together with Forrest Kendall, who had joined the Heidelberger lab, the protein content of immune precipitates was determined by measuring total nitrogen, using the Kjeldahl procedure that came to be the hallmark of laboratories carrying out Heidelberger-type quantitative immunochemistry."

https://www.google.com/url?sa=t&source=web&rct=j&url=https://www.nasonline.org/publications/biographical-memoirs/memoir-pdfs/heidelberger-michael.pdf&ved=2ahUKEwjS_eHep5LwAhWRW80KHbpiBkYQFjADegQIDxAC&usg=AOvVaw0k4UkxWaHA7b3x5vqFuJuO

Since they assumed the pneumococci bacteria was a polysaccharide, that meant any nitrogen left over was the antibody content. Based on the 1923 paper, this seems to be a rather falicious premise to build from. In any case, Heidelberger carried on with his assumption and it can be seen by this second paper from 1929 how Heidelberger came to his conclusion using the precipitin test and mathematics as proof that antibodies exist. I edited out the long mathematical sections with his equations so if you are interested in Heidelberger showing his work, I recommend reading the full paper. Highlights below:

A QUANTITATIVE STUDY OF THE PRECIPITIN REACTION BETWEEN TYPE III PNEUMOCOCCUS POLYSACCHARIDE AND PURIFIED HOMOLOGOUS ANTIBODY*

"Of all the reactions of immunity the precipitin test is perhaps the most dramatic and striking. While other immune reactions are more delicate, the precipitin test is among the most specific and least subject to errors and technical difficulties. Attempts at its quantitative interpretation and explanation have been hampered either by the difficulty of finding suitable analytical methods or by the failure to separate the reacting substances from closely related, non-specific materials with which they are normally associated.

With the aid of recent work it has been found possible to avoid these difficulties to some extent. The isolation of bacterial polysaccharides which precipitate antisera specifically and possess the properties of haptens has not only afforded one of the components of a precipitin reaction in a state of comparative purity, but has greatly simplified the analytical problem. Since many of these polysaccharides contain no nitrogen, and antibodies presumably are nitrogenous, the latter may be determined in the presence of any amount of the specific carbohydrate. Moreover, Felton's method for the separation of pneumococcus antibodies from horse serum not only permits the isolation of a high proportion of the precipitin, freed from at least 90 percent of the serum proteins and much of the serum lipoid, but is also applicable on a sufficiently large scale to furnish the amounts of antibody solution needed to make quantitative work possible. It is realized that antibody solutions of this type do not contain pure antibodies–indeed, only 40 to 50 percent of the nitrogen is specifically precipitable–but since so small a proportion of the original serum protein remains with the antibody a far-reaching purification actually has been effected. It should thus be possible with the aid of antibodies purified by Felton's method to obtain data of a preliminary character which should point toward the mechanism of the reaction. The present paper is concerned with such data obtained in a quantitative study of the precipitin reaction between the soluble specific substance of Type III pneumococcus and Type III pneumococcus antibody solution.

EXPERIMENTAL

1. Materials and Methods.–a. Solutions of Soluble Specific Substance, Type Ill
Pneumococcus.–The soluble specific substance of Type III pneumococcus used was kindly supplied by Drs. O. T. Avery and W. F. Goebel of The Rockefeller Institute for Medical Research. It was ash-free, contained 0.04 percent of nitrogen, and showed a/d = -32 °. A weighed amount of anhydrous substance was suspended in 0.9 percent saline, dissolved with the aid of 0.1 normal sodium hydroxide, and the solution was diluted with saline, adjusted to pH 7.6 and made up to volume with saline to yield a 1 percent solution. This was sterilized in the autoclave and used as a stock solution for making up other dilutions. These were prepared with sterile saline under aseptic precautions, and were kept in the ice-box.

b. Type III Pneumococcus Antibody Solution.–The antibody solutions used were prepared essentially according to Felton's procedure (loc. cit.) from Type III antipneumococcus horse serum containing no preservative and supplied by the New York State Department of Health through the courtesy of Dr. A. B. Wadsworth and Dr. Mary B. Kirkbride. 100 to 200 cc. of serum were stirred slowly into 20 volumes of ice-cold water containing 9.5 cc. of molar potassium dihydrogen phosphate and 0.5 cc. of molar dipotassium hydrogen phosPhate per liter. The final pH varied from 5.6 to 6.3. After standing over night in the cold the supernatant was decanted and the precipitate was centrifuged off in the cold and dissolved in a volume of chilled 0.9 percent saline equal to that of the serum taken. 0.1 normal hydrochloric acid was then added until a precipitate no longer formed on dilution of a test portion with two volumes of water, after which 0.1 normal sodium hydroxide solution was added until a slight precipitate again formed on dilution. In general, 5 cc. of acid and 1.5 cc. of alkali per 100 cc. of serum were satisfactory, although as Felton emphasizes, different lots vary and no absolutely definite procedure can be given. In the present work the process of purification was followed either by testing the agglutinating power of the fractions against a heat-killed Type III pneumococcus vaccine, or by the precipitin reaction, or by both methods. After addition of the alkali the opalescent solution was diluted with 2 volumes of water and centrifuged in the cold. The almost inactive precipitate was discarded and the supernatant poured into 6.7 volumes of the chilled buffer solution previously used, (equivalent to 20 times the volume of saline employed), also adding enough 0.1 normal sodium hydroxide to neutralize the remaining acid. The resulting precipitate was collected and dissolved in a volume of 0.9 percent saline equal to that of the serum taken, and the pH was adjusted to 7.6. The solution was sterilized by passage through a Berkefeld N grade filter which previously had been washed with saline containing a drop of normal sodium hydroxide, followed by saline alone.

Antibody solutions prepared in this way were found to be rather unstable under the usual conditions of the precipitin test, and it therefore was necessary to subject them to a preliminary "ageing" treatment in order that control solutions might be relied upon to remain clear. This consisted in immersing the solution in a water bath at 37 ° for 2 hours, letting stand in the ice-box over night, centrifuging off the precipitate which usually formed, readjusting the pH if necessary, and filtering through a Berkefeld candle prepared as above. This treatment was repeated as many times as necessary, but the solutions usually remained clear after the second incubation at 37 °. Much time was lost and very inconstant results were obtained until "ageing" was resorted to.

The relative antibody content of the resulting solutions was estimated by determining the agglutination titer against a single heat-killed Type III pneumococcus suspension.

It will be seen from Table I that the agglutination titer and the
maximum amount of protein precipitable by the type III polysaccharide ([total N–N in supernatant] X 6.25) are approximately proportional. The latter may therefore be taken as a more definite, though not necessarily more accurate, measure of the actual antibody content of the solutions.

It is also evident that the antibody in all of these solutions has been purified to approximately the same extent, since the ratios of protein precipitable by SSS III to total protein are not very different."

DISCUSSION

"For purposes of discussion it will be assumed with Felton (lot. cir.) that antibody is ,modified protein, and that, in order to provide a uniform method of measurement, it may be expressed as nitrogen precipitable by specific polysaccharide, multiplied by 6.25. Since only relative values are under consideration, the actual magnitude of the factor used is of little significance so long as it be used throughout. Moreover, Table I shows a correspondence between this measure of antibody content and the agglutination titer, so that its use as a relative measure is independent of the nature of Type III pneumococcus antibodies.

doi: 10.1084/jem.50.6.809.

The Precipitin Reaction
In Summary:
  • Michael Heidelberger teamed up with Oswald Avery to characterize a "soluble specific substance" found in pneumococcal bacteria that fell out of solution when incubated with type-specific antisera
  • When it was virtually nitrogen-free, recalled Heidelberger in a 1979 article, Avery ventured a guess: "Could it be a carbohydrate?"
  • Chemical analysis confirmed its sugary character, and subsequent studies of other pneumococcal serotypes revealed that each bacterial capsule had a distinct polysaccharide signature
  • It was this signature that dictated the serological specificity of the organism
  • Their results were met with considerable skepticism, as it was then thought that only proteins could incite a specific immune response
  • According to polysaccharide-based vaccine specialist Emil Gotschlich: "Nobody believed it. It took them a lot of effort to convince people that the polysaccharide was the immunoreactive component."
  • Heidelberger and Avery's discovery came at a time when antibodies were regarded—by those who believed they existed at all—as mysterious substances that floated around in serum
  • "It appeared to me that there was a crying need to determine the true nature of antibodies," wrote Heidelberger in 1979, "and that until this was done there could be no end to the polemics and uncertainties that were plaguing immunology"
  • Heidelberger and his postdoctoral fellow Forrest Kendall later quantitated the precipitin reaction, bringing much-needed mathematics to the study of antibody–antigen interactions and lifting antibodies even further out of the realm of the mysterious
  • In 1917 Dochez and Avery showed that whenever pneumococci are grown in fluid media, there is present in the cultural fluid a substance which precipitates specifically in antipneumococcus serum of the homologous type
  • It was assumed that the formation of this soluble specific material by pneumococci on growth in vitro suggested the probability of an analogous substance being formed on growth of the organism in the animal body
  • Examination of the urine of patients with pneumococci showed the substance in only approximately 2/3rds of the samples
  • Zinsser and Parker found similar substances with other bacteria and believe that the substances are the same as that of the pneumococci
  • In spite of the fact that these "residue antigens" are precipitable by homologous sera produced by immunization with the whole bacteria, Zinsser and Parker failed to produce antibodies in animals by injecting the residues.
  • The process for the isolation of the soluble specific substance consisted in:
    1. Concentration of the broth
    2. Precipitation with alcohol
    3. Repeated re-solution and reprecipitation
    4. A careful series of fractional precipitations with alcohol or acetone after acidification of the solution with acetic acid
    5. Repeated fractional precipitation with ammonium sulfate and dialysis of the aqueous solution of the active fraction
  • For a complete step-by-step breakdown of the numerous chemical-altering procedures done to the sample, see the highlighted tan section of the paper provided above
  • Even with the numerous "purification" steps, the obtained soluble specific substance formed an almost colorless varnish-like mass
  • The residual material for which no claim of purity was made, as efforts at its further purification were still under way, contained, on the ash-free basis, 1.2 percent of nitrogen.
  • It was considered essentially a polysaccharide
  • The method of purification given had not been fully worked out for many of the preparations
  • Attempts to stimulate antibody production by the immunization of animals with the purified substance yielded negative results
  • While it had long been known that the capsular material of many microorganisms consists, at least in part, of carbohydrates, any connection between this carbohydrate material and the specificity relationships of bacteria remained unsuspected
  • While it could not be said that their work established this relationship, they felt it certainly pointed in that direction
  • The small amounts of substance available hindered the solution of the problem, and it was hoped that efforts at further purification of the soluble specific substance with larger amounts of material would definitely settle the question
  • Whether the soluble specific substance is actually the polysaccharide, or occurs merely associated with it, was left undecided
  • Heidelberger acknowledged that the precipitin test he used during this experiment has 2 drawbacks:
    1. Quantitative interpretation/explanation is difficult due to lack of a suitable analytical method
    2. Failure to separate out the reacting substances from non-specific material which these substances are closely related to and associated with
  • He stated that it was possible to avoid these failures to some extent
  • It is presumed that antibodies are nitrogenous
  • Only 90% of the precipitin can be freed from serum proteins and "much" of the lipoid
  • Heidelberger admitted that these are not pure antibodies and that only 40-50% of nitrogen is precipitable while small amounts of serum remain
  • The antibody solutions used were prepared essentially according to Felton's procedure from Type III antipneumococcus horse serum containing no preservative and supplied by the New York State Department of Health through the courtesy of Dr. A. B. Wadsworth and Dr. Mary B. Kirkbride
    1. 100 to 200 cc. of serum were stirred slowly into 20 volumes of ice-cold water containing 9.5 cc. of molar potassium dihydrogen phosphate and 0.5 cc. of molar dipotassium hydrogen phosphate per liter
    2. The final pH varied from 5.6 to 6.3
    3. After standing over night in the cold the supernatant was decanted and the precipitate was centrifuged off in the cold and dissolved in a volume of chilled 0.9 percent saline equal to that of the serum taken
    4. 0.1 normal hydrochloric acid was then added until a precipitate no longer formed on dilution of a test portion with two volumes of water, after which 0.1 normal sodium hydroxide solution was added until a slight precipitate again formed on dilution
    5. In general, 5 cc. of acid and 1.5 cc. of alkali per 100 cc. of serum were satisfactory, although as Felton emphasized, different lots vary and no absolutely definite procedure can be given
    6. After addition of the alkali the opalescent solution was diluted with 2 volumes of water and centrifuged in the cold
    7. The almost inactive precipitate was discarded and the supernatant poured into 6.7 volumes of the chilled buffer solution previously used, (equivalent to 20 times the volume of saline employed), also adding enough 0.1 normal sodium hydroxide to neutralize the remaining acid
    8. The resulting precipitate was collected and dissolved in a volume of 0.9 percent saline equal to that of the serum taken, and the pH was adjusted to 7.6
    9. The solution was sterilized by passage through a Berkefeld N grade filter which previously had been washed with saline containing a drop of normal sodium hydroxide, followed by saline alone
  • Antibodies were found to be unstable during testing so they were put through preliminary "ageing" processes as many times as needed until they got the result they wanted
  • Much time was lost and very inconstant results were obtained until "ageing" was resorted to.
  • The relative antibody content of the resulting solutions was estimated by determining the agglutination titer against a single heat-killed Type III pneumococcus suspension
  • For purposes of discussion it was assumed with Felton that antibody is modified protein, and that, in order to provide a uniform method of measurement, it may be expressed as nitrogen precipitable by specific polysaccharide, multiplied by 6.25
  • There is no need to spend any more time on the rest of Heidelberger's paper as he admitted he assumed antibodies were protein and could be expressed as nitrogen thus he did not prove anything
Why would monoclonal antibodies not form a precipitate?

It is rather obvious that many assumptions were made about a substance (antibodies) for which the researchers could not see. Michael Heidelberger assumed that antibodies are modified proteins and nitrogenous. He assumed that it may be expressed as nitrogen precipitable by specific polysaccharide, multiplied by 6.25. He assumed that the failure of the precipitin test to separate out the reacting substances from non-specific material which these substances are closely related to and associated with could be somewhat avoided to some extent. He assumed that his earlier work with the pneumococcus bacteria was accurate and that he had proved the antigen component was a carbohydrate even though he was unable to produce any antibody response upon immunizing animals using his supposed antigen. Maybe this lack of any antibody response to his "antigen" has to do with the fact that, according to the WHO, the pneumococcus bacteria is regularly found in healthy people?

"Infection is acquired mainly through pneumococci contained in respiratory droplets. There are many healthy, asymptomatic carriers of the bacteria but no animal reservoir or insect vector."

https://www.who.int/ith/diseases/pneumococcal/en/

https://web.archive.org/web/20200818101511/https://www.who.int/ith/diseases/pneumococcal/en/

If an antigen is a toxin or foreign substance which produces an immune response creating antibodies, the pneumococci bacteria doesn't meet that definition at all. If it isn't an antigen, then the pneumococcus "antigen" would not be carbohydrates as described in Heidelberger's 1923 paper. This would mean that Heidelberger's 1929 paper measuring any of the remaining protein content, calculating the amount, and claiming the resulting protein mass as antibodies is essentially meaningless. Can you see the problem with assuming things to be true without ever proving this to be the case?

The conclusions drawn by Heidelberger were born out of chemistry experiments and reactions using the precipitin test which have no bearing on reality while using mathematical equations attempting to quantify the unquantifiable. Whether or not these indirect experiments and assumptions provide proof that antibodies exist and are proteins, I leave up to the reader. However, keep in mind that no antibodies had ever been seen nor proven to exist by proper purification and isolation up to that time and that still holds true to date. This work is based off of theoretical explanations of immunity for which nothing could be observed. Heidelberger's indirect chemical reactions and equations provided no direct evidence for the existence of anything other than non-specific precipitate.

ViroLIEgy
5 May 2022 | 12:17 pm

Paul Ehrlich’s Side-Chain Antibody Theory (1900) Part 2: The Complement System


For of course it would be absurd to imagine that the mechanical diagrams have any representation in the world of fact. They are figments of the imagination, and may serve some useful purpose as picture books serve in teaching a child the alphabet. But as the time comes when the child puts aside the blocks and takes in hand the pen, so pathologists must ultimately lay aside the crude mechanism of haptophores and amboceptors and learn to deal with the phenomena of immunity in terms of the protein molecule and the chemical atom.

Williams and Beveridge, "Mechanism of Immunization" (cit. n. 27), pp. 623-624.

In part one of Paul Ehrlich's address to the Royal Society, we looked at the outline for his "Side-Chain" theory of immunity. Ehrlich laid out the main components of his vision such as the side-chains, the haptophore group, the toxophore group, the antigen-antibody relationship, the lock and key mechanism, and the "tentacles" aiding in digestion. He built his concepts off of Emil Von Behring's diphtheria toxin research and the suggestion of something present in the blood. From there, Ehrlich abandoned the law of parsimony and used his "lively imagination" to dream up what he considered to be the most plausible and easy explanation for immunity.

This last part of Ehrlich's presentation to the Royal Society centered around the idea of the complement system. In short, this is a part of the immune system that complements the ability of antibodies to clear out the damaged cells and the inhabiting microbes through inflammation in order to attack the pathogen and eliminate it from the body. Here is a brief overview of Ehrlich's role in the creation of this theory:

The complement system: history, pathways, cascade and inhibitors

"Paul Ehrlich described the side-chain theory of antibody formation, especially the mechanisms of antibody neutralisation by toxins that induced bacterial lysis with the help of complement (which has replaced the historical term alexin). According to his theory, the immune cells contained receptors that could recognise antigens, and following immunisation, these receptors multiplied and were shed into the circulation as 'amboceptors' (now called antibodies). These antibodies attached not only to specific antigens but also to a heat-labile antimicrobial component called 'complement' [89]. Ehrlich's theory proposed that the antibody and complement combined to form a complex enzyme capable of attacking and killing cells and micro-organisms. In the ensuing years, this concept had a protagonist in the form of Bordet who argued that the antigen-antibody union was reversible, contradicting Ehrlich's view that the antigen-antibody union was a firm and based on stereo chemical specificity [10]. Ehrlich's concept emphasised the presence of multiple antigens and complements in the serum, while Bordet's view revolved around a 'single complement' component that bound non-specifically to the antigen."

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3956958/

It looked as if there was a war brewing in the early 20th century between Paul Ehrlich and Jules Bordet's competing antibody theories. While Ehrlich envisioned antibodies and their complements that were specific to the antigen, Bordet viewed the relationship as non-specific. Fortunately, as virology and Immunology love to have their cake and eat it too, both Ehrlich and Bordet were deemed correct when it was "understood" that the complements can be both specific and non-specific, thus averting the war:

"Ehrlich believed that each antigen-specific amboceptor had its own specific complement, while Bordet believed that there is only one type of complement. In the early 20th century, this controversy was resolved when it was understood that complement can act in combination with specific antibodies, or on its own in a non-specific way."

https://www.bionity.com/en/encyclopedia/Complement_system.html

Below are the rest of the highlights from the remainder of Ehrlich's report on his theoretical investigation into immunity which details the complement system. I provided some additional insight afterwards regarding how this theory was utilized and the controversy surrounding Ehrlich's "beautfil drawings:"

On Immunity with Special Reference to Cell Life.

"I have now laid before you the fundamental facts which up to the present constitute our knowledge in the field pertaining to immunity,
and which can be most easily and successfully explained through the agency of "the side-chain theory." I wish in a few words to dispel some erroneous ideas which have been advanced in opposition to this theory.

Roux has shown that very small quantities of tetanus toxine, if injected directly into the brain, cause the death of the animal. Roux assumes that such an occurrence is not compatible with my theory. Roux is of opinion that according to my theory the brain must be quite immune against tetanus toxine, as the toxophile side-chains of the brain-cells must be identical with the antitoxine, and therefore must exercise an immediate protective action. Experiment showing quite the reverse, the theory is overthrown.

Roux came to this incorrect conception through an erroneous conception of antitoxine. The toxophile side-chains of the brain cells draw directly to themselves the toxine molecules, and, according to my theory, are thus a necessary preliminary condition of the illness. The toxophile groups are therefore really inducers of the action of the poison, and not its preventives.

Those toxophile groups which, like the antitoxines present in the serum, are able to lay hold of toxine immediately on its entry into the blood, and so to divert it from organs essential to life, can alone be regarded as being possessed of any antitoxic action in the true sense of the word. I may be allowed to call to mind Weigert's excellent simile of iron and the lightning conductor. Iron attracts electricity, and is therefore used as a lightning conductor. Great masses of iron present in buildings give rise to, or increase, the risk of their being struck by lightning, and the metal only becomes protective against lightning when it is so employed that the electricity is conducted away outside the building. It would never occur to anyone to speak of great masses of iron machinery present in buildings as if they were lightning conductors. It is equally unreasonable to speak of
the antitoxic property of the brain cortex, in which the toxophile groups are present in great quantity, but also retain their relations with the nerve-cells. When this really considerable misunderstanding is eliminated from Roux's results these become entirely confirmatory of my views, and it is difficult to understand how, subsequent to Weigert having placed the matter in so clear a light, the beautiful experiments of Roux can be utilised by another eminent authority as a means of combating my theory.

Much more complex than in the cases hitherto discussed are the conditions when, instead of the relatively simple metabolic products of microbes, the living micro-organisms themselves come to be considered, as in immunisation against cholera, typhoid, anthrax, swine fever, and many other infectious diseases. There then come into existence alongside of the antitoxines, produced as a result of the action of the toxines, manifold other reaction products. This is because the bacterium is a highly complicated living cell, of which the solution in the organism yields a great number of bodies of different nature, in consequence of which a multitude of "Antikorper" are called into existence. Thus we see, as a result of the injection of bacterial cultures, that there arise alongside of the specific bacteriolysines, which dissolve the bacteria, other products, as, for example, "coagulines" (Kraus, Bordet), i.e., substances which are able to cause the precipitation of certain albuminous bodies contained in the culture fluid injected; also the so-much discussed "agglutinines"
(Durham, Gruber, Pfeiffer), the antiferments (von Dungern), and no doubt many other bodies which we have not yet recognised.

It is by no means unlikely that each of these reaction products finds its origin in special cells of the body; on the other hand, it is quite likely that the formation of any single one of these bodies is not of itself sufficient to confer immunity. Thus in case of the introduction of bacteria into the body we have to do with a many-sided production of different forms of "Antikorper," each of which is directed only against one definite quality or metabolic product of the bacterial cell. Accordingly, in recent times, the practice of using for the production of immunisation definite toxic bodies isolated from the bacterial cells has been more and more given up, and for this purpose it is now regarded as important to employ the bacterial cells as intact as possible. The beautiful results obtained for plague by Haffkine, and quite recently by Wright in your own country for typhoid fever, have been arrived at in this way.

The most interesting and important substances arising during such an immunising process are without doubt the bacteriolysines, in the investigation of which Pfeiffer has done such yeoman's service. How really wonderful it is that after the introduction of the cholera-vibrio into the animal body a substance is formed endowed with the power of dissolving the cholera vibrio, and that vibrio only!

This seemingly purposeful and novel phenomenon seems at first sight to have nothing to do with those forces which are normally at the disposal of the organism. It was of the greatest importance to explain the origin of these substances from the standpoint of cellular physiology. The solution offered very considerable difficulties, and was first attained when instead of bacteriolysines, hsemolysines came to be employed in experiments. Hsemolysines are peculiar toxic bodies, which destroy red blood corpuscles by dissolving them. Hsemolysines may occur in a normal blood when they exercise a solvent action on the red blood corpuscles of other species, or they may be artificially produced, in which case, after an animal has undergone a process of immunisation against the blood corpuscles of another species, there appear in the serum hsemolysines which destroy the kind of blood corpuscles employed in the production of the immunity. In their essential characters they are absolutely comparable with the bacteriolysines: but they possess over them the great advantage that they admit of being employed in test-tube experiments, and thus afford opportunity for exact quantitative work altogether independent of the variability of the animal body.

Belfanti and Carbone first discovered the remarkable fact that horses which have been treated with the blood corpuscles of rabbits contain in their serum constituents which are poisonous for the rabbit, and for the rabbit only. While the serum of the normal horse, to the quantity of 60 c.c., could be intravenously injected without harm to the rabbit, a very few c.c. of serum from horses previously so treated with rabbit's blood, proved fatal.

Bordet showed shortly thereafter, that in the case quoted there was present in the serum a specific hsemolysine which dissolved the blood corpuscles of the rabbit. He also proved that these hsemolysines as had already been shown by Buchner and Daremberg in the case of similarly acting bodies which are present in normal blood lost their solvent property on being maintained during half an hour at a temperature of 55° C. Bordet added, further, the new fact, that the blood-solvent property of these sera which had been deprived of solvent power by heat, the solvent action could be restored if certain normal sera were added to them.

By this important observation an exact analogy was established with the facts of bacteriolysis as elicited by the work of Pfeiffer, Metchnikoff, and Bordet. In the work on the Pfeiffer phenomenon of bacteriolysis, it had already been ascertained that the solution of bacteria by specific bacteriolysines was brought about by the combined action of two different bodies: one which was specific, arose during the immunisation and was stable; and another, a very unstable body, which was present in normal serum.

In collaboration with Dr. Morgenroth, I have sought in regard to this question, for which haemolysis offered prospects favourable to experimentation, to make clear the mechanism concerned in the action of these two components—the stable, which may be designated "immune body," and the unstable, which may be designated "complement"—which, acting together, effect the solution of the red blood corpuscles. For this purpose, in the first place, solutions containing either only the "immune body" or only the "complement" were brought in contact with suitable blood corpuscles, and after separation of the fluid and the corpuscles by centrifugalising, we investigated whether these substances had been taken up by the red blood corpuscles or remained behind in the fluid. The proof of its location in the one position or in the other was readily forthcoming, since to restore to the hsemolysine its former activity, it was only necessary to add to the "immune body" a fresh supply of "complement," or to the "complement" a fresh supply of "immune body," in order that the presence of the hsemolysine in its integrity might be shown by the occurrence of solution of the blood-cells.

The experiments proved that, after centrifugalising, the "immune body" is quantitatively bound to the red blood corpuscles, and that the "complement," on the contrary, remains entirely behind in the fluid. The presence of the two components in contact with blood corpuscles only occasions the solution of these at higher temperatures, and not at 0° C. And an active haemolytic serum (with "immune body" and "complement" both present) having been placed in contact with red blood corpuscles and maintained for a while at 0° C., it was found after centrifugalising that, under these circumstances also, the "immune body" had united with the red blood corpuscles, but that the "complement" remained in the serum. This experiment showed that both components must, at a temperature of 0° C., have existed alongside of one another in a free condition.

But when analogous experiments were undertaken at a higher temperature it was found that both components were retained in the sediment.

These facts can only be explained by making certain assumptions regarding the constitution of the two components, i.e., of the "immune body" and the "complement." In the first place, two haptophore groups must be ascribed to the "immune body," one having a great affinity for a corresponding haptophore group of the red blood corpuscles and with which at lower temperatures it quickly unites, and another haptophore group of a lesser chemical affinity, which at a higher temperature becomes united with the "complement" present in the serum. Therefore, at the higher temperature, the red blood corpuscles will draw to themselves those molecules of the "immune body" which in the fluid have previously become united with the "complement." In this case the "immune body" represents in a measure the connecting chain which binds the complement to the red blood corpuscles, and so brings them under its deleterious influence. Since under the influence of the "complement"—at least, in the case of the bacteria— appearances are to be observed (for example, in the Pfeiffer phenomenon) which must be regarded as analogous to digestion, we shall not seriously err if we ascribe to this "complement" a ferment-like character.

It is obvious that when the normal serum of one animal possesses haemolytic action on the blood of another, the component of the hsemolysine which here unites with the red blood corpuscle and forms the connecting link between it and the "complement" which is essential to the occurrence of solution, cannot, in the absence of any preceding process of immunisation, be designated "immune body." In its
characteristics and action, however, it only differs from this in occurring naturally, and may well be designated "intermediate body" (Zwischenkorper). It may here be stated that the constitution of a haemolysine is graphically represented in fig. 7, Plate 7.

Very important for the conclusion that only with the assistance of the
"intermediate body" or of the "immune body" can the "complement," which leads to the solution, become united with the blood corpuscle, is the following experiment. The serum of the dog has very considerable solvent action upon guinea-pig's blood, but loses this property if warmed. If dog's serum, thus rendered inactive by warming, is brought into contact with suspended corpuscles of guinea-pig's blood, these are not dissolved; but, if to such a mixture there be also added guinea-pig serum, i.e., the serum normal to these red blood corpuscles,
the erythrocytes are at once dissolved. Here the only explanation is that the "intermediate body," which possesses a specific affinity for guinea-pig erythrocytes, and is present in the inactive dog's serum, is able to seize on one of the many "complements" present in guinea-pig's serum, with the result that the "complement" which cannot normally attach itself to the corpuscles, comes now to exercise its destructive influence.

We see at the same time from this experiment that the hsemolysines occurring naturally, obey the same laws as those produced through the process of immunising. In fact, for them also, in a great number of instances, precisely similar behaviour has been demonstrated.

The character of the specific union made it possible to find solutions for a number of important questions. In the first place, regarding the multiplicity of the heemolysines, which occur normally in serum, it is well known that numerous sera are able to dissolve blood corpuscles of different species. For example, serum of the dog dissolves blood corpuscles of the rabbit, guinea-pig, rat, goat, sheep, &c. The complex nature of these haemolysines has been already indicated.

Another question arises whether in a serum that is capable of such manifold action there is present one single haemolysine that destroys different red blood-cells, or whether a whole series of hsemolysines come into action, of which one is adapted to guinea-pig blood, another to rabbit blood, &c. The solution of this question may be approached in another way. The serum may be rendered inactive by heat, and then placed in contact with red blood corpuscles of a given kind. Then, supposing, for example, that rabbit blood has been employed, it is found that if the fluid is freed from the erythrocytes by centrifugalisation and the "complement" afterwards added, it is no longer in a position to dissolve rabbit blood, but has not suffered any impairment of its action on other kinds.

By this method of elective absorption it is proved that the normally occurring hsemolysines which chain the blood corpuscles of the rabbit to themselves, are specifically adapted to this purpose. If with suitable adjustment of conditions similar experiments be conducted with other kinds of blood, results are obtained which force us to the conviction that in such a serum acting on various kinds of blood there are present absolutely different "intermediate bodies" (analogues of the "immune bodies"), of which each one is specific for one kind of blood, i.e., one is adapted for rabbit's blood, a second for calf's blood, &c. Dr. Morgenroth and I have in some cases, indeed, succeeded in proving that the "complements" which are adapted to fit themselves to these "intermediate bodies," and occur in normal sera, differ among themselves. If we reflect that in normal blood, in addition to these different hsemolysines, there are besides a long series of analogous bodies, agglutinines of very different kinds, bacteriolysines, enzymes, anti-enzymes, we are brought more and more to the conviction that the blood serum is the carrier of substances innumerable as yet little known or conceived of.

Having obtained a precise conception of the method of action of the lysines of the serum—of the hsemolysines, and thereby also of the bacteriolysines—it becomes possible for us to attempt to solve the mystery of the origin of these bodies. I have in the beginning of this lecture fully developed the "side-chain theory," according to which the antitoxines are merely certain of the protoplasm "side-chains," which have been produced in excess and pushed off into the blood.

The toxines, as secretion products of cells, are in all likelihood still relatively uncomplicated bodies; at least, by comparison with the primary arid complex albumins of which the living cell is composed. If a cell of the organism has, with the assistance of an appropriate "side-chain," fixed to itself a giant molecule, as the proteid molecule really is, then, with the fixation of this molecule, there, is provided one of the conditions essential for the cell nourishment. Such giant molecules cannot at first be utilised by the cells, and are only made available when, by means of a ferment-like process, they are split into smaller fragments. This will be very effectually attained if, figuratively speaking, the "tentacle" or grappling arm of the protoplasm possesses a second haptophore group adapted to take to itself ferment-like material out of the blood fluid. Through such complex organisation, by which the "tentacle" acts also as the bearer of a ferment-functioning group, this group is brought into close relation with the prey destined to be digested and assimilated.

For such appropriate arrangements, in which the "tentacular" apparatus also exercises a digestive function—if it be permissible to pass from the abstract to the concrete—we find analogies in the different forms of insectivorous plants. Thus it has been known since the famous researches of Darwin that the tentacles of Drosera secrete a proteid-digesting fluid.

If we now recognise that the different lysines only arise through absorption of highly complex cell material—such as red blood corpuscles or bacteria—then the explanation, in accordance with what I have said, is that there are present in the organism "side-chains" of a special nature, so constituted that they are endowed not only with an atomic group by virtue of the affinities of which they are enabled to pick up material, but also with a second atomic group, which, being ferment-loving in its nature, brings about the digestion of the material taken up. Should the pushing-off of these "side-chains" be forced, as it were, by immunisation, then the "side-chains" thus set free must possess both groups, and will therefore in their characteristics entirely correspond to what we have placed beyond doubt as regards the "immune-body" of the hsemolysine.

In this manner is simply and naturally explained the astonishingly specialised arrangement that, through the introduction of a definite bacterium into the body, something is produced which is endowed with the power of destroying by solution the bacterium which was administered and no other. This contrivance of the organism is to be regarded as nothing more than a repetition of a process of normal cell-life, and the outcome of primitive wisdom on the part of the protoplasm.

In conclusion, I wish hastily to touch on only a few points. First, to direct attention to the fact that the immunising sera produced by the
administration of bacteria are sometimes limited in their operation to certain animal species, and are much more inconstant in their action than are the antitoxines. Sobernheim, in the laboratory of C. Fraenkel, found that the anthrax serum obtained by immunising German marmots (Hamster) protected this species, even in small doses; but was absolutely without action for rabbits. Kitt had a precisely similar experience with symptomatic anthrax. This circumstance is easy to understand, if the complex nature of the lysines be borne in mind. The lysine, be it bacteriolysine or hsemolysine ( i.e. "immune body + "complement"), possesses altogether three haptophore groups, of which two belong to the "immune-body" and one to the "complement." Each one of these haptophore groups can be bound by an appropriate "anti-group." Three anti-groups are thus conceivable, any one of which, by uniting with one of the haptophore groups of the lysine, can frustrate the action of the lysine. To my mind, of these three possible "Antikorper," that one which can lay hold of the haptophore group of the "complement," and so prevent this from uniting with the "immune body," is the most important. Dr. Morgenroth and I have experimentally succeeded in producing such bodies by processes of immunisation, and in proving that they unite with the "complement" (anticomplement).

Dr. Neisser at the Steglitz Institute sought to find an explanation of Sobernheim's experiments. He was able to determine that anthrax serum failed in mice, even if great quantities of fresh sheep's serum (i.e., containing excess of "complement") were at the same time introduced. The failure in this case appears to be due, on the one hand, to the destruction, in the body of the mouse, of the "complement" present in the sheep's serum, and, on the other hand, to the fact that the "immune body" yielded, by the sheep does not find in mouse serum an appropriate new "complement."

From this it appears, that in the therapeutic application of anti-bacterial sera to man, therapeutical success is only to be attained if we use either a bacteriolysine with a "complement" which is stable in man ("homostabile complement"), or at least a bacteriolysine, the "immune body" of which finds in human serum an appropriate "complement." The latter condition will be the more readily fulfilled the nearer the species employed in the immunisation process is to man. Perhaps the non-success which as yet has attended the employment of typhoid and cholera serum will be converted into the contrary if the serum be derived from apes and not taken from species so distantly removed from man as the horse, goat, or dog. However this may be, the question of the provision of the appropriate "complement" will come more and more into the foreground, for it really represents the centre round which the practical advancement of bacterial immunity must turn.

A second and at present much-discussed question is the immunising of the organism against elements standing biologically much higher in the scale than erythrocytes and much less foreign to the body than those exceedingly lowly organisms, the bacteria. I refer here to the production of "Antikorper" against cells of the higher animal organisation, e.y., ciliated epithelium (v. Dungern), spermatozoa (Landsteiner, Metchnikoff, Moxter), kidney cells, and leucocytes. These "Antikorper" are also of a complex nature. They obey the already described law of elective absorption, and their origin is in keeping with the "side-chain" theory. It is to be hoped that such immunisations as these, which are of great theoretical interest, may also come to be available for therapeutic application. The idea has already been mooted by v. Dungern, of attacking epithelial new formations, particularly carcinoma, by means of specific "antiepithelial sera," and Metchnikoff' has expressed the somewhat bold hope of being able to delay old age by means of a serum directed against phagocytes
(macrophages). But even if in the immediate future no great practical success is attained, we must remember that we are only at the very beginning of a rational investigation of properties of cells which hitherto have been far too lightly regarded.

The sifting of the material obtained by observation is rendered more difficult by the occurrence under normal conditions of a great number of quite unlooked for bodies furnished with haptophore groups and arising from diverse organs, and which we may designate collectively as haptines. It is to be expected that the study of these haptines will not only throw light on the more minute details of cellular metabolism, but also prove fruitful in the fields of pathology and therapeutics. By the fact that we can cause the individual haptines of the cells to pass out into the blood serum by a process of specific immunisation, it becomes possible in the test-tube to analyse more accurately the mode of operation of their binding groups than is possible in the case of the complicated conditions which present themselves in the animal body. The importance, for the study of immunity, of considering the circumstances from a purely cellular standpoint is evident from all that I have said.

I trust, my lords and gentlemen, that from what I have said you may have obtained the impression, to allude again to my quotation from Bacon, that we no longer find ourselves lost on a boundless sea, but that we have already caught a distinct glimpse of the land which we hope, nay, which we expect, will yield rich treasures for biology and therapeutics.

I desire to express my indebtedness to Dr. E. F. Bashford, McCosh Scholar of the University of Edinburgh, now working with me in my Institute, for his kindness in undertaking the translation of my lecture into English, a task to which he has devoted much time and trouble.

https://doi.org/10.1098/rspl.1899.0121

This process of binding serum complement was yet another indirect method used in an attempt to prove antibodies exist. If a reaction occurs, it is assumed the antibody-antigen exist in the mixture. The complement system described by Ehrlich formed the basis for the complement fixation test which has been used as an indirect method to discover "novel viruses" and/or to determine someone positive for a known "virus." You can find this test used in numerous virology papers as "evidence" that a new "virus" exists as well as whether or not it is related to other "viruses." Here is a brief description of what this test entails:

Complement Fixation

"Complement fixation is a classic method for demonstrating the presence of antibody in patient serum. The complement fixation test consists of two components. The first component is an indicator system that uses combination of sheep red blood cells, complement-fixing antibody such as immunoglobulin G produced against the sheep red blood cells and an exogenous source of complement usually guinea pig serum. When these elements are mixed in optimum conditions, the anti-sheep antibody binds on the surface of red blood cells. Complement subsequently binds to this antigen -antibody complex formed and will cause the red blood cells to lyse.

The second component is a known antigen and patient serum added to a suspension of sheep red blood cells in addition to complement. These two components of the complement fixation method are tested in sequence. Patient serum is first added to the known antigen, and complement is added to the solution. If the serum contains antibody to the antigen, the resulting antigen-antibody complexes will bind all of the complement. Sheep red blood cells and the anti-sheep antibody are then added. If complement has not been bound by an antigen-antibody complex formed from the patient serum and known antigens, it is available to bind to the indicator system of sheep cells and anti-sheep antibody. Lysis of the indicator sheep red blood cells signifies both a lack of antibody in patient serum and a negative complement fixation test. If the patient's serum does contain a complement-fixing antibody, a positive result will be indicated by the lack of red blood cell lysis."

https://bio.libretexts.org/Bookshelves/Microbiology/Book%3A_Microbiology_(Boundless)/12%3A_Immunology_Applications/12.2%3A_Immunoassays_for_Disease/12.2G%3A_Complement_Fixation

If you are wondering how the combining of human, sheep, and guinea pig blood proves not only the presence of antibodies but also a "virus," you are not alone. This lab-created concoction has no relevance to reality whatsoever. There are even some noted drawbacks for the complement fixation test beyond the combining of human blood with that from sheep/guinea pigs:

Disadvantages of Complement Fixation Test
  1. Not sensitive – cannot be used for immunity screening.
  2. Time-consuming.
  3. Often non-specific e.g. cross-reactivity between Herpes Simplex Virus and Voricella Zoster Virus.

Complement Fixation Test- Steps, Advantages and Disadvantages

As can be seen, the complement fixation test, based on the imaginary story as proposed by Ehrlich, is not sensitive nor is it specific and often leads to cross-reactivity with other "viruses." Sensitivity and specificity are the two most important criteria in determining whether a test is accurate or not:

What are sensitivity and specificity?

"Whenever we create a test to screen for a disease, to detect an abnormality or to measure a physiological parameter such as blood pressure (BP), we must determine how valid that test is—does it measure what it sets out to measure accurately? There are lots of factors that combine to describe how valid a test is: sensitivity and specificity are two such factors. We often think of sensitivity and specificity as being ways to indicate the accuracy of the test or measure.

In the clinical setting, screening is used to decide which patients are more likely to have a condition. There is often a 'gold-standard' screening test—one that is considered the best to use because it is the most accurate. The gold standard test, when compared with other options, is most likely to correctly identify people with the disease (it is specific), and correctly identify those who do not have the disease (it is sensitive). When a test has a sensitivity of 0.8 or 80% it can correctly identify 80% of people who have the disease, but it misses 20%. This smaller group of people have the disease, but the test failed to detect them—this is known as a false negative. A test that has an 80% specificity can correctly identify 80% of people in a group that do not have a disease, but it will misidentify 20% of people. That group of 20% will be identified as having the disease when they do not, this is known as a false positive. See box 1 for definitions of common terms used when describing sensitivity and specificity."

https://ebn.bmj.com/content/23/1/2

How can a test be used when it is neither sensitive nor specific? Obviously, without these two components, the complement fixation test is not an accurate meaaure of anything whatsoever. The reason for this lack of accuracy is that in order to know which antibody reacts and is specific for a "virus" so that a test can be used to be able to detect it, one would have to have first purified/isolated an actual "virus" directly from a human sample. Without this, sensitivity and specificity can not be concluded. This applies to antibodies themselves as before antibody tests could be considered accurate, antibodies must also be purified and isolated from human fluids. As this has never been done for either "viruses" nor antibodies, the sensitivity and specificity will remain unknown and the tests will remain inaccurate.

This is the problem with basing tests on theoretical concepts and experimental reactions. To date, the antibody-antigen relationship is still nothing but an unproven theory conjured up in the mind of a man with a "lively imagination." The existence of these invisible entities is still in doubt as they have never been properly purified nor isolated. Ehrlich did nothing more than create a story about what he believed occurred inside of lab-created chemical reactions, which even he himself apparently did not believe. This was brilliantly summarized in the highlights from the following 1993 paper:

Ehrlich's "Beautiful Pictures" and the Controversial Beginnings of Immunological Imagery

ARE "ANTIBODIES" MATERIAL SUBSTANCES?

"The term antibody today refers to discrete biochemical entities present in the blood. The belief that antibodies are such entities is held not only by scientists and physicians, but also by the lay public, who learns about "antibodies," if not in school, then at least in the course of routine medical practices such as vaccination and, increasingly, through the so-called popularization of science. In addition, some people will readily associate the term antibody with the characteristic Y-shaped structure found not only in textbooks and specialized scientific articles but also, more recently, in advertisements and even as the logos of pharmaceutical and biotechnology companies. In order to understand the beginnings of immunological imagery at the turn of the century, we have to discard our present-day notions about the reality and structure of antibodies.

Indeed, it is relatively easy to slide into anachronism. For instance, it is now commonplace to claim that in 1890 Emil von Behring and Shibasaburo Kitasato discovered "antitoxins" and further to describe this discovery as one of the inaugurating events of humoral immunology, and thus of antibody research. Even such a sophisticated historian as Arthur Silverstein speaks of this landmark contribution as the "discovery of antitoxic antibodies." However, as Jean Lindenmann has pointed out, Behring and Kitasato carefully avoided the use of the term antitoxins, which they considered inappropriate, precisely because they regarded their findings as pointing to the action and properties of sera, and not to the presence of a substance. The contemporaries of Behring and Kitasato did not miss this distinction. In a letter to an Australian colleague written circa December 1898, Ehrlich noted that "two years ago Behring still believed that antitoxins should be conceptualized as forces rather than chemical substances."

Following Behring and Kitasato's contribution, scientists engaged in a number of discussions concerning the action of immune sera. While some researchers had reservations about what they saw as simplistic explanations of immune phenomena, others jumped to the conclusion that "antitoxins"-that is, specific, discrete substances-accounted for the property of certain immune sera to neutralize the effects of diphtheria and tetanus toxins. Along the same line, "agglutinins" and "precipitins" were deemed responsible, respectively, for agglutination and precipitation reactions, and "cytotoxins" for the lysis of cells, such as red blood cells and bacteria-in which case one spoke, respectively, of "hemolysins" and "bacteriolysins."

The term antibody, used for the first time by Ehrlich in 1891 in a somewhat vague sense, was thus used concurrently with a variety of other terms that referred to specific immune reactions observed under laboratory conditions. Antibodies was by no means an unproblematic general term covering the more specialized ones, since the question of the possible (chemical) identity of these various entities and events was left open. In fact, while it could be argued that the term antibodies had, by the beginning of the 1900s, acquired a generic meaning, this was true only in a superficial sense. As late as 1929 Harry G. Wells, a defender of the "unitarian hypothesis" according to which "antibodies" should be considered as a single entity, independent of the variations in the experimental procedures by which they were "recognized," could still lament that the hypothesis "currently accepted as if it were an established fact" was that the various precipitins, agglutinins, antitoxins, and so on were indeed "different and distinct substances."

In addition to "experiment-laden" terms, "theory-laden" terms also circulated. For instance, following experiments examining the properties of immune sera heated to 55° C, cytotoxins were deemed to comprise two components. The first one, relatively heat resistant, was called by Ehrlich "immune body" (Immunkorper), "amboceptor" (Ambozeptor), or "intermediate body" (Zwischenkorper), while Bordet called it "sensitizing substance" (substance sensibilisatrice). Other researchers resorted to such terms as "copula" or "desmon." The second, non-heat-resistant component of cytotoxins was called "complement" by Ehrlich, "alexin" by Bordet, who adopted the Greek-derived term (aleksein = protecting substance) proposed in 1889 by Hans Buchner, and "cytase" by other researchers. Lexicologists have noted that during periods characterized by the emergence of new referents, many terms compete for the status of designator. Thus, the presence of a plurality of terms is indicative of the presence of objects and practices that have yet to be stabilized. In our case, the different terms were "theory laden" both because they implied different mechanisms of action of the putative substances that they named and because they ascribed a different ontological status to those same substances. To speak of "intermediate body" or "amboceptor" was to postulate the existence of a chemical substance, composed of distinct chemical groups, that would literally insert itself, as a bridge, between the cell and the complement, thus allowing the complement to lyse the cell. To speak of "sensitizing substance" was to refer to a more diffuse model of action, metaphorically equated to the action of dyes on tissues, whereby no specific atom groups were entrusted with the capacity of establishing links with the cells or the alexin (Ehrlich's complement). In particular, Bordet compared the action of the substance sensibilisatrice to the action of "certain fixing agents or mordants which confer to certain substances (or to cells, as is the case in histological techniques) the property to absorb colors they previously refused."'l

The debate over the existence, nature, and-properties of "antibodies" lasted for decades. Very few people shared the extreme position of Felix Le Dantec, who denied the physical existence of what he called "phenomenines"-that is, mythical substances that, like Moliere's "vertu dormitive," were being used tautologically to account for empirically observed phenomena:

While making fun of the physicians of his time, our great Moliere had foreseen Ehrlich's system. Ehrlich has added nothing to the explanation of the imaginary invalid. Rather than saying that chloral causes sleep because it contains a soporific power ("vertu dormitive"), we would today say, according to the German scholar, that chloral contains a soporine ("dormitine"); well, it is the same thing! . . . Here then is a therapeutic serum which produces, when inoculated into the rabbit, a given phenomenon. How will you explain this particular activity? It is very simple: put on your glasses and your doctor's cap and gravely say: "The serum produces this phenomenon because it contains a phenomenine which has that power." Nobody will laugh. Point out timidly that the same serum has a different action on the snake; that it produces a second phenomenon; that will be because it contains a second phenomenine. If the same substance produces a thousand different phenomena in a thousand different species, then it contains a thousand different phenomenines; and there you go! Why had we not thought of this earlier? Henceforth we have the explanation for everything!

While Le Dantec's remarks in this passage were addressed exclusively to Ehrlich, since they figured in one of the many anti-German pamphlets published by French scientists during World War I, they could equally have been directed at the "French" scientist Bordet, as indeed they had been in a prewar pamphlet. Moreover, other researchers, while not sharing Le Dantec's extreme position, did share his hostility to "terminology-based" explanations. Henry Dean, for instance, noted that "agglutinins, precipitins, amboceptors are mere words, and a passive belief in the existence of such bodies tends to impede rather than advance our understanding of what is actually taking place"; he added that "ignorance, however aptly veiled in an attractive terminology, remains ignorance."

So, despite various professions of faith in the material nature of "antibodies," their ontological status remained uncertain, a situation ascribed by some scientists to the failure to purify chemically the elusive entities and thus to ascertain whether they were indeed material substances. This, of course, begged the question, because in order to base an argument on the possible chemical purification of antibodies one had first to assume that antibodies were indeed discrete chemical substances, which is precisely what Ehrlich's opponents contested. In a 1902 letter nominating Ehrlich for the Nobel Prize, Bernhard Naunyn noted that the German researcher could be credited with the introduction of a stereochemical approach into biology and could thus be compared with the great scientists (August Kekule, Adolf von Baeyer) who had done the same in their own disciplines; but he added that Ehrlich's contribution should still be regarded as tentative or premature, since the isolation and purification of the relevant substances (namely, "antibodies") would long remain a chimera. Those same substances were treated, in a 1910 German textbook, as didactic devices:

In order to learn the nature of these antibodies attempts have been made to isolate them chemically. Thus far all such trials have been unsuccessful. It is even uncertain whether these so-called antibodies are definite chemical entities. Only the effects of the serum as a whole are known, and the ingredients in it to which these activities are attributed are thought of as antibodies. For didactic purposes antibodies, as antitoxins, agglutinins,
etc., will be spoken of in this book when the antitoxin or agglutinating properties, exclusively, are meant.

Julius Citron's 1910 statement was echoed nineteen years later by Wells, according to whom:

We attribute this altered reactivity [of sera] to the presence of "antibodies," despite the fact that we have absolutely no knowledge of what these antibodies may be, or even that they exist as material objects. Like the enzymes, we recognize them by what they do without discovering just what they are. We do not know whether they are specific molecular aggregates or merely physical forces dependent on altered surface energy of the same substances present in the blood before the process of immunization was begun.

REPRESENTATIONAL PRACTICES AND THE ORIGINS OF EHRLICH'S IMAGERY

"Early twentieth-century serological manuals, such as the one published in France by Paul-Felix Armand-Delille in 1911, resorted to three kinds of illustrations, corresponding to three types of representational practices. A first kind referred to the macroscopic observation of the behavior of blood in test tubes following manipulation with immune sera (Figure 3). In the case of hemolytic sera, a plate would
typically represent a series of test tubes containing a red liquid (blood): in some tubes the liquid would appear to be opaque, while in others-where, according to the caption, hemolysis had taken place-the liquid would appear to be transparent. In yet other test tubes, described as having undergone centrifugation, a red pellet could be observed, which would be lacking in the case of hemolysis. A second kind of illustration aimed at showing microscopic pictures of bacterial cultures mixed with immune sera (Figure 4). According to the descriptions accompanying the figures, bacteria, immobilized and agglutinated by the immune serum, could be seen. Finally, a third kind of illustration, derived from Ehrlich's imagery, depicted the interactions of the invisible entities allegedly causing the macroscopic and microscopic phenomena represented in the previous two kinds of illustration (Figure 5). Notice that all three kinds of illustration function by contrast: they presuppose a comparison between a "before" and an "after," which corresponds to the order in which they are supposed to be read, from left to right or from top to bottom.

This third kind of illustration belongs to what has been termed the "domain of invisible specimen behavior" or the "molecular realm," a virtual space devoted to molecular events, construed as lying along a visibility continuum stretching from microscopic to macroscopic laboratory bench operations. There is sharp distinction between the first two types of illustrations and the third. Even though scientists might, and indeed often do, entertain a realistic, mirror-of-nature conception of the first two types of illustration, this distinction does not lie so much in the fact that they are held to be more "real" than the third type. Although, from the scientists' point of view, the reality issue can indeed be relevant and, as we shall see, controversial, it is, so to speak, derivative. The primary distinction lies, rather, in the fact that the first two types of illustration deal with representations of elements that can in principle, as well as in practice (depending on the availability of the necessary instruments, reagents, and so on), be seen or made visible, while the third type is representative of elements that are, at a given time, by definition invisible. Lest the reader believe that, in establishing this distinction, we have abandoned our participant-centered perspective, consider Figure 6, taken from a 1930 article, in which a "threshold of visibility" is presented as an explicit dimension of immunological practice and simultaneously situated along a visibility-invisibility continuum. Moreover, Ehrlich himself, in his Croonian Lecture, had hinted at the distinction between a visible and an in-principle invisible domain when he pointed out that "we may regard the cell quite apart from its familiar morphological aspects, and contemplate its constitution from the purely chemical standpoint." As we shall see, it is precisely in the establishment of a "domain of invisible specimen behavior" for immunology that Ehrlich's controversial contribution lay."

"It is a telling indication of the disregard in which visual elements were held that the second possibility-images as heuristic, in a strong, constitutive sense-was used by Ehrlich's opponents as an argument against his theories: yes, images were constitutive of Ehrlich's science, and that is why his science was flawed. As Bordet was to argue: "By its abusive use of quite puerile graphical representations, which simply translate the exterior aspects of phenomena without at all penetrating their intimacy, Ehrlich's theory has created a deceiving taste for facile but illusory explanations." Ehrlich's reaction to these accusations was, to a large extent, to distance himself in principle from a literal interpretation of his images while in practice making liberal use of them in both his articles and his experimental work. In other words, the argument that the diagrams were fictions could be (and was) interpreted in two ways: the drawings were not a faithful image of reality, and thus they should be discarded because they were fundamentally misleading; while the drawings did not correspond to anything "out there," immune reactions happened as if the various entities they portrayed did actually exist, and thus the diagrams were an important heuristic tool. Ehrlich's attitude approximated the latter interpretation; it is well summarized by a statement he reportedly made to Karl Fliigge: "Those stupid people think that I really do represent the things to myself in that way."

DOI:10.1086/356636

In Summary:
  • Ehrlich felt immunity could be most easily and successfully explained through the agency of "the side-chain theory" (haptophore, toxophore, side-chains, complement, etc…obviously he did not believe in Occam's Razor which states the simplest explanation is preferred)
  • Roux showed that very small quantities of tetanus toxine, if injected directly into the brain, causes the death of the animal which was not compatible with Ehrlich's theory
  • Ehrlich claimed that Roux came to this incorrect conception through an erroneous conception of antitoxine
  • He stated that the toxophile groups are therefore really inducers of the action of the poison, and not its preventive
  • Yet Ehrlich immediately contradicted himself by claiming that those toxophile groups which, like the antitoxines present in the serum, are able to lay hold of toxine immediately on its entry into the blood, and so to divert it from organs essential to life, can alone be regarded as being possessed of any antitoxic action in the true sense of the word
  • Upon immunisation, many other reaction products come into existence alongside of the antitoxines which are produced as a result of the action of the toxines
  • As a result of the injection of bacterial cultures, there arise:
    1. Specific bacteriolysines, which dissolve the bacteria
    2. Other products such as "coagulines," substances which are able to cause the precipitation of certain albuminous bodies contained in the culture fluid injected
    3. The much discussed "agglutinines"
    4. The antiferments
    5. And no doubt many other bodies which have not yet recognised
  • He believed it was quite likely that the formation of any single one of these bodies is not of itself sufficient to confer immunity
  • Thus in case of the introduction of bacteria into the body they have to do with a many-sided production of different forms of "Antikorper," (i.e. antibodies) each of which is directed only against one definite quality or metabolic product of the bacterial cell
  • The practice of using for the production of immunisation definite toxic bodies isolated from the bacterial cells had been more and more given up, and for this purpose it was regarded as important to employ the bacterial cells as intact as possible
  • Ehrlich stated that there are two different bodies acting in combination for bacteriolysines (the rupture of a bacterial cell by antibodies): a stable one brought about by immunization and an unstable one already present in the blood
  • The stable element was called the "immune body" while the unstable element was referred to as the "complement"
  • After centrifugation, the "immune body" stays with the red blood cells while the "complement" is left behind
  • Through his experiments, Ehrlich determined that temperature had an effect on whether or not the substances were left behind after centrifugation
  • Ehrlich claimed that these facts can only be explained by making certain assumptions regarding the constitution of the two components, i.e., of the "immune body" and the "complement"
  • When the blood of a non-immunized animal has haemolytic action on the blood of another animal, this can not be considered the action of the "immune body"
  • Ehrlich termed this phenomena the "intermediate body" even though it had exactly the same action and characteristics of the "immune body" and the only difference was it occurred naturally
  • He stated that the process of hsemolysines (lysis of the red blood cells) is the same when it occurs naturally or through immunizations
  • The question arose whether in a serum that is capable of such manifold action if there is present one single haemolysine that destroys different red blood-cells, or whether a whole series of hsemolysines come into action, of which one is adapted to guinea-pig blood, another to rabbit blood, &c
  • Ehrlich stated that if experiments are carried out on various blood types, and there are multiple "intermediate bodies" specific for each kind of blood (rabbits blood, calf blood, Guinea pig blood, etc.), then he succeeded in proving that there are different complements that fit these "intermediate bodies"
  • His experiments brought more and more to the conviction that the blood serum is the carrier of substances innumerable as yet little known or conceived of
  • Having obtained a precise conception of the method of action of the lysines of the serum—of the hsemolysines, and thereby also of the bacteriolysines—Ehrlich felt it became possible for them to attempt to solve the mystery of the origin of these bodies
  • The toxines, as secretion products of cells, were in all likelihood still relatively uncomplicated bodies
  • Ehrlich believed that this will be very effectually attained if, figuratively speaking, the "tentacle" or grappling arm of the protoplasm possesses a second haptophore group adapted to take to itself ferment-like material out of the blood fluid
  • Through such complex organisation, by which the "tentacle" acts also as the bearer of a ferment-functioning group, this group is brought into close relation with the prey destined to be digested and assimilated
  • This tentacle business where it aids in the process of digestion/fermentation to eliminate toxins is a perfect example of Ehrlich's "lively imagination" for which he was criticized for
  • In order to add proof for his tentacle creation, Ehrlich decided to pass from the abstract to the concrete—to find analogies in the different forms of insectivorous plants
  • He claimed that there are present in the organism "side-chains" of a special nature that are endowed not only with an atomic group by virtue of the affinities of which they are enabled to pick up material, but also with a second atomic group, which, being ferment-loving in its nature, brings about the digestion of the material taken up
  • He stated that should the pushing-off of these "side-chains" be forced, as it were, by immunisation, then the "side-chains" thus set free must possess both groups
  • Ehrlich felt that the inconsistencies of immunising sera of animals in different experiments could be easily be explained by his theory
  • Ehrlich's "easy" explanation:
    1. This circumstance was easy to understand, if the complex nature of the lysines be borne in mind
    2. The lysine, be it bacteriolysine or hsemolysine ( i.e. "immune body + "complement"), possessed altogether three haptophore groups, of which two belong to the "immune-body" and one to the "complement"
    3. Each one of these haptophore groups can be bound by an appropriate "anti-group"
    4. Three anti-groups are thus conceivable, any one of which, by uniting with one of the haptophore groups of the lysine, can frustrate the action of the lysine
    5. To his mind, of these three possible "Antikorper," that one which can lay hold of the haptophore group of the "complement," and so prevent this from uniting with the "immune body," was the most important
  • Thus, the "easy" explanation involves the immune body, the complement, three haptophore groups, and three conceivable anti-groups (antibodies)…Occam's Razor anyone?
  • Dr. Neisser at the Steglitz Institute was able to determine that anthrax serum failed in mice, even if great quantities of fresh sheep's serum (i.e., containing excess of "complement") were at the same time introduced
  • The failure in this case appeared to be due, on the one hand, to the destruction, in the body of the mouse, of the "complement" present in the sheep's serum, and, on the other hand, to the fact that the "immune body" yielded, by the sheep does not find in mouse serum an appropriate new "complement" (in other words, it is explained by Ehrlich's lively imagination but not by any observed processes)
  • In the therapeutic application of anti-bacterial sera to man, therapeutical success is only to be attained if they use either a bacteriolysine with a "complement" which is stable in man ("homostabile complement"), or at least a bacteriolysine, the "immune body" of which finds in human serum an appropriate "complement"
  • The latter condition would be the more readily fulfilled the nearer the species employed in the immunisation process is to man
  • Ehrlich felt that perhaps the non-success for typhoid and cholera serum would be fixed if the serum was derived from apes and not taken from species so distantly removed from man as the horse, goat, or dog (why not take from a human instead of an animal…?)
  • According to Ehrlich, the "Antikorper" (antibodies) are of a complex nature
  • He claimed his unseen theoretical creations obey the already described law of elective absorption, and the origin of his imaginary substances is in keeping with his own "side-chain" theory (go figure that his dreamt up entities fit in with his dreamt up theory)
  • It was hoped that immunisations with antibodies, which were of great theoretical interest, may come to be available for therapeutic application
  • The sifting of the material obtained by observation was rendered more difficult by the occurrence under normal conditions of a great number of quite unlooked for bodies furnished with haptophore groups and arising from diverse organs, and which he designated collectively as haptines (i.e. he found numerous other unknown substances and threw them all under the same category he created)
  • Complement fixation is a classic method for demonstrating the presence of antibody in patient serum
  • The first component is an indicator system that uses combination of sheep red blood cells, complement-fixing antibody such as immunoglobulin G produced against the sheep red blood cells and an exogenous source of complement usually guinea pig serum
  • When these elements are mixed in optimum conditions, the anti-sheep antibody binds on the surface of red blood cells
  • The second component is a known antigen and patient serum added to a suspension of sheep red blood cells in addition to complement
  • If the patient's serum does contain a complement-fixing antibody, a positive result will be indicated by the lack of red blood cell lysis
  • Disadvantages of complement fixation tests:
    1. Not sensitive – cannot be used for immunity screening.
    2. Time-consuming.
    3. Often non-specific e.g. cross-reactivity between Herpes Simplex "Virus" and Voricella Zoster "Virus"
  • As can be seen, the complement fixation test is neither sensitive nor specific
  • Sensitivity and specificity are the ways to indicate the accuracy of the test or measure
  • The gold standard test, when compared with other options, is most likely to correctly identify people:
    1. With the disease (it is specific)
    2. Those who do not have the disease (it is sensitive)
  • The term antibody today refers to discrete biochemical entities present in the blood
  • The belief that antibodies are such entities is held not only by scientists and physicians, but also by the lay public, who learns about "antibodies," if not in school, then at least in the course of routine medical practices such as vaccination and, increasingly, through the so-called popularization of science
  • In order to understand the beginnings of immunological imagery at the turn of the century, we have to discard our present-day notions about the reality and structure of antibodies
  • Behring and Kitasato carefully avoided the use of the term antitoxins, which they considered inappropriate, precisely because they regarded their findings as pointing to the action and properties of sera, and not to the presence of a substance
  • In a letter to an Australian colleague written circa December 1898, Ehrlich noted that "two years ago Behring still believed that antitoxins should be conceptualized as forces rather than chemical substances."
  • The term antibody, used for the first time by Ehrlich in 1891 in a somewhat vague sense, was thus used concurrently with a variety of other terms that referred to specific immune reactions observed under laboratory conditions
  • As late as 1929 Harry G. Wells, a defender of the "unitarian hypothesis" according to which "antibodies" should be considered as a single entity, independent of the variations in the experimental procedures by which they were "recognized," could still lament that the hypothesis "currently accepted as if it were an established fact" was that the various precipitins, agglutinins, antitoxins, and so on were indeed "different and distinct substances."
  • The different terms for "antibodies" were "theory laden" both because they implied different mechanisms of action of the putative substances that they named and because they ascribed a different ontological status to those same substances
  • According to Felix Le Dantec, 'Ehrlich has added nothing to the explanation of the imaginary invalid."
  • Henry Dean noted that "agglutinins, precipitins, amboceptors are mere words, and a passive belief in the existence of such bodies tends to impede rather than advance our understanding of what is actually taking place"; he added that "ignorance, however aptly veiled in an attractive terminology, remains ignorance."
  • Despite various professions of faith in the material nature of "antibodies," their ontological status remained uncertain, a situation ascribed by some scientists to the failure to purify chemically the elusive entities and thus to ascertain whether they were indeed material substances
  • This, of course, begged the question, because in order to base an argument on the possible chemical purification of antibodies one had first to assume that antibodies were indeed discrete chemical substances, which is precisely what Ehrlich's opponents contested
  • In a 1902 letter nominating Ehrlich for the Nobel Prize, Bernhard Naunyn stated Ehrlich's contribution should still be regarded as tentative or premature, since the isolation and purification of the relevant substances (namely, "antibodies") would long remain a chimera
  • In a 1910 German textbook, it was stated: "In order to learn the nature of these antibodies attempts have been made to isolate them chemically. Thus far all such trials have been unsuccessful. It is even uncertain whether these so-called antibodies are definite chemical entities. Only the effects of the serum as a whole are known, and the ingredients in it to which these activities are attributed are thought of as antibodies."
  • Accirding to Wells in 1929: "We attribute this altered reactivity [of sera] to the presence of "antibodies," despite the fact that we have absolutely no knowledge of what these antibodies may be, or even that they exist as material objects. Like the enzymes, we recognize them by what they do without discovering just what they are."
  • Ehrlich's imagery depicted the interactions of the invisible entities allegedly causing the macroscopic and microscopic phenomena
  • His imagery belonged to what has been termed the "domain of invisible specimen behavior" or the "molecular realm," a virtual space devoted to molecular events, construed as lying along a visibility continuum stretching from microscopic to macroscopic laboratory bench operations
  • The images are representative of elements that are, at a given time, by definition invisible
  • It is precisely in the establishment of a "domain of invisible specimen behavior" for immunology that Ehrlich's controversial contribution lay
  • The argument that Ehrlich's diagrams were fictions could be (and was) interpreted in two ways: the drawings were not a faithful image of reality, and thus they should bediscarded because they were fundamentally misleading; while the drawings did not correspond to anything "out there," immune reactions happened as if the various entities they portrayed did actually exist, and thus the diagrams were an important heuristic tool
  • Ehrlich's attitude approximated the latter interpretation; it is well summarized by a statement he reportedly made to Karl Fliigge: "Those stupid people think that I really do represent the things to myself in that way."

Ehrlich provided a theory that weaved together various disparate elements like the best fiction writers often do. He defined the concepts of antibody, antigen, the complement system, and provided a framework for how immunity could work. This led to indirect non-specific and non-sensitive complement fixation tests (among others) used as evidence for the existence of "viruses" and/or as proof that the vaccines are effective against them. The problem is that Ehrlich's theories were just mere words with nothing physical backing them. He first assumed any such entities existed in the blood based on lab-created chemical reactions and then developed a framwork around his imagination. He created concepts and ideas for that which he could not physically see. His creations remain in the "domain of the invisible." They belong to the realm of fantasy and fairy tales.

As Henry Dean stated: "Ignorance, however aptly veiled in an attractive terminology, remains ignorance."

ViroLIEgy
1 May 2022 | 2:27 pm

Paul Ehrlich’s Side-Chain Antibody Theory (1900) Part 1


In 1900, Paul Ehrlich, considered the "Father of Chemotherapy," took a shot at creating a theoretical explanation for how immunity occurs inside the human body. According to Ehrlich's Nobel Prize biography, while working with Behring's anti-diphtheria serum, Ehrlich was said to have standardized this serum in units related to a fixed and invariable standard. Previously, his work had shown that the anti-toxin in the sera varied so much due to numerous factors that it could not be measured. This unit of standardization became the basis for all future standardization of sera. Ehrlich's work on these substances eventually led him to create the Side-Chain theory of immunity which introduced the idea of unseen antibodies, antigens, and the concept of the lock and key mechanism of action:

The Contributions of Paul Ehrlich to Pharmacology: A Tribute on the Occasion of the Centenary of His Nobel Prize

"With the aim of proposing a plausible explanation for the process of immunity, it was in 1897 when Ehrlich formulated his 'side-chain theory', which became the basis for his immunological research at the time. This theory postulated that cells present on their surface a set of side-chains to which Ehrlich attributed functions related with the assimilation of metabolic products. A side-chain from a given cell might have, by simple coincidence, a molecular structure that allowed it to bind with a specific toxin corresponding to diphtheria, tetanus, or some other microorganism. This strictly specific binding between the toxin and the side-chain, in a manner similar to the 'lock-and-key' model for enzymes and their substrates described in 1894 by Hermann Emil Fischer (1852–1919), would mean the cell lost its normal function, a phenomenon which in turn would trigger the production of additional side-chains. A large part of these newly produced excess side-chains would be released into the blood stream, where they would act as antibodies or antitoxins upon binding to the toxin present in the blood, and would thus prevent the toxin from binding to other cells in the organism. Thus a small amount of toxin could produce a large amount of antitoxin able to neutralize the toxin's own effect [13].

In 1900, Ehrlich introduced the term 'receptor' as a substitute for the term 'receptive side-chain'. This idea led to a more functional concept that could be applied to the field of pharmacology. John Newport Langley (1852–1925) (table (table1),1), who studied the effect of alkaloids on muscle cells, proposed the existence of receptors that could be blocked by antagonists or activated through the action of agonists. Although they came from very different research backgrounds, the interaction between Ehrlich's and Langley's thinking was highly productive. Together they developed the receptor theory and extended the concept of the receptor to 'chemoreceptors' to describe the interaction between drugs and cells. Thus the side-chain theory, although not entirely accurate, gave rise to concepts which were to become basic tenets in immunology [6, 13, 17].

In addition to the side-chain and receptor theory, Ehrlich also established the concepts of active and passive immunity, as well as the mechanisms of transmission of immunity from mother to fetus. However, he was not without detractors who criticized him for his excessively lively imagination and lack of self-criticism. He himself compared his side-chain theory to the cellular theory of Rudolf Virchow (1821–1902), which had also met resistance initially."

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2790789/

Ehrlich aimed to create a "plausible explanation" for the phenomena that he and other researchers were unable to observe. He worked with them to dream up and formulate the concepts and even though his ideas were not "entirely accurate," these inaccurate fantasies became the foundation for Immunology. Ehrlich was criticized by his peers for his lively imagination and upon reading his theory, you will see why. Depending on where you look, there are differences of opinion on just how accurate Paul Ehrlich's Side-Chain theory of immunity truly was.

It was either entirely correct and laid the foundation for what we know about antibodies today:

"Ultimately all aspects of Ehrlich's theory would be proven correct with the minor exception that the "receptor" exists as both a soluble antibody molecule and as a cell-bound receptor; it is the soluble form that is secreted rather than the bound form released."

https://en.m.wikipedia.org/wiki/Side-chain_theory

Or was somewhat correct:

"Two of Ehrlich's postulates, that antibodies are identical to the antigen receptors and that antigen binding triggers the synthesis of antibody with the same specificity as the receptor, are now known to be essentially correct."

https://www.ufrgs.br/imunovet/molecular_immunology/historyi.html

Or was wrong and completely abandoned:

"But this theory was abandoned when Landsteiner demonstrated that antibodies are also produced against various synthetic chemicals besides natural antigens."

https://ecoursesonline.iasri.res.in/mod/page/view.php?id=61812

Whatever the case may be, it is clear Ehrlich's 27-page presentation brought about many of the concepts used today when discussing immunity. However, it is also clear that Ehrlich created a fictional account of what occurs inside the body based on indirect evidence from multiple researchers over multiple years to explain an unobservable phenomena. As it is a long paper covering many aapects of his theory, I have broken it down into two parts. This first section deals with the first 18 pages which contain the bulk of Ehrlich's Side-Chain theory. The second part will be in a separate post and will look more specifically at the complement system. The first 18 pages are presented below with a summary at the end:

On Immunity with Special Reference to Cell Life.

"Honoured President, my lords and gentlemen,—It is to me the very greatest honour that I have been summoned here by your most highly esteemed Society, which for more than two centuries has represented and still represents the centre of the scientific life of England, in order that I may deliver the Croonian Lecture. I consider I am not so much personally concerned in the honour that you bestow on me, and that I shall not err if I see in it a recognition of the scientific path which I, in company with many others, have sought to follow, and which in your eyes suffices to place the field in which I work on a footing alongside of exact science. It is an extreme pleasure for me to have the privilege of addressing so many medical colleagues with whom for so many years I have been bound in close ties of friendship, and who have always been the first to welcome and to give recognition to the results of my work.

Since Jenner made his great discovery of the protective action of vaccinia against small-pox, a century has passed away. During these years that terrible scourge of mankind has been almost completely eradicated from the civilised world. The beneficial consequences of Jenner's discovery are so evident to all who have any wish to properly appreciate them, that one wonders why, during so great a portion of the long period of 100 years, they were allowed to stand alone, without any endeavour being made to induce an artificial immunity in the case of other infectious diseases. This is all the more remarkable because Jenner's discovery demonstrated in their entirety those essential principles which, in later times, have been established for other infectious diseases.

In the first place, it was shown that by the use of an attenuated virus, which of itself was non-injurious to the organism, it was possible to ward off the disease caused by the virulent virus. Jenner also established—what is most important from the practical
point of view—that by the inoculation of the weakened poison there was produced not only an immediate, but also an enduring, protection. That Jenner's discovery remained so isolated was due essentially to the fact that the theoretical conceptions of the cause and nature of infectious diseases made no advance during the subsequent decades; indeed, it would be an interesting topic for some
historian of medicine to trace step by step the gradual advance in the knowledge of infectious diseases during the past century. Schwann's
classical investigations must be regarded as the first link in the long chain. Schwann it was who, in an unsually brilliant manner, first demonstrated that the decomposition of organic bodies in the processes of fermentation and putrefaction was never spontaneous, but constantly arose through the agency of micro-organisms coming from without. This line of investigation reached its zenith in the fundamental work of Pasteur, of which the first and the greatest result—Lister's method of wound treatment—worked a revolution in surgery. Then followed the profound investigations of Koch on Anthrax, and the pure cultivation of the most important pathogenic bacteria.

The work of Pasteur and of Koch afforded the first basis on which the study of artificial immunity could be again undertaken. The possibility of voluntarily producing a number of the most important infectious diseases of men and animals, and of modifying at will pure cultivations of bacteria, either, according to Jenner's precedent, by passage through the animal body, or otherwise in artificial culture media, laid the foundation on which advancement could proceed. Pasteur himself was the first, after Jenner, to produce an artificial immunity by using an attenuated virus; and he was also able to introduce the procedure to some extent into practice with most beneficial results. Still the theoretical explanations of all these facts lagged far behind their practical effects. The very able investigations of Metchnikoff and his theory of phagocytosis were, to many investigators, inconclusive.

With Behring's discovery, that in the blood serum of animals immunised against diphtheria and tetanus, there were contained bodies which were able to specifically protect other animals against the toxines of these diseases, an altogether new factor was introduced into the question. This remarkable discovery seemed at one stroke to open up an entirely new and" extremely promising prospect of immunising mankind against the majority of the infectious diseases. It was, therefore, somewhat disappointing when there did not follow, on the successful practical application of diphtheria antitoxic serum, a rapid succession of similar achievements. It may with truth be said, that during recent years there has been somewhat of a standstill in the further following-out of a work at first so enthusiastically received. By purely empirical methods, e.g., by the production and use of sera of very great antitoxic value, the results attained showed no improvement. Better success was only to be hoped for when by an accurate knowledge of the theoretical considerations underlying the question of immunity, explanations of the previous ill-success were forthcoming. Impelled by these considerations I laboured for years trying to shed some light into the darkness that shrouded the subject.

In all exact work with chemical bodies—for only as such can we regard the toxines produced by the living bacteria—the first desideratum in the investigation is the exact numerical determination of action and counteraction. The words of the gifted natural philosopher Clerk Maxwell, who said that if he were required to symbolise the learning of our time he would choose a metre measure, a clock, and a kilogramme weight, are equally apposite in reference to progress in the field of inquiry in which we are at present interested. And so at the very beginning of my theoretical work on immunity I made it my first task to introduce measures and figures into investigations regarding the relations existing between toxine and antitoxine. From the outset it was clear that the difficulties to be overcome wrere extremely great. The toxines, i.e., the poisonous products of bacteria, are unknown in a pure condition. So great is their potency, that we are obliged to assume that the strongest solid (feste) poisons which are obtained by precipitating toxic bouillon with ammonium sulphate, represent nothing more than indifferent materials, peptones and the like, to which the specific toxine attaches itself in mere traces beyond the reach of weighing; for up to the present time, by the purely chemical methods of weighing and measuring, it has been impossible to ascertain anything as to their presence or the intensityof their action.

Their presence is only betrayed by the proof of their specific toxicity on the organism. For the exact determination, e.g., of the amount of toxine contained in a culture fluid, the essential condition was that the research animals used should exhibit the requisite uniformity in their susceptibility to the poison. Uniformity is not to be observed in the reaction of the animal body to all toxines. Fortunately in the case of one important body of this nature, viz., the diphtheria toxine, the conditions are such that the guinea-pig affords for investigations the degree of accuracy necessary in purely chemical work. For other toxines this accuracy in measuring the toxicity cannot be attained. It was necessary for me to try to eliminate, as far as possible, the varying factor of the animal body, and bring the investigations more nearly into line with the conditions necessary for experiments of a chemical nature. In the course of these endeavours it was shown that it was possible to obtain in a comparatively simple manner an insight into the theoretical considerations necessary to a proper understanding of immunity, by means of test-tube experiments with suspended animal tissues. The relations were simplest in the case of red blood corpuscles. On them, outside the body, the action of many blood poisons, and of their antitoxines, can be most accurately studied, e.g., the actions of ricin, eel-serum, snake-poison, tetanus toxine, &c. In an experiment of this kind, in which are employed a series of test-tubes containing definite quantities of suspended blood corpuscles, each test-tube represents as it were a research animal, uniform in any one series, and one that can be reproduced at will. By means of these test-tube experiments, particularly in the case of ricin, I was able, in the first place, to determine that they yielded an exact quantitative representation of the course of the processes in the living body. The demonstration of this fact formed the basis of a more extended application of experiments of this nature. It was shown that the action of toxine and antitoxine took place quantitatively as in the animal body. Further, these experiments yielded a striking series of facts of importance for the theoretical valuation of the reaction between toxine and antitoxine. It was proved in the case of certain toxines—notably tetanus toxine—that the action of antitoxines is accentuated or diminished under the influence of the same factors which bring about similar modifications in chemical processes—warmth accelerates, cold retards the reaction, and this proceeds more rapidly in concentrated than in dilute solutions. These facts, first ascertained by means of test-tube experiments, have since been confirmed by Behring and Knorr for tetanus within the animal body, and by Martin and Cherry in the case of snake-venom.* The knowledge thus gained led easily to the inference that to render toxine innocuous by means of antitoxine was a purely chemical process, in which biological processes had no share. Yet again insurmountable obstacles seemed to present themselves to this conclusion.

It must be postulated that in chemical processes the bodies sharing m the action react with one another in definite equivalent quantities. This proposition appeared, however, not to hold in the case of the action of antitoxine on diphtheria toxine. When, in the case of diphtheria toxines of different stocks, that quantity of toxine bouillon which is exactly neutralised by a certain definite quantity of diphtheria
antitoxine (the official German immunity unit, as laid down for the control examination of sera), was determined, so that every trace of toxic action was abolished, the figures obtained were not in accord. Of one toxine bouillon 0'2 c.c., of another 2-5 c.c., were so neutralised by one immunity unit. Such a relation need not have given rise to surprise, because it was well known that the diphtheria bacillus, according to outside circumstances, yields in the bouillon very different quantities of toxine. It was therefore allowable to infer that the different quantities of toxine bouillon, which were saturated by one immunity unit, were exact expressions of the toxicities of the
various bouillons, or, to use other words, indifferently whether the bouillon was strongly or feebly toxic, the same multiple of the minimal lethal dose would be constantly neutralised by one immunity unit, so that in every case the law of equivalent proportions would hold good.

But when looked into more closely, the relations showed themselves to be by no means so simple. In what manner could one obtain a satisfactory estimation of the strength of a toxine? As the constant factor in such an estimation, it was only possible to proceed from a previously determined standard reaction in the case of a definite species of animal, and so we came to regard as the "toxic unit" that
quantity of toxic bouillon which exactly sufficed to kill, in the course of four days, a guinea-pig of 250 grammes weight.

When we employed this standard unit, or "simple lethal dose," to estimate the amount of toxic bouillon neutralised by one. "immunity unit," the facts which presented themselves were far more surprising than it was possible to have foreseen at the outset. These results were, that of one toxine, perhaps 20, of a second, perhaps 50, and of yet a third, it might be 130 simple lethal doses were saturated by one immunity unit. Since, however, we had previously assumed that the simple lethal dose alone afforded a standard on which reliance could be placed in determining the combining relations of toxine and antitoxine, it appeared from these results that the neutralisation of toxines by antitoxines did not follow the law of equivalent proportions, and, notwithstanding all earlier work in agreement with such a conception of the action, we were obliged to conclude that between toxine and antitoxine a purely chemical affinity did not exist. The seemingly inexplicable contradiction between the results just stated and previous work was very soon explained. When the neutralisation point of toxine and antitoxine was investigated for one and the same sample of poison, the following results were obtained. Immediately on its preparation, fresh from the incubator, it was found that one immunity unit neutralised a c.c. of toxic bouillon, and this quantity represented (3 simple lethal doses. When the same toxic bouillon was examined after a considerable interval, the remarkable fact was discovered that exactly a c.c. of the toxic bouillon were again neutralised by one immunity unit; but that these a c.c. now represented only simple lethal doses. It therefore followed that the toxic bouillon had retained exactly the same combining affinity, but possessed feebler toxicity. From this it was evident that the toxic action on animals and the combining capacity with antitoxine represented two different functions of the toxine, and that the former of these had become weakened, while the latter had remained constant.

Treated from the chemical standpoint, this circumstance was most simply explained by assuming that the toxine was characterised by the possession of two different combining groups: one, which may be designated haptophore, conditions the union with antitoxine, while the other group, which may be designated toxopliore, is the cause of the toxic action. From the constancy of the combining capacity, and the diminution in the toxicity, it was to be inferred that the toxophore group was very unstable, but the haptophore group more stable, and also that the deterioration of the toxophore group proceeded of necessity quite independently of any relation to the haptophore group.

If we now designated a toxine molecule, of which the toxophore group is destroyed, but its haptophore group retained, as "toxoid," then the above-described process will represent the quantitative progress of the conversion of the toxine molecules into toxoid molecules. Such a toxoid molecule has the same quantitative combining affinity for antitoxine as the original toxine molecule, in spite of the disappearance of toxicity to the animal body. In other words, the affinity of the haptophore group for the antitoxine is absolutely independent of the existence of a toxophore group. Also, in the original toxine molecule, both groups must be to such a degree non-related or independent of one another, that a mutual reaction between them does not take place. This conception of the constitution of diphtheria toxine, after more extensive, very exact, and much varied experimentation, based on its partial neutralisation by antitoxine, has been confirmed in the completest manner possible. At this time it would be superfluous for me to enter into all the details pertaining to these investigations. It need only be remarked that in principle the same relations have been established for tetanolysine by Madsen, for snake-poison by Meyers, and for the milk-curdling ferment by Morgenroth.

The separation of the characteristic atom groups of the toxine molecule into a haptophore and a toxophore group, afforded not merely a satisfactory chemical explanation of the process of neutralisation: the possession of the knowledge of the existence of these groups yielded us, at the same time, the key to the nature of the toxic property of toxines, and to the mystery of the origin of the antitoxines themselves. After it had been established by the already described method, that the toxine molecule was possessed of a definite haptophore group, which accounted for its capacity to enter into combination with other bodies, it was immediately necessary to inquire into the question whether, and if so to what degree, this group entered into the causation of the symptoms of illness. That chemical substances are only able to exercise an action on the tissue elements with which they are able to establish an intimate chemical relationship is a conception of a general nature, which has been entertained since the birth of scientific medicine.

It is astonishing, almost astounding, that this axiom, of which the theoretical importance has been so long recognised, and which has served indeed as the first ground for certain therapeutical procedures, 
should as a matter of fact have played in the building up and furtherance of scientific pharmacology a role so insignificant in proportion to its great importance. In glancing through the modern text-hooks of pharmacology, with rare exceptions, as, e.Stokvis, one finds absolutely no mention of the distribution of drugs in the organism, a matter which is of so much moment for arriving at a true comprehension of the relations existing between pharmacological action, location in the organism, and chemical constitution. As a matter of fact, the methods for obtaining any knowledge of the exact distribution of drugs in the body are as yet very imperfect. Even if we can prove that certain alkaloids are again recognisable as being, e.g., present in the brain, we are but little further advanced in our knowledge of the process, because we cannot determine in which cells and which system of fibres the alkaloid is localised.

I may say, indeed, that as yet the investigation of the laws pertaining to the minute distribution of a chemical substance in the body is only possible when, as in the case of coloured bodies, these are at once recognisable by the eye. But that it is possible at once to draw conclusions of therapeutic importance from the laws governing the distribution was shown in the case of methylene-blue, in which I was able, knowing its distribution in the body, to anticipate for it certain antineuralgic and antimalarial properties which were both established by subsequent investigation. It may be permitted me to call to mind, that in malaria methylene-blue is especially of service in the case of persons who, on account of susceptibility, cannot be treated with quinine, and that in the hands of Koch it has shown itself of eminent value in hsemoglobinuric fever, since as opposed to quinine it exercises no destructive action on the erythrocytes. If we are not able to discover the principles governing the localisation of common chemical bodies, which can be used in suitable quantities in chemical purity, and which chemical and other reactions render perceptible, it was a priori very unlikely that efforts directed to locating the toxines, which are potent in the slightest traces, and which are bodies we have no means of rendering perceptible to our senses, would be anything else than absolutely without result.

But that this is not so, has been shown by experiments carried out by Professor Donitz, in the Steglitz Institute, to which, on account of their great importance, I shall refer somewhat extensively. When a rabbit receives a suitable dose of diphtheria or tetanus toxine injected directly into the circulation, the animal remains for many hours well, and then begins to show symptoms of illness, which gradually increase till they end in death. In order to arrive at an explanation of the incubation period, Donitz determined the amount of antitoxine which, injected intravenously immediately after the toxine, absolutely neutralised the latter. This neutralising dose is able to render all the toxine circulating in the blood innocuous. When, however, the neutralising dose so determined was injected not immediately, but seven or eight minutes after the injection of the toxine, death occurred from tetanus exactly as if no antitoxine had been given. Part of the toxine, equal at least to the minimal lethal dose, must within this time have disappeared from the blood, in which it would have been neutralised, and passed over to the tissues, especially to the brain. The experiments of Donitz were afterwards confirmed by an investigation conducted in quite a different manner by Heymans, who showed that a research animal from which the blood had been removed immediately after the injection of the minimal lethal dose of tetanus toxine, and replaced by transfusion of fresh blood, succumbed from typical tetanus. In this case, therefore, in that brief interval of time the minimal lethal dose of toxine had passed through the walls of the vessels and been taken up by the tissues.

Regarding the nature of the processes here concerned, a satisfactory explanation was also afforded by the experiments of Donitz. It admitted of demonstration that the toxine held in the tissues could still be withdrawn from them, if not the simple neutralising dose were injected but larger quantities of the same.

The quantity necessary was greater in proportion as the interval elapsing after the injection of the toxine was longer. However, after a definite period was exceeded, all possible doses of antitoxine, even the very greatest, were impotent, notwithstanding that the animal at the time of the injection of the antitoxine had not developed any symptoms. Since a very great number of other chemical substances, narcotics, alkaloids, and other neurotropic bodies, were not in a position to withdraw the toxine once deposited in the central nervous system, and as the property to do so was solely the characteristic of the specific antitoxine, one was obliged to come to the conclusion that the union between the toxine and the tissues, which could only be overcome by means of a specific chemically-related antagonising agent, must itself depend on a chemical combination. One was therefore forced to accept the idea that the central nervous system, that is to say certain ganglion cells in it, possessed atom groups resembling those of the antitoxine, in having a maximum affinity for tetanus poison. The predilection of the nervous system for tetanus toxine, the rapid union of the toxine with the nervous tissue, the gradual onset of the symptoms and their long duration could only be explained by the existence of such toxophil groups. The statement of Donitz that the tetanophile atom groups are in the guinea-pig essentially confined to the central nervous system, whereas in the case of other species, especially rabbits, these are also present in other organs, is one of prominent importance.

The beautiful experiments of Roux on intracerebral injection of toxine have yielded absolute confirmation of the statement of Donitz. Roux found in guinea-pigs that the same dose of tetanus toxine was lethal, whether given by intracerebral or by subcutaneous injection for rabbits, however, the lethal dose was twenty times greater subcutaneously than it was in intracerebral injection. This can only be explained in the terms of Donitz's observation, viz., that in the case of direct injection of the toxine into the brain, the toxophile atom groups there present at once seize on all the toxine, while when the toxine is administered through the blood stream, the toxophile groups present in other organs also take up the toxine in equivalent quantities. In the case of rabbits the absorption of the toxine in this way is very considerable: indeed of twenty parts only one part finds its
way into union with the nervous system.

We now come to the important question of the significance of the toxophile groups in organs. That these are in function specially designed to seize on toxines cannot be for one moment entertained. It would not be reasonable to suppose that there were present in the organism many hundreds of atomic groups destined to unite with toxines, when the latter appeared, but in function really playing no part in the processes of normal life, and only arbitrarily brought into relation with them by the will of the investigator. It would indeed be highly superfluous, for example, for all our native animals to possess in their tissues atomic groups deliberately adapted to unite with abrin, ricin, and crotin, substances coming from the far distant tropics.

One may therefore rightly assume that these toxophile protoplasmic groups in reality serve normal functions in the animal organism, and that they only incidentally and by pure chance possess the capacity to anchor themselves to this or that toxine.

The first thought suggested by this assumption was that the atom groups referred to must be concerned in tissue change; and it may be well here to sketch roughly the laws of cell metabolism. Here we must in the first place draw a clear line of distinction between those substances which are able to enter into the composition of the protoplasm, and so are really assimilated, and those which have no such capacity. To the first class belong a portion of the food-stuffs par excellence; to the second almost all our pharmacological agents, alkaloids, antipyretics, antiseptics, &c.

How is it possible to determine whether any given substance will be assimilated in the body or not. There can be no doubt that assimilation is in a special sense a synthetic process—that is to say, the molecule of the food-stuff concerned enters into combination with the protoplasm by a process of condensation involving loss of a portion of its water. To take the example of sugar, in the union with protoplasm, not sugar itself as such but a portion of it comes into play, the sugar losing in the union some part of its characteristic combining reactions. The sugar behaves here as it does, e.g., in the glucosides, from which it can only be obtained through the agency of actual chemical cleavage. The glucoside itself yields no trace of sugar when extracted in indifferent solvents. In a quite analogous manner the sugar entering into the constitution of albuminous bodies (glycoproteids) cannot be obtained by any method of extraction; at least, not until chemical decomposition has previously taken place. It is therefore generally easy, by means of extraction experiments, to decide whether any given combination in which cells take part is or is not a synthetic one. If alkaloids, aromatic amines, antipyretics, or aniline dyes be introduced into the animal body it is a very easy matter, by means of water, alcohol, or acetone, according to the nature of the body, to remove all these substances quickly and easily from the tissues. This is most simply and convincingly demonstrated in the case of the aniline dyes. The nervous system stained with methylene blue, or the granules of cells stained with neutral red, at once yield up the dye in the presence of alcohol. We are therefore obliged to conclude that none of the foreign bodies just mentioned enter synthetically into the cell complex; but are merely contained in the cells in their free state. The combinations into which they enter with the cells, and notably with the not really living parts of them (Kupffer's paraplastic portions), are very unstable, and correspond usually only to the conditions obtaining in solid solutions, while in other cases only a feeble salt-like formation takes place. I myself in 1887 placed on a sure footing the fact that the nervous system and the fatty tissues allow of alkaloids and aniline dyes being mechanically shaken out of them, as in the poison-detection process of Stas and Otto.

Hence with regard to the pharmacologically active bodies in general, it was not allowable to assume that they possessed definite atom groups, which entered into combination with corresponding groups of the protoplasm. This corresponds, as I may remark beforehand, with the incapacity of all these substances to produce antitoxines in the animal body. We must therefore conclude, that only certain substances, food-stuffs par excellence, are endowed with properties admitting of their being, in the previously defined sense, chemically bound by the cells of the organism. We may regard the cell quite apart from its familiar morphological aspects, and contemplate its constitution from the purely chemical standpoint. We are obliged to adopt the view, that the protoplasm is equipped with certain atomic groups, whose function especially consists in fixing to themselves certain food-stuffs, of importance to the cell-life. Adopting the nomenclature of organic chemistry, these groups may be designated side-chains. We may assume that the protoplasm consists of a special executive centre (Leistungs-centrum) in connection with which are nutritive side-chains, which possess a certain degree of independence, and which may differ from one another according to the requirements of the different cells. And as these side-chains have the office of attaching to themselves certain food-stuffs, we must also assume an atom-grouping in these food-stuffs themselves, every group uniting with a corresponding combining group of a side-chain. The relationship of the corresponding groups, i.e., those of the food-stuff, and those of the cell, must be specific. They must be adapted to one another, as, male and female screw (Pasteur), or as lock and key (E. Fischer). From this point of view, we must contemplate the relation of the toxine to the cell.

We have already shown that the toxines possess for the antitoxines an attaching haptophore group, which accords entirely in its nature with the conditions we have ascribed to the relation existing between the food-stuffs and the cell side-chains. And the relation between toxine and cell ceases to be shrouded in mystery if we adopt the view that the haptophore groups of the toxines are molecular groups, fitted to unite not only with the antitoxines but also with the side-chains of the cells, and that it is by their agency that the toxine
becomes anchored to the cell.

We do not, however, require to suppose that the side-chains, which fit with the haptophore groups of the toxines, the side-chains which are toxophile, represent something having no function in the normal cell economy. On the contrary, there is sufficient evidence that the toxophile side-chains are the same as those which have to do with the taking up of the food-stuffs by the protoplasm. The toxines are, in opposition to other poisons, of highly complex structure, standing in their origin and chemical constitution in very close relationship to the proteids and their nearest derivatives. It is, therefore, not surprising if they possess a haptophore group corresponding to that of a food-stuff. Alongside of the binding haptophore group, which conditions their union to the protoplasm, the toxines are possessed of a second group, which, in regard to the cell, is not only useless but actually injurious. And we remember that in the case of the diphtheria toxine there was reason to believe that there existed alongside of the haptophore group another and absolutely independent toxophore group.

Now for certain cellular elements of the body it can be proved in the test-tube that between these tissues and certain toxines an "anchoring" process takes place exactly similar to that between toxine and antitoxine. Wassermann first demonstrated this in the case of the brain substance. In a mixture of tetanus toxine and broken-down fresh guinea-pig brain the latter so bound or "anchored" the toxine that not only was the surrounding fluid toxine-free, but the brain substance laden with the tetanus toxine had also lost its own toxic action, and so the mixture when injected into an animal was borne without any harm.

The deduction is, that in this case, a chemical union between the brain substance and the tetanus toxine had taken place, and this was of so firm a nature that on introduction into the body the union was not broken up and therefore the toxine remained innocuous. The brain of the normal animal had, in keeping with my theory, acted exactly like a real antitoxine. There are present in the brain, i.e., in the ganglion cells, tetanophile protoplasmic groups, which unite themselves with the toxine. The presence of such groups is the necessary preliminary and cause of the poisonous action of the tetanus toxine in the living animal. That the process here was not one of simple absorption is proved by the fact that, if the group concerned was destroyed by heat, the brain substance became as incapable of removing the toxine as an emulsion of any other organ of the guinea-pig.

As has been said, the possession of a toxophile group by the cell is the necessary preliminary and cause of the poisonous action of the toxine. This can be most sharply demonstrated in the case of certain blood poisons, viz., the hsemolysines, which exercise a solvent action only on such red blood corpuscles as are able to unite chemically with them. The union with the red corpuscles can be proved, and one has here the great advantage of dealing with living and intact red blood cells instead of broken-down cellular material. Under these conditions it is easy to determine the quantitative relations of the union. If we now regard the action of the toxines with which we are concerned in accordance with the views we have just been discussing, we are obliged to conclude that these are only in a position to act prejudicially on the organism if they are able, by means of their haptophore groups, to anchor themselves to the side-chains of the cells of organs essential to life. If the cells of these organs lack side-chains fitted to unite with them, the toxophore group cannot become fixed to the cell, which therefore suffers no injury, i.e., the organism is naturally immune. One of the most important forms of natural immunity is based upon the circumstance, that in certain animals the organs essential to life are lacking in those haptophore groups which seize upon definite toxines. If, for example, the ptomaine occurring in sausages, which for man, monkeys, and rabbits is toxic in excessively minute doses, is for the dog harmless in quite large quantities, this is because, the binding haptophore groups being wanting, the ptomaine cannot, in the dog, enter into direct relation with organs essential to life. We see, then, that the haptophore groups act especially in bringing definite areas of the cell within the sphere of influence of the toxophore group. In the behaviour of the haptophore and toxophore groups there exists a difference essentially great, as we have already pointed out when referring to the work of Donitz and Heymans. The haptophore group exercises its activity immediately after injection into the organism, while in all toxines—with the, perhaps,solitary exception of snake-venom—the toxophore group comes into activity after the lapse of a longer or shorter incubation period, which may, e.g.,in the case of diphtheria toxine, extend to several weeks. It is in the highest degree interesting that it is possible, by voluntarily influencing certain of the outside conditions, to exclude absolutely the action of the toxophore group. Courmont has shown that frogs, when kept at a temperature lower than 20° C., manifest no sign of tetanus, even after very large doses of tetanus toxine, but they succumb to fatal tetanus if they are placed in surroundings of a higher temperature. Dr. Morgenroth, working in my Institute, has thrown light on this behaviour by proving that in frogs maintained in cold surroundings the tetanus toxine is fixed in their central nervous system, and that the absence of action at lower temperatures can only be explained by the toxophore group of tetanus toxine having its action restricted within a certain temperature minimum, while independent of this the haptophore group exercises its action on the nervous system at all temperatures.

The theory above developed allows of an easy and natural explanation of the origin of antitoxines. In keeping with what has already been said, the first stage in the toxic action must be regarded as being the union of the toxine by means of its haptophore group to certain "side-chains" of the cell protoplasm. This union is, as animal experiments with a great number of toxines show, a firm and enduring one. The side-chain involved, so long as the union lasts, cannot exercise its normal physiological nutritive function—the taking up of definite food-stuff's. It is as it were shut out from participating, in the physiological sense, in the life of the cell. We are therefore now concerned with a defect which, according to the principles so ably worked out by Professor Carl Weigert, is repaired by regeneration. These principles, in fact, constitute the leading conception in my theory. If, after union has taken place, new quantities of toxine are administered at suitable intervals and in suitable quantities, the side-chains, which have been reproduced by the regenerative process, are taken up anew into union with the toxine, and so again the process of regeneration gives rise to the formation of fresh side-chains. In the course of the progress of typical systematic immunisation, as this is practised in the case of diphtheria and tetanus toxine especially, the cells become, so to say, educated or trained to reproduce the necessary side-chains in ever-increasing quantity. As Weigert has confirmed by many examples, this, however, does not take place as a simple replacement of the defect; the compensation proceeds far beyond the necessary limit; indeed, over-compensation is the rule. Thus the lasting and ever-increasing regeneration must finally reach a stage at which such an excess of side-chains is produced that, to use a trivial expression, the side-chains are present in too great a quantity for the cell to carry, and are, after the manner of a secretion, handed oyer as needless ballast to the blood.

Regarded in accordance with this conception, the antitoxines represent nothing more than side-chains reproduced in excess during regeneration, and therefore pushed off from the protoplasm; and so coming to exist in a free state. With this explanation the phenomena of antitoxine formation lose all their strange, one might say miraculous, characters. I have deemed it advisable to represent by means of some purely arbitrary diagrams (Plates 6 and 7) the views I have expressed regarding the relations of the cell considered in the manner I have been describing. Needless to say, these diagrams must be regarded quite apart from all morphological considerations, and as being merely a piictorial method of presenting my views on cellular metabolism, and the method of toxine action and antitoxine formation during the process of immunisation.

In the first place our theory affords an explanation of the specific nature of the antitoxines, that tetanus antitoxine is only caused to be produced by tetanus toxine, and diphtheria antitoxine through diphtheria toxine. This very specific nature of the affinity between toxine and cell is the necessary preliminary and cause of the toxicity itself. Further, our theory makes it easy to understand the long-lasting character of the immunity produced by one or several administrations of toxine, and also the fact that the organism reacts to relatively small quantities of toxine by the production of very much greater quantities of antitoxine. By the act of immunisation, certain cells of the organism become converted into cells "secreting" antitoxine at the same rate as this is excreted. New quantities of antitoxine are constantly produced, and so throughout a long period the antitoxine content of the serum remains nearly constant. The secretory nature of the formation of antitoxines has been very strikingly illustrated by the beautiful experiments of Salmonson and Madsen, who have shown that pilocarpine, which augments the secretion of most glands, also occasions in immunised animals a rapid increase' in the antitoxine content of the serum.

The production of antitoxines must, in keeping with our theory, be regarded as a function of the haptophore group of the toxine, and it is therefore easy to understand why, out of the great number of alkaloids, none are in a position to cause the production of antitoxines. Conversely, indeed, I recognise in this incapacity of the alkaloids, in opposition to the toxines, to produce antitoxines, a further and salient proof of the truth of the deduction I have previously based on chemical grounds, that the alkaloids possess no haptophore group which
anchors them to the cells of organs. To formulate a general statement, the capacity of a body to cause the production of antitoxine stands in inseparable connection with the presence of a haptophore atomic group. In the formation of antitoxine the toxophore group of the toxine molecule is, on the contrary, of absolutely no moment. But the toxoid modifications of the toxines, in which the haptophore group of the toxine is retained, while its toxophore group has ceased to be active, possess the property of producing antitoxines.

Indeed, in some cases of extremely susceptible animals, immunity can only be attained by means of the toxoids, and not by the too strongly acting toxines. The toxoids are certainly able to cause the production of antitoxines. To quote an example, it is hardly possible in an animal, which, like the guinea-pig, has all the tetanophile groups confined to the cells of the central nervous system, to produce immunity by means of the unaltered tetanus toxine, whereas this is attained with extraordinary rapidity and ease by means of its toxoids.

The symptoms of illness due to the action of the toxophore group, therefore, play no part in the production of antitoxine. On the contrary, we may consider that the severe symptoms, which indicate injury to the cell-life, disturb the regenerative functions, and thus hinder or entirely frustrate the course of the immunisation process. I have from the first adopted this view, and it was simply a misunderstanding when Knorr, who has been all too soon taken from the field of his labours, affirmed that, according to my theory, sickness of the cell constituted the necessary condition precedent to the new formation and pushing-off of side-chains.

If I am not altogether deceived, the toxoids, where it is a question of producing an active immunisation (and this will always be the case when the immunisation concerns human beings), are destined to play an important role in practical medicine.

In their theoretical relations the toxoids are also of far-reaching interest, in that they provide a transition to that immunisation which can be called forth by substances which would a priori be considered entirely devoid of toxic character, and which are sometimes, like the autochthonous ferments (i.e., those normally present in blood), products of normal cell-life, and in some cases food-stuffs proper. Thus Dr. Morgenroth, working in my laboratory, has proved that the rennet ferment, if introduced in great quantities into the organism, behaves exactly like a real toxine, in that it causes the production of a typical anti-rennet, which up to a certain limit accumulates in proportionally greater quantity, the greater the injected doses. Here, however, we have to do with processes which are altogether within the region of the normal, as is most clearly shown in certain animals, the horse, in the blood serum of which there is normally present a quantity of anti-rennet, equal to that attained in the goat only after a systematic immunisation carried on for months. The rennet ferment present naturally in the body of the horse is the cause of this great formation of anti-rennet. According to Bordet's experiments, if injections of milk be given to animals, their serum acquires thereby the capacity to cause flocculent curdling. This action is seemingly rigidly specific because (according to Morgenroth's experiments) the body produced by the injection of goat's milk, coagulated goat's milk, but not human or cow's milk.

The behaviour is also similar when different kinds of albumin, e.g., the sera of different animals or the white of egg, are injected. There appear constantly in the serum of the animal so treated new substances—specific coagulines—which act only in a specific manner, i.e., precipitate only the form of albumin injected. Thus there are produced, by the injection of common food-stuffs, typical "Antikorper," which unite with the substances used to occasion their production, and form with them insoluble combinations.

From all these considerations I think myself warranted in concluding that the formation of antitoxines lacks all the characters of that purposeful, intelligently directed, and remarkable process which it at first seemed to be, and that it is to be regarded merely as a process analogous to those constituting an essential portion of the normal metabolism of the organism. We must admit that the majority of the food-stuffs and of the intermediate products of tissue-change must be able to cause the production and throwing-off of nutritive side-chains. It may be that the new formation only takes place to a limited extent, and that the replacement of any side-chains which have been shut out from their physiological function is all that is accomplished; but the formation may occur in greater proportions, may become excessive, and therefore lead to the presence of "Antikorper" in the blood.

In this way is easily explained the fact of the occurrence in the normal blood serum of antitoxines and of bodies inimical to bacteria, without the animals having ever been brought into relation with the corresponding toxines or bacteria. Here I need only refer to the fact that diphtheria antitoxine is not uncommonly present in normal horses and in men who have never suffered from diphtheria. Particularly weighty in this connection are the observations that have been made on horses, because, on the one hand, these animals never suffer from diphtheria, and, on the other hand, Cobbett has brought forward experimental proof that this normally occurring antitoxine corresponds absolutely as to its properties with the antitoxine produced by artificially immunising. The conclusion, therefore, is that in the body of the normal horse certain substances may be present which possess side-chain affinities similar to those of the diphtheria toxine, and which, therefore, are quite as capable as the latter are of taking possession of the cell side-chains, and occasioning the regeneration and pushing off of these from the cells; in other words, of causing the presence of an actual diphtheria antitoxine in a normal animal.

Such occurrences direct attention to the possibility of producing immunity in some cases by the administration of definite food-stuff's. Perhaps we have in some such peculiarity of feeding and tissue-change the explanation of the fact so difficult to understand, viz., that individuals of the same race and species react in such diverse manners to the same infection. Certainly we are very far removed from the solution of this important question, which, as yet, has scarcely assumed a tangible form. Still it is our duty to strive with tenacity to overcome the difficulties which surround this point, bearing in mind the words of your illustrious countryman, Francis Bacon: "Sunt certe ignavi regionum exploratores, qui, ubi nil nisi coelum et pontus videtur, terras ultra esse prorsus negant."

https://doi.org/10.1098/rspl.1899.0121

In Summary:
  • Ehrlich praised the work of Edward Jenner and his smallpox inoculations but wondered why no progress had been made with his findings for over 100 years to induce an artificial immunity in the case of other infectious diseases
  • Jenner's work remained isolated due to there being no theoretical explanation for the cause and nature of infectious disease
  • Ehrlich gave credit to Pasteur and Koch for allowing the work on artificial immunization to continue
  • The artificial immunity could be done by passaging through an animal body or through artificial culture methods
  • The theoretical explanations of all these facts lagged far behind their practical effects
  • Ehrlich credited Behring for the "discovery" of unseen bodies in the blood creating immunity against diphtheria/tetanus but he was disappointed by the lack of progress in regards to expanding Behring's research
  • It was disappointing when there did not follow, on the "successful" practical application of diphtheria antitoxic serum, a rapid succession of similar achievements
  • By purely empirical methods, e.g., by the production and use of sera of very great antitoxic value, the results attained showed no improvement
  • Better success was only to be hoped for by an accurate knowledge of the theoretical considerations underlying the question of immunity
  • Ehrlich began his theoretical investigation by looking at the toxins
  • He stated that the toxines, i.e., the poisonous products of bacteria, were unknown in a pure condition
  • So great is their potency, it had to be assumed that the strongest solid poisons which were obtained by precipitating toxic bouillon with ammonium sulphate, represent nothing more than indifferent materials, peptones and the like, to which the specific toxin attaches itself in mere traces beyond the reach of weighing
  • Up to that time, by the purely chemical methods of weighing and measuring, it had been impossible to ascertain anything as to their presence or the intensity of their action
  • In other words, there was no chemical way to ascertain the presence or intensity of action of toxins
  • The presence of the toxin is only betrayed by the proof of their specific toxicity on the organism (i.e. the only proof is the effect as the causal agent can not be seen)
  • For the exact determination of the amount of toxin contained in a culture fluid, the essential condition was that the
    research animals used should exhibit the requisite uniformity in their susceptibility to the poison, however, uniformity is not observed in the reaction of the animal body to all toxins
  • For many toxins, accuracy in measuring the toxicity cannot be attained
  • By means of test-tube experiments with animal blood, Erlich claimed he was able to determine that they yielded an exact quantitative representation of the course of the processes in the living body
  • The demonstration of this "fact" formed the basis of a more extended application of experiments of this nature
  • The knowledge gained led easily to the inference that to render toxin innocuous by means of antitoxin was a purely chemical process, in which biological processes had no share
  • However, insurmountable obstacles presented themselves to this conclusion
  • Ehrlich stated that it must be postulated that in chemical processes the bodies sharing m the action react with one another in definite equivalent quantities
  • This proposition appeared, however, not to hold in the case of the action of antitoxin on diphtheria toxin
  • He tried to establish a unit of measurement through inference by looking at diphtheria but the numbers did not hold
  • He attempted to make it so that in every case the law of equivalent proportions would hold good
  • Ehrlich openly pondered how one can actually measure the strength of a toxin accurately
  • He came to regard as the "toxic unit" that quantity of toxic bouillon which exactly sufficed to kill, in the course of four days, a guinea-pig of 250 grammes weight
  • Erhlich created estimated units of measurement: the "simple lethal dose" and the "immunity unit"
  • The conclusions led him to determine that neutralization of toxin by antitoxin did not follow the law of equivalent proportions
  • He was "obliged to conclude" that the chemical affinity for toxin-antitoxin did not exist due to contradictory results obtained
  • The results could only be explained by ASSUMING that there were two different groups that characterized the toxin:
    • Haptophore: conditions the union with antitoxin
    • Toxophore: the cause of the toxic action
  • Ehrlich inferred that the toxophore group was unstable whereas the haptophore group was more stable
  • He claimed that his conception of the constitution of diphtheria toxin, after more extensive, very exact, and much varied experimentation, based on its partial neutralisation by antitoxine, had been confirmed in the completest manner possible
  • However, at that time, he felt it would be superfluous (unnecessary, especially through being more than enough) to enter into all the details pertaining to these investigations
  • Ehrlich stated his theory about these two groups allowed him to progress as it provided a satisfactory chemical explanation for neutralization but also the key to the toxic nature of toxins as well as the mystery of the origin of antitoxins
  • That chemical substances are only able to exercise an action on the tissue elements with which they are able to establish an intimate chemical relationship is a conception of a general nature, which has been entertained since the birth of scientific medicine
  • Ehrlich felt it was astonishing, almost astounding, that this axiom, of which the theoretical importance had been so long recognised, and which served as the first ground for certain therapeutical procedures, should as a matter of fact have played in the building up and furtherance of scientific pharmacology a role so insignificant in proportion to its great importance
  • The methods for obtaining any knowledge of the exact distribution of drugs in the body were very imperfect
  • The investigation of the laws pertaining to the minute distribution of a chemical substance in the body is only possible when these are at once recognisable by the eye
  • He felt that if they are not able to discover the principles governing the localisation of common chemical bodies, which can be used in suitable quantities in chemical purity, and which chemical and other reactions render perceptible, it was a priori very unlikely that efforts directed to locating the toxins, which are potent in the slightest traces, and which are bodies they have no means of rendering perceptible to the senses, would be anything else than absolutely without result
  • In other words, Ehrlich was making the case that chemical reactions needed to stand in as evidence for the invisible particles
  • He shared evidence that the length of time, by a matter of minutes, determines if an antitoxin will counteract a toxin
  • If a certain period of time had elapsed and even if an animal showed no symptoms, not even the greatest amount of antitoxin would save it
  • Through these observations with tetanus toxins, Ehrlich was "forced" to accept that the central nervous system has similar atomic bodies as to the antitoxin
  • He felt that the predilection of the nervous system for tetanus toxin, the rapid union of the toxin with the nervous tissue, the gradual onset of the symptoms and their long duration could only be explained by the existence of such toxophil groups (i.e. his imaginary creations were the only possibility)
  • Ehrlich felt that the fact that the toxicity in guinea pigs being greater when injected subcutaneously rather than intracerebrally could only be explained when injecting toxin directly into the brain, the toxophile atom groups there present at once seize on all the toxin, while when the toxin is administered through the blood stream, the toxophile groups present in other organs also take up the toxin in equivalent quantities
  • Ehrlich stated that it can not be entertained that these unseen atmoic bodies exist only to remove toxins and must have other cellular functions
  • Accordingly, he felt one may rightly assume that these toxophile protoplasmic groups in reality serve normal functions in the animal organism, and that they only incidentally and by pure chance possess the capacity to anchor themselves to this or that toxin
  • The first thought suggested by this assumption was that the atom groups referred to must be concerned in tissue change
  • Ehrlich then formulated many assumptions such as:
    1. The protoplasm has certain atomic bodies that fix to food-stuff important to cellular life called side-chains
    2. That they are free and independent to varying degrees and may differ due to requirements by different cells
    3. That every atom-group has a corresponding side-chain, i.e. lock and key
  • Ehrlich believed that the mystery of immunity ceases when looking through his view that the haptophore fit with antitoxins/side-chains and they bind the toxin to the cell
  • Discussing experiments using tetanus and guinea-pig brains, Ehrlich made the deduction that an "anchoring" took place as an unseen chemical union between the brain substance and the tetanus toxin made it non-toxic with later use
  • He stated that the possession of a toxophile group by the cell is the necessary preliminary and cause of the poisonous action of the toxin
  • This is demonstrated in the case of certain blood poisons, like the hsemolysines, which exercise a solvent action only on such red blood corpuscles as are able to unite chemically with them
  • The union with the red corpuscles can be proved, and one has here the great advantage of dealing with living and intact red blood cells instead of broken-down cellular material (as is the case in virology studies)
  • Ehrlich stated that he was obliged to conclude that these toxins are only in a position to act prejudicially on the organism if they are able, by means of their haptophore groups, to anchor themselves to the side-chains of the cells of organs essential to life
  • If the cells of these organs lack side-chains fitted to unite with them, the toxophore group cannot become fixed to the cell, which therefore suffers no injury, i.e., the organism is naturally immune
  • Ehrlich believed his theory allowed an easy and natural explanation for the origin of antitoxins
  • His theory proposed that side-chains regenerate to a point where they break off the cell into the bloodstream
  • Ehrlich stated that with his explanation of the phenomena of antitoxin formation, it loses all the strange and miraculous characters
  • Ehrlich presented drawings which he admits are not morphologically accurate but are a representation of his thoughts on how immunity occurs
  • He claimed his theory affords an explanation of the specific nature of the antitoxins
  • He also felt his theory made it easy to understand the long-lasting character of the immunity produced by one or several administrations of toxin
  • In order to keep with his theory, Ehrlich stated that the production of antitoxins must be regarded as a function of the haptophore group of the toxin
  • To formulate a general statement, the capacity of a body to cause the production of antitoxin stands in inseparable connection with the presence of a haptophore atomic group (i.e. the body can not make antibodies without the antigen)
  • In some cases of extremely susceptible animals, immunity can only be attained by means of the toxoids, (i.e. vaccination) and not by the too strongly acting toxins (i.e. natural immunity)
  • Ehrlich admitted that SEVERE SYMPTOMS associated with immunization is against the very nature of immunity and hinders/disrupts the process
  • He said he adopted this view from the start, and it was simply a misunderstanding when Knorr affirmed that according to Ehrlich's theory, sickness of the cell constituted the necessary condition precedent to the new formation and pushing-off of side-chains
  • He stated that if he is not altogether deceived, the toxoids (i.e. antigens), where it is a question of producing an active immunisation, are destined to play an important role in practical medicine
  • The theoretical relations of the toxoids were also of far-reaching interest to Ehrlich
  • He claimed that "Antikorper" (a.k.a. antibodies) were produced by the injection of common food-stuffs, which unite with the substances used to occasion their production, and form with them insoluble combinations
  • Ehrlich felt that they must admit that the majority of the food-stuffs and of the intermediate products of tissue-change must be able to cause the production and throwing-off of nutritive side-chains
  • He referred to the fact that diphtheria antitoxin is not uncommonly present in normal horses and in men who have never suffered from diphtheria
  • The conclusion was that in the body of the normal horse certain substances may be present which possess side-chain affinities similar to those of the diphtheria toxin, and which, therefore, are quite as capable as the latter are of taking possession of the cell side-chains, and occasioning the regeneration and pushing off of these from the cells; in other words, of causing the presence of an actual diphtheria antitoxin in a normal animal
  • In other words, horses and men with no exposure to diphtheria had antibodies to diphtheria similar to if they had been vaccinated

As can be see from this section of the lengthy highlights of Ehrlich's report, he did nothing more than create a story around a possible way immunity occurs inside the body. Nothing he said was ever physically seen nor proven by his theoretical investigation. It is all conjecture and inference which was later taken to be fact due to further indirect evidence. Ehrlich used his lively imagination to conjure up a narrative for how immunity works which became the very foundation for all of Immunology today. It does not matter that his theory was based on nothing but chemical reactions and invisible entities nor that his theory turned out to be less than accurate. Ehrlich established the bottom layer of this house of cards known as Immunology. Like virology, it is a house of cards ready and willing to come tumbling on down.

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