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=http://citeseerx.ist.psu.edu/viewdoc/download%3Fdoi%3D10.1.1.856.5258%26rep%3Drep1%26type%3Dpdf&ved=2ahUKEwiwz6XAltD3AhV1RDABHUx5C-AQFnoECAMQAQ&usg=AOvVaw1xA888tO37GEWbXdFG_HWI

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/

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.”

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

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

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.