David Pressman

David Pressman, 1937

[Part 6 of 6 in our series exploring Linus Pauling’s work on the serological properties of simple substances, and the colleagues who assisted him in this work.]

After a meeting with Karl Landsteiner in 1936, Linus Pauling began serious investigations into the link between antibodies and antigens, compiling notes for what would eventually become his serological series, a collection of fifteen papers published during the 1940s. Landsteiner had specifically piqued Pauling’s curiosity on the question of the human body’s specificity mechanism – e.g., how could the body produce antibodies tailored to lock onto and fight specific antigens?

Pauling ultimately surmised that the answer lie in the shape of the molecules, and in the type and number of bonding sites. He described this as a “lock and key” mechanism, otherwise termed as molecular complementarity. Throughout this project, which made a significant impact on the modern study of immunology, Pauling enlisted the help of many undergraduate, graduate, and doctoral students, including a promising young scholar named David Pressman.

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David Pressman was born in Detroit, Michigan in 1916. He attended Caltech as an undergraduate, studying under Pauling and completing his degree in 1937. He stayed in Pasadena for his doctorate, earning it in 1940. During this time, he became a part of Pauling’s quest to unravel the structure of proteins, and was particularly involved with the antibody and antigen work.

By this point, Pauling and his colleague Dan Campbell felt confident enough in what they had learned about antibody specificity to attempt creating artificial antibodies. Pauling was enthusiastic about the practical application that such an endeavor might promise for physicians. Warren Weaver, Pauling’s primary contact at the Rockefeller Foundation, which was funding the work, cautioned Pauling against becoming overconfident, but still granted him enough money to hire Pressman full-time. Thus began Pressman’s career in immunology.

At Pauling’s request, Pressman stayed on at Caltech as a post-doc, and during this time the two became friends. In 1943, after failing to prove that they could synthesize antibodies, Pauling’s research team changed their focus from understanding the structural components of antibodies and antigens, to looking for the binding mechanism that allowed antibodies to attach to specific antigens through Van der Waals bonds. One outcome of this was their development of the theory of complementarity, a “lock and key” model in which molecules fit together because of the high levels of specificity that they show for one another.

Pressman authored three papers with Pauling during this phase, including a very important one titled “The Nature of the Forces between Antigen and Antibody and of the Precipitation Reaction,” published in Physiological Reviews. In this paper, the researchers discussed the historical significance of immunology within the context of structural chemistry. Speaking of the tradition in which they worked, Pauling and his colleagues wrote that “two of the most important advances in the attack on the problem of the nature of immunological reactions were the discovery that the specific precipitate contains both antigen and antibody, and the discovery that antibodies, which give antisera their characteristic properties, are proteins.”  In this paper, they also theorized that the immune system depends on structural and chemical forces to function.

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Pressman (at right) in the lab, ca. early 1960s.

In 1947, Pressman decided to pursue an interest in cancer research and moved on to the Sloan Kettering Institute in New York City to investigate the use of radioactive tracers as they pertained to cancer treatment.  The West Coast was never far from his thoughts however, and he often wrote back to friends comparing the two regions and asking for information about life in Pasadena. Of his new arrangements he observed, “The mechanics of living take a much greater part of the time in New York, so that I do not have as much time to do as much as I would like to or could do in Pasadena.”

Pressman’s first few years at Sloan-Kettering were difficult, not only because of the nature of the research that he was conducting – a continuation of the research that he started with Pauling – but because he was frequently forced to move both his lab and his residence, a source of continuous disruption for himself and his family. Sloan Kettering had just been established in the early 1940s and wasn’t formally dedicated until the year after Pressman moved there. Though it eventually became one of the nation’s leading biomedical research institutions, Pressman’s early experiences there coincided with institutional growing pains.

Eventually, as the environment at Sloan-Kettering became more stable, Pressman settled in to his position and provided Pauling with regular updates on his progress. The two often traded manuscripts back and forth, and each solicited technical advice from one another on their specific endeavors, which gradually grew further afield as time moved forward. At Kettering, Pressman continued to study antibody specificity and explored the potential use of radioactive antibodies for tumor localization to develop immunotoxins. In 1954, he left New York City for the Roswell Park Institution in Buffalo, remaining there until his death.

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60th birthday greetings sent to Pauling by David and Reinie Pressman, February 1961.

Pauling and Pressman remained in frequent contact for many years, focusing their voluminous correspondence primarily on work that Pressman continued to do as an outgrowth of their time together in Pasadena.  In July 1961, Pressman wrote that he and a colleague, Oliver Roholt, had potentially made a breakthrough with regard to the sequencing of the polypeptide chain associated with the region of specific binding sites in antibodies. He sent his manuscript, “Isolation of Peptides from an Antibody Site,” to Pauling for review prior to submission to Proceedings of the National Academy of Science. Pauling felt that the manuscript had been put together too quickly and challenged Pressman to do better. He annotated the manuscript with numerous suggestions, most of which Pressman adopted. Less than a week later, Pressman sent the manuscript back to Pauling with the corrections and Pauling transmitted it in to PNAS, where it was received favorably.

The late 1960s were a period of great activity and advancement for Pressman. In 1965, he received the Schoellkopf Medal, a prestigious award granted by the Western New York section of the American Chemical Society. In 1967, he became assistant director at Roswell and, in 1968, he published a book, The Structural Basis of Antibody Specificity. By all outside indications, Pressman’s life was going well.

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In 1977 however, tragedy struck when Jeff Pressman, David and Reinie Pressman’s son, committed suicide at the age of 33. Jeff was an up-and-coming professor of political science at MIT, where he was well-liked by faculty and students. Up until a few months before his death, Jeff had seemed happy, both with his career and his life at home. In a letter to Pauling, Pressman described Jeff’s descent into depression as sudden, severe, and uncharacteristic. He also documented the events leading up to his son’s suicide, conveying that he and his wife had become increasingly convinced that the responsibility for the tragedy lay at the feet of a rheumatologist to whom Jeff had been seeking assistance for back pain.

Believing Jeff’s back pain to be primarily muscular in cause, the rheumatologist had prescribed Indocin in January 1977. According to multiple sources that Pressman later consulted, Indocin was a mood-changer, so much so that other patients had reported sudden depressive symptoms and, in severe cases, committed suicide a few months after starting the medication. To complicate matters, the rheumatologist had increased Jeff’s dose to a level that few patients could tolerate well, and had done so more rapidly than was advisable. When Jeff began complaining of insomnia, the rheumatologist prescribed two additional medications, both of which had the potential to worsen his depression. Jeff finally stopped taking Indocin, but the effects lingered. Jeff’s wife, Katherine, reported that Jeff had felt increasingly hopeless about his depression, even though he continued to work at MIT up until his death.

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David Pressman’s former secretary, Cheryl Zuber, posing with a plaque mounted in Pressman’s honor at the Cancer Cell Center, Roswell Memorial Institute, 1981.

In the wake of Jeff Pressman’s death, his colleagues at MIT published a collection of political essays dedicated in his honor. The dedication specifically called out Jeff’s commitment to his students and his impact as a teacher. In it, his colleagues wrote, “He cared deeply about public affairs and immersed himself in them because he genuinely felt that government at its best could improve peoples’ lives.”

Nonetheless, the loss took its toll and, for David Pressman, the only source of solace that he could identify was a return to work. In 1978, his focus in the laboratory was on localizing radio-iodinated antitumor antibodies. He later wrote to Pauling about chronic shoulder pain that he was experiencing, as he was aware of Pauling’s vitamin research and was in search of an alternative to the shoulder replacement surgery that had been recommended by his physician. Pauling put forth an argument for a megadose of vitamins, but Pressman was eventually diagnosed with osteoarthritis. By the end of the year, he was slowing down, both in his work and in his correspondence.

Two years later, in June 1980, Pauling received the news that David Pressman had jumped from the roof of Roswell Park Memorial Institute. In a letter to Pauling informing him of her husband’s death, Reinie Pressman cast about for answers. She wrote at length about the health problems that he had been experiencing, including partial hearing loss, prostate trouble, and chronic problems associated with the osteoarthritis in his right shoulder. She also confided that “You were a significant part of Dave’s happier past.” Pauling replied in kind, stating

I was very fond of David. Also, I owe much to him, because of the vigor and effectiveness with which he tackled scientific problems during the eight years that he worked with me. Much of the success of our program in immunochemistry was due to his contribution.

A Master of Many Fields

Linus and Ava Helen Pauling, Oxford, 1948.

[The serological properties of simple substances – part 4 of 6]

By the Spring of 1946, having published no fewer than twelve articles – over a little more than three years – on the serological properties of simple substances, Linus Pauling’s busy life began to get in the way of continued advancement of his research program. Perhaps chief among competing interests was a separate fifteen-year joint research program, funded by a $300,000 grant, that Pauling and George W. Beadle, the head of Biology at Caltech, were in the midst of setting up.

Pauling had also returned to studies of sickle cell anemia with the arrival of Dr. Harvey Itano in the fall of 1946. He was likewise engaged with new inquiries in inorganic chemistry that reached a crescendo with a famous article, “Atomic Radii and Interatomic Distances in Metals,” published in March 1947. From there, the dawn of 1948 saw Pauling moving to England, where he served as George Eastman Professor at Oxford University. Not long after, he received the Presidential Award for Merit for work done during World War II. Clearly there was much going on in Pauling’s world.

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Drawings of antibodies and antigens made by Linus Pauling in the 1940s.

Nonetheless, consequential progress continued to be made in the serological program with the thirteenth paper – an important one – coming into print in April 1948, while Pauling was still in England. This article, written by Pauling along with David Pressman and John Bryden, marked a continuation of the precipitation experiments that had been carried out in the previous two papers, but this time with a different antiserum and antigen substitute. The Paper XIII experiments determined that antibodies are rigid and cannot change shape to bond to a different antigen.

Significantly, these data also confirmed that structural complementarity was responsible for the reaction’s specificity, affirming Pauling’s early notions of a “hand in glove” fit. Furthermore, the paper’s findings established that the principal forces involved in the complementary bonds were Van der Waals interactions – very weak bonds induced by sheer proximity. In short, the experiments verified the importance of intermolecular interaction in the specificity of serological reactions, a significant breakthrough.

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With Pauling now having returned stateside, the year 1949 saw the publication of the final two serological articles, one released in January and another during the summer. Paper XIV, written by Pauling and Arthur Pardee, was fashioned as a response of sorts to disagreements that had been expressed by other scientists concerning Pauling’s interpretations of his experimental results.

The paper specifically focused on experiments utilizing simple antigens and purified antibodies, rather than the antisera that Pauling had been using. These trials found that, although the behavior of simple antigens was different when matched with purified antibodies rather than antisera, “…the earlier work, carried out with serum, is presumably reliable.” In making this statement, Pauling and Pardee cited the non-specific combination of dye molecules along with other components of the serum for past results that had varied slightly.

Illustration of the antibody-antigen framework, 1948.

The last article in the serological properties series, Paper XV, appeared in the Journal of the American Chemical Society in August 1949; Pauling and Pressman were its authors. The article detailed the results of experiments using an antiserum with two or more positive charges. This experimental set-up, Pauling hoped, would allow him to determine the difference in combining power between antibodies containing only one negative charge as well as those containing two negative charges. The duo discovered that the antibody would only combine strongly with antigens that contained two negatively charged groups in specific positions. From this, Pauling concluded that the attraction between the negative charges of the antigen and the positive charges of the antibody are very strong.

After completing the fifteenth paper, Pauling largely left immunology behind in favor of the work that he and Itano were doing on sickle cell anemia. In 1950 and 1951, Pauling and several collaborators also published multiple articles delineating protein structures. In addition, it was during this time that Pauling began to really ramp up his peace work, delivering more and more lectures on the topic as the years went by.

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The fifteen articles that comprise Pauling’s serological properties series were published over a span of seven years. During that period, Pauling worked with twelve collaborators, several of whom were graduate students. By the conclusion the project, hundreds of experiments, using dozens of compounds, had been run.

Particularly given the fact that he lacked any sort of formal background in immunology, the massive impact that Pauling made on the field is truly impressive. By the time that he moved on to other topics, Pauling’s work had served to raise the level of immunological knowledge by orders of magnitude. He is credited now with having discerned a relatively complete understanding of both antibody structure as well as the reaction mechanics underlying the interplay between antigens and antibodies. He also applied the vast collection of data that he had compiled to develop a theory of antibody formation. Of this, biographer Tom Hager wrote

For fifteen years…until a new, more powerful theory of antibody formation was put forward, Pauling’s idea led the field. His antibody work again expanded his growing reputation as a master of many fields.

Pauling himself believed that this work had solved “the general problem of the nature of specific biological forces” and that this understanding would “permit a more effective attack on the many problems of biology and medicine.”

Indeed, Pauling’s work with antibodies was influential even outside of the field of immunology. In 1990, journalist Nancy Touchette declared, “In his 1946 paper [“Molecular Architecture and Biological Reactions”], Pauling prophesied about the future of biology and medicine and why understanding the nature of complementarity is so important to the future of the field.” Five years later, at a Pauling symposium held at Oregon State University just a few months after Pauling’s death, molecular biologist Francis Crick stated flatly that Pauling “was one of the founders of molecular biology.” Once again, Linus Pauling had revolutionized a scientific field while following his curiosity and intuition.

A Period of Rapid Advancement in Pauling’s Immunological Work

Dan Campbell and Linus Pauling in a Caltech laboratory, 1943.

[Part 3 of 6 in a series investigating Pauling’s work on the serological properties of simple substances.]

In April 1943, only four months after releasing his first four papers on the serological properties of simple substances, Linus Pauling was ready to publish more. His fifth paper in the series reported out on the results of hapten inhibition experiments that his lab had conducted using two different antibodies. In the experiments, “measurements were made of the inhibitory effect of each of twenty-six haptens on one antigen-antibody reaction, and interpreted to give values of the bond-strength constant of the haptens with the antibody.”

The results of the experiments, with particular attention paid to the twenty-six hapten molecules, were then discussed in the context of their possible molecular structure. In this discussion, Pauling pointed out that some of the polyhaptenic molecules did not produce participates, a detail that was explained as having been caused by steric hindrance, or the inability for a reaction to take place due to molecular structure.

David Pressman was again a co-author of the paper, as were two graduate students, John T. Maynard and Allan L. Grossberg. Grossberg would stay with Pauling’s lab until 1946 – two years after completing his war-time master’s degree – and was involved with three more papers from the series. He later went on to work with Pressman at the Roswell Park Memorial Institute and eventually became associate chief of cancer research there.

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Pauling’s immunological work was quickly producing exciting new results, momentum that was recognized by The Rockefeller Foundation, which awarded Pauling another grant in June 1943. Pauling also began delivering lectures on his serological research, notably including the Julius Stieglitz Memorial Lecture in January 1944.

Articles six, seven, and eight of the serological series were each published a few months apart from one another, beginning in March 1944. Pauling co-authored these papers with previous collaborators Pressman, Campbell and Grossberg, and also with Stanley Swingle, a research fellow and instructor who had earned his Ph.D. at Caltech in 1942.

Paper VI put forth more evidence for the Marrack-Heidelberger framework theory, for which Pauling had first announced his support in Paper I. The experiments specified in Paper VI made use of fifty different substances possessing either one, two, or three haptenic groups. The results of these trials indicated that a substance containing two different haptenic groups would only form a precipitate when antisera binding to both of those two groups were present. Of this finding the article states, “this provides proof of the effective bivalence of the dihaptenic precipitating antigen, and thus furnishes further evidence for the framework theory of antigen-antibody precipitation.”

In the seventh paper, published in May 1944, Pauling returned to the simple theory for calculating the inhibition of precipitation that he had developed in Paper II, published at the end of 1942. In his discussion, Pauling reported that his laboratory’s experiments found general qualitative agreement with the theory, but the numbers tended to be off. In seeking a more reliable equation, Pauling worked to improve the theory, accounting now for the fact that a single antiserum can contain slightly different antibody molecules with assorted combining powers.

This new and improved theory, and the equation that accompanied it, agreed with experimental results much better than had the original proposal. Indeed, by accounting for variations in the antibodies, Pauling and his colleagues had succeeded in developing a “quantitative theory of the inhibition by haptens,” which would prove important to much of the work that was to come.

Paper VIII, “The Reactions of Antiserum Homologous to the p-Azobenzoic Acid Group,” appeared in October 1944 and shared the results of experiments done with a new type of antibody. Previously, experiments had been conducted with antisera homologous to two different acid groups. However, in these new investigations, the Caltech researchers used antisera homologous to another type of acid group. In doing so, Pauling and his colleagues were attempting to gauge optimum acidity levels for serological reactions; to identify the types of antigens that most readily cause precipitation; to likewise identify haptens that inhibit precipitation; and to measure the strength of their inhibiting power.

Despite Pauling’s extensive involvement in studying reactions of antibodies and antigens, he still had time for other research interests. In February 1945, Pauling and Campbell announced that they had created a usable substitute for blood plasma, the result of three years of work supported by military contracts. Shortly thereafter, Pauling learned a few key details about sickle cell anemia while meeting with the other members of the Medical Advisory Committee. He immediately thought that hemoglobin was involved and went on to experimentally prove that the disease located its source on the molecular level; a first in the history of science.

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Arthur Pardee, 1980

June, July, and September of 1945 each saw the publication of another serological article: Papers IX, X, and XI respectively. The final two of this set featured the addition of a pair of new collaborators. John Bryden, a co-author for Paper X, completed his master’s degree around the time that the article was published, and Arthur Pardee was in the middle of his doctoral program when he worked on Paper XI. Pardee also worked on the experiments described in Paper XIV, although the article was published after he had completed his Ph.D. and returned to Berkeley. Pardee later went on to enjoy a hugely successful career as the Chief of the Division of Cell Growth and Regulation of the Dana Farber Cancer Institute at Harvard Medical School.

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Karl Landsteiner

Papers IX and X shared the results of still more inhibition experiments. The experiments reported on in Paper IX largely confirmed Karl Landsteiner’s discovery on the combining of antiserum and antigen, or antiserum and hapten. Landsteiner had found that less bonding occurred between antibody and antigen or antibody and hapten if the substituent groups on the binding molecule were different from the antigen that created the antibody. The Pauling group confirmed this theory and, in addition, described the forces that affect hapten inhibition. Pauling believed that it had to do with intermolecular forces “including electronic van der Waals attraction…the formation of hydrogen bonds, and steric hindrance,” a supposition that would play a crucial role in later papers in which Pauling explained the incredible specificity that governs the behavior of these molecules.

Paper X studied the effect of molecular asymmetry on serological reactions. In this series of experiments, Pauling and two collaborators, David Pressman and John Bryden, had prepared an antiserum with an optically inactive immunizing antigen; e.g., a molecule that does not rotate plane polarized light. However, even though the immunizing antigen was not optically active, the antibodies in the serum combined more strongly with one configuration over an optically active hapten, which does rotate light, than in the other configuration. Pauling and his colleagues hypothesized that this was due to the presence of optically active amino acid residues in the antibody molecules.

Paper XI, published in September 1945, discussed reactions of antisera with various antigen substitutes. In this instance, the Pasadena group measured the precipitate formed by these reactions to gauge the inhibiting power of the haptens. They then correlated hapten-inhibiting power to molecular structure, suggesting that if a substance mixed with antisera more readily, then the structure of the molecule might be smaller. They ultimately discovered that if a hapten structure matched an immunizing azoprotein structure, the haptenic group exhibited a strong inhibitory effect.

In February 1946, Pauling and co-authors Pressman, Grossberg, and Leland Pence published the twelfth serological article. This was Grossberg’s fourth and final contribution; ultimately, he served as co-author on more of the series than did any other collaborator, save David Pressman and Dan Campbell. New to the series was Leland Pence, an assistant professor of organic chemistry at Reed College who had been collaborating with Pauling since 1942.

Prior to Paper XII, all previous experiments carried out by the lab had used negatively charged or neutral compounds. Paper XII presented the results of experiments that used a positively charged antibody. Pauling and his collaborators found that, even when using positively charged antibodies, hapten inhibition occurred the same way, with the same factors, as was the case with a negative or neutral compound. That said, one important difference that was observed was the ideal acidity for maximizing precipitates; when using a positively charged antibody, the pH required for the optimum amount of precipitate was much lower.

Analyzing Precipitation Reactions Between Simple Substances

Linus Pauling, 1942

[Part 2 of 6 in a series investigating Pauling’s work on the serological properties of simple substances.]

The first four papers published by Linus Pauling and his Caltech colleagues on the serological properties of simple substances described general aspects of the precipitation reactions that occur between antibodies and antigens. This work was spurred by a fundamental conundrum: Pauling and many others knew that antibodies and antigens would react to form solid precipitates. However, because the chemical structures of these precipitates were, at the time, so difficult to determine, scientists had been unable to decipher crucial details about the antibodies and the antigens that combined to form them.

Pauling’s solution to this problem was to investigate the products of a reaction that utilized, in part, a chemical compound whose structure he already knew. The constituents of these products were a simple organic compound consisting of carbon, oxygen, and hydrogen, combined with one or more haptenic groups – small molecules that spur the formation of antibodies when coupled with a larger molecule. Employing this methodology would, Pauling felt, allow him to better approximate the make-up of the antibody, because the experiment now involved only one unknown structure.

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In order to run the experiments, Pauling set up a standard protocol for preparing the compounds that he needed. Each experiment required three types of compounds: simple antigens used in the precipitation reactions; immunizing antigens used to create antibodies; and antisera, which are liquids containing antibodies formed through the coagulation of blood. Pauling used this method for all of his serological reaction experiments.

Pauling and his collaborators obtained the antisera by injecting rabbits (some of them housed in Pauling’s yard and cared for by his children) with immunizing antigens. The rabbits then produced antibodies to combine with and neutralize the immunizing antigens. Once the last injection was carried out, the scientists drew blood from the rabbits, allowed it to clot, and collected the antiserum.

The reactants for Pauling’s experiments – immunizing antigens and simple antigens – were either purchased or prepared by Pauling and his collaborators, typically the graduate students.

For each precipitation test, equal portions of antiserum and a saline solution containing a simple antigen were mixed together. Typically, four to six different concentrations of antigen were used. The mixtures stood at room temperature for one hour, then were refrigerated overnight. The next day, a centrifuge was used to separate out the precipitates, which were then washed with saline solution and analyzed. Pauling’s method of analysis involved measurements of nitrogen, arsenic, carbon, and hydrogen. From there, the amount of a given antibody in the precipitate was determined using the nitrogen measurements.

The initial set of experiments used twenty-seven different compounds as the antigen, each containing between one and four haptenic groups. All of the polyhaptenic substances – those that had more than one haptenic group per molecule – formed precipitates, but none of the monohaptenic substances did. This finding supported the framework theory, devised by the British chemist John Marrack in 1934, that postulated that multivalent antibody molecules could combine with polyhaptenic molecules to form large aggregates, which would become precipitates. On the other hand, Marrack suggested, if multivalent antibody molecules combined with monohaptenic molecules, only small complexes would form and these would not precipitate.

Pauling summarized this work in a set of four papers that were published in the December 1942 issue of the Journal of the American Chemical Society.

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John Richardson Marrack

Pauling’s first article, “Precipitation Reactions between Antibodies and Substances Containing Two or More Haptenic Groups,” served primarily to provide support for Marrack’s framework theory. Eight years before, Marrack had stated that antibodies were multivalent; in other words, they can bond to more than one antigen molecule. In order for them to bind in this way, the molecules must be properly oriented such that the binding sites fit together. This causes the formation of a lattice-like structure which grows until it is too large to stay in solution and precipitates out.

As noted above, Pauling’s experiments found that “simple antigens containing two or more haptenic groups per molecule were found to give precipitates with the antisera, whereas the seven monohaptenic substances failed to precipitate,” a discovery that confirmed the validity of the Marrack-Heidelberger framework, or lattice theory.

The second paper in this installment was titled “The effects of changed conditions and of added haptens on precipitation reactions of polyhaptenic simple substances.” The alterations to conditions that were tested by Pauling included allowing the mixture to rest longer, changing its temperature, and altering its pH. Having confirmed his own belief, in Paper I, that antibodies are multivalent, Pauling used Paper II to first note his assumption – and provide evidence for – bivalence.

In addition, Pauling used this paper to publish an equation that could be employed to find the amount of a precipitated compound in a given solution based on solubility, equilibrium constant, and total amount of hapten. Notably, the equation led Pauling to deduce “that in each case the maximum amount of precipitate is produced by an amount of antigen approximately equal to the amount of antibody,” an idea that unfolded more fully in the following paper.

The equation published by Pauling in Paper II.

Paper III, “The composition of precipitates of antibodies and polyhaptenic simple substances; the valence of antibodies,” further explores the supposition of bivalence through an examination of the ratio of antibody to antigen in precipitates.

While the bulk of Pauling’s experiments focused on dihaptenic antigens, some used trihaptenic antigens, and others used tetrahaptenic antigens. Through careful analyses of the different precipitates that resulted, Pauling was able to determine that the ratio of antibody to antigen in any given precipitate was approximately 1:1.

This finding suggested that most antigens could have only two antibody molecules attach to them, even if they possessed more than two haptenic groups, since the antibody molecules were relatively large and interfered with one another’s attachment. Pauling also used the one-to-one ratio to conclude that most antibody molecules possess two binding sites. The major development of this paper – the near one-to-one ratio – was “taken to indicate bivalence of most of the antibody molecules.”

The last paper of the first installment, Paper IV, reported the results of initial experiments on the inhibition of precipitation in the presence of hapten. Pauling and his colleagues had tested precipitate inhibition in three basic ways: by altering temperature, by augmenting the amounts of hapten present in their mixtures, and by isolating the effects of twenty-four specific haptens. These experiments found that adding haptens to a mixture of antibodies and antigens inhibited the precipitation of the antibody-antigen complex.

Furthermore, Pauling concluded that the structure of the haptens correlated with their inhibition power and detailed the relative values of each hapten’s bond strength. He then used the hapten inhibition data from these experiments to update his earlier equation for finding the amount of antibody precipitated.

Next week, we’ll examine eight more papers that Pauling published on the topic over the next three years and explore the ways in which this body of research evolved and expanded during that time.

The Serological Properties of Simple Substances

Linus Pauling, 1935

[Part 1 of 6]

Today, Linus Pauling is most commonly known for unraveling the chemical bond, working for peace, and promoting vitamin C. However, this short list barely scratches the surface of Pauling’s work in any number of fields. Beginning today, we will explore a lengthy program of research that Pauling oversaw on the serological properties of simple substances, a title that he appended to fifteen publications authored from 1942 to 1949. Post one in this series will focus primarily on Pauling’s background in biology and the work that led up to his first set of serological publications.

One of Pauling’s first major forays into the world of biology came about through his study of hemoglobin, the molecule responsible for transporting oxygen in the blood. Specifically, in 1934, he launched a study hemoglobin partly as a means to begin a larger inquiry into the structure of proteins.

An investigation of hemoglobin, Pauling quickly decided, would require more than one year to obtain results. Consequently, in November 1934, he applied for a grant from the Rockefeller Foundation to “support researches on the structure of Haemoglobin and other substances of biological importance.”

At the time, the Rockefeller Foundation was keenly interested in funding studies of “the science of life,” and Pauling’s grant request was promptly approved, with the first injection of funds received in July 1935. Although Pauling had originally intended for the grant money to go specifically toward his work on hemoglobin, as he corresponded with his funders he expressed an openness to studying other “interesting biochemical problems,” and indeed this quickly became the case.

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A few months later, in 1936, Pauling met Karl Landsteiner, whose ideas would help to shape the course of Pauling’s research for the next several years. Landsteiner was an Austrian biologist and physician best known for discovering the human blood groups. By the time that he met Pauling, he was also actively engaged with topics in immunology.

Over the course of their conversations, Landsteiner passed this interest on to Pauling, who became fascinated by the specificity of antigens (foreign substances that enter into the body) and antibodies (proteins that neutralize antigens and prevent them from causing harm). The human immune system is capable of building thousands of antibodies, each of which reacts with a specific antigen. This specificity is seen in few other physical or chemical phenomena. However, one area in which it is found is crystallization, an area of chemistry with which Pauling was very familiar. This body of knowledge set Pauling down a path to making important contributions to the study of antigen-antibody behavior.

As he sought to learn more, Pauling read Landsteiner’s recently published book, The Specificity of Serological Reactions, finishing it shortly after their initial meeting. The following year, 1937, Pauling and Landsteiner met again and spent several days discussing the most current ideas in immunology. For Pauling, immunology presented two particularly compelling questions: First, what were the forces that enabled the combination of an antibody and its homologous antigen, but no other molecule? Second, how were antibodies produced and how did this means of production allow antibodies and antigens to combine so specifically?

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Dan Campbell and Linus Pauling in a Caltech laboratory, 1943.

In 1939, Pauling decided to shift the bulk of his research focus to the interaction dynamics of antigens and antibodies. As his work moved forward, Pauling came to theorize that the specificity shown by antibodies when combining with antigens depended on how well-matched the shapes of the two molecules were, a theory called molecular complementarity. In other words, antibodies and antigens were able to come together because their shapes complemented one another, like a hand in a glove.

From there, Pauling developed a plan to perform a broad range of experiments that would, he hoped, strengthen this theory and prompt it forward as the accepted explanation for the specificity of serological reactions. To assist in this promising line of inquiry, Pauling hired Dan Campbell, at the time a research fellow at the University of Chicago, to come to Caltech and serve as the Institute’s first faculty member in Immunochemistry. Campbell arrived in January 1940 and remained at Caltech until his death in 1974.

Once relocated to Pasadena, Campbell starting out by working on structural studies of hemoglobin – Pauling’s old research project dating back to 1934. A few months later however, a key shipment of serum antigens arrived from Karl Landsteiner’s laboratory, and both Campbell and Pauling began experimenting on the issue of the day. Initially, the duo encountered only disappointment as they uncovered no results of interest. However, the early setbacks did not stop Pauling. He persevered and, in October, published a landmark article, “A Theory of the Structure and Process of Formation of Antibodies,” which detailed his ideas on molecular complementarity.

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In 1941, Pauling began an experimental program on serological reactions focusing on simpler organic compounds whose structure he already knew. In so doing, he also began to add more collaborators. Besides Campbell, the first of these was David Pressman, who earned his doctorate under Pauling and then stayed on at Caltech to support the nascent immunology program until finally leaving in 1947.

In addition to the simple substances work, this trio of researchers also continued other lines of study pertaining to Pauling’s antibodies projects. In early 1942, one of these produced what seemed to be an incredible result: that March, through a press release rather than a conventional journal article, Pauling, Campbell and Pressman announced that they had created artificial antibodies. A wide array of newspapers and magazines picked up the story and interest rapidly grew. However, other scientists could not replicate the trio’s results and skepticism of the group’s claim began to mount. Pauling, however, continued to believe that his team had truly created artificial antibodies, though subsequent efforts found only dead ends.

Undaunted, Pauling continued his experiments on serological reactions in simple substances and, in December 1942, published the first four papers of what would ultimately become a fifteen-paper series. This body of scholarship was the culmination of several years of work conducted by many people including Pauling, his two main collaborators, David Pressman and Dan Campbell, as well as one other non-student colleague. Several graduate students also supported the effort by helping to prepare the necessary compounds and running the experiments; as the publication series ran its course, eight were eventually listed as co-authors. Three graduate students, Carol Ikeda, Miyoshi Ikawa, and David H. Brown, were involved in the first four papers. Beginning next week, we will take a closer look at the details of what this group published.

Campbell, Pressman, Pauling and the Binding of Antibodies

Drawings of the interaction between an antibody and azoprotein by Linus Pauling. 1940s.

Dan Campbell first collaborated with Linus Pauling on a fellowship at Caltech in 1940, during which time the duo tried to explain how antibodies are formed. At the time, Pauling believed that antibodies were proteins in-the-making that needed to bind to antigens in order to fold and complete their structure. If this principle was correct, Pauling thought, it might be possible to create artificial antibodies by simply denaturing proteins and allowing them to bind and refold in the presence of antigens.

Despite the fact that Campbell’s initial test results cast doubt on his collaborator’s theories, Pauling went ahead and published his ideas on how antibodies work, hoping that further research could support his paper. Thus began a lengthy study of antigen-antibody binding in which Pauling and Campbell attempted to develop a complete theory. Along the way, Dan Campbell’s research at Caltech would become very important to the Institute as well as to Pauling.

In 1943 a Caltech research fellow named David Pressman agreed to join Campbell and Pauling in their study of immunology. Starting with work that had previously been published, Pressman, Pauling and Campbell refocused their studies to explain how antigens and antibodies bind, a change in focus from Campbell and Pauling’s earlier inquiries into how antibodies and antigens are formed. The decision to focus on previous research was made after Pauling had mistakenly announced that antibodies had successfully been synthesized at the Gates and Crellin Laboratories. As it turned out, attempts to create synthetic antibodies using Pauling’s proposed methods were completely unsuccessful. Pauling thus decided to start from scratch by developing a theory of antigen-antibody binding, which he would use to further investigate the chemistry of this interaction.

In July 1943, the three men published “The Nature of the Forces Between Antigen and Antibody and of the Precipitation Reaction,” appearing in the journal Physiological Reviews. The paper attempted to make more educated predictions about antigen-antibody binding.  In doing so, the article begins by referencing the concept of structural complementarity, which posits that antigen-antibody binding is driven by the close complementary physical shapes of the two molecules, which fit together like two adjoining pieces in a jigsaw puzzle. Commonly referred to as “the lock and key mechanism,” this idea was developed in the early 1930s, and served as Pauling and Campbell’s starting point in their initial investigations.

The 1943 study also drew from outside theories, such as the framework theory of precipitation, to suggest that antigen-antibody binding results in the formation of a precipitate; that is, that the two structures react to form an insoluble compound. Using these points as their foundation, the three researchers developed a new theory of antigen-antibody binding.

Pauling and Campbell, 1943.

Campbell, Pressman and Pauling’s breakthrough came by way of their proposal that structural complementarity is an especially important feature for reactions that depend on Van der Waals forces. Van der Waals forces are weak forces of attraction that bind together molecules located in close proximity to one another. The Caltech researchers believed that the close complementary geometry of antibodies and antigens was significant enough to enable these molecules to fit together using the weak Van der Waals attraction as a binding force. In other words, the summation of Van der Waals forces present along the binding site of an antibody worked to bind it to its antigen, specifically because the shapes of antibodies and antigens complimented each other so closely. This theory explained much of what had been observed by immunologists across the discipline in multiple investigations of antigen-antibody reactions.

From here, the three researchers also asserted that two propositions placed forth in Pauling’s 1940 paper should still be considered for further study: the multivalence of antigen-antibody interactions and the probability of hydrogen bonds acting between the two molecules. The trio also concluded that the antigen-antibody mechanism would require at least two supplementary types of forces: Coulomb attraction and polar attraction.

Of the conclusions published by Campbell, Pressman and Pauling in 1943, the multivalence of antigen-antibody interactions and the three proposed forces (Van der Waals, Coulomb and polar) between the two molecules are still considered to be contributing factors to the functioning of the human immune system. With this publication, Campbell, Pauling and Pressman also showed that the immune system relies heavily on both structural and chemical features to carry out its processes.

The important conclusions derived from research conducted by Campbell, Pauling and others established Caltech as a leader in the field of immunology. Over the years that followed, Campbell and Pauling continued to develop their theory of antibody formation, which remained widely accepted until the 1950s. Even when the duo’s work began to be disproven by findings in the genetics field, the understanding of antigen-antibody interactions suggested by research done at Caltech remained undisputed.

Dan Campbell and Linus Pauling went on to publish more than twenty articles relating to immunology, exchanging ideas on the topic until the end of Pauling’s tenure at Caltech in the early 1960s. The attention that their work brought to the Gates and Crellin Laboratories at Caltech prompted the creation of a separate department, one that was entirely dedicated to immunochemistry. (The first of its kind on the west coast.)

For thirty years, Campbell headed Caltech’s immunochemical research and his fame as an immunologist grew to the point where, in 1972, he was named president of the American Association of Immunologists. Two years later, in 1974, Campbell passed away at the age of 67, the victim of a heart attack.  Over the course of his career, he published more than 200 papers as well as several books, and he served on editorial boards of four scientific journals related to immunology.

The Arrival of Dan Campbell at Caltech

Dan Campbell, ca. 1940s.

[Part 1 of 2]

As a scientist, Linus Pauling is remembered by many for combining his expertise in chemistry with other fields. Often times Pauling would start off thinking about a problem from a chemical perspective and end up learning about a field entirely new to him, like cellular biology or medicine. Though this sort of cross-disciplinary work is more commonplace today (partly because of the example that Pauling provided), in the 1930s it was fairly rare for scientists to combine different fields of study. This given, pioneers of the cross-disciplinary approach often found it difficult to identify like-minded researchers with whom to collaborate. Fortunately for Pauling, a man with a very wide network, other researchers often found him.

After delivering a talk about hemoglobin in 1936, Pauling was pleasantly surprised to be consulted by Austrian medical researcher Karl Landsteiner. For many years, Landsteiner had been trying to understand how antibodies in the immune system work, and he believed that Pauling’s knowledge of medicine and chemistry could help him in his investigations. An antibody is a disease-fighting macromolecule that targets and rids the body of unwanted foreign substances, such as viruses and incompatible blood types. Landsteiner wanted to know how antibodies can target specific foreign substances with such precision. This encounter drew Pauling’s attention to the field of immunology, which would eventually become an important part of his research and would remain so for many years to come.

Pauling’s communications with Landsteiner spurred an interest in looking into the chemistry of antibodies and their substrates, antigens. At the time, however, most of Pauling’s focus was necessarily occupied with finishing up his previous program of grant-funded research on protein structures. Furthermore, Pauling was not an immunologist and the demands on his time were such that he could do little more than keep immunology in the back of his mind.

It wasn’t until 1939 that Landsteiner once again brought Pauling’s full attention back to antigens when he used Pauling’s theory of protein structure in a discussion about antibodies. Reading Landsteiner’s article sparked several ideas for Pauling which quickly led to his drafting a rudimentary theory of antibody chemistry. Six months later he found the perfect opportunity to test some these ideas.

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Image extracted from a glass plate display, “Pictures of Antibodies,” prepared for the First International Poliomyelitis Conference, New York, 1948. The caption accompanying this image reads: “…[An] antibody-antigen framework which may precipitate from a solution or be taken up by phagocytic cells.”

In January 1940, immunologist Dan Campbell first visited Caltech on a fellowship. Campbell was an Ohio native who had been trained at Wabash College in Indiana and George Washington University in St. Louis, before receiving a doctoral degree from the University of Chicago, where he was subsequently hired as an assistant professor. During his tenure at Chicago, Pauling invited Campbell to spend a fellowship period at Caltech.  Campbell was only scantly familiar with the Institute, but was aware of the reputation of its chemistry department and accepted Pauling’s offer largely on this basis.

Due to his unfamiliarity with the institution, by the time of his arrival in Pasadena Campbell had still not yet identified a research project on which to collaborate. Pauling advised Campbell to consider different researchers before making his final decision on where and with whom he might work. In the end, after asking around, Campbell chose to collaborate with Pauling on his theory of immunology.

This was a fortuitous decision, for several reasons.  First, in addition to immunology, Campbell had a background in biophysics and chemistry, which made him a perfect candidate to test and develop Pauling’s antigen theory. More importantly, as Campbell began his initial investigations, it became apparent that Pauling’s ideas were flawed and that Pauling’s knowledge of chemistry alone would not be sufficient to make further progress in immunological research.

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Campbell and Pauling, 1943.

Pauling had alleged that antibodies were similar to denatured proteins; that is, a protein that has lost its secondary and tertiary structures and has unfolded into an amino acid chain. Pauling’s theory anticipated that antibodies were an unfinished protein that required specific antigens in order to fold into the proper secondary and tertiary structures.

According to this model, antibodies would only form hydrogen bonds and thus would coil around chemically complementary antigens. As such, the theory explained how antibodies are able to bind unambiguously to their complementary molecules. However, Campbell’s results did not support all of Pauling’s ideas. Though his research showed that antibodies were in fact proteins, their physical structure before and after binding to antigens remained unclear.

Pauling’s lack of evidence for his theory of antibody structure and composition limited him to publishing only a single theoretical paper in which he explained his ideas about antibodies. In July 1940 the Journal of the American Chemical Society featured Pauling’s “A Theory of the Structure and Process of Formation of Antibodies.” The article received much attention and, despite the lack of evidence, was widely acclaimed, though it failed to provide a definitive explanation for antibody structure.

After the publication of the piece, Campbell once again tested Pauling’s theory, and this time his results were much more confusing, to say the least. Initially, it appeared that Campbell had succeeded in creating artificial antibodies by simply denaturing beef globulins (a protein found in blood) and later allowing them to refold around an antigen.

Word of these results greatly excited Pauling, who began to envision the mass production of antibodies using Campbell’s method. Reality turned out to be not so simple; when students and postdoctoral fellows tried to replicate Campbell’s experiment, they were unable to obtain the same results. Looking back now, it seems most likely that Campbell’s research assistants had misinterpreted the results of his experiment.

Pauling knew that he would need more time with Campbell to refine his theory, but that could only happen if Campbell’s position at Caltech was secured. In 1942 Pauling arranged for the Institute to offer Campbell an assistant professorship, which he accepted. By 1950 Campbell had become a full professor.

Combining immunology and chemistry proved to be a commendable approach for tackling many health concerns of the time. Likewise, Campbell’s presence was crucial to the development of Caltech’s immunochemistry department, which over a span of five years grew from a single office (Campbell’s) to a space occupying most of the third floor of Caltech’s Church Laboratory. Students and professors alike flocked to the growing department to discuss questions and engage in research on immunology, using chemistry as the basis of their approach. From the outset, both Pauling and Campbell benefited from one another’s expertise while colleagues at Caltech, and their partnership would continue to yield fruit for many years.

Rebecca Mertens, Resident Scholar

Rebecca Mertens

Rebecca Mertens of Bielefeld University, located in northwest Germany, is the latest visitor to complete a term as Resident Scholar in the Oregon State University Libraries Special Collections and Archives Research Center.  A Ph.D. candidate in the philosophy and history of science, Mertens spent a month stateside, visiting both the OSU Libraries as well as the Caltech Archives.

During her stay she braved both a major (and unusual) snow event in Corvallis as well as torrential rains in southern California.  Despite these obstacles, Mertens enjoyed a fruitful visit to the west coast as she pursued her research on Linus Pauling’s contributions to the lock-and-key model of biological specificity and the influence that this model imparted upon the sweep of modern biochemistry.

The conditions that awaited Mertens upon her arrival at OSU.

An outgrowth of his research on antibodies and antigens, Linus Pauling’s work on biological specificity comprised a major contribution to contemporary thinking on biochemical topics.  Pauling biographer Thomas Hager gives us this primer on what is meant by by the term, “biological specificity.”

Pauling demonstrated that the precise binding of antigen to antibody was accomplished not by typical chemical means – that is, through covalent or ionic bonds — but solely through shape. Antibodies recognized and bound to antigens because one fit the other, as a glove fits a hand. Their shapes were complementary. When the fit was tight, the surfaces of antibody and antigen came into very close contact, making possible the formation of many weak links that operated at close quarters and were considered relatively unimportant in traditional chemistry — van der Waals’ forces, hydrogen bonds, and so forth. To work, the fit had to be incredibly precise. Even a single atom out of place could significantly affect the binding.

In her Resident Scholar presentation, Mertens described the thrust of her research, which focuses on how one should interpret the contributions that Pauling made in this particular arena.

In the course of his research on antibodies, Linus Pauling postulated that the complementary structure of two molecules or two parts of a molecule determined the specificity of reactions in the living organism. However, the idea that molecular complementarity and biological specificity are deeply connected was already mentioned by Emil Fischer at the end of the 19th century. Thus, Pauling’s novel contribution was not the initial articulation of the model, but rather his emphasis on the importance of molecular complementarity for all biological phenomena.

Through examination of the Ava Helen and Linus Pauling Papers, as well as the institutional records held at Caltech, Mertens is pursuing the idea that “Pauling’s interdisciplinary reputation, his public presence and his engagement in the organization of scientific institutions led to the popularity of the lock-and-key model and to its standardization in the second half of the twentieth century.”  These forces of Pauling’s status and personality in turn made an impact on questions of “financial support, networking and science popularization within the administration of scientific projects.”

Beyond uncovering and detailing the history of Pauling’s role in the development of the lock-and-key model, Mertens is also using her research to “suggest an approach to the study of analogical models that considers social and political factors on successful model usage…[and] the formation and consolidation of model-based research programs.” Mertens returned to Germany with a large volume of content to sift through and absorb as she continues to develop her thinking on these issues.

Now entering its seventh year, the Resident Scholar Program at OSU Libraries provides research stipends of up to $2,500 to support work conducted in the Special Collections and Archives Research Center.  Applications for the 2014 class of scholars are being accepted now – the deadline for entry is April 30, 2014.  For more details, please see the program homepage.

The End of Artificial Antibodies

Illustration of bivalent antibodies attaching to complementary antigen molecules. Image extracted from a glass plate display, “Pictures of Antibodies,” prepared for the First International Poliomyelitis Conference, New York. The caption accompanying this image reads: “In vitro or in vivo bivalent antibodies may become attached to complementary portions of antigen molcules.”

[Part 3 of 3]

Though highly controversial, Linus Pauling’s claim that he had created artificial antibodies gave him a boost in funding. Many of his backers realized that if Pauling was correct he had just revolutionized modern medicine and they were just as eager as he was for his project to succeed. Since he now had more money, Pauling hoped he could expand his staff, though the war greatly prohibited this effort. Pauling lamented:

Unfortunately the amount of war work which is being done now here is so great that the usual seminars and informal discussions of science have decreased somewhat in number, but still a good bit of work in pure science is being carried on.

To help remedy this situation, Pauling corresponded frequently with William C. Boyd of Boston University’s School of Medicine, trying to persuade him to transfer to Caltech over the summer. Boyd refused, stating that he had too many other medical defense projects, though suggesting that “perhaps it will not be too late when the war is over, unless it goes on for 10 years or more, as some pessimistic writers predict.” Pauling also failed to hire two other well-known researchers, Henry F. Treffers, and A.M. Pappenheimer. Both declined because he was only able to offer a one year position. Indeed, from 1942-1943, Pauling actively tried to find staff for his lab, offering a $3,500 one-year position and draft deference. Despite this, most of the competent researchers he wanted were otherwise employed doing war work. He was able to hire Leland H. Pence in December of 1942, and in 1945 Frank Johnson visited from Princeton and worked at the lab in Caltech a bit. These individuals were, however, the exceptions, and Pauling was generally unsuccessful with his offers of employment.

Pauling was also facing other staff issues that were relics of the era. Among his group were two employees who were born and raised in the United States, but whose families were Japanese, Carol Ikeda and Miyoshi Ikawa. All too cognizant of the forthcoming policy of internment, Pauling began corresponding with Michael Heidelberg of Columbia University, hoping that he could temporarily trade employees as a method of getting Ikeda, at least, to a less hostile location. The plan did not work out, and Heidelberger ended one of his letters to Pauling bemoaning the fact that “…unfortunately a lot of wholly patriotic people are going to suffer.” Pauling was eventually able to get both Ikeda and Ikawa into graduate programs, though doing so took a substantial amount of work.

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Dan Campbell and Linus Pauling, 1943.

Even though William Boyd had refused a job, he and Pauling continued to correspond frequently. Boyd was critical of Pauling’s theories on antibodies, warning his colleague that “preconceived notions evidently play a big role in the field [of immunochemistry.]” Boyd told Pauling to be careful with his research and his declarations, as he had often made arguments that he felt infallible only to have his colleagues inform him that they were unconvinced.

In August 1942, five months after issuing his controversial press release, Pauling finally published his research on artificial antibodies in an article titled “The Manufacture of Antibodies in vitro,” which appeared in the Journal of Experimental Medicine. As with the press release, Pauling’s paper was somewhat lacking in detail and many scientists found it hard to replicate his experiments. Those who did, such as Pauling’s valued colleague Karl Landsteiner, were unable to obtain the same results that Pauling had reported. Despite this, Pauling remained convinced that his research was valid and worth pursuing. However, Pauling’s collaborator Dan Campbell seemed to be the only one who could successfully produce the antibodies, and even those were weak and ineffective.

In early 1943 the Rockefeller Foundation assigned Frank Blair Hanson to assume some of the work that Warren Weaver had been conducting, and right away it was clear that Hanson was notably “less entranced than Weaver with Pauling’s work.” Despite the fact that he agreed to continue funding Pauling’s artificial antibody research, he was skeptical of its worth and began polling immunologists across the country to that end. They did not respond favorably – even Landsteiner believed that there was a less than 50% chance that Pauling had actually created artificial antibodies. As a result of these lackluster opinions, Hanson cut Pauling’s funding by half.

Pauling proceeded nonetheless, his enthusiasm still strong. His next move was to submit a patent application, “Process of Producing Antibodies.” The response that he received was not what he wanted to hear:

The claims are again rejected for lack of utility as no evidence has been presented to show that the antibodies alleged to be produced by the claimed have any utility at all… The claims are rejected as being too broad, functional, and indefinite…the claims are rejected for lack of invention…all the claims are rejected.

Pauling was disheartened by this response but still confident that he could salvage the project. However, shortly afterward he received a letter from the Rockefeller Foundation informing him that they did not approve of researchers using their funds to apply for medical patents. Because of the letter, and the general ineffectiveness of the research, Pauling opted not to pursue his patent application any further.

Illustration of the antibody-antigen framework. The caption accompanying this image reads: “…[An] antibody-antigen framework which may precipitate from a solution or be taken up by phagocytic cells.”

As 1943 progressed, Pauling continued to piece together a better understanding of how antibodies adhere to antigens. Later that year, he and Campbell published a paper in Physiological Review which further elaborated on their idea that shape was the primary determinant of antibody functions. They wrote that while many of their colleagues at other institutions felt that antibody formation adhered to a “lattice theory,” they did not, because their research showed that the structures created by antibody/antigen precipitation were not regular enough. Instead, they coined the phrase “framework theory” to describe their idea.

By the winter of 1943-1944, Pauling had at last concluded that the artificial antibody research was going nowhere. This was a difficult admission for Pauling because, despite the fact that progress in the research was still slow, he was convinced that he could make it work if given more time. Unfortunately for him, the war effort demanded quicker results and actively prohibited greater focus on antibodies. In the end, he decided to abandon the artificial antibodies research. Pauling never retracted his support for the work, though many years later Dan Campbell admitted that a laboratory technician had “shaded” the results to fit what he thought Pauling wanted to see.

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The failure of the artificial antibodies project allowed Pauling to move on to more productive lines of research. He continued to build his ideas on how exactly antibodies function, and by 1945 he was able to prove that shape was indeed what caused antibodies to adhere to antigens. In Pauling’s description, the antibodies would fit to the antigens like a glove, at which point they adhered, not due to orthodox chemical bonds, but because of another weak, poorly understood force.

The force that causes antibodies to bind with antigens is called the van der Waal’s force. It is a very weak, almost imperceptible subatomic bond between two molecules existing in extremely close proximity. Historically, due to their weakness, van der Waal’s forces had been ignored as viable components of biochemical reactions. However, Pauling was able to show that the extremely tight fit between antibodies and antigens exposed a large surface area across which the van der Waal’s force could become a factor. The fit had to be precise, as even one or two atoms being out of place would effectively break the hold that the van der Waal’s force put into place. This concept, known variously as molecular complementarity or biological specificity, cast a great deal of light on a central mystery of molecular biology. With it, Pauling was additionally able to confirm his earlier hypothesis that antibodies are bivalent.

As World War II drew to a close, Pauling shifted his focus away from antibodies and back towards a more general study of the shape, creation, and function of proteins. Pauling’s focus on proteins was long lived, stretching at least twenty-five years from 1933-1958. His foray into antibodies was notably shorter, a nine-year interlude from 1936-1945. Yet in this time, he had managed to dramatically impact immunochemistry with his discovery that antibodies are bivalent and his insights into how they work. These accomplishments are made even more impressive when considering that Pauling was neither an immunochemist nor an immunobiologist by trade. As was so often the case during his life, he threw himself into the challenge with characteristic enthusiasm, and managed to make major contributions to the field.

The Tantalizing Prospect of Artificial Antibodies

Linus Pauling and Dan Campbell, 1943.

[Part 2 of 3]

By late 1939, Linus Pauling had thrown himself wholeheartedly into the study of antibodies, specifically how they work and how they are made. He’d already developed a few memorable and unique hypotheses, though by 1940 they were still yet to be proven correct or otherwise.

In January 1940, a researcher named Dan Campbell arrived at Caltech with the intent of working on problems in immunochemistry. He and Pauling began collaborating, and ended up co-authoring several papers together. Later in 1940, after a first, erroneous paper, Pauling published another in which he claimed:

all antibody molecules contain the same polypeptide chains as normal globulin, and differ from normal globulin only in the configuration of the chain; that is, in the way that the chain is coiled in the molecule.

In other words, shape was what determined the effect of antibodies. Pauling acknowledged the potential for flaws lying within his hypothesis, but adopted it because of his inability to otherwise “formulate a reasonable mechanism whereby the order of amino-acids residues would be determined by the antigen.”

Nobody knew about the genetic basis of amino acids at the time. As a result, while Pauling’s hypothesis on how antibodies worked eventually turned out to be correct, his coupled hypothesis on how they were formed was still wrong. Regardless, Pauling’s theory “ruled the roost amongst immunochemists” for almost 20 years. It wasn’t even until 1949 that people began to seriously question Pauling’s model, and when that finally happened, it was because the model failed to account for the development of immunity to antibodies.

In July a colleague at Caltech, Max Delbrück – a German researcher who had arrived in the US on a study abroad trip and never went home – showed Pauling a paper written by Pascual Jordan, another German scientist. Jordan claimed that identical molecules were attracted to each other, and opposite molecules repelled from one another, because of quantum mechanical resonance. Pauling declared the idea “baloney,” and decided to write a paper debunking Jordan’s theory. He asked Delbrück to co-author it, which he did with hesitation, as the young scientist was nervous about signing his name to a paper attacking the theories of the famous and well-respected Jordan. The duo finished the paper and published it in Science, but unfortunately for them, the work went largely unnoticed.

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Since the start of the Battle of Britain in 1940, Pauling had been doing some side research on explosives, propellants, and other “war work” in anticipation of United States involvement in the war raging across Europe and the Asia. In 1941, U.S. engagement finally came about with the attack on Pearl Harbor. Pauling and his laboratory at Caltech switched their main priority to war work, as did just about every other research laboratory in the country. As part of this realignment of efforts, Pauling received a grant to begin a research project with the goal of creating gelatin-based blood plasma artificial antibodies, to be used for military medical purposes.

Pauling hypothesized that if a generic protein, such as beef globulin, was very carefully denatured, then placed in the presence of an antigen and revitalized, it would grow and mold itself to the antigen. If this worked, medical technicians would someday be able to create antibodies “made to order.” The potential import of the idea was clear: “the end result of a million years of evolution” would become available “by the quart.” Successful implementation would represent a massive breakthrough in medical technology, and Pauling was convinced that he was just the man to do it. He picked Dan Campbell and David Pressman to be his primary assistants and the group got to work.

The process was slow and complicated; it involved a mixture of 1-2% beef globulin, dissolved in 0.9% sodium chloride, then very slowly heated to 57˚F. Once at that temperature, alkali would be carefully added to bring the mixture to a pH of 11, before very delicately and slowly returning the mixture to pH 0. To further complicate issues, the process produced a large amount of nitrogen, which had to be removed, but the process of removing the nitrogen created carbon monoxide and other “undesirable substances.” The Pauling group tested their antibodies on rabbits, mice, and guinea pigs predominantly, as their labs lacked the space for more extensive animal research.

In March 1942, instead of submitting his ideas to a peer-reviewed scholarly journal, Pauling issued a press release to Science News, claiming that his lab had developed artificial antibodies.

Text of Pauling’s artificial antibodies press release, March 1942.

The scientific community was vexed by this highly unusual action. Numerous scientists questioned the strength of the data, saying that the reports released by Pauling and Campbell were vague and weak. Pauling asserted that while the data they had gathered was not definitive, it was strong enough to make the statement that artificial antibodies had been produced. Pauling said that his artificial antibodies, though weak, were still real, and that he was going to continue improving on them. Specifically, he claimed that the antibodies had been used to prevent mice from getting pneumonia.

Pauling’s reputation helped to coax certain colleagues in the direction of his arguments. And after some skillful debating, the Rockefeller Foundation offered $31,000 to continue the research, while the federal Office of Science Research and Development offered $20,000.

Despite the skepticism of his peers, Pauling was enthusiastic and eager to continue research on this line of work. He had the funding and the desire, and he was convinced it was only a matter of time before he could start mass-producing artificial antibodies. However, he was about to run into some issues outside of his laboratories.