The Nature of Specificity 190050

In the nineteenth century, the startling success of the serum treatment of diphtheria had given rise not only to the practical problems of standardization and their solution, and to the international organization to coordinate the work, but also to a theoretical interest in the antigen-antibody reaction and the nature of specificity. The earliest formulation of these problems came with Ehrlich's side-chain theory. According to the theory, the antibody had a unique chemical affinity for the haptophore on the toxin. Ehrlich suggested that the union of antitoxin with its toxin tore the antibody molecule free of the cell that carried it on its surface. This trauma caused an overproduction of identical replacement molecules, rather as healing of an injury caused overproduction of new tissue (Ehrlich 1887-8; Mazumdar 1976; Silverstein 1982).

The side-chain theory had two implications that drew criticism. First, the side chains were supposed to bind to their receptors firmly and irreversibly according to the law of definite proportions, by the type of linkage now called covalent. Second, the theory required that the body be provided with preexisting antibodies to match every conceivable antigen, even those artificial molecules that could have had no possible role in the evolution of the species.

Madsen, who had learned the technique of diphtheria serum assay from Ehrlich himself, but who had also been in contact with Jules Bordet at the Institut Pasteur, was the first to develop an alternative interpretation of Ehrlich's stepped diagram. Working with the physical chemist Svante Arrhenius, he suggested that the antigen—antibody reaction was a reversible equilibrium like the neutralization of acid by base, a type of reaction that gave a smooth exponential curve rather than a series of steps. There was no need to postulate a whole series of different substances (Arrhenius and Madsen 1902; Rubin 1980). The theory had an indifferent reception, but Ehrlich's enemies, led by Max von Gruber in Vienna, hoped that what they saw as a splendid refutation meant the end of Ehrlich's ascendancy.

For Bordet, however, the outstanding fact was that an agglutinable substance absorbed different amounts of its agglutinin according to the relative proportions of the reacting substances: The reaction was not chemical at all, but physical. He compared the phenomenon to that of dyeing: If one dipped a series of pieces of paper in a dye solution, the first few would be strongly colored, and the later ones paler and paler as the dye was exhausted. As early as 1896, he had suggested that "serum acts on bacteria by changing the relations of molecular attraction between bacteria and the surrounding fluid" (Bordet 1896).

Bordet's physical point of view was taken up by Karl Landsteiner in Vienna, who had been trained in both medicine and structural organic chemistry. He had joined Gruber's serological laboratory in 1896 and, like other Gruber students, followed the Gruber line of anti-Ehrlich argument. Landsteiner proposed a physicochemical model for the antigen-antibody reaction: the precipitation of inorganic colloids. The model was particularly apt because the form of antigen-antibody reaction that Landsteiner usually worked with was a precipitin reaction, in which a soluble antigen was precipitated out of solution by an antibody.

Colloid chemistry dealt with the behavior of materials that formed very large particles, so large that their reactions depended on the physical properties of their surfaces rather than on their chemical nature. In the early part of the twentieth century, the physical chemistry of colloids seemed to hold great promise for explaining the reactions of living tissue and its major constituent, protein. It seemed particularly appropriate for the antigen-antibody reaction, because it was thought that only proteins were antigenic. Working with the Viennese colloid chemist Wolfgang Pauli, who was one of the most enthusiastic proponents of the "chemistry of life," Landsteiner pointed out that it was colloids with opposite charges that precipitated each other. The antigen-antibody reaction might be an electrochemical surface adsorption: Subtle surface charge effects might account for antibody specificity (Landsteiner 1909). He and Pauli developed an apparatus for comparing the charge on proteins by following their movements in an electric field. Their apparatus was adopted by Leonor Michae-lis and his group in Berlin, who thought the same features might account for the activities of enzymes. Landsteiner then entered into a long program of research, aiming to define the antigenic specificity of proteins carrying known substituent groups, a project that made use of his skill in structural chemistry. The final proof of his position came in 1918, when he demonstrated that antigenic specificity was determined mainly by the charge outline of the antigen (Landsteiner and Lampl 1918). This conclusion united the colloid or physical concept of charge with the structural concept of the chemical nature of the molecule (Mazumdar 1976; Silverstein 1982).

Landsteiner's demonstration of the importance of charge outline had several implications. The first was that specificity need not be absolute, as Ehr-lich's theory would have it. Cross-reactions could take place between similarly charged groups similarly placed on the antigens. A huge number of different specific antibodies was not, therefore, necessary. The second was that the antigen-antibody reaction was not a firm chemical binding; the two were linked not by valency bonds, but by the so-called short-range forces that surrounded a molecule, as in the model of a crystal lattice (Marrack 1934). The third implication, that of the generation of antibody diversity, was not made until 1930, by the Prague biochemist Felix Haurowitz.

Haurowitz's training, like that of Landsteiner, had included both colloid chemistry and structural chemistry. He spent some time with Michaelis working on the physical chemistry of charge in relation to enzyme activity. But after the war Prague had lost its links with Vienna; Haurowitz heard nothing about the work of Landsteiner until 1929, when the serologist Fritz Breinl told him about it. Breinl and Haurowitz, then working together, suggested that antibody might be assembled on the charge outline of its antigen, appropriately charged amino acids lining up by adsorption onto the antigen to form a specific antibody globulin. There was no need for Ehrlich's innumerable preformed specificities. The organism made its antibody as required. It was a simple and economical solution to the problem of antibody diversity; it made use of the most advanced thinking in both immunology and chemistry (Mazumdar 1989). In English, it became known as the template theory, and in one form or another, it was generally accepted for the next 20 years or more.

Landsteiner had been a rather isolated theoretical thinker at a time when theory was of little interest to the practical serologist. In addition, criticism of Ehrlich was not popular in Vienna. Thus, after the political and economic collapse of Austria at the end of World War I, Landsteiner was eager to leave, and in 1922 was invited to joing the Rockefeller Institute in New York. There, in a sympathetic environment, he was able to continue his work on artificial antigens. He also turned again to the blood groups, which he had not worked on since 1901-9, when he discovered them (Landsteiner 1901). It was for this discovery, and not the work on artificial antigens, that he was given the Nobel Prize in 1932.

Landsteiner's interest in the blood groups began in 1900, when he and many others tried to explain the presence of antibodies in human blood that agglutinated the red cells of other human bloods. Most proposals linked them to some common disease, such as tuberculosis and malaria. It was Landsteiner's suggestion that these were "natural antibodies" whose presence was independent of infection. Landsteiner himself showed curiously little interest in this pregnant finding, perhaps because the "natural antibodies" and the sharply defined specificity of the different groups seemed to support Ehrlich's theory. Although Landsteiner had suggested in 1901 that a knowledge of blood groups would be useful in blood transfusion, transfusion was used very little for many years afterward. It was reported on occasionally during World War I, and although a large literature accumulated, it did not become a routine hospital procedure until the establishment of blood banks shortly before World War II. In Britain, fear of civilian casualties on a large scale stimulated the organization of a blood collection and distribution system.

Landsteiner and his colleagues at the Rockefeller Institute discovered several more sets of inherited antigens on red cells, the MN and P systems, and in 1940 the rhesus, or Rh, system. These workers found rhesus incompatibility between mother and fetus to be the cause of hemolytic disease of the newborn, a discovery that led in the 1960s to an immunologic method of specifically suppressing antibody formation in the mother and so preventing the disease.

In an era when human genetics was represented by the eugenics movement, blood groups were the only normal human trait that was clearly inherited according to Mendelian laws. They seemed to be direct products of the genes, untouched by any environmental effect. They could be analyzed mathematically as the Göttingen statistician Felix Bernstein had shown (Bernstein 1924; Mazumdar 1992), and as a model genetic system, they might come to do for human genetics what the fruit fly Drosophila had done for genetic theory in the 1920s. To critics of the eugenist's social biases, such as the left-wing scientists of the 1930s, blood groups represented a means of making human genetics the genuinely "value-free science" that it had so far grotesquely failed to be (Hogben 1931). In practice, the complex genetics of blood group antigens was to provide a model for the genetics of tissue types, essential in the choice of organs for grafting and for the genetics of proteins (Grubb 1989).

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