href="#ulink_d7c16af5-da12-552e-8320-77d3002aab4b">52).
The theoretical basis for the activity of antibodies was developed by Paul Ehrlich in his side chain theory (31). With the stimulation of an antigenic challenge, host cells produced side chain receptors that specifically combined with the antigen. With sufficient stimulation, side chain receptors could be released into the blood as circulating antibodies (12). Perhaps more to the point in the current context, Ehrlich established principles of quantitation (8). Paul Ehrlich and Elie Metchnikoff shared the Nobel Prize in 1908, an early recognition of the importance of both humoral and cellular immunity (1).
The above-described studies, which were of great importance to establishing mechanisms of host defense, were heavily dependent on microscopic observations of the infecting organism. There was little direct application to submicroscopic viruses until the studies of Jules Bordet (Fig. 5), another Nobel Prize winner (1919); he was recognized for his work on complement fixation, which became one of the key mechanisms with which to document antiviral immunity. During the period in which this work was done, Bordet worked in the laboratory of Elie Metchnikoff at the Pasteur Institute in Paris. There were two preliminary steps which Bordet addressed in an 1895 paper in Annales de l’Institut Pasteur (translated and condensed in reference 7). Building on the work of R. F. J. Pfeiffer, he confirmed that the granulation followed by lysis of Vibrio cholerae bacteria exposed to serum of immunized rabbits is strain specific (Pfeiffer phenomenon). Testing several strains of vibrios in vitro, he showed that the greatest destruction was of bacterial strains against which the rabbits had been vaccinated. Such immunological specificity is a cornerstone of serological diagnosis.
Figure 5 Jules Bordet. With his brother-in-law, Octave Gengou, Bordet demonstrated the fixation of complement by reacting with bacteria and immune serum. The complement was then no longer available to participate in a hemolysis reaction. The assay, known as complement fixation, became a mainstay of diagnostic virology by demonstrating the development of antibodies in serum after infection. Bordet received the Nobel Prize in 1919. (Courtesy of the National Library of Medicine.)
doi:10.1128/9781555818586.ch3.f5
Bordet demonstrated that there were separate immunizing and bacteriolytic components. While the bactericidal property is destroyed by heating, he built on the work of C. Fraenkel and G. Sobernheim, who showed that the immunizing component is resistant to heating. In a system in which counts of Vibrio cholerae were the markers, he demonstrated that neither heated immune serum from goats nor fresh serum from nonimmune guinea pigs alone would inhibit growth. However, the combination of heated immune goat serum and unheated guinea pig serum abolished bacterial growth. This phenomenon occurred whether or not cells were present in the guinea pig serum. From a diagnostic perspective, two features deserve emphasis: (i) the source of complement, then known as alexine, need not be the species tested for immunity, and (ii) complement, but not the immune function of serum, is destroyed by heating to 60°C. Therefore, the components can be added separately. The dissection of the bacteriolytic system into two components was a remarkable accomplishment on its own merits. The experimental challenge had been made further complicated by at least two other factors. Some nonimmunized animals possessed bacteriolytic capacity in their serum, as shown in Nuttall’s work. In addition, not all bacteria were equally susceptible to immune-mediated bacteriolysis. Hence, the clarity of Bordet’s experimental design is all the more remarkable.
Next came the crucial paper in the scientific foundation of the complement fixation reaction. With his brother-in-law Octave Gengou, Bordet demonstrated the deletion or fixation of complement, as currently understood, by a combination of immune serum and the target bacilli (reference 6, translated in reference 22). With this combination, the complement was unavailable to facilitate hemolysis as a marker system. As Bordet and Gengou pointed out, the work was dependent on the previous demonstration of two concepts. First, red cells and microbes could each delete alexine (complement). Second, the same alexine could participate in either hemolysis or bacteriolysis. With appropriate controls, in a two-step experimental setup, they first mixed plague antiserum and a suspension of plague bacilli with alexine-containing serum from animals. In the second step, into the first mixture they introduced heated guinea pig serum immunized against rabbit’s blood together with rabbit’s blood. Hemolysis occurred in all tubes except those containing the bacilli, the specific antiserum, and alexine (Fig. 6). Hence, the antiserum conferred the capacity to fix alexine (complement), making it unavailable to participate in hemolysis of sensitized red cells. At a stroke, Bordet and Gengou had demonstrated a marker system that did not require microscopic examination for the presence of the pathogen. Complement fixation to detect viral antibodies became the cornerstone of viral diagnosis for many years.
Figure 6 Complement fixation. In stage 1, complement, antigen, and antibodies are mixed together. If antibody is present for the antigen, complement will be bound (fixed). In stage 2, if the complement has been fixed in the first stage, it will be unavailable to combine with antibody-coated erythrocytes. Therefore, a bull’s eye pellet of cells will appear in the bottom of the tube, as shown on the bottom left. However, if complement has not combined with antigen and antibody in the first stage, it will be available to lyse antibody-coated red cells. In that case, no bull’s eye pellet will be seen at the bottom of the tube, as shown on the bottom right (22a). (From Diagnostic Virology, courtesy of the author, Diane S. Leland, Indiana University School of Medicine.)
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There was an interesting parallel of scientific reports in 1901, each of importance to the beginnings of clinical virology. In that year, Bordet and Gengou demonstrated the experimental basis of the complement fixation reaction, while W. Reed and J. Carroll demonstrated that the submicroscopic pathogen for yellow fever passed through a filter which blocked bacteria (33).
Antiviral Neutralization and Protection
In years to come, neutralization tests, with complement fixation assays, would become serological mainstays of diagnostic virology. Neutralization of vaccinia virus was perhaps the earliest demonstration of principle. In the context of reviewing advances in bacteriological research in 1892, George Sternberg, a pioneer American bacteriologist, laid particular stress on antitoxin and its therapeutic implications (45). He also reported an experiment on vaccinia virus neutralization that he had conducted with William Griffiths, an expert in the production of vaccine in calves.
The intent was a preliminary step in antiserum production, but its usefulness was as a prototype for a diagnostic neutralization test. Vaccinia virus-containing lymph was incubated with serum from a recovered calf; separately, a lesion crust from a child was incubated with immune serum. Each mixture was inoculated into the skin of a nonimmunized calf.
