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. No lesions developed. In another controlled experiment, nonimmune serum was mixed with vaccinia virus-containing lymph and was compared with a mixture of vaccinia virus and immune serum. Skin lesions appeared at the site of inoculation of the mixture of virus and nonimmune serum but not at the site of inoculation of virus plus immune serum. While preliminary in scope, the prototype of neutralization was established. The marker system here was the skin of the calf, and in years to come it would be broadly implemented in experimental animals, in embryonated eggs, and in tissue culture. At the end of the published discussion, Sternberg commented, “I believe that there is something in the blood of the immune calf that neutralizes the vaccine virus.” The year of Sternberg’s paper, published in 1892, is usually marked as the start of the science of virology. Coincidentally, in a paper published in the same year in St. Petersburg, Dmitri Ivanowski described the passage of tobacco mosaic disease infectivity through a bacterial filter (20). These landmark events signify that virology and viral serology had their birth in the same year, albeit on different continents. Protection by neutralization was soon demonstrated in other virus-host systems. In April 1910 in Paris, A. Netter and C. Levaditi demonstrated protection of monkeys against experimental polio with serum from human subjects who had experienced the illness from 6 weeks to 3 years earlier (28). The monkeys revealed no signs of illness. In comparison, a monkey that received virus mixed with serum from a healthy person became paralyzed and died. The investigators noted that these studies demonstrated the identity of the human disease and the experimental disease in monkeys. In a following paper in May 1910, they also demonstrated antiviral activity from a subject who had not been paralyzed but had had symptoms of abortive illness (29). Thus, they identified the virus causing the abortive form of polio as well as the paralytic form. This supported the epidemiological findings of Ivar Wickman on the role of the abortive form in disseminating the illness (51). Also in May 1910, Simon Flexner and Paul A. Lewis in New York City demonstrated that serum from monkeys that had recovered from experimental poliovirus infection, when mixed with active virus, prevented paralysis in intracerebrally inoculated monkeys (14).

Similar findings were later reported for yellow fever. In their 1928 report from Nigeria on the experimental transmission of yellow fever to Macacus rhesus monkeys, Adrian Stokes et al. demonstrated that convalescent-phase human serum protected monkeys against experimental infection (46). Max Theiler greatly facilitated further laboratory studies of yellow fever infection by demonstrating the susceptibility of white mice to intracerebral inoculation of the virus (47). Virus passed in mice was neutralized by convalescent-phase serum from humans or monkeys. Parenthetically, in a later study of the protection test in mice, Theiler distinguished the terms “protection” as in vivo survival after inoculation of virus mixed with immune serum, whereas “neutralization” occurred in vitro (48) (Fig. 7). This seemed a useful distinction at the time, but it was frequently ignored in the literature. For example, in tabulating various serological reactions, Joseph Edwin Smadel listed “neutralization” for in vivo tests for many viruses (42). In the present context, Theiler’s assay was termed the mouse protection test, and it was an important step in laboratory diagnosis. The application of serology to the epidemiology of yellow fever was emphasized by W. A. Sawyer and W. Lloyd. They wrote of mapping “large areas with respect to endemicity, epidemicity, or absence of yellow fever . . .” (38). They pointed out the difficulty of adapting monkeys to such studies because of the great number of animals required. Instead, they modified Theiler’s use of white mice and found the technique useful for epidemiological studies.