Another major step in the laboratory measurement of antiviral immunity was the development of the hemagglutination inhibition assay. George Hirst of the Rockefeller Foundation first observed and reported hemagglutination of red cells by allantoic fluid of influenza virus-infected embryonated chicken eggs. He reported, “When the allantoic fluid from chick embryos previously infected with strains of influenza A virus was being removed, it was noted that the red cells of the infected chick, coming from ruptured vessels, agglutinated in the allantoic fluid” (17). The observation was also made by L. McClelland and R. Hare in the same year (24). M. Burnet had observed the phenomenon but rued that he had not followed up. “Then came a discovery which I should have made but did not” (9).
Inhibition of agglutination by serum from a recovered individual would be a demonstration of immunity that could be measured. In the first publication, Hirst described hemagglutination inhibition as an efficient and immune-specific method to determine antibody titers. Compared to the more cumbersome mouse neutralization tests, hemagglutination results were “of the same order of magnitude.” In a subsequent report, Hirst wrote, “. . . the mouse test is very complicated and involves the interplay of many forces over a period of 10 days, while the in vitro test is relatively simple. Because of the complexity of the mouse test, it seems probable that the agglutination inhibition test gives a more accurate picture of the in vitro combining ratios of virus and antibody” (18).
An assay related to hemagglutination, hemadsorption (Fig. 8), was later developed by Alexis Shelokov and his colleagues at the U.S. National Institutes of Health (39). Several viruses, particularly influenza virus, were isolated and identified by “selective attachment of erythrocytes onto the monolayer surface of tissue culture cells.” In the absence of cytopathic effect, infection was recognized by the specific attachment of red cells to the infected, intact cell monolayer. One of the methods reported by the investigators to verify the type of viral isolates was hemadsorption-inhibition.
Figure 8 Hemadsorption. Tissue culture cells infected with certain viruses produce receptors with an affinity for red blood cells which attach to the surface and are visible microscopically. In this illustration, influenza B virus has infected rhesus monkey kidney cells, facilitating the specific attachment of red blood cells which outline only the virus-infected cells. (Collection of Marilyn J. August.)
doi:10.1128/9781555818586.ch3.f8
Standardization of Reagents for the First Diagnostic Laboratories
By the 1940s, the technical advances made it possible to establish viral diagnostic laboratories. Evidence is marked by the appearance of first editions of two books that would run through several editions: Rivers’s Viral and Rickettsial Infections of Man (37) and Francis’s Diagnostic Procedures for Virus and Rickettsial Diseases, published by The American Public Health Association (15). Because the techniques used were often the same for both types of infectious agents, early labs were set up for viral and rickettsial diseases. The development of diagnostic capacities occurred none too soon, because the world was to embark on the massive conflict of World War II. The U.S. Army established the first general diagnostic virology and rickettsiology lab. It was set up at Walter Reed Army Medical Center in Washington, DC, in January 1941 under Colonel Harry Plotz. Plotz’s investigative background was in rickettsial and viral diseases.
In 1940, commenting on serological techniques, C. E. van Rooyen and A. J. Rhodes wrote, “The principal difficulty, in fact, involves preparation of suitable antigens” (50). When the U.S. Army diagnostic lab was established, there was an urgent need to develop diagnostic reagents and standardize procedures. The Army did this as Smadel described in 1948 concerning the hemagglutination inhibition assay for the diagnosis of influenza: “. . . the United States Army adopted a single procedure, and, in addition, supplied its laboratories with standard antigens and antisera” (42). Smadel, who had worked at the Rockefeller Institute developing complement fixation assays (41) and who directed the viral and rickettsial diagnostic lab in the European Theater of Operations in World War II, returned to assume leadership of the diagnostic lab at Walter Reed in 1946 (Smadel Archives, Walter Reed Army Institute of Research, organized by Andrew Rogalski). Possessed of a keen analytic temperament and a personality at once forceful and supportive (D. Carleton Gajdusek and M. B. A. Oldstone, personal communications), Smadel was to remain a major figure in virology for several years.
The foundation was in place for serology in diagnostic virology laboratories with Sternberg’s neutralization of vaccinia virus on the skin of a calf in 1892, by Bordet and Gengou’s description of complement fixation in a hemolytic assay in 1901, and, to a lesser extent, by Hirst’s 1941 study of the inhibition of hemagglutination of influenza virus. By 1956, in a consideration of diagnostic procedures in laboratory diagnosis, Edwin H. Lennette was to write, “By far the greatest proportion of examinations conducted in a diagnostic laboratory consists of serologic tests. These comprise the complement fixation, agglutination, hemagglutination, hemagglutination-inhibition, and in vitro neutralization techniques. The complement fixation method finds the greatest application, with hemagglutination and neutralization tests next, and the agglutination tests the least commonly used” (23).
References
1 American Medical Association. 2009. The Nobel Prize for 1908: Metchnikoff-Ehrlich. JAMA 301:335. (Editorial.)
2 Bailey, I. 2011. Edward Jenner, benefactor to mankind, p. 21–25. In S. A. Plotkin (ed.), History of Vaccine Development. Springer, New York, NY.
3 Behring, E. 1890. Untersuchungen uber das Zustandekommen der Diphtherie-Immunitat bei Thieren. Dtsch. Med. Wochenschr. 16:1145–1148.
4 Behring, E., and S. Kitasato. 1890. Uber das Zustandekommen der Diphtherie-Immunitat und der Tetanus-Immunitat tei Thieren. Dtsch. Med. Wochenschr. 16:1113–1114.
5 Blake, J. B. 1953. Smallpox inoculation in colonial Boston. J. Hist. Med. Allied Sci. 8:284–300.
6 Bordet, J., and O. Gengou. 1901. Sur l’existence de substances sensibilisatrices dans la plupart des sérums antimicrobiens. Ann. Inst. Pasteur 15:289–302.