SEROLOGY
The specific detection of hepatitis A infection was first accomplished in 1973 using immune electron microscopy of fecal extracts to visualize virus-like particles 78. Antibody in convalescent-phase serum samples from persons with experimentally or naturally acquired infection aggregated the virus and permitted its visualization by electron microscopy. When serum collected before infection or early in the disease was used, few or no virus particles were identified. This research technique was successfully used to investigate the period of infectivity but could not be employed for routine diagnosis of large numbers of clinical samples. The next step was the development of complement fixation 200 and immune adherence 172 tests for detection of serum antibody to HAV antigens in 1974. This required a source of HAV antigen, supplied by liver extracts from marmosets infected with a Costa Rican strain of HAV, CR326.
Despite being cumbersome, the immune adherence test quickly provided a wealth of important data about hepatitis A infection. With the original description of the test came demonstration of simultaneous infection with both HAV and HBV; evidence that HAV antibodies were acquired early in life in areas of high prevalence; association of low socioeconomic status with seropositivity in areas of low incidence; persistence of antibody for at least 7 years; an antigenically related or identical virus infection in chimpanzees, grivets, and rhesus monkeys not experimentally infected; and detection of various quantities of antibody in lots of immune serum globulin 172.
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Both the complement fixation test and the immune adherence test were used to examine seroconversion following experimental infection with the MS-1 strain of hepatitis 137. The complement fixation test was not as specific or as sensitive as the immune adherence test. Using the immune adherence test, seroconversion was demonstrated in 20 of 20 infected persons. Antibody was detectable soon after the onset of clinical hepatitis but was present within the first week in only 45%; in 20% detectable seroconversion was delayed for at least 2 weeks after disease onset. Nevertheless, a test that could be used for diagnosis of acute hepatitis A infection was now available. Hemagglutination assays for HBV surface antigen and antibody to HBV core and surface proteins were developed in 1970. Consequently, by 1975, acute viral hepatitis could be ascribed to either HAV or HBV, permitting the recognition of viral non-A, non-B hepatitis (hepatitis C and hepatitis E).
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In 1975, solid-phase RIAs developed for the detection of HAV antigen were modified to measure antibody 203. A comparison between immune adherence, immune electron microscopy, and RIA demonstrated that each test was able to detect seroconversion following inoculation with MS-1 virus 69. Antigen partially purified from stool was equivalent to marmoset liver-derived viral antigen in these assays. RIAs were also modified to use minimal quantities of viral antigen and to assay anti-HAV IgM antibody 38.
A competitive binding assay (HAVAB; Abbott Laboratories, North Chicago, Ill.) was developed by 1978 to improve sensitivity 37. In this assay, antibody in patient serum competes with radiolabeled antibody for HAV. The assay was also adapted to measure anti-HAV IgM, which was detected in acute-phase sera but not in convalescent-phase sera 37. However, the absorption of IgG from samples to measure IgM reactivity was difficult to perform, and the resultant assay lacked reliable specificity. In 1980, an alternative technique was developed (HAVAB-M; Abbott Laboratories) in which IgM antibody was directly selected and anti-HAV activity was then measured 63. With these improvements, anti-HAV IgM antibody was detected at the time of onset of symptoms in most patients. IgM titers decreased in the weeks after onset and then became undetectable. This assay could thus be used to diagnose acute hepatitis A at the time of clinical symptoms.
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