Specific Immune Adherence Assay for Human Hepatitis A Antibody. Application to Diagnostic and Epidemiologic Investigations

Abstract

Studies of human hepatitis A (infectious hepatitis) have been severely hampered by the lack of a simple assay for specific antibody against hepatitis A virus.In 1973, we reported (1) the development of a specific serum neutralization test for human hepatitis A antibody using the CR326 virus isolated from a case of human hepatitis. The test, though highly specific, is carried out in marmosets and is too laborious and costly for routine purpose. More recently, we developed (2) a specific complement-fixation (CF) test for hepatitis A antibody employing, as antigen, liver extract of a marmoset infected with the CR326 virus. The test provided a workable in vitro assay for serodiagnostic and seroepidemiologic investigations of hepatitis A in man.The present report describes the development of yet another test for hepatitis A antibody. This is an immune adherence (IA) assay employing, as for the CF tests, extract of liver of marmosets infected with CR326 virus. Data relating to the development of IA antibody against the virus in the course of hepatitis A infection and to the seroepidemiology of the disease are given. Additionally, data relative to the content of hepatitis A antibody in commercial human immune globulin and in various subhuman primate species are presented.Materials and Methods. Virus. The CR326 strain (3) of human hepatitis A virus isolated in marmosets from a case of hepatitis A in Costa Rica was used. Preparation of hepatitis A immune adherence ( I A ) test antigen. The antigen was prepared from the liver of a marmoset (Saguinus mystax) infected intravenously with CR326 virus.The liver was taken 27 days after virus inoculation, at a time when the serum enzymes (SGOT and SICD) were elevated. The liver was perfused with phosphate buffered saline (PBS) at pH 7.2, minced with scissors, and ground with sterile alundum in a mortar to give a final 10% suspension by weight in PBS. The extract was clarified by low speed centrifugation, applied to cesium chloride gradients (4), and fractions in the density range of 1.32-1.36 were collected. The fractions were dialyzed against PBS and were used as antigen in the IA assays. Control antigen was similarly prepared from liver of an uninfected marmoset. Immune adherence (IA) test. The procedure was similar to that of Mayumi et al. (5) for assay of hepatitis B antigen and antibody. The sera were not heated. The commercial human immune globulins were diluted 1:lO and absorbed with an equal volume of 25% kaolin before they were assayed for antibody content. The antigens were standardized in grid titrations in which serial dilutions of antigen were assayed with serial dilutions of human hepatitis A convalescent serum. Four units of viral antigen were employed in the tests and the control antigen was used at identical dilution. Complement-jixation (CF) and serum neutralization tests. The techniques for the CF tests for hepatitis A antibody for hepatitis B surface antigen (HB,Ag or Australia antigen), and for neutralizing antibody were described previously (1, 2).Patients' ...

Epidemiological and serological study of hepatitis A virus outbreaks in France: a comparison between immunoadherence and radioimmunoassay

“…Anti-HAV was detected by RIA as soon as the first clinical signs of illness appeared. On the contrary, anti-HAV was detectable later (one week) by IAHA (Krugman, Friedman & Lattimer, 1975;Miller et al 1975;Moritsugu et al 1976). Thus, RIA could be used to make a very early diagnosis of the disease providing it is improved by separation of the IgM fraction .…”

“…This historical technique was time consuming and limited 0022-1724/81/0012-1980 $01.00 © 1981 Cambridge University Press by interpretation errors (Almeida, Gay & Wreghitt, 1974;Gravelle et al 1975;Dienstag et al 1976). Since then, other serological methods have been developed such as complement fixation test ) and immunoadherence hemagglutination assay (IAHA) (Miller et al 1975). The latter method was able to detect anti-HAV in the early convalescence period of HAV infection.…”

Application of immune adherence hemagglutination test in the screening of blood donors carrying a high titer antibody to varicella-zoster virus.

“…Recently, the IAHA test has been commonly used for examining the immune status to viral infection (Ito and Tagaya 1966;Krugman et al 1975 ;Miller et al 1975;Dienstag et al 1976;Gershon et al 1976;Kalter et al 1977;Wong et al 1978;Gillani and Spence 1978;Yamada et al 1979), since the IAHA test is Fig . 3.…”

Hepatitis A virus infections in Voyvodina

“…By means of sensitive serological tests (immune-adherence and radioimmune assay) it was established that actively acquired anti-HAV antibodies persist in the host for years or for life (Miller et al 1975;Purcell et al 1976).…”