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' ...

A previous report ( I ) from these laboratories described the recovery in S. mystax marmosets of CR326 and related strains of human hepatitis A virus from cases of hepatitis A that occurred in Costa Rica. The etiologic relationship of the CR326 virus to hepatitis in man was established based on the demonstration of physical-chemical properties considered characteristic of human hepatitis A virus (viz., small size and heat, ether and acid stability) (2), and on the development in patients with hepatitis A of neutralizing (2), and complement fixing (3) antibodies against the virus. The present report extends these findings to include electron microscopic demonstration of the virus intracellularly and in purified form, and a definition of the reaction of purified virus to heat (60" and loo"), ultraviolet irradiation, and formalin. These data, together with current evidence for RNA composition, fix hepatitis A as a likely enterovirus.Materials and Methods. Virus and host system. The origin, history, and marmoset propagation of CR326 strain human hepatitis A virus were described previously ( I , 2). These same reports described the tests for serum enzymes, and for infectious virus, and the conduct of the serum neutralization test.Isopycnic banding of hepatitis A virus and viral density determination. Three ml of CR326-infected serum from marmosets containing about 104-5 ID5o/ml was applied to 36 ml of cesium chloride gradient formed by conventional procedure and covering the density range as given in Fig. 1. Thirteen 3 ml fractions were collected after centrifugation at 23,000 rpm for 18 hr in a Beckman SW-27 rotor. The density of each fraction was measured by refractive index and the densities of certain of the fractions were also determined by direct weighing. Each gradient fraction was diluted 1:3000, a dilution at which the fraction tested would contain about 10 ID50/ml if the total amount of virus were present in that fraction. For testing gradient samples, groups of six marmosets were given 1.0 ml each, intravenously, and these were followed for serum enzyme elevations that indicated infection with the virus.Effect of heat, ultraviolet irradiation and formalin on purijed CR326 virus. Fractions 3 and 4 from a cesium chloride gradient (see Fig. 1) were pooled and successively dialyzed against phosphate buffered saline solution (PBS) and distilled water. The infectivity titer, in marmoset titration, was about 104.5 IDS0/ml and the protein content (4) was less than 6 pg/ml. This material was used to carry out the stability tests. Infectivity determinations were carried out in marmosets given 1 ml of test material intravenously. Appropriate positive and negative control animals were included. Heat, 60". The purified virus was diluted 1: 100 in distilled water and heated 1 hr in a sealed glass ampoule that was fully immersed in water at 60". A total of 24 S. mystax marmosets were given the heated preparation. A similar experiment had been carried out employing unpurified virus in marmoset serum diluted 1: 100 (2). Hea...

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Studies of human hepatitis A (infectious hepatitis) have been hampered by the lack of a simple assay for antibody against hepatitis A virus. Such a test has been developed as the present report will show.Historically, Deinhardt et al.(1) first presented evidence for propagation of hepatitis A virus in Saguinus nigricollis and Saguinus oedipus marmosets. Provost et al.(2) in our laboratories developed an assay for neutralizing antibody against hepatitis A virus in S. mystax marmosets and provided definitive proof of etiologic relationship between human hepatitis A isolate CR326 and human hepatitis A. The serum neutralization findings were confirmed by Holmes et al. (3). Using another approach, Feinstone et al. (4) later demonstrated a 27 nm diameter particle in the stool filtrate of a case of hepatitis A and the development of an antibody against it that was detected by immune electron microscopy.The present report describes the development of a specific complement-fixation (CF) test for human hepatitis A antibody employing, as antigen, liver extract of marmosets infected with the CR326 strain (2, 5 ) of human hepatitis A virus. Data relating to the development of CF antibody against the virus in hepatitis A cases are presented and discussed.Materials and Methods. Virus. The CR326 strain (5) of human hepatitis A virus isolated in marmosets from a case of hepatitis A in Costa Rica was used to prepare CF antigen. Preparation of CF antigen. The hepatitis A CF antigen preparations were made from livers of S. mystax marmosets infected intravenously with CR326 virus (2, 5). The livers were collected from the animals at a time when the serum enzyme values (SGOT and SICD) were ele-vated. The control CF antigens were made from livers of uninfected marmosets. All livers were perfused thoroughly with phosphate buffered saline (PBS), pH 7.2, minced with a scissors, and ground in a mortar with sterile alundum to give a 10% suspension by weight in PBS. The antigens consisted of the supernatant fluids obtained after low speed clarification. Both viral and control antigens were heated at 56" for 2 hr prior to use in the CF tests. Hepatitis A complement-fixation (CF) tests. Grid titrations were performed using serial dilutions of human hepatitis A convalescent serum and serial dilutions of antigen. The CF antigen titers were low and it was usually necessary to employ the infected liver preparation at a 1 :2 dilution (2 units of antigen) in the tests for hepatitis A antibody. The normal control CF antigen was used at the same dilution. Two and one half full units of guinea pig complement were employed in the tests. Incubation with complement was overnight at 4". The hemolytic system consisted of a 1 % suspension of sensitized sheep red blood cells. Antibody titers were expressed as the highest initial dilution of serum giving 50% or greater fixation of complement. Hepatitis B CF tests. Tests for the surface antigen (HB,Ag, Australia antigen) were performed according to Purcell et al. (6).Patients' sera. Natural hepatitis A and B ca...

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