Complement Fixation and Immunodiffusion Tests for Assay of Hepatitis-Associated “Australia” Antigen and Antibodies

Abstract

A serologic comparison was made of “Australia” (Au) antigens and antisera from different individuals using complement fixation (CF) and immunodiffusion tests. Titers of Au antigens varied widely in block CF titrations and in immunodiffusion tests against different antisera. CF titers of the antisera varied to a lesser extent in tests against different antigens. The importance of using optimal proportions of reagents for assay of Au antigen or antibody was demonstrated in both CF and immunodiffusion tests. In CF tests an excess of antigen produced an extensive “prozone” of inhibitory activity, and it was essential to examine a wide range of serum dilutions for antigen in order to avoid “false negative” results. An excess of either antigen or antibody was also shown to be inhibitory in immunodiffusion tests. The sensitivity of immunodiffusion assays for Au antigen or antibody could be enhanced by testing sera adjacent to wells containing positive reagents. Thus, of 204 sera from hepatitis patients which were positive for Au antigen by CF, 197 (approximately 98%) were positive by immunodiffusion; of 280 sera negative by CF, only 1 gave a positive immunodiffusion reaction. In contrast to findings reported by others, only a low proportion of sera from hepatitis patients in this study showed anti-complementary activity, and the activity was not reversed by an excess of Au antigen, although in some instances it was reduced by treatment with either antigen-containing or negative human sera. Sera from a few hepatitis patients in whom Au antigen had disappeared showed low titers of CF activity against their homologous antigens, which suggested that the convalescent-phase sera might contain very low levels of free Au antibody.