Specificity of the Unlabeled Antibody Hemocyanin Bridge Method to Label Virion And Cell Surface Antigens Using Hyperimmune Serum and Monoclonal Antibodies

Abstract

The unlabeled antibody hemocyanin technique (UAHT) was evaluated for specificity of detection of retrovirus antigens gp70 and p15(E). UAHT used infected cell monolayers incubated stepwise with primary hyperimmune or monoclonal antibodies, secondary bridging antisera, tertiary anti-hemocyanin sera, and hemocyanin (hey) and was amplified by additional anti-hcy sera and hcy incubations. Further, the detection of p15(E) and gp70 by UAHT was compared with the antibody binding (AB) assay. UAHT using hyperimmune sera detected gp70 viral antigens at dilutions >10³ and was increased five-fold with amplification steps. Surprisingly, the detection of these same antigens decreased to <10³ using monoclonal antibodies in place of the hyperimmune serum. The AB assay however detected viral gp70 and p15(E) using monoclonal reagents: 16-11C1, 19-F8 and 19-IIIA2. The only positive UAHT detection of gp70 was with the monoclonal antibody 16-11C1 and 19-F8 for p15(E). The presentation of the antigenic sites may therefore be different in the AB and UAHT assays. Finally, detection of gp70 and p15(E) was determined by the UAHT assay during virus maturation. Hcy labeling was observed in the stages of virus morphogenesis in retrovirus-infected cell monolayers but not in the NSI/1 myeloma parent cell pellets although these cells contained intracisternal type A and extracellular type C viruses.