Immunoassay of baculovirus structural polypeptides

Abstract

The study has demonstrated the feasibility of using Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) and Heliothis zea single nuclear polyhedrosis virus (HzSNPV) structural polypeptides purified under denaturing conditions as antigens. Structural polypeptides purified on polyacrylamide slab gels in the presence of sodium dodecyl sulfate (SDS-PAGE) were shown to retain immunoreactivity when examined in two sensitive immunoassays, radioimmunoassay (RIA) and [^125]I-labeled protein binding to antibodies attached to polypeptides transferred from SDS-PAGE slab gels to diazobenzyloxymethyl (DBM)-paper. Competition RIA using four AcMNPV viral polypeptides and their homologous antisera with SDS-disrupted baculovirus enveloped nucleocapsids demonstrated a specificity sufficient to distinguish between all competing antigens except for the closely related viruses, AcMNPV and Rachiplusia ou (RoMNPV). Competition RIA using HzSNPV vp28 and its homologous antiserum with SDS-disrupted baculovirus enveloped nucleocapsids as competitors was also specific enough to discriminate between all the antigens except HzSNPV and Heliothis armegira SNPV. These same homologous systems, when used in competition RIA with AcMNPV or HzSNPV viral polypeptides as competitors, were characterized by extensive cross-reactivity. Two methods for the transfer of polypeptides from SDS-PAGE slab gels to DBM-paper and nitrocellulose were compared. Diffusion transfer was found to be slightly more efficient than electrophoretic transfer and nitrocellulose membranes were found to be slightly more efficient in polypeptide binding than was DBM-paper. However, since DBM-paper was shown to be reusable following removal of [^125]I-labeled protein A and bound antibodies, it was the method of choice, and antigen detection to the same level (0.1 to 0.2 ng) as nitrocellulose membranes was shown. Polypeptides transferred from Coomassie blue stained and dried SDS-PAGE slab gels were shown to retain immunoreactivity when reacted with AcMNPV vp37 antiserum. When reacted with AcMNPV vp18.5 antiserum, however, significant immunoreactivity was lost...