Development and characterization of monoclonal antibodies against avian reovirus sigma C protein and their application in detection of avian reovirus isolates

Abstract

Avian reovirus (ARV) is a non-enveloped virus with a segmented double-stranded RNA genome surrounded by a double icosahedral capsid shell. ARVs are associated with viral arthritis, immunosuppression, and enteric diseases in poultry. The sigma C protein was involved in induction of apoptosis and neutralization antibody. In the present study, sigma C-His protein was expressed in Sf9 insect cells and purified by immobilized metal affinity chromatography. Eight monoclonal antibodies (mAbs) against sigma C-His and three mAbs against His were screened from hybridoma cells produced by fusion of splenocytes from immunized mice with NS1 myeloma cells. Among the eight mAbs against sigma C protein, all belonged to the IgG isotype except three for IgM. It was discovered that all anti-His mAbs were mixtures of IgG and IgM isotypes. mAbs reacted with sigma C-His protein in a conformation-independent manner based on dot blot and western blotting assays. The competitive binding assay indicated that all mAbs recognized the same epitope on sigma C protein that was conserved in different isolates. Compared with the commercial anti-ARV S1133 polyclonal antibody, mAb (D15) had universal reactivity to all serotypes or genotypes of ARVs tested. This monoclonal antibody may therefore be useful for the development of an antigen-capture enzyme-linked immunosorbent assay for rapid detection of field isolates.