Isolation and characterization of monoclonal antibodies directed against two subunits of rabbit poxvirus-associated, DNA-directed RNA polymerase

Abstract

A library of monoclonal antibodies directed against individual proteins of the rabbit poxvirus (RPV) virion within a complex immunogenic mixture has been generated through the use of in vivo and in vitro immunization regimens. The relative efficacies of the two procedures were compared. Based on immunoblot analysis, the in vitro immunization regimen led both to a wider variety of monoclonal antibodies to different proteins and to a larger number of antibodies directed against proteins of higher molecular weights. Each method, however, has advantages, and the two procedures appear to be complementary. A simple method to recognize antibodies directed against the virion DNA-directed RNA polymerase was developed. Monoclonal antibodies directed against two subunits (137 and 34 kilodaltons [kDa]) of the RNA polymerase were identified and used to study the biogenesis of the enzyme and to map the two corresponding genes within the viral genome by using an RPV DNA library cloned into the lambda gtll expression vector. Both proteins are synthesized late in the infectious cycle and are restricted totally to the cytoplasm. Preliminary mapping data place the genes encoding the 137-kDa protein within the HindIII H fragment, whereas the gene for the 34-kDa protein is located within the left most region of the HindIII A fragment.