Isolation of Monoclonal Antibodies Specific for Rous Sarcoma Virus Structural, Polymerase and Transforming Proteins and Their Use for the Study of Mutant Virus-infected Cells

Abstract

SUMMARYMonoclonal antibodies were developed that are specific for Rous sarcoma virus structural, polymerase (reverse transcriptase) and transforming proteins. The monoclonal antibodies were shown to bind to purified virus proteins in an indirect ~ :5I-labelled Protein A binding assay suitable for screening even very large numbers of hybridomas. Additional tests for specificity included radioimmunoprecipitation of purified virus structural proteins P 12 and P27, of reverse transcriptase subunits ~ and/~, and of the transforming protein pp60 ..... . Pilot immunofluorescence and protein kinase assays of the expression of virus proteins in avian and mammalian cells infected by wild-type virus as well as by temperature-sensitive, transformation-defective virus mutants revealed that synthesis of virus structural and transforming proteins is hardly affected by changes in temperature, whereas the pp60"-~c-associated kinase activity is temperature-sensitive in cells infected by most, but not all the virus mutants.

Analysis of retroviral pol gene products with antisera raised against fusion proteins produced in Escherichia coli

“…Sera specific for the pol gene products, however, are difficult to prepare because the antigens are present in small quantities in the virions and are thus more difficult to purify. Several sera reactive with various RTs, however, have been prepared (19,24,31,39,56,57). Sera specific for the R-MuLV RT are available but show considerable reactivity with gag proteins (see references 24 and 58 and below) and are not helpful in detection of the other pol proteins.…”