Identification of Marek's disease virus‐specific antigens in Marek's disease lymphoblastoid cell lines using monoclonal antibody against virus‐specific phosphorylated polypeptides

Abstract

For identification of the antigens specific to Marek's disease virus (MDV) in virus-non-producer lymphoblastoid cell lines established from a tumor of Marek's disease (MD), hybridomas producing monoclonal antibodies (MAbs) against the antigens were isolated. Immunogens for preparation of the hybridomas were purified from the lysate of an MD-lymphoblastoid cell line, MSB1, by affinity chromatography coupled with chicken anti-MDV serum immunoglobulin G. Three of the MAbs obtained, MB1, MB2 and MB3, were specific to MDV by immunofluorescence test. An immunofluorescence test using MB2 antibody showed that immunofluorescence-positive cells in non-producer MD-lymphoblastoid line cells became detectable when the culture temperature was shifted from 41 degrees C to 33 degrees C or when treatment with 5-iodo-2-deoxyuridine (IUdR) was performed, indicating that the antigen reactive with MB2 antibody is an MDV-specific early antigen. This temperature shift or IUdR-treatment did not induce other MDV-specific antigens, such as late gene products of MDV, detected with MAbs. MB1 and MB2 antibodies immunoprecipitated 4 MDV-specific phosphorylated polypeptides with molecular weights (MWs) of 43,000 (43kd), 39kd, 36kd and 24kd from chick embryo fibroblasts productively infected with virulent MDV. In the place of 43kd, phosphorylated 44kd polypeptide was precipitated from avirulent MDV-infected fibroblasts. However, MB3 antibody did not precipitate any MDV-specific polypeptides from infected fibroblasts. These results suggest that the phosphorylated polypeptides are MDV-specific polypeptides predominantly expressed in non-producer MD-lymphoblastoid cell lines.

Analysis of Marek's Disease Virus Serotype 1-specific Phosphorylated Polypeptides in Virus-infected Cells and Marek's Disease Lymphoblastoid Cells

“…1 a). In the present study, pp39 and pp36 were not separated so well as in the previous report (Ikuta et al, 1985a) and were, therefore, named pp39/36. In addition to pp39/36 and pp24, a polypeptide of 41K was found in immunoprecipitates from cells infected with the other MDVI strains, JM and C2(A).…”

“…Usually, the virus-specific antigen can not be detected in MD lymphoma cells and virus non-producer MD lymphoblastoid cell lines by an immunofluorescence (IF) test with conventional anti-MDV sera (Payne, 1982). Recently, we demonstrated the presence of an MDVl-specific early antigen in the cytoplasm of MD lymphoblastoid cell lines by an IF test with monoclonal antibodies against MDVl-specific phosphorylated polypeptides, when the culture temperature was shifted from 41 °C to 33 °C or when the cells were treated with 5-iodo-2-deoxyuridine (IUdR; Ikuta et al, 1985a). Under these conditions, none of the non-producer MD cell lines reacted with monoclonal antibodies against other kinds of MDV-specific proteins, such as glycoproteins gA (Ikuta et al, 1983 b) or gB (Ikuta et al, 1984a), major virus-specific polypeptides (Ikuta et al, 1984b), or DNA-binding proteins , suggesting that the phosphorylated polypeptides are predominantly expressed in non-producer MD cell lines.…”

“…wt. 43000 (43K), 39K, 36K and 24K were identified in chicken embryo fibroblasts (CEF) productively infected with MDV1 by immunoprecipitation with the monoclonal antibodies used for detection of virus-specific cytoplasmic antigen in MD cell lines, followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE; Ikuta et al, 1985a). It is not known whether these four polypeptides share the same epitope or amino acid sequences.…”

“…The specificities of M21 mouse monoclonal antibodies against the MDV1-specific phosphorylated proteins have been described previously (Ikuta et al, 1985a).…”

Analysis of transcriptional and translational activities of Marek's disease (MD) virus genes in MD central nervous system lesions in chickens

“…For detecting the MDV antigens, we used M21 monoclona l antibody (MAb) combinabl e mainly with MDV early phosphorylate d antigen, pp38 (Ikuta et al, 1985;Cho et al, 1997) , M51 MAb against MDV late antigen, gB (Ikuta et al, 1984) , and M26 MAb against MDV late antigen, gA (Ikuta et al, 1983), respectively. The MAb against major histocompatibility (MHC) class II chicken Ia antigen was kindly provided by C. H. Chen (University of Alabama, Birmingham, USA) and polyclona l antibody against chicken IgG was purchased from Vector Laboratories (Burlingame, CA, USA).…”