Identification of two independent MHC class II antigens in a bovine lymphoblastoid cell line

Bovine major histocompatibility complex (MHC) class II antigens were investigated using monoclonal antibodies (MoAbs) with known MHC class II specificities in other species. Thirty-four MoAbs were tested for reactivity with bovine peripheral blood mononuclear (PBM) cells and the bovine lymphoblastoid cell line, BL3, by flow cytometry. Twenty-seven of 31 MoAbs tested, reacted with BL3 cells, and 22 of 25 MoAbs tested with PBM cells were reactive. MoAbs that reacted with BL3 cells were used to immunoprecipitate class II molecules from BL3 lysate labeled with [35S]methionine. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography, many MoAbs were found to immunoprecipitate a single band of approximately 31,000 relative mass (Mr). MoAbs yielding successful immunoprecipitations and with known antigen specificity in other species were then used in sequential immunoprecipitations and two dimensional (2-D) non-equilibrium pH gradient electrophoresis (NEPHGE). The HLA-DR specific MoAb H4 and the predominantly HLA-DQ specific MoAb CC11.23 were used to identify the presence of two independent antigens in BL3 cell lysate. These class II molecules consist of alpha and beta chains.