The overall goal of this project is to develop xenogeneic monoclonal antibody reagents recognizing unique surface antigenic receptors on murine lymphocytes and their precursors. Female Wistar rats were immunized with single cell preparations of whole spleen from New Zealand Black Mice. Hybridoma cell lines were generated by fusing immune rat spleen cells with non secreting NS-1, myeloma cell line. A hybridoma clone VMM-2 was found to secrete an lgG2, antibody recognizing antigenic receptors on cells from bone marrow, spleen but not from thymus by flow microfluorometry. VMM-2 binds specifically to IgM+ B cells, but the antigen recognized does not relate to any lineage specificity. Further, the antibody was found to be specific for the cells of the H-2d haplotype. Genetic mapping using a panel of recombinant inbred and congenic mice, revealed that the antigenic site is located at the K end of the MHC complex. Further studies, using a series of transfected cell lines expressing chimeric H-2Kd gene products on their membranes has confirmed that the antigenic determinant is the region between the residues D152 to S184, located at the C terminal end of the a2 domain or the molecule. A comparative Western analyses of lysates from T, B and monocyte cell lines (H-2d) has indicated that in addition to binding to the monomeric from (45000 m.w), of the class I molecules from all the cell types (similar to a conventional anti-Kd), Vmm-2 specifically binds to a protein of approximately 180-200,000 m.w. Experiments are in progress to further determine whether the high molecular weight protein recognized by VMM-2 is really the tetrameric form of the class I molecule.
