Production of monoclonal antibody specific to HLA by utilizing HLA transgenic mice.

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The matching of HLA is very important to obtain good results in organ transplantation of bone marrow and kidney. Serological HLA typing is a well established and easy method for HLA typing. Therefore it is very important to supply enough anti-HLA antisera for HLA typing of donors and recipients in organ transplantation. But anti-HLA antisera are collected by a laborious and time consuming screening of sera from women having child and the amount of antisera was obviously limitted. In order to overcome this problem, we attempted to produce good monoclonal antibodies specific to polymorphic determinants of HLA class 11 molecules by immunizing HLA transgenic mice with human cells. For this aim, we generated HLA-DRA, DQw4 and DQw6 transgenic mice successfully. HLA molecules expressed in these transgenic mice were immunologicaly functional because 1)HLA molecules expressbd on spleen cells of trasgenic mice stimulated definite mixed lymphocyte reaction in lymph node T cells of non-transgenic mouse. 2)Transgenic mice acquired a tolerance to HLA molecules encoded for by transgene. 3)Transgenic mice acquired or lost immune responsiveness to specific soluble antigens. These transgenic mice were immunized intraperitoneally with human B lymphoblastoid cell lines expressing allogeneic HLA class 11 molecules and immune spleen cells were fused with mouse myeloma cell line P3XAg8 by standard cell fusion with polyethylene glycol. Hybridomas were selected by HAT medium and the specificity of monoclonal antibodies produced in culture supernate were determined by an investigation of binding capacity of antibodies to panel of cells utilizing ELISA and flow cytometry. Four monoclonal antibodies which distinguish DR2 subgroups were established and the frequency to obtain mabs specific to polymorphic determinants of HLA molecules seemed to be higher than the previous method in which non-transgenic mice were immunized with human cells.