Preparation of anti-human OX40L functional monoclonal antibody and characterization of its biological activities
Objective To prepare an anti-human OX40L functional monoclonal antibody and to characterize its biological activities. Methods A stable human OX40L transfected cell line L929/OX40L was used as an antigen to immunize BALB/c mice. By means of the cell fusion hybridoma technique and multiple cell subcloning and repeated screening with L929/OX40L as the antibody screening positive cell while L929/mock as the negative control, the hybridoma cell lines specifically secreting anti-OX40L monoclonal antibodies were selected. Then their biological activities were investigated by Western blot, fast-strip murine Ig subclass typing method, indirect immunofluorescence, competitive inhibition test and 3H-TdR incorporation assay. Results Three hybridoma cell lines, 9H10, 4C12 and 1G1, with the property of secreting specific anti-OX40L monoclonal antibody continuously and steadily were successfully obtained. Their biological activity study showed that these monoclonal antibodies could bound to human OX40L epitopes on activated B cells and maturate DCs and inhibit the proliferation of T lymphocytes costimulated by DCs. Furthermore, three anti-OX40L monoclonal antibodies also synergize with the anti-B7-1 monoclonal antibody to block the DC-inducing T cell proliferation in vitro. Conclusion Three hybridoma cell lines which secrete anti-OX40L monoclonal antibodies continuously and steadily have been established. These monoclonal antibodies could specifically recognize OX40L molecule and block OX40/OX40L costimulatory signals to suppress the proliferation of T lymphocytes costimulated by DCs.