Human monoclonal autoantibodies to human sperm will be immortalized through construction of human X human and mouse X human hybridomas. Lymphocytes, derived from the peripheral blood of vasectomized donors, will be stimulated in vitro with sperm antigens to undergo blastogenesis prior to fusion with the HGPRT deficient fusion partners UC-729-6 and MHFP-1. In addition, murine anti-human sperm monoclonal antibodies will be constructed following intrasplenic injection of human sperm membrane proteins. A panel of human and murine monoclonal antibodies currently available (HAS and MHS series) and newly derived monoclonals will be employed to characterize and isolate human sperm autoantigens and cell surface antigens. A combination of biochemical, immunological and immunocytochemical techniques, specifically Western blotting, PAGE, high resolution IEF, vectorial labelling, affinity chromatography, immunoprecipitation, competitive inhibition, Cleveland digestion and indirect immunofluorescence microscopy will be employed in antigen characterization, mapping and purification. The cross reactivity of monoclonal anti-human sperm monoclonals with human seminoma, embryonal carcinoma, choriocarcinoma, normal testis, epididymis, prostate, and seminal vesicle will be studied with immunohistochemistry. Cross reactivity with fresh tumor specimens, when available, will be studied by ELISA. Monoclonal antibodies to human sperm surface antigens will be conjugated with cytotoxins. The effect of monoclonal antibody-cytotoxin conjugates on sperm motility and viability will be assessed vitro. In addition, studies will be made of the effect of toxin conjugated and unconjugated monoclonal antibodies on fertilization of zona free hamster eggs by human sperm. It is expected that information derived from these studies will provide a better understanding of the character of the antibody response to sperm autoantigens and the nature of these autoantigens. Further, human sperm surface antigens recognized by murine monoclonals will be characterized and topographical antigenic domains determined. These studies are relevant to identification of potential immunogenic determinants for contraceptive vaccines; to the feasibility of a monoclonal antibody based spermicide, and may identify monoclonal antibodies which will serve as markers for testicular tumors or as agents inhibiting or enhancing fertilization.
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