Production and immunolocalization of monoclonal antibodies against human sperm

In order to help identify and characterize antigens involved in sperm functions and immune infertility, monoclonal antibodies (mAbs) were raised against human sperm surface antigens. The IgG fraction of serum from a donor with a spontaneous high titer of IgG-positive antisperm reactivity (as determined by immunobead binding) was purified by ammonium sulfate precipitation. This IgG preparation was coupled to Sepharose 4B, and used for immunoaffmity purification of antigens from a detergent-solubilized extract of pooled normal human sperm. The affinity-purified antigens were used to immunize female mice, and resultant spleen cells were fused with SP 2/0 mouse myeloma cells to generate hybridomas. A single-step semi-solid methylcellulose method was used to isolate hybridomas for selection of positive clones, which were determined by ELISA. Thirty-two positive hybridoma lines were selected for immunolocalization and crossreactivity studies using avidin-biotin complex (ABC) assay. Distinctive staining patterns and distribution of sperm surface antigens were observed for ten monoclonal antibodies. Among them, the cross-reactivity with human lymphocytes was not observed for four monoclonal antibodies.