Obtaining and characterization of anti-testis monoclonal antibodies: Invaluable tools toward the identification of testis antigens involved in fertilization.

Abstract

Generation and utilization of the specific monoclonal antibodies against testis antigens is reported to identify the antigens that are important in reproductive field. Current study aimed to introduce a hybridoma that producing a specific anti-testis monoclonal antibody to identify the testis antigens that can be important in the reproduction field. To make hybridoma against testis antigens, mice were immunized with testis cell lysate. After cell fusion, resulted hybridomas were screened by indirect ELISA, then cloned by limiting dilution and finally the produced monoclonal antibody were characterized by some of the molecular laboratory techniques such as immunohistochemistry, immunocytochemistry and western blot. By using hybridoma technique, cell fusion was performed and ten (8A11, 8D6, 8D7, 9F6, 9G11, 10C3, 10B3, 10B2, 10C2 and 10H7) antibodies specific to the testis antigens were produced finally. Among the produced antibodies, 10C3 was found to cross-react with testis, but not detected in other tissues. mAb 10C3 recognized the sperm and testis antigens, specifically the intertestitial tissue of testis, spermatogonia, primary and secondary spermatocyte antigens, so they were most likely the target of generated mAb. Also our mAb could totally detect the mouse sperm antigens and the specific antigens of head and tail of human sperm. In western blotting analysis, mAb 10C3 could recognize the specific protein bands of sperm and testis extracts. Also in this study the testis specific genes that were target of generated mAb, were selected according to the mouse EST profile available at UniGene part of NCBI. So this stable anti-testis mAb has a potential for laboratory researches and also for diagnostic procedures in fertilization. Thus it could be exploited as a suitable tool for target-specific diagnosis and research in several diseases.