Monoclonal antibodies to rabbit sperm autoantigens

Rabbits were immunized with homologous spermatozoa to investigate their autoimmunogenic nature. Major sperm autoantigens that elicit antisperm antibodies were analyzed molecularly by the sodium dodecylsulfate (SDS) gel/protein blot radioimmunobinding method. IgG fractions of the autoimmune sera were purified by a protein A-Sepharose column, immobilized on Sepharose as affinity ligands, and utilized to purify major sperm autoantigens from rabbit testes. The autoimmunogenicity of the purified autoantigens was verified by reimmunizations in rabbits. BALB/c mice were immunized with the affinity-purified autoantigens to raise monoclonal antibodies by modified hybridoma techniques. Following fusions and clonings, we have established more than 100 hybrid cell lines that were shown to secrete antibodies to purified autoantigens and to rabbit sperm. A variety of techniques was employed to characterize these monoclonal antibodies. By the SDS gel/protein blot radioimmunobinding method, some were found to react with unique proteins of rabbit spermatozoa. By indirect immunofluorescent assay, about one third were shown to bind different cytologic regions of spermatozoa from rabbit, man, and/or mouse. Six were found to inhibit rabbit sperm binding to rat ova in vitro. In addition, agglutinating and immobilizing activities of these antibodies on live sperm were observed.