Production and characterization of monoclonal antibodies to cross-reactive antigens of human and porcine zonae pellucidae

Three monoclonal antibodies (Mabs), 3A4-2G1, 1D5-2B7 and 1F2-1B8 were produced against heat-solubilized porcine zona pellucida (ZP). Each Mab stained intact ZP but no other pig tissues using immunofluorescence staining. All three Mabs stained selectively zonae pellucidae (ZPe) from pigs and humans but not from hamsters, rats or mice, and showed no inhibitory effect on sperm binding to human oocytes. When goat antiserum to mouse gamma-globulin was added to human oocytes pre-treated with 3A4-2G1 or 1D5-2B7, sperm binding to oocytes was completely blocked with formation of immune precipitates around them. SDS-PAGE analysis of the immune precipitates of 125I-labeled porcine zona proteins and Mab showed that the antigen binding 3A4-2G1 was mainly composed of components with approximate molecular weights of 92,000, 65,000 and 23,000 and the antigen binding 1D5-2B7 contained two components with approximate molecular weights of 57,000 and 49,000, respectively. The epitope of ZP antigen, corresponding to 3A4-2G1, was found to be present in the molecule of 92,000 daltons as demonstrated by enzyme immunostaining of the proteins after blotting to nitrocellulose membrane from SDS-PAGE gels.