DELINEATION OF EPITOPES ON PORCINE ZONA-PELLUCIDA RELEVANT FOR BINDING OF SPERM TO OOCYTE USING MONOCLONAL-ANTIBODIES

Seven monoclonal antibodies (MAs) generated against porcine zona pellu cida glycoprotein, ZP3 (comprising both ZP3alpha and ZP3beta) were cha racterized for their specificities to ZP3alpha (MA 7 and MA 28) or ZP3 beta (MA 1, MA 2, MA 10, MA 27 and MA 30) and their relative affinitie s in competitive ELISA. Among the seven MAs tested, MA 28 showed the h ighest affinity for ZP3 and ZP3alpha and MA 30 for ZP3beta. All the an tibodies bound to the zona pellucida in an indirect immunofluorescence assay, but only four (MA 7, MA 28, MA 10 and MA 30) were able to inhi bit the binding of boar sperm to the porcine oocyte. Reduction followe d by carboxyamidomethylation of the antigen or its chemical deglycosyl ation reduces reactivity to MA 7 and MA 10, suggesting that these anti bodies read conformational or discontinuous determinants. The epitope recognized by MA 28 is sequential or conformational, stabilized by dis ulfide bonds while MA 30 reads a sequential determinant. ZP3alpha dige sted with alpha-chymotrypsin, trypsin and V8 protease, respectively, r evealed fragments in the range of 27-20 kDa with MA 28 in immunoblots. Proteolytic digests of ZP3beta show that MA 30 recognizes approximate ly 14 kDa fragment of an alpha-chymotrypsin digest and a approximately 6 kDa fragment of a tryptic digest. These studies will help in deline ation of smaller determinants of ZP involved in sperm binding.