Characterization of antigenic determinants on porcine zona pellucida-3 β(ZP3 β) glycoprotein by monoclonal antibodies

Abstract

Out of 11 monoclonal antibodies(MAbs) developed against porcine zona pellucida-3 β(ZP3 β) glycoprotein, 6 (MA-451, -454, -455, -462, -467 and -470) reacted with ZP2 β, deglycosylated ZP3 β(DGZP3 β), and reduced and carboxyamidomethylated ZP3 β(RCMZP3 β) in ELISA and Western blot suggesting that these monoclonals recognise a linear protein epitope. Five MAbs(MA-452, -456, -459, -474 and -476) showed reduced binding with DGZP3 β and RCMZP3 β as compared to ZP3 β in ELISA suggesting that these may recognize either carbohydrate moities or conformation dependent epitopes. Moreover, these MAbs failed to recognize these antigens on Western blot. Based on competitive inhibition studies in ELISA for the ability of MAbs to inhibit the binding of biotinylated ZP3 β to solid phase antibody, 6 distinct domains were discernible. Domain V recognised by MA-467 partially overlaps with the domain corresponding to MA-454 and MA-455. All 11 MAbs reacted with zona pellucida of intact oocytes as revealed by indirect immunofluorescence but only 3 antibodies (MA-454, -455 and -467) delayed the zona lysis by trypsin. In immunoblots MA-467 recognised 18kDa fragment of RCMZP3β digested with endoproteinase lys-C and 37 and 30kDa bands of elastase digest of ZP3 β. The results will help in delineation of functionally relevant domains and design of contraceptive vaccine based on zona pellucida aiming for pre-fertilization block.