Characterization of antigenic determinants on porcine zona pellucida-3 beta(ZP3 beta) glycoprotein by monoclonal antibodies.
Out of 11 monoclonal antibodies(MAbs) developed against porcine zona pellucida-3 beta(ZP3 beta) glycoprotein, 6 (MA-451, -454, -455, -462, -467 and -470) reacted with ZP2 beta, deglycosylated ZP3 beta(DGZP3 beta), and reduced and carboxyamidomethylated ZP3 beta(RCMZP3 beta) 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 beta and RCMZP3 beta as compared to ZP3 beta 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 beta 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 RCMZP3 beta digested with endoproteinase lys-C and 37 and 30kDa bands of elastase digest of ZP3 beta. The results will help in delineation of functionally relevant domains and design of contraceptive vaccine based on zona pellucida aiming for pre-fertilization block.
Gupta
M
M
Gamete Antigen Laboratory, National Institute of Immunology, New Delhi, India.
Gupta
S K
SK
D000818:Animals; D000911:Antibodies, Monoclonal; D004527:Egg Proteins / Q000276:immunology*; D000939:Epitopes; D005260:Female; D008562:Membrane Glycoproteins / Q000276:immunology*; D011956:Receptors, Cell Surface; D013552:Swine; D015044:Zona Pellucida / Q000276:immunology*; D000072216:Zona Pellucida Glycoproteins
D000911:Antibodies, Monoclonal; D004527:Egg Proteins; D000939:Epitopes; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D000072216:Zona Pellucida Glycoproteins