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 pellucida glycoprotein, ZP3 (comprising both ZP3 alpha and ZP3 beta) were characterized for their specificities to ZP3 alpha (MA 7 and MA 28) or ZP3 beta (MA 1, MA 2, MA 10, MA 27 and MA 30) and their relative affinities in competitive ELISA. Among the seven MAs tested, MA 28 showed the highest affinity for ZP3 and ZP3 alpha and MA 30 for ZP3 beta. All the antibodies 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 inhibit the binding of boar sperm to the porcine oocyte. Reduction followed by carboxyamidomethylation of the antigen or its chemical deglycosylation reduces reactivity to MA 7 and MA 10, suggesting that these antibodies read conformational or discontinuous determinants. The epitope recognized by MA 28 is sequential or conformational, stabilized by disulfide bonds while MA 30 reads a sequential determinant. ZP3 alpha digested with alpha-chymotrypsin, trypsin and V8 protease, respectively, revealed fragments in the range of 27-20 kDa with MA 28 in immunoblots. Proteolytic digests of ZP3 beta show that MA 30 recognizes approximately 14 kDa fragment of an alpha-chymotrypsin digest and a approximately 6 kDa fragment of a tryptic digest. These studies will help in delineation of smaller determinants of ZP involved in sperm binding.
Bagavant
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Gamete Antigen Laboratory, National Institute of Immunology, Shaheed Jit Singh Marg, New Delhi, India.
Yurewicz
E C
EC
Sacco
A G
AG
Talwar
G P
GP
Gupta
S K
SK
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D004527:Egg Proteins; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D005260:Female; D008297:Male; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D011487:Protein Conformation; D011956:Receptors, Cell Surface; D013084:Sperm-Ovum Interactions; D013552:Swine / Q000276:immunology*; D015044:Zona Pellucida / Q000276:immunology*; D000072216:Zona Pellucida Glycoproteins
D000911:Antibodies, Monoclonal; D004527:Egg Proteins; D000939:Epitopes; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D000072216:Zona Pellucida Glycoproteins; C000606521:Zp3 protein, mouse