MAPPING OF IMMUNOGENIC DOMAINS ON PORCINE ZONA-PELLUCIDA 3-ALPHA-GLYCOPROTEINS AND BETA-GLYCOPROTEINS BY MURINE MONOCLONAL-ANTIBODIES

PROBLEM: Immunization with zona pellucida (ZP) leads to block in ferti lity with variable degree of ovarian dysfunctions. To design an immuno contraceptive vaccine based on synthetic peptides of zona pellucida, i t is imperative to identify and define epitopes involved in sperm bind ing. METHOD: Epitopic domains recognized by monoclonal antibodies (MAb s) specific to either porcine ZP3alpha or ZP3beta glycoproteins were d elineated in an enzyme-linked immunosorbent assay (ELISA) based on the ability of a MAb in solution to inhibit the binding of biotinylated Z P3 to another MAb coated on a microtitration plate. Immunoblot studies were carried out to determine the nature of reactive determinants. Po rcine oocytes preincubated with MAbs were tested for sperm binding in vitro. RESULTS: Out of 23 MAbs generated, 10 had specificity for ZP3al pha and 13 for ZP3beta. By using these antibodies, eight epitopic doma ins on both ZP3alpha and ZP3beta were discernible. On ZP3beta, epitopi c domain DI partially overlaps with DII and DV with DVI, whereas on ZP 3alpha domains DI to DV were in close proximity with a partial overlap , suggesting the dominance of this region. All 10 MAbs against ZP3alph a, and 10 out of 13 against ZP3beta recognized deglycosylated forms of antigens. Seven antibodies having specificities for ZP3alpha and ZP3b eta respectively recognized linear epitopes. MA-30, having specificity for ZP3beta and MA-420 for ZP3alpha and recognizing linear epitopes s ignificantly inhibit the binding of boar sperm to porcine oocytes in v itro. CONCLUSIONS: Collectively, these studies indicate the value of u tilizing MAbs for identifying and characterizing functionally signific ant ZP determinants. MAbs recognizing sequential epitopes will help in the elucidation of the amino acid sequence of the epitopes, which wil l subsequently help in design of synthetic immunocontraceptive vaccine s.