Mapping of Immunogenic Domains on Porcine Zona Pellucida 3 α and β Glycoproteins by Murine Monoclonal Antibodies

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Abstract
Immunization with zona pellucida (ZP) leads to block in fertility with variable degree of ovarian dysfunctions. To design an immunocontraceptive vaccine based on synthetic peptides of zona pellucida, it is imperative to identify and define epitopes involved in sperm binding. Epitopic domains recognized by monoclonal antibodies (MAbs) specific to either porcine ZP3 alpha or ZP3 beta glycoproteins were delineated in an enzyme-linked immunosorbent assay (ELISA) based on the ability of a MAb in solution to inhibit the binding of biotinylated ZP3 to another MAb coated on a microtitration plate. Immunoblot studies were carried out to determine the nature of reactive determinants. Porcine oocytes preincubated with MAbs were tested for sperm binding in vitro. Out of 23 MAbs generated, 10 had specificity for ZP3 alpha and 13 for ZP3 beta. By using these antibodies, eight epitopic domains on both ZP3 alpha and ZP3 beta were discernible. On ZP3 beta, epitopic domain DI partially overlaps with DII and DV with DVI, whereas on ZP3 alpha domains DI to DV were in close proximity with a partial overlap, suggesting the dominance of this region. All 10 MAbs against ZP3 alpha, and 10 out of 13 against ZP3 beta recognized deglycosylated forms of antigens. Seven antibodies having specificities for ZP3 alpha and ZP3 beta respectively recognized linear epitopes. MA-30, having specificity for ZP3 beta and MA-420 for ZP3 alpha and recognizing linear epitopes significantly inhibit the binding of boar sperm to porcine oocytes in vitro. Collectively, these studies indicate the value of utilizing MAbs for identifying and characterizing functionally significant ZP determinants. MAbs recognizing sequential epitopes will help in the elucidation of the amino acid sequence of the epitopes, which will subsequently help in design of synthetic immunocontraceptive vaccines.