Identification of Epitopes of Monoclonal Antibodies to Porcine Zona Pellucida 3β Glycoprotein, a Homologue of the Mouse/Human Sperm Receptor

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Abstract
PROBLEM: Immunization with zona pellucida (ZP) glycoproteins leads to a block in fertility with a variable degree of ovarian dysfunction. To avoid autoimmmune oophoritis, synthetic peptides corresponding to B cell epitope(s) and devoid of oophoritogenic T cell epitopes as immunogens have been proposed. The main objective of the present study is to define the epitopes recognized by monoclonal antibodies (mAbs) generated against porcine ZP3β, a homologue of the designated primary sperm receptor in mice and humans.METHODS: A multipin synthetic peptides approach has been used to map the epitopes recognized by mAbs. Dodecapeptides with an overlap of 6 amino acids corresponding to a precursor pZP3β‐deduced amino acid sequence (excluding the signal sequence) were synthesized on polypropylene pins and were tested for their reactivity with mAbs by enzyme‐linked immunoadsorbent assay (ELISA). The ability of synthetic peptides corresponding to the identified epitopes to inhibit the binding of mAbs to pZP3β in a competitive inhibition ELISA was investigated to confirm the above findings.RESULTS: Reactivity of the mAbs with the pin‐bound peptides in ELISA‐identified epitopes for mAb‐451 to EEKLVF (166–171) and mAb‐462/470 to FKAPRP (250–255) amino acid residues. mAb‐30 recognized QPVWQDEGQRLR (23–34) and VICRCC (316–321) amino acid residues. Competitive inhibition with synthetic peptides encompassing the motifs corresponding to 23–34 and 316–321 for binding of mAb‐30 to pZP3β revealed the epitopic domain to be 23–34 amino acids. Synthesis of overlapping octapeptides further identified WQDE as the minimum motif for binding of mAb‐30, and the replacement of one amino acid at a time with glycine revealed tryptophan as the critical residue.CONCLUSIONS: Collectively, these results describe peptide epitopes that will help in the design of an immunocontraceptive vaccine based on synthetic peptides corresponding to pZP3β or its homologues in other species.

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