Characterization of Porcine Zona Pellucida Antigens by Immunoaffinity Chromatography and by High-Pressure Liquid Chromatography

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Abstract
In the present work, 500 and 50,000 porcine zonae pellucidae were solubilized using Lithium‐3,5‐diiodosalicylate. The zona antigens were purified by immunoaffinity chromatography (IAC) on immobilized antizona immunoglobulin G (IgG). The antizona‐IgG was raised by immunization of female rabbits with 500 heat‐solubilized porcine zonae. Four antigens could be detected following IAC: ZP I/1 (Mr = 42,000), ZP II/1 (Mr = 67,000), ZP II/2 (Mr = 32,000), ZP III/1 (Mr = 17,000). In a parallel experiment, 50,000 zonae were solubilized in a similar manner and the mixture was analyzed by high‐pressure liquid chromatography (HPLC) using a protein column. Altogether, 9 protein peaks that contained the antigens ZP I/1, ZP II/1, ZP II/2, and ZP III/1 could be detected following HPLC. The carbohydrate composition is characteristic for O‐glycosidic‐glycoproteins. ZP II/1 and ZP II/2 are probably in close association within the zona. Based on the reaction of the antigens with antibodies induced by intact and heat‐solubilized zonae, it is postulated that only ZP I/1 and ZP II/l are expressed on the surface in intact zonae.