Use of Specific Monoclonal and Polyclonal Antibodies to Define Distinct Antigens of the Porcine Zonae Pellucidae1

Abstract

Specific monoclonal and polyclonal antibodies to solubilized porcine and rabbit zonae pellucidae (ZP) and to purified ZP glycoprotein components have been used to define distinct ZP antigens. These studies demonstrate that the individual ZP glycoproteins contain both unique and shared determinants. One monoclonal antibody (R5) has been used to demonstrate that the major porcine ZP glycoprotein, which has multiple charge species ranging in molecular weight from 42,000 to 120,000, is composed of two distinct polypeptide antigens unique to this glycoprotein class. These distinct antigens can be differentiated by immunoblotting after high-resolution two-dimensional polyacrylamide gel electrophoretic separation of trypsin-treated or deglycosylated glycoproteins. The two polypeptides also differ in their staining properties with the silver-based color stain and in their susceptibility to proteolysis. A second monoclonal antibody (PSI) has been used to define a determinant shared by all three major porcine ZP glycoprotein classes. This determinant appears to involve either a carbohydrate moiety or some other molecular feature related to post-translational modification, since the antibody recognizes only the acidic species of each glycoprotein class, and does not recognize the deglycosylated forms of the proteins. This work demonstrates that there are both unique and shared antigenic determinants present in the individual components of the ZP, but that the immunodominant determinants appear to be unique to each glycoprotein.