Monoclonal antibody G3 epitope location on Guinea pig sperm membrane protein, sperad.

Abstract

A protein isolated from guinea pig testes was purified, characterized as having a molecular weight of 34 kDa, and subjected to amino acid sequencing. A monoclonal antibody to the protein (G11) was found to cross-react with a second monoclonal antibody (G3). Two peptide sequences derived from the purified protein, labeled as 34 kDa G11, show sequence homology with sperad. Sperad is a transmembrane protein present in the peri-acrosomal plasma membrane of guinea pig spermatozoa. Both G11 and G3 monoclonal antibodies recognise antigens on the equatorial segment plasma membrane of guinea pig spermatozoa following the acrosome-reaction. Therefore, experiments were designed to check whether monoclonal antibody G3 epitope is present on the sperad. The intra-cytoplasmic domain and the extra-cytoplasmic domain of sperad was amplified from a guinea pig testes cDNA expression library by polymerase chain reaction and cloned into pGEM T-Easy vector. The recombinant pGEM T-Easy plasmids were subjected to in vitro transcription and translation by rabbit reticulocyte lysate system. The resulting translated products were immunoprecipitated with monoclonal antibody G3. The results obtained from this study confirmed that monoclonal antibody G3 epitope is located on the extra-cytoplasmic domain but not on the intra-cytoplasmic domain of sperad.