EPITOPE ANALYSIS OF A SPERM ACROSOMAL ANTIGEN DEFINED BY HSA-5 MONOCLONAL-ANTIBODY

Among the monoclonal antibodies recommended by the WHO Sperm Antigen W orkshop for immunocontraceptive vaccine development, HSA-5 showed a hi gh degree of sperm specificity and significantly inhibited in vitro fe rtilization in both humans and mice. Using a Western blot assay, HSA-5 was found to recognize a sperm antigen designated as HSAg-5 (human) o r MSAg-5 (mouse) which ranged in molecular weight from 18 to 100 kDa. This monoclonal antibody was used as the probe for the immunoscreening of mouse testis cDNA libraries constructed in the lambda gt-11 expres sion vector. One of the positive cDNA clones was shown to have a cDNA insert of approximately 1 kb and to encode a recombinant fusion protei n containing 77 amino acid residues in the C-terminal region of MSAg-5 , This 1 kb cDNA insert was engineered in a pGEX vector to express a r ecombinant glutathione S-transferase fusion protein (GST-5). Using an enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, b oth anti-GST-5 sera and the monoclonal antibody were shown to react wi th GST-5. The Northern blot of a mouse testis RNA preparation revealed that the isolated cDNA probe hybridized with a 4.0 kb mRNA. Several o ligopeptides were synthesized based on the predicted C-terminal hydrop hilic regions of the recombinant fusion protein. Using ELISA and a dot blot assay, peptide regions containing the immunogenic epitopes recog nized by HSA-5 monoclonal antibody were identified.