Production and characterization of monoclonal antibodies to the sperm acrosome stabilizing factor (ASF): utilization for purification and molecular analysis of ASF.
Utilizing hybridoma technology and highly purified acrosome stabilizing factor (ASF), six monoclonal antibodies (mAbs) specific for ASF were produced and characterized. Specificity and binding properties of each clone were examined by immunoperoxidase labeling of electrophoretic blots of rabbit serum, seminal plasma, cauda epididymal fluid and vasectomized seminal plasma separated on native and sodium dodecyl sulfate (SDS)-polyacrylamide gels. All mAbs recognize ASF in seminal plasma and cauda epididymal fluid but do not bind components in serum or vasectomized seminal plasma. Purification of ASF by affinity chromatography utilizing the mAbs, has shortened the 6-day isolation procedure for ASF used previously to less than 2 h and has increased the yield from 2 micrograms to 300 micrograms of ASF obtained per ml of seminal plasma. Three mAbs were used in conjunction with Cleveland digest with Staphylococcus aureus V8 protease, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoperoxidase labeling of Western Blots, to identify peptides containing specific determinants recognized by each mAb. At least five separate determinants were recognized by the six mAbs. The sensitivity of the Western blotting technique in conjunction with the specificity of the mAbs was exploited to detect polymeric forms of ASF in seminal plasma and cauda epididymal fluid. ASF is shown to be a 360-kd dimer consisting of two identical 180-kd monomers. Tools are now available to develop sensitive qualitative and quantitative assays for ASF, thus providing rapid, extremely sensitive methods for evaluating experiments designed to probe the molecular mechanism of capacitation.
D000177:Acrosome / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D004822:Epididymis / Q000276:immunology*; D006023:Glycoproteins / Q000276:immunology* / Q000302:isolation & purification; D008297:Male; D011817:Rabbits; D013075:Sperm Capacitation; D013094:Spermatozoa / Q000276:immunology*
C041608:ASF protein, Oryctolagus cuniculus; D000911:Antibodies, Monoclonal; D006023:Glycoproteins