Comparative studies of the antigens recognized by sperm-immobilizing monoclonal antibodies.
Characteristic properties of the antigens recognized by sperm-immobilizing monoclonal antibodies (SI-mAbs) from different sources were compared by ELISA competitive inhibition assay, Western blot analysis, chromatographic analysis, and enzymatic digestion studies. Among 9 SI-mAbs, human mAb H6-3C4 and three mouse mAbs--2C6, 2B6, and 2E5--also possessed strong sperm-agglutinating activity. Binding of human mAb H6-3C4 to sperm was strongly inhibited by the three mouse mAbs (2C6, 2B6, and 2E5), but not by the rat or the other four mouse mAbs. SDS-PAGE revealed that mAb H6-3C4 and three mouse mAbs recognized the same antigen molecules of 15-25 kDa present in both sperm extracts and seminal plasma. Chemical treatments with trifluoromethanesulfonic acid and sodium metaperiodate destroyed the antigen determinants recognized by the above four mAbs, as detected by both ELISA and antibody absorption tests. Western blot analysis revealed that the antigens were susceptible to treatments with papain, proteinase K, and N-glycanase, but resistant to trypsin, V8 protease, and thermolysin. These results indicate that one of the major antigens recognized by mAbs with sperm-immobilizing action may be a sperm membrane-associated glycoprotein of 15-25 kDa and the epitope may involve N-linked oligosaccharides.
Kameda
K
K
Department of Obstetrics and Gynecology, Hyogo Medical College, Nishinomiya, Japan.
Tsuji
Y
Y
Koyama
K
K
Isojima
S
S
D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000941:Antigens / Q000276:immunology*; D001667:Binding, Competitive; D015153:Blotting, Western; D002236:Carbohydrate Conformation; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000276:immunology; D006801:Humans; D007163:Immunosorbent Techniques; D008297:Male; D008565:Membrane Proteins / Q000276:immunology; D051379:Mice; D010206:Papain / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D051381:Rats; D012697:Serine Endopeptidases / Q000378:metabolism; D013081:Sperm Motility; D013094:Spermatozoa / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000941:Antigens; D006023:Glycoproteins; D008565:Membrane Proteins; D012697:Serine Endopeptidases; D019286:Endopeptidase K; D010206:Papain; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase