Characterization of the fertilization antigen 1 for the development of a contraceptive vaccine.
A fertilization antigen, FA-1, was purified from either deoxycholate- or lithium diiodosalicylate-solubilized murine testes by immunoaffinity chromatography using a monoclonal antibody, MA-24, which inhibited fertilization in vitro. The FA-1 was recovered at high (11.4) or low (2.8) pH using stepwise elution procedures of the deoxycholate or lithium diiodosalicylate extracts, respectively. Both of these fractions showed a single band of 47 kDa when analyzed by NaDodSO4/PAGE and silver staining. Following removal of the detergent and extensive dialysis at pH 5.8 or treatment with 0.15 M NaCl, even in the presence of detergent, a monomer of 23 kDa was detected. Two-dimensional PAGE of FA-1 showed, four or five polypeptides in the 47-kDa or 23-kDa range. The dialyzed FA-1 contained a major 23-kDa and a minor 48-kDa band when separated on both sucrose and cesium chloride gradients. High performance size-exclusion chromatography showed a major peak at 23 kDa and a minor peak at 50 kDa. Further analysis of the 23-kDa peak by reverse-phase chromatography resolved the antigen into three peaks, which gave similar two-dimensional gel patterns as the native FA-1. Lectin affinity chromatography on a lens culinaris column demonstrated that a part of the antigen was bound to the lectin while the rest was not. The FA-1 revealed a positive reaction with periodic-Schiff reagent and contained glucose and mannose, which together constituted 18.8% of the total antigen mass. Amino acid analysis showed a high percentage of aspartic acid, glutamic acid, serine, and glycine. As a single injection of MA-24 significantly reduced fertilization rates in vivo, the purified FA-1 is an attractive candidate for the development of contraceptive vaccine.
Naz
R K
RK
Phillips
T M
TM
Rosenblum
B B
BB
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000941:Antigens / Q000302:isolation & purification*; D002241:Carbohydrates / Q000032:analysis; D002417:Cattle; D005260:Female; D005306:Fertilization; D008253:Macaca mulatta; D008297:Male; D051379:Mice; D008970:Molecular Weight; D011817:Rabbits; D013045:Species Specificity; D013737:Testis / Q000276:immunology*
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D000941:Antigens; D002241:Carbohydrates