Isolation and biological characterization of boar sperm capacitation-related antigen.
OBJECTIVE
Monoclonal anti-capacitated sperm antibody has been used as a probe to identify, isolate, and characterize specific, fertilization-related antigen with some characteristic features that point to its possible significance in immunocontraception.


METHODS
Fast protein liquid chromatography (FPLC), enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and isoelectrofocusing were used for isolation, immunochemical and physicochemical characterization of a monoclonal antibody (mAb) 1F10 cognate antigen. Sperm-zona pellucida binding and hemizona assay were used for testing the biological roles of mAb 1F10 and Ag 1F10 in boar and human fertilization processes.


RESULTS
The Ag 1F10 was found to be eluted in the eighth protein peak of FPLC-fractionated Nonidet P40 (NP40) extracts of capacitated boar spermatozoa. The SDS-PAGE and immunoelectrofocusing experiments showed that Ag 1F10 is a protein composed of a single peptide chain with a relative molecular mass of 68/70 kDa and an isoelectric point of 3.5. It was demonstrated that the zona binding activity of spermatozoa preincubated in the presence of mAb 1F10 was significantly inhibited both in porcine and human in vitro fertilization (IVF) systems. A dose-dependent manner of inhibition of the sperm/ligand activities of porcine and human zona pellucida was observed when the effect of purified Ag 1F10 was investigated by its preincubation with zona pellucida.


CONCLUSIONS
It is assumed that the protein bearing the epitope recognized by mAb 1F10 may be accepted as one of the molecules with receptor function in sperm-zona pellucida interaction during fertilization.
Mollova
M
M
Department of Immunobiology of Reproduction, Institute of Biology and Immunology of Reproduction, Bulgarian Academy of Sciences, Sofia.
Djarkova
T
T
Ivanova
M
M
Stamenova
M
M
Kyurkchiev
S
S
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000941:Antigens / Q000276:immunology / Q000302:isolation & purification* / Q000502:physiology*; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D007525:Isoelectric Focusing; D008297:Male; D013075:Sperm Capacitation; D013094:Spermatozoa / Q000737:chemistry* / Q000276:immunology*; D013552:Swine; D015044:Zona Pellucida / Q000502:physiology
D000911:Antibodies, Monoclonal; D000941:Antigens