Epitope analysis of a sperm acrosomal antigen defined by HSA-5 monoclonal antibody.
Among the monoclonal antibodies recommended by the WHO Sperm Antigen Workshop for immunocontraceptive vaccine development, HSA-5 showed a high degree of sperm specificity and significantly inhibited in vitro fertilization in both humans and mice. Using a Western blot assay, HSA-5 was found to recognize a sperm antigen designated as HSAg-5 (human) or 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 expression vector. One of the positive cDNA clones was shown to have a cDNA insert of approximately 1 kb and to encode a recombinant fusion protein 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 recombinant glutathione S-transferase fusion protein (GST-5). Using an enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, both anti-GST-5 sera and the monoclonal antibody were shown to react with 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 oligopeptides were synthesized based on the predicted C-terminal hydrophilic regions of the recombinant fusion protein. Using ELISA and a dot blot assay, peptide regions containing the immunogenic epitopes recognized by HSA-5 monoclonal antibody were identified.
Lee
C Y
CY
Department of Obstetrics and Gynaecology, University of British Columbia, Vancouver, Canada.
Yoshiki
T
T
Chui
L
L
McChesney
P
P
Herr
J C
JC
Hwang
E S
ES
Huang
E S
ES
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000941:Antigens / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D015152:Blotting, Northern; D018076:DNA, Complementary / Q000302:isolation & purification; D000939:Epitopes; D005982:Glutathione Transferase / Q000276:immunology; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013094:Spermatozoa / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000941:Antigens; D018076:DNA, Complementary; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase