Assay for recombinant and native human intraacrosomal antigen SP-10.
OBJECTIVE
To develop a method to measure a recombinant sperm protein, SP-10, during scale up and purification for a contraceptive vaccine formulation.


METHODS
A quantitative assay method for the human intraacrosomal protein SP-10 was developed utilizing the format of indirect capture enzyme-linked immunosorbent assay (ELISA). A SP-10 specific monoclonal antibody mAb, MHS-10, was used as the capture antibody. Two recognition reagents, a rabbit polyclonal anti-SP-10 antisera (pAb) and a biotin-labeled mAb, MHS-10, were used as the recognition antibodies, respectively. A SP-10 recombinant fusion protein consisting of 125 SP-10 amino acids linked to glutathione transferase was used as a working SP-10 standard. The coefficient of variance for the assay system using the rabbit pAb was in the range of 0.099 to 0.157, and for the assay system using the biotinylated mAb MHS-10 was in the range of 0.081 to 0.084.


RESULTS
Employing biotinylated MHS-10 as the recognition antibody, it was found that the native SP-10 molecule had more than one MHS-10 epitope. The concentration of SP-10 in a pool of human sperm extracts was found to be approximately 1% of the total proteins, assayed by both of the recognition antibody systems.


CONCLUSIONS
The assay system described is useful to monitor the yield of recombinant SP-10 during scale-up production of the SP-10 vaccine.
Shen
M
M
Department of Chemical Engineering, University of Virginia, Charlottesville 22908.
Wright
R M
RM
Carta
G
G
Herr
J C
JC
D000177:Acrosome / Q000276:immunology*; D000595:Amino Acid Sequence; D000941:Antigens / Q000032:analysis* / Q000235:genetics; D001709:Biotechnology; D003269:Contraception, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000706:statistics & numerical data; D000939:Epitopes; D012739:Gonadal Steroid Hormones / Q000032:analysis* / Q000235:genetics; D006801:Humans; D008297:Male; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology; D012680:Sensitivity and Specificity
C512729:ACRV1 protein, human; D000941:Antigens; D000939:Epitopes; D012739:Gonadal Steroid Hormones; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins