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Assay for Recombinant and Native Human Intraacrosomal Antigen SP‐10
Author(s) -
SHEN MINCHENG,
WRIGHT RICHARD M.,
CARTA GIORGIO,
HERR JOHN C.
Publication year - 1993
Publication title -
american journal of reproductive immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.071
H-Index - 97
eISSN - 1600-0897
pISSN - 1046-7408
DOI - 10.1111/j.1600-0897.1993.tb00592.x
Subject(s) - polyclonal antibodies , biotinylation , recombinant dna , monoclonal antibody , microbiology and biotechnology , antibody , antiserum , epitope , fusion protein , antigen , biology , bicinchoninic acid assay , chemistry , biochemistry , immunology , gene
PROBLEM: To develop a method to measure a recombinant sperm protein, SP‐10, during scale up and purification for a contraceptive vaccine formulation. METHOD: 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.