M Shen1, R M Wright, G Carta, J C Herr. 1. Department of Chemical Engineering, University of Virginia, Charlottesville 22908.
Abstract
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.
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 rabbitpAb 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.
Authors: M A Coppola; K L Klotz; K-a Kim; H Y Cho; J Kang; J Shetty; S S Howards; C J Flickinger; J C Herr Journal: Hum Reprod Date: 2010-02-05 Impact factor: 6.918
Authors: Kenneth L Klotz; Michael A Coppola; Michel Labrecque; Victor M Brugh; Kim Ramsey; Kyung-ah Kim; Mark R Conaway; Stuart S Howards; Charles J Flickinger; John C Herr Journal: J Urol Date: 2008-10-19 Impact factor: 7.450