CORE B - FSH Glycoform Production, Purification, and Analysis

项目总结:

Project Summary: Core B: FSH Glycoform Production, Purification and Analysis Core B provides critical and requisite reagents, analyses, and expertise to support all of the Projects in fulfilling their Specific Aims. Specifically, Core B will continue to produce recombinant hFSH glycoforms. Procedures have been established to produce milligram quantities of glycoforms from the conditioned medium of cultured GH3 cells transfected with the human FSH α and ß subunit genes. In conjunction with Project 3, Core B will generate mutant hFSH glycoforms, which mimic known recombinant hypoglycosylated hFSH glycoforms and supply them to all 3 Projects. Core B will continue to work with Project 1 to generate monoclonal antibodies that recognize specifically intact hFSH, hFSH α subunit, as well as individual glycoform ß subunits, for use in glycoform isolation and detection. Core B will provide a variety of cells for use in Projects 1 and 2 to assess glycoform receptor binding and signal transduction and glycoform impact upon cell differentiation. Cells to be furnished include primary porcine granulosa cells, which naturally express the FSH receptor, KGN cells, a human granulosa cell line that expresses the hFSH receptor, CHO cells that express the human FSH receptor, and GH3 cells transfected with hFSH genes for use in the production of recombinant hFSH. Core B will provide a variety of analyses for Projects 1, 2, and 3 including FSH, LH and TSH RIA, FSH and LH receptor binding, E2ß and P4 RIA, and ELISAs for urinary FSH and ELISA development for monoclonal antibody screening. Core B will assist Project 1 in the analysis of recombinant hFSH glycoform circulating half-life following administration to FSHß null mice. Core B will assist Project 1 in the analysis of FSH glycoform abundance change as it relates to the menstrual cycle and the impact of aging. In addition, the relationship between glycoform abundance and infertility will be studied through the continued development of rapid and direct methods to determine hFSH glycoform abundance in urine. The goal is to develop urinary assessment of hFSH glycoforms diagnostic as a clinical diagnostic tool.