Production of a monoclonal antibody directed against rat liver glutathione-insulin transhydrogenase |
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Authors: | D B Dawson R A Smith P T Varandani |
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Institution: | 1. Biomedical Sciences Program Wright State University, Dayton, Ohio 45435 USA;2. the Endocrinology Section of Fels Research Institute, Wright State University, Dayton, Ohio 45435 USA;3. Department of Biological Chemistry School of Medicine, Wright State University, Dayton, Ohio 45435 USA;4. Department of Microbiology & Immunology, School of Medicine, Wright State University, Dayton, Ohio 45435 USA;5. College of Science & Engineering, Wright State University, Dayton, Ohio 45435 USA |
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Abstract: | A hybridoma cell line secreting monoclonal antibody specific for glutathione-insulin transhydrogenase has been produced by fusing mouse myeloma cells with spleen cells from mice immunized to purified rat liver glutathione-insulin transhydrogenase. The secreted antibody isotypes were found to be: Ig gamma 1 heavy chains and kappa light chains. This monoclonal antibody has been used to screen glutathione-insulin transhydrogenase in various rat tissue extracts (liver, fat, heart, testis, spleen, lung and kidney) following separation on NaDodSO4/urea polyacrylamide disc-gel electrophoresis and electrophoretic transfer to nitrocellulose. Screening with the monoclonal antibody showed the presence of one immunoreactive protein band equal in molecular weight to that of purified rat liver GIT (Mr 53,000) in extracts of all tissues studied and a second immunoreactive protein band of lower molecular weight (Mr 49,000) in spleen and lung tissue extracts. Separation of these two proteins by HPLC using a TSK-DEAE column demonstrated that both proteins exhibit insulin degrading activity. These data indicate that GIT may occur in multiple forms in some tissues. |
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Keywords: | GIT glutathione-insulin transhydrogenase sodium dodecyl sulfate PBS phosphate-buffered saline BSA bovine serum albumin McAb monoclonal antibody ELISA enzyme-linked immunosorbent assay PBS-T PBS containing 0 05% Tween 20 and 0 05% BSA HPLC high performance liquid chromatography |
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