Generation of a mouse monoclonal antibody against tilapia ( Oreochromis nilotica) insulin: Application to histology and enzyme-linked immunosorbent assay.

摘要

Our laboratory transplants tilapia islets as a treatment for experimental diabetes and has generated transgenic tilapia expressing a “humanized” tilapia insulin gene for possible future clinical application. Our ultimate goal is to knockout the native tilapia insulin gene in our transgenic fish. To better understand the secretion of insulin, both in the native and the transgenic tilapia, we need to develop an assay to measure tilapia insulin concentrations. Our approach has been to develop monoclonal antibodies by immunizing BALB/c mice with purified tilapia insulin. A mouse spleen-mouse myeloma fusion was screened using an antibody-capture ELISA and 23 cultures secreting anti-tilapia insulin antibodies were identified. One hybridoma was found to secrete antibody that bound to tilapia insulin but not to human insulin. This hybridoma was cloned and the monoclonal antibody was expanded in cell culture. The antibody was tested in immunohistology and shown to bind to tilapia and sculpin islet cells, but not to human, mouse, rat, Atlantic salmon, or pacu islet cells. In Western blotting using purified tilapia insulin, the monoclonal antibody did not bind to either the A chain or the B chain when separated, but did bind to tilapia insulin under non-reducing conditions, suggesting that the antibody binds to non-contiguous residues. The preliminary work on a quantitative two-site ELISA as well as a time-resolved fluoroimmunoassay (TR-FIA) for insulin measurement was completed and both assays can measure tilapia insulin levels from approximately 16–500 ng/ml.