| Summary: | 碩士 === 國立臺灣大學 === 微生物與生化學研究所 === 96 === Hybridoma technology is the most frequently used method for preparing monoclonal antibody. It is produced by fusing antibody secreting splenic B cells with myeloma cells. This technology was basically unchanged since its first introduction in 1975 by Kohler and Milstein. The concept of monoclonal antibody bank was proposed by our lab in 2006, and totally 192 specific monoclonal antibodies was collected by immunizing mice with the total water-soluble proteins from bamboo shoot, and then following an improved fusion and screening procedure. In this project, we further proposed the concept of differential antibody bank, in which the antibodies recognizing those proteins that were specific to variuos growing stages of bamboo shoots were screened out. First attempt to construct this bank by immunoaffinity column was not successful, since we cannot produce the protein banks with significant difference. Flow cytometer which could differentially sort out the cells labeled with specific proteins was utilized. In preliminary tests, we confirmed that hybridma cells could produce membrane-bound antibodies as detected by anti-mouse IgG-FITC antibody; and several established hybridoma lines (H7c, C7a, and D5b) could be sorted successfully by antigen labeling. Then we immunize mice with total proteins from bamboo shoots (BS0TP or BS60TP), and fuse the B cells with myeloma cells. After 3 wk cultured in T80-flask, the specific antibody producing lines were sorted out by negative selection and subsequently positive selection. The antibody produced was then checked by Western blotting after limiting dilution. Finally, we get some monoclonal antibody, but it can’t recognize BS0TP or BS60TP specifically. Therefore, we have to improve this technique and the procedure.
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