| Summary: | 碩士 === 國立臺灣大學 === 微生物與生化學研究所 === 92 === The heart lactate dehydrogenase (LDH) is taken as the goal antigen to produce its monoclonal antibody. The Balb/c female mouse was immunized with LDH for four times. Their spleen cells were collected and fused with NS-1 myeloma cells under the presence of PEG1500. The fused cells were than selected with HAT medium. The anti-LDH antibody-secreting hybridoma cells were screened with enzyme-linked immuno-adsorbent assay (ELISA) The hybridomas were subcloned by limited dilution. Three hybridoma producing anti-LDH monoclonal antibodies (mAbs) were obtained and designated as A1C, B7G, C9C. The isotype of these mAbs were identified as IgG1 heavy chain and κ light chain. Among then A1C hybridoma can secrete higher titer of anti-LDH antibodies, and its growth condition is better than others. DEAE 650M ion exchange and Protein G columns were used to purify IgG of the anti-LDH mAbs. With ELISA assay, antibodies diluted 5^5 times were used to detection LDH. The detection range of LDH was from 0.2 to 5 μg/mL at r2 = 0.997.
These antibodies were also applied to the development of a LDH immunosensor. The anti-LDH mAb is immobilized on the gold electrode chips by 2-IT direct immobilizing method. A frequency shift from 60 to 300 Hz was observed in accordance with a concentration range of LDH from 1 to 6 μg/mL. Through Cystamine-glutaraldehyde immobilizing method, the frequency shifts were 95 to 360 Hz in accordance with LDH concentrations from 1 to 6 μg/mL. The successive work of this study is to establishing a quartz crystal immunosensor of LDH with the characteristics of high sensitivity, rapidly detection, low cost and easy operation.
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