Production of Anti Lactate Dehydrogenase Monoclonal Antibody and Its Application to Develop a Quartz Crystal Immunobiosensor

• Main Authors: Hui-Chi Hsu, 許惠綺
• Other Authors: 陳建源
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/83632122417695402046

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.