抗乳酸脫氫酶單株抗體之產製及其應用於石英晶體免疫感測器之開發

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

許惠綺(Hui-Chi Hsu)

指導教授 ： 陳建源

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.01442

摘要

本研究以乳酸脫氫酶作為目標抗原，進行Balb/c小鼠免疫。利用PEG 1500 使Balb/c小鼠脾細胞與NS-1骨髓癌細胞融合，以HAT (Hypoxanthine Aminopterin Thymidine)培養液進行選擇性培養，以酵素免疫分析法(ELISA)篩選具分泌抗乳酸脫氫酶抗體之融合瘤(Hybridoma) 細胞，結果共獲得A1C、B7G、C9C三株抗乳酸脫氫酶之單株抗體。分型結果顯示三者之重鏈均為IgG1、輕鏈均為

關鍵字

石英晶體免疫感測器 ； 乳酸脫氫酶單株抗體

並列摘要

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.

並列關鍵字

quartz crystal immunosensor ； lactate dehydrogenase ； monoclonal antibody

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