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標題: 重組抗-氯黴素單鏈抗體之製備與分析
Preparation and characterization of recombinant single-chain variable fragment (scFv) against chloramphenicol
作者: 黃郁涵
Yu-Han Huang
關鍵字: 氯黴素;單鏈抗體;chloramphenicol;single-chain variable fragment;scFv
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在食品安全議題受到重視的現今,利用有效且不昂貴的免疫方法快速檢測抗生素殘留以取代昂貴費時的LC-MS分析應更具有發展價值。 因此開發一個高感度的抗體並研製成檢測套組成為確保食品安全的重要方針。
本研究自表現CAP抗體之融合瘤細胞中成功地取得其可變區基因並進一步建構成融合His-tag之單鏈抗體之表現質體,在最適化之大腸桿菌表現宿主BL21(DE3)pLysS表現後可利用Ni-resin親和性管柱純化並獲得CAP之單鏈抗體。利用ELISA方法分析可證實CAP單鏈抗體可以專一識別CAP,在抗體競爭性試驗同時也證實了CAP單鏈抗體與其單株抗體均會競爭相同結構。在未來更可利用易錯聚合酶鏈反應(error prone PCR)來增加靈敏度並輔以融合不同螢光蛋白或結合其他偵測技術以增加免疫檢測的偵測極限與多元應用性。

Chloramphenicol (CAP) is a potent and efficient antibiotic widely used in pharmacological treatments in veterinary and human. Despite being highly effective, it shows severe toxicity as the residual CAP results in Aplastic anemia (AA) and bone marrow suppression. Due to the potential risk in public health of CAP utilization, the use in food-producing animal's therapy has been banned. Taiwan government has established a zero tolerance policy to CAP in livestock products.
Recently, the food crises have grabbed huge public attention in Taiwan. Replacing the expensive LC-MS analysis by cheap and efficient rapid immunoassay to detect the residual illegal compound can better fit to public interest. Therefore, to generate high sensitivity antibodies for immunoassay kits play a key role in improving the food safety.
This study has successfully cloned CAP-scFv gene from hybridoma and constructed CAP-scFv gene onto an expression vector fused with a His-tag gene. The expression of this protein in E. coli BL21(DE3) pLysS was optimized and the expressed recombinant CAP-scFv protein was purified with Ni-IMAC. We confirmed the specificity between CAP-scFv and chloramphenicol via ELISA assay. Furthermore we also demonstrated that CAP-scFv recognizes the same molecular structure as CAP monoclonal antibody does by performing the competition assay with CAP monoclonal antibody. In the future, using the error-prone PCR approach further improve the CAP-scFv sensitivity and fusion of fluorescent to the protein flank on scFv or combination with various detection methods will be the next target to enhance the detection limit of immunoassay and create novel applications to numerous aspects.
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