Development of an Immunoassay for Chloramphenicol Based on the Preparation of a Specific Single-Chain Variable Fragment Antibody

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• 作者：Xin-jun Du ; Xiao-nan Zhou ; Ping Li ; Wei Sheng ; Frédéric Ducancel ; Shuo Wang
• 刊名：Journal of Agricultural and Food Chemistry
• 出版年：2016
• 出版时间：April 13, 2016
• 年：2016
• 卷：64
• 期：14
• 页码：2971-2979
• 全文大小：551K
• 年卷期：0
• ISSN：1520-5118

文摘

Specific antibodies are essential for the immune detection of small molecule contaminants. In the present study, the heavy and light variable regions (V_H and V_L) of the immunoglobulin genes from a hybridoma secreting a chloramphenicol (CAP)-specific monoclonal antibody (mAb) were cloned and sequenced. In addition, the light and heavy chains obtained from the monoclonal antibody were separated using SDS–PAGE and analyzed using Orbitrap mass spectrometry. The results of DNA sequencing and mass spectrometry analysis were compared, and the V_H and V_L chains specific for CAP were determined and used to construct a single-chain variable fragment (scFv). This fragment was recombinantly expressed as a soluble scFv-alkaline phosphatase fusion protein and used to develop a direct competitive ELISA. Compared with the parent mAb, scFv exhibits lower sensitivity but better food matrix resistance. This work highlights the application of engineered antibodies for CAP detection.