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

作者：Du, Xin-jun; Zhou, Xiao-nan; Li, Ping; Sheng, Wei; Ducancel, Frederic; Wang, Shuo^*

来源：Journal of Agricultural and Food Chemistry, 2016, 64(14): 2971-2979.

DOI：10.1021/acs.jafc.6b00639

• chloramphenicol
• single-chain variable fragment
• recombinant expression
• direct competitive ELISA

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.

• 出版日期2016-4-13
• 单位天津科技大学; 中国地震局