期刊
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
卷 64, 期 14, 页码 2971-2979出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.jafc.6b00639
关键词
chloramphenicol; single-chain variable fragment; recombinant expression; direct competitive ELISA
资金
- National Science Foundation for Distinguished Young Scholars of China [31225021]
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.
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