title: Development of an Immunoassay for Chloramphenicol Based on the Preparation of a Specific Single-Chain Variable Fragment Antibody.
abstract: 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.
authors.affiliation: Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Tianjin University of Science and Technology , Tianjin 300457, China.
authors.forename: Xin-jun
authors.initials: XJ
authors.lastname: Du
authors.affiliation: Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Tianjin University of Science and Technology , Tianjin 300457, China.
authors.forename: Xiao-nan
authors.initials: XN
authors.lastname: Zhou
authors.affiliation: Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Tianjin University of Science and Technology , Tianjin 300457, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Li
authors.affiliation: Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Tianjin University of Science and Technology , Tianjin 300457, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Sheng
authors.affiliation: Pharmacology and Immune Analysis Department, CEA/Saclay , F-91191 Gif-sur-Yvette, France.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Ducancel
authors.affiliation: Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Tianjin University of Science and Technology , Tianjin 300457, China.
authors.forename: Shuo
authors.initials: S
authors.lastname: Wang
mesh_terms: D000911:Antibodies, Monoclonal / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D002701:Chloramphenicol / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D057127:Single-Chain Antibodies / Q000032:analysis* / Q000235:genetics / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D057127:Single-Chain Antibodies; D002701:Chloramphenicol
keywords: chloramphenicol; direct competitive ELISA; recombinant expression; single-chain variable fragment