A receptor-based chemiluminescence enzyme linked immunosorbent assay for determination of tetracyclines in milk

This study for the first time reported a receptor based chemiluminescence immunoassay for analysis of tetracyclines. During the experiments, the gene of TetR protein was transformed into Rosetta-gami(DE3) to express a receptor, and its recognition mechanism for 5 tetracyclines was studied by using molecular docking technique. Results showed that hydrophobic interaction and Pi-Pi bond were the main intermolecular forces responsible for TetR-tetracyclines binding, and the D ring was the main receptor binding position in tetracyclines molecules. Then the receptor was used as recognition reagent to develop a direct competitive enzyme linked immunosorbent assay for determination of the 5 drugs in milk, and the light signal was induced with 4-(imidazol-1-yl)-phenol enhanced luminol-H2O2 system. The limits of detection for the 5 drugs in milk were in the range of 5-16 pg/mL, and the recoveries from the standards fortified blank milk were in the range of 71.7%-95.8%. Therefore, this method could be used as a simple, rapid, and ultra-sensitive tool to monitor the residues of tetracyclines in milk.