Development and application of an immunoaffinity column clean-up for enrofloxacin determination in food samples

Enrofloxacin (ENR) is a synthetic fluoroquinolone antibiotic used for the treatment of infection in animals. The use of ENR has been strictly regulated because of its harmful effect on consumers of animal products. In this study the generation of polyclonal antibody (pAb) against ENR, the preparation of an immunoaffinity chromatography column and its potential application to the selective extraction of ENR residues from food samples were described. The produced pAb exhibited good sensitivity to ENR with an IC₅₀ value of 5.4?ng/mL. The cross-reactivity values of the antibody with ENR structurally related compounds were lower than 0.5%. An immunoaffinity column was constructed by coupling antibody covalently with chitosan microspheres. 0.8% NaOH/0.01?mol/L phosphate-buffered saline (PBS) (pH 7.4) and 85% methanol were selected as loading and eluting solution by careful optimization. Further characterization indicated that the binding capacity of the column for ENR was approximately 3780?ng/mL gel. The immunoaffinity columns were then challenged with ENR-fortified pork, and the recoveries of ENR were found to be in the range of 79.8–94.9%, demonstrating the feasibility of the prepared immunoaffinity column for sample clean-up in ENR residue determination.