Development of an enzyme-linked immunosorbent assay for the detection of florfenicol in fish feed

A sensitive enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody was developed for routine screening of florfenicol (FF) in fish feed. FF succinate was synthesised and conjugated to human serum albumin by N-hydroxysuccinimide-activated ester method as immunogen, while it was conjugated to ovalbumin by mixed anhydride reaction as coating antigen. The ELISA for FF showed an IC50 value of 2.5 ng/mL, and negligible cross-reactivities with other amphenicol family of antibiotics. The inter-assay recoveries of FF from fortified fish feed at levels of 2-20 mg/kg ranged from 98.8 to 117.6% with coefficients of variation (CV) of 9.3-14.2%, intra-assay recoveries ranged from 102.5 to 121.2% with CV of 10.9-13.8%. The developed ELISA were then validated by liquid chromatography method and liquid chromatography-tandem mass spectrometry method, the results indicated this ELISA could be used as convenient method for detection of FF in fish feed.