Development of a monoclonal antibody–based time-resolved fluorescence immunochromatographic assay strip for sensitively detecting florfenicol residues in milk and eggs: Theoretical chemical insights into unexpected high specificity

Florfenicol (FF), with its broad-spectrum antibacterial activity, is frequently abused in the livestock and poultry industries and has aroused the growing public concern. Owing to structural similarities and varying maximum residue limits between florfenicol and other chloramphenicol (CAP)-type antibiotics, including thiamphenicol (TAP) and chloramphenicol (CAP), there is an urgent need for a rapid and effective immunoassay method to distinguish them, in order to minimize the risk of false positives. Fortunately, a highly specific monoclonal antibody (mAb), named as SF11, has been developed using hybridoma technology. Molecular simulations have revealed that the mAb SF11's specificity in recognizing florfenicol stems from the π-π stacking interaction between florfenicol and the mAb SF11 binding pocket. Using this highly specific mAb, a sensitive time-resolved fluorescence immunochromatographic assay (TRFICA) strip for rapid florfenicol detection has been developed. Under optimal conditions, this TRFICA demonstrated good analytical performance for the detection of florfenicol in milk and eggs samples, with the half-maximal inhibition concentration (IC50) values of 1.89 and 2.86 ng mL−1, the limit of detection (LOD) of 0.23 and 0.48 ng mL−1, the cut-off values of 62.50 and 31.25 ng mL−1, and the testing time of approximately thirteen minutes. Spiked recoveries in the milk and eggs samples ranged from 104.7 % to 112.3 % and 95.3 % to 116.4 %, respectively, with no obvious cross-reactions with the other analogues observed. The TRFICA results correlated well with those of high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) for real samples, indicating that the developed TRFICA method was sensitive, accurate and adapted for the rapid determination of florfenicol in milk and egg samples. A highly specific monoclonal antibody (mAb) has been achieved using hybridoma technology and systematically analyzed for its recognition mechanism with CAP-type antibiotics. The Anti-FF-mAb was easily modified with time-resolved fluorescent microspheres, leading to the development of the time-resolved fluorescence immunochromatographic assay (TRFICA) method for rapid and specific qualitative and quantitative detection of florfenicol in milk and egg samples, in good agreement with HPLC-MS/MS. [Display omitted] •The binding mode between the mAb SF11 and florfenicol was analyzed through molecular simulation.•The TRFICA exhibited high sensitivity (IC50:1.89–2