Fluorescence Polarization Immunoassays For Determination Of Quinolones Residue In Animal-origin Foods

In recent years, the residues of veterinary drug have been considered as an issue of worldwide concern due to its potential threat to human health by entering the food chain and contributing to bacterial resistance. Quinolones (QNs) represent a large group of synthetic antibacterial agents with a broad spectrum of activities, which have been extensively applied in both human medicine and veterinary treatment. To minimize the risk of QNs exposure to humans via products from food-producing animals, maximum residue limits (MRLs) have been established for several QNs in different edible samples by a number of countries and organizations. So it is necessary to monitor these chemical residues in food samples in order to obtain a better food safety assurance. Fluorescence polarization immunoassay (FP1A) is an antibody-based homogeneous analytical method and is usually performed in competitive format involving the competition between an unlabelled analyte and a labelled tracer for the antibody binding. In this work, we describe the synthesis of QNs fluorescent conjugates (tracer) for and development of FPIA based on antibodies for determination of Orbifloxacin (ORB), Enrofloxacin (ENR) and simultaneous determination of multiple QNs antibiotics in food samples for the first time.QNs tracers were synthetized in two different modes. Some tracers were prepared by linking amino fluorescein derivatives with the carboxylic group at position3of QNs, including ORB tracers (ORB-EDF, ORB-BDF and ORB-HDF), LOM tracers (LOM-EDF, LOM-BDF and LOM-HDF) and ENR tracers (ENR-5-AF, ENR-4’-AMF and ENR-BDF). While some tracers were prepared by linking carboxylic fluorescein derivatives with QNs through the piperazine moiety at position7, including five FQs-FITC (CIP-FITC, NOR-FITC, ENO-FITC, LOM-FITC and SAR-FITC) and four SAR tracer (SAR-5-FAM, SAR-6-FAM, SAR-DTAF and SAR-GAF).A FPIA based on ORB MAb and tracer was developed for determining ORB residue in milk., the influence of tracer structures on the sensitivity of assay was studied. Heterogeneous tracer, LOM-BDF, was selected due to high sensitivity and stability obtained. Optimized FPIA displayed a detection limit (LOD) of3.9ng mL-1and IC50of24.5ng mL-1with Z’ factor of0.81. A simple pretreatment with saturated (NH4)2SO4precipitation was applied for milk sample due to high tolerance to elevated-ionic strength in the developed FPIA. Mean recoveries were74.3to112%at the adding levels of10,20and40ng mL-1.A FPIA based on ENR MAb and tracer ENR-4’-AMF was developed for determining ENR residue in chicken muscle. Optimized FPIA displayed a LOD of2.4ng mL-1and IC50of22.9ng mL-1with working range of3.3～120ng mL-1. Analysis of ENR fortified milk samples by the FPIA showed average recoveries from74.4～79.5%with CV ranged from8.6～15.6%.This paper also describes a rapid one-step FPIA for the simultaneous determination of multiple QNs in food samples based on a broad-specificity monoclonal antibody toward QNs. Several fluorescent tracers were synthesized and evaluated in FPIA method The heterogeneous tracer, SAR-5-FAM, was considered as the optimal choice to prepare the immunocomplex single reagent, which allows a rapid and sensitive displacement reaction by addition of analytes. Optimized single-reagent FPIA exhibited broad cross-reactivities in the range of7.8～172.2%with16QNs tested, and was capable of determining most of QNs at the level of maximum residue limits. Recoveries for5QNs fortified milk and6QNs fortified chicken muscle samples were from77.8～116%, with relative standard deviation lower than17.4%. Therefore, this method could be applicable in routine screening analysis of multiple QNs residues in food samples.Quad-fluorophore FPIA platform was developed by combination of fluorescent probes with different wavelengths and corresponding specific antibodies for simultaneous analysis of multiple antibiotics, including Streptomycin (STR), Sulfathiazole (ST), ENR and Cephalexin (CEP). Optimized quad-fluorophore FPIA exhibited a satisfactory compatibility for the multiple analysis, and showed a LOD of7.3ng mL-1for STR,2.7ng mL-1for ST,2.5ng mL-1for ENR and3.8ng mL-1for CEP. All four antibiotics are highly soluble in water and could be simultaneously extracted from milk and determined in one assay system. Recoveries for4antibiotics fortified milk were from59.8～88.1%, with relative standard deviation lower than25.5%.