Detection of quinolones in milk and groundwater samples using an indirect immunofluorescent magneto assay

Abstract

A simple magneto immunofluorescence assay for quinolone family detection in 96-well microplate was developed and applied to milk and groundwater samples. Norfloxacin (NOR) conjugated to albumin (BSA) was immobilised on magnetic beads (MBs) in a first step. The strategy was based on an indirect competitive assay between free and immobilised quinolone for anti-quinolone IgG antibody. Then, a peroxidase tracer was added as developer reagent. The fluorescence responses at 440 and 540 nm - excitation and emission wavelengths, respectively–were obtained by adding o-phenylenediamine and peroxide hydrogen as peroxidase substrates. The fluorescence response was indirectly proportional to the concentration of quinolone in the sample. Ciprofloxacin was selected as model quinolone with cut-off and limit of detection (LOD) of 4 and 8 μg L−1, respectively. Bovine milk and groundwater blank samples were spiked with ciprofloxacin at 5, 25 and 50 µg L−1 obtaining good recoveries (90–100%) with coefficient variation lower than 20% in all cases, in concordance with the results obtained by applying a reference method. Therefore, the method developed would be applicable for the detection of quinolones in bovine milk from dairy farms and in groundwater from sites with intensive livestock activities.