Multiresidue Determination of Sarafloxacin, Difloxacin, Norfloxacin, and Pefloxacin in Fish using an Enzyme-Linked Immunosorbent Assay

Abstract

An indirect competitive ELISA (icELISA) method for multiresidue determination of Fluoroquinolones (FQs) residues in fish samples has been developed. For this purpose, Sarafloxacin (SAR) was employed to synthesize the immunogen and coating antigen through EDC conjugation method, and cell fusion technology was used to produce anti-SAR monoclonal antiobdy. Based on the square matrix titration, an icELISA method was established. The dynamic range for Sarafloxacin in assay buffer was from 0.004 to 18 ng/mL, with LOD and IC50 value of 0.002 ng/mL and 0.32 ng/mL, respectively. After optimization, the physiological pH (7.4) was selected for the immunoassays, and this assay could tolerate up to 10% acetonitrile. The results of this assay showed a high cross- reactivity to Difloxacin (85.5%), Norfloxacin (61.7%), and Pefloxacin (34.8%). Under the 10-fold dilution in authentic fish samples, the regression curve equations for Sarafloxacin, Difloxacin, Norfloxacin and Pefloxacin were y = 1.0114x - 0.4003, R2 = 0.9901; y = 0.9782x + 0.2754, R2 = 0.9807; y = 0.9892x +0.0489, R2 = 0.9843; and y = 0.9797x +0.8017, R2 = 0.9844, respectively. The results suggest this immunoassay can be used for simultaneous detecting four kinds of FQs in fish samples.