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A multiresidue immunoassay method for determination of Fluoroquinolones (FQs) residues has been developed. For this purpose, NHS ester technology was employed to synthesize the immunogen and coating antigen of Norfloxacin (NFLX). SDS-PAGE, UV-visible spectra and Infrared spectra identification showed that the artificial antigen was conjugated successfully. Based on the square matrix titration, an icELISA method was established. The dynamic range in assay buffer was from 0.038 to 112.8 ng/mL, with LOD and IC50 value of 0.02 ng/mL and 1.2 ng/mL, respectively. This assay showed a high cross-reactivity to Ciprofloxacin (86%), Enrofloxacin (75%), Difloxacin (63%), Sarafloxacin (57%) and Pefloxacin (33.8%). The chemical effects on assay performance showed that the physiological pH (7.4) in assay buffer pursued the maximum absorbance (Amax) and the most sensitive IC50 values. The results suggest the artificial antigen was synthesized successfully, and the established immunoassay could be used for simultaneous detecting of Norfloxacin, Ciprofloxacin, Enrofloxacin, Difloxacin, Sarafloxacin and Pefloxacin residues in animal-original food samples.

A rapid indirect competitive ELISA (icELISA) for the determination of enrofloxacin (ENR) residue has been developed. EDC method was employed to synthesize the artificial antigen of ENR-BSA, and anti-serum produced from rabbits was selected. Based on the square matrix titration, an icELISA method was developed with the polyclonal antibody. The Linear range was from 0.006 to 31.5 ng/mL, with LOD and IC50 values of 0.003 ng/mL and 0.45 ng/mL, respectively. Except for a high cross-reactivity (105.2%) to ciprofloxacin, negligible cross-reactivity to other compounds was observed. The recoveries of ENR were in the range of 98.3-127.5%, 85.7-112.5% and 97.4-103.8% for chicken muscle, liver and kidney, respectively. Therefore, this assay has the potential to be incorporated into a quantitative monitoring program for the rapid screening of ENR residue in food.

An icELISA immunoassay for determination of marbofloxacin (MAR) residue has been developed. For this purpose, modified EDC technology was employed to synthesize the immunogen and coating antigen of MAR. UV-vis spectrum and SDS-PAGE identification showed that the artificial antigen was conjugated successfully. New Zealand white rabbits were used to produce anti-MAR polyclonal antibody. Based on the square matrix titration, an icELISA method was established. The dynamic range in assay buffer was from 0.18 to 153.6 ng/mL, with LOD and IC50 value of 0.11 ng/mL and 6.2 ng/mL, respectively. Except for a slight cross-reactivity (16.4%) to ofloxacin, this assay showed negligible cross-reactivity to other FQs tested. To reduce the matrix effects, the least dilutions for muscle extractions were expected to be 10- and 20-fold, for beef and pork, respectively. The results suggest the established immunoassay could be used for detecting of marbofloxacin residue in animal samples.