Development and Optimization of an Indirect Competitive ELISA for Detection of Norfloxacin Residue in Chicken Liver

Abstract

An indirect competitive ELISA has been developed for the determination of Norfloxacin (NFLX) residue in chicken liver. For this reason, carbodiimide active ester method was employed to synthesize the artificial antigen of NFLXBSA, and coating antigen of NFLX-OVA, and rabbits were used to produce the anti-NFLX polyclonal antibody. Based on the square matrix titration, an icELISA method was developed for the quantitative detection of NFLX. The dynamic range was from 0.006 to 38 ng/mL, with LOD and IC50 value of 0.004 ng/mL and 0.48 ng/mL, respectively. Except for a high cross-reactivity to Ciprofloxacin (38%) and Sarafloxacin (33%), negligible cross-reactivity to the other compounds was observed. After optimization, 10-fold dilution in chicken liver extract gave an inhibition curve almost the same as that in PBS buffer. When spiked in liver extract at 8, 20, and 50 ng/mL, the recovery rate was 90- 117.8% for intra-assay, and 97.5-114.4% for inter-assay, respectively. The relative standard deviation ranged from 5.6-9.2% for intra-assay and 5.9-11% for inter-assay. Therefore, the developed icELISA method could be used for the routing screening of NFLX residues in chicken edible tissues.