Fluorescence Polarization Immunoassay for Determination of Enrofloxacin in Pork Liver and Chicken.

Xing Shen,Jiahong Chen,Boris B Dzantiev,Sergei A Eremin,Jianfa Xu,Yuanming Sun,Hongtao Lei,Xiulan Sun,Anatoly V Zherdev,Shuwei Lv

doi:10.3390/molecules24244462

Abstract

Enrofloxacin (ENR) is a widely used fluoroquinolone (FQ) antibiotic for antibacterial treatment of edible animal. In this study, a rapid and highly specific fluorescence polarization immunoassay (FPIA) was developed for monitoring ENR residues in animal foods. First, ENR was covalently coupled to bovine serum albumin (BSA) to produce specific polyclonal antibodies (pAbs). Three fluorescein-labeled ENR tracers (A, B, and C) with different spacers were synthesized and compared to obtain higher sensitivity. Tracer C with the longest arm showed the best sensitivity among the three tracers. The developed FPIA method showed an IC50 (50% inhibitory concentration) of 21.49 ng·mL−1 with a dynamic working range (IC20–IC80) of 4.30–107.46 ng·mL−1 and a limit of detection (LOD, IC10) of 1.68 ng·mL−1. The cross-reactivity (CR) of several structurally related compounds was less than 2%. The recoveries of spiked pork liver and chicken samples varied from 91.3% to 112.9%, and the average coefficients of variation were less than 3.83% and 5.13%, respectively. The immunoassay took only 8 min excluding sample pretreatment. This indicated that the established method had high sensitivity, specificity, and the advantages of simplicity. Therefore, the proposed FPIA provided a useful screening method for the rapid detection of ENR residues in pork liver and chicken.

Highlights

Fluoroquinolone (FQ) drugs are widely used in the practices of veterinary clinics due to their effective and broad-spectrum antibacterial activity [1]
For the preparation of artificial antigen, the carbonyl group of enrofloxacin was coupled to bovine serum albumin (BSA) and the resulting enrofloxacin-BSA immunogen was confirmed by using UV spectroscopy (Figure 1)
Two rabbits were injected with enrofloxacin-BSA immunogen, and both produced the final antisera of high titer values after the fifth injection

Summary

Introduction

Fluoroquinolone (FQ) drugs are widely used in the practices of veterinary clinics due to their effective and broad-spectrum antibacterial activity [1]. Gram-negative pathogens by affecting DNA gyrase [2]. As a third-generation fluoroquinolone only available in veterinary medicine, enrofloxacin is used in many species with few adverse effects. In parallel to the exposure to low levels of these antibiotics, an increase has been observed in human. Molecules 2019, 24, 4462 pathogen resistance constituting a public health hazard, primarily through the increased risk of treatment failures [4,5]. Enrofloxacin residues can adversely affect the human digestive system and nervous system as well as cause allergic reactions [6]. The European Union and the Ministry of Agriculture of the People’s Republic of China have set the maximum residue limits (MRLs) of enrofloxacin in muscle tissue to 100 μg·kg−1 , and the MRLs in the kidneys to 200 μg·kg−1

Methods

Results

Conclusion