Preparation of Monoclonal Antibody and Development of Indirect Competitive Enzyme-Linked Immunosorbent Assay and Fluorescence-Linked Immunosorbent Assay for Detecting 3-Amino-5-methylmorpholino-2-oxazolidinone (AMOZ) in Edible Animal Tissue.

Yong Xie,Xueling Yan,Yarong Wang,Tao Le,Lu Gan

doi:10.3390/molecules26144243

Abstract

To monitor the illegal used of furaltadone, a highly sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and fluorescence-linked immunosorbent assay (FLISA) based on a monoclonal antibody (mAb) were developed for the detection of 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ), the major metabolite of furaltadone in animal tissues. The highly specific mAb, which was very sensitive to a nitrophenyl derivative of AMOZ (2-NP-AMOZ) with IC50 values of 0.11 and 0.09 ng/mL for ic-ELISA and FLISA, respectively, was selected for the development of immunoassays. For both the ic-ELISA and FLISA for AMOZ-spiked experiments, acceptable recovery rates of 81.1–105.3% and coefficients of variation of 4.7–9.8% were obtained. In addition, results from both ic-ELISA and FLISA methods for spiked samples’ data showed excellent correlation coefficients ranging from 0.9652 to 0.9927. Meanwhile, the proposed ic-ELISA and FLISA for thirty spiked samples were confirmed by standard LC-MS/MS with high correlation coefficients of 0.9911 and 0.9921, respectively. These results suggest that the developed ic-ELISA and FLISA are valid and cost-effective tools for high-throughput monitoring methods for AMOZ residues in animal tissues.

Highlights

Nitrofuran antibiotics, such as furaltadone, furazolidone, nitrofurantoin, and nitrofurazone (Figure 1), are widely used in gastrointestinal and dermatological microbiological infections that often occur in farmed bees, swine, cattle, poultry, fish, and shrimp [1].this family of drugs and their metabolites are known to be carcinogenic and mutagenic [2,3], and as a result, the nitrofurans have been listed as prohibited veterinary drugs in farmed animals for food production by many countries and regions, including the European Union (EU), USA, and China [4]
As the furaltadone antibiotic is highly unstable in vivo, and its metabolite can bind to tissue proteins to form stable compounds with a long residence time, the detection of illegal uses of furaltadone in farmed animal feed generally involves the detection of furaltadone metabolite concentrations in animal tissues
An excellent correlation between ic-ELISA and LC-MS/MS (R2 = 0.9911) and fluorescence-linked immunosorbent assay (FLISA) and LCMS/MS (R2 = 0.9921) can be seen from the side-by-side comparison. These results suggest that the new ic-ELISA and FLISA measurements are reliable tools for the determination of AMOZ in real food samples

Summary

Introduction

Nitrofuran antibiotics, such as furaltadone, furazolidone, nitrofurantoin, and nitrofurazone (Figure 1), are widely used in gastrointestinal and dermatological microbiological infections that often occur in farmed bees, swine, cattle, poultry, fish, and shrimp [1]. This family of drugs and their metabolites are known to be carcinogenic and mutagenic [2,3], and as a result, the nitrofurans have been listed as prohibited veterinary drugs in farmed animals for food production by many countries and regions, including the European Union (EU), USA, and China [4]. These metabolites of nitrofurans have been recognized as a marker residue in edible tissues for evaluation of nitrofurans [10,11]

Methods

Results

Conclusion