Development of a Competitive ELISA for the Detection of a Furaltadone Marker Residue, 3-Amino-5-Morpholinomethyl-2-Oxazolidinone (AMOZ), in Cultured Fish Samples

Abstract

ABSTRACT. This report describes an enzyme-linked immunosorbent assay (ELISA) for tissue-bound metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) and the application to residue analysis in cultured fish samples. The residue is monitored as a marker for the drug furaltadone. The assay enables the detection of protein bound AMOZ in the form of a 2-nitrophenyl derivative (2-NP-AMOZ) in sample supernatant or extract after acid hydrolysis and derivatization with o-nitrobenzaldehyde. Polyclonal rabbit antibodies were produced with a new immunogen hapten, 2-NP-HXA-AMOZ. The new ELISA had adequate analytical sensitivity (IC 50 value 0.325 µg kg −1 ; limit of detection 0.1 µg kg −1 ) to determine a trace of AMOZ residue and had a high selectivity. Recoveries of AMOZ fortified at the levels of 0.1, 0.5 and 1.0 µg kg −1 ranged from 89.8 to 112.5% with coefficients of variation of 12.4−16.2% over the range of AMOZ concentrations studied. The results obtained with the ELISA correlated well with those obtained by commercial test kits for 150 tested samples (r=0.984). The results suggest that the developed ELISA is a highly specific, accurate, and sensitive method suitable for high throughput screening for AMOZ residues.

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

“…In this study, AMOZ, the stable metabolite of furaltadone, was used as the desired hapten was to generate monoclonal antibodies. However, AMOZ has only one amino group available for coupling the hapten to a protein carrier, which were designed by derivatizing furaltadone metabolites with 3-carboxybenzaldehyde or 2-(3-formylphenoxy)acetic acid to generate immunogens [ 7 , 19 , 20 , 34 ]. In the present study, AMOZ was reacted directly with 4-carboxybenzaldehyle, instead of 3-carboxybenzaldehyle and 2-(3-formylphenoxy) acetic acid, to form novel hapten CPAMOZ.…”

“…developed the polyclonal antibody (pAb)-based ic-ELISAs for the detection of 2-NP-AMOZ in tissue samples. However, the disadvantages of polyclonal antibodies are mainly its low sensitivity and high cross-reactivity, which limits its application [ 21 , 23 , 34 ]. It is well known that mAb can overcome these shortcomings.…”

Scientific Opinion on nitrofurans and their metabolites in food

“…A number of ELISA methods have been reported for the determination of NPAMOZ in shrimps and fish samples with reported LOD values of 0.1-0.3 µg/kg and LOQ or CCβ values of 0.3-0.36 µg/kg (Pimpitak et al, 2009;Shen et al, 2012;Sheu et al, 2012;Yang et al, 2012;Liu et al, 2013). Other ELISA methods were developed and applied to the determination of NPAMOZ in chicken, pork, fish and shrimp samples, reporting LOD values ranging from < 0.1 to 0.34 µg/kg (Luo et al, 2012;Xu et al, 2013;Shu et al, 2014).…”

Development of Antibodies and Immunoassays for Monitoring of Nitrofuran Antibiotics in the Food Chain

“…Similarly, 2-NP-HXA-AMOZ was used as a novel hapten for the production of polyclonal antibodies and, when applied in an antigen capture ELISA coupled to HRP, achieved a LOD of 0.1 µg/kg for AMOZ in fish. No cross-reactivity to other nitrofuran compounds was observed [44]. Two novel haptens derivatised with 2-(4-formylphenoxy) acetic acid or 2-(3-formylphenoxy) acetic acid (Fig.…”

“…Shu et al modified AMOZ with 4-CBA in methanol at 60°C for 3 h. The product was washed with methanol and verified by NMR; a monoclonal antibody was developed with optimum sensitivity to NPAMOZ. [8] for AOZ, was employed by Sheu et al [44] to produce antisera with good sensitivity to NPAMOZ.…”