A sensitive and specific polyclonal enzyme-linked immunosorbent assay (ELISA) for the determination of tissue-bound metabolite 3-amino-2-oxazolidinone (AOZ) is described. The procedures allow for the detection of protein-bound AOZ in the form of a 2-nitrophenyl derivative (2-NP-AOZ) in the sample supernatant or extract after acid hydrolysis and derivatization with o-nitrobenzaldehyde. The polyclonal rabbit antibodies were produced with the immunogen hapten, 2-NP-HXA-AOZ, and the 50% inhibition values (IC(50)) of 0.14 microg kg(-1) of AOZ was achieved with the most sensitive antibody A0505. The mean lower detection limit of the ELISA method is about 0.025 microg kg(-1). According to the test preparation record, the detection limit is 0.1 microg kg(-1), which is well below the minimum required performance limits (MRPLs) for tissue-bound residues of AOZ at 1 microg kg(-1) in the European Communities. In the present study, we investigated the use of homemade ELISA, a new immunoassay, to monitor the presence of the furazolidone marker residue in 370 samples of cultured fish. Adopting 0.3 microg kg(-1) AOZ as a cutoff value, the ELISA has a sensitivity of 100% and a specificity of 98.5% versus high-performance liquid chromatography-mass spectrometry (HPLC-MS) at a cutoff of 0.3 microg kg(-1) and gives no false-negative rate results. From the practical point of view, the homemade kit could be advantageously used for the screening of large groups of animal-edible tissue samples and the kit employed has good reliability even in routine application for the control of the illegal use of the drug.

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.

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