Development and residue screening of the furazolidone metabolite, 3-amino-2-oxazolidinone (AOZ), in cultured fish by an enzyme-linked immunosorbent assay.
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.
Cheng
Chu-Chen
CC
Taiwan Advance Bio-Pharmaceutical, Incorporated, Taipei County 221, Taiwan.
Hsieh
Kuan-Huei
KH
Lei
Yi-Chih
YC
Tai
Yung-Te
YT
Chang
Tong-Hsuan
TH
Sheu
Shi-Yuan
SY
Li
Wen-Ren
WR
Kuo
Tzong-Fu
TF
D000818:Animals; D000900:Anti-Bacterial Agents / Q000032:analysis*; D017756:Aquaculture; D004350:Drug Residues / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005399:Fishes; D023303:Oxazolidinones / Q000032:analysis*; D017747:Seafood / Q000032:analysis*; D012680:Sensitivity and Specificity
D000900:Anti-Bacterial Agents; D023303:Oxazolidinones; C000209:3-amino-2-oxazolidinone