关键词: 呋喃它酮；呋喃它酮代谢物；单链抗体；间接竞争酶联免疫分析

Abstract: In this study, the genes of heavy chain and light chain variable fragment were cloned from hybridoma cells secreting a monoclonal antibody against a derivative of the furaltadone metabolite 5-morpholinomethyl-3-amino-2-oxazolidinone (AMOZ). The gene of single-chain variable fragment (scFv) antibody against this derivative was constructed by splicing overlap extension-polymerase chain reaction (SOE-PCR) through the ligating peptide (G1y4Ser)3, inserted into the expression vector pET-22b(+) and expressed in E. coli BL21(DE3). The expressed product was purified by Ni affinity column chromatography. The concentration of coating antigen and the dilution factor of scFv antibody were optimized by chessboard titration to establish and evaluate an indirect competitive enzyme-linked immunosorbent assay (icELISA) based on this scFv antibody for AMOZ residues. The results showed that the optimal concentration of coating antigen and the dilution factor of scFv antibody were 0.062 5 μg/mL and 20, respectively. The IC50 value, linear range and limit of detection (LOD) of the established icELISA method were 8.65 μg/L, 2.90–53.28 μg/L and 1.48 μg/L, respectively. In addition to the cross-reactivity with the original drug furantadone, the cross-reactivity rates of the scFv antibody with other nitrofuran antibiotics and their metabolites were lower than 0.1%. The average recoveries of AMOZ from the spiked shrimp samples ranged from 74.3% to 86.6%. Good correlation (R2 = 0.999 2) was obtained between the results of icELISA and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The icELISA method established in this study is suitable for the rapid screening of AMOZ residues in shrimp samples and other aquatic products.

Key words: furaltadone; 5-morpholinemethyl-3-amino-2-oxazolidinone; single-chain variable fragment antibody; indirect competitive enzyme-linked immunosorbent assay