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Chemiluminescence competitive indirect enzyme immunoassay for 20 fluoroquinolone residues in fish and shrimp based on a single-chain variable fragment

Tao, Xiaoqi, Chen, Min, Jiang, Haiyang, Shen, Jianzhong, Wang, Zhanhui, Wang, Xia, Wu, Xiaoping, Wen, Kai
Analytical and bioanalytical chemistry 2013 v.405 no.23 pp. 7477-7484
acetic acid, antibodies, chemiluminescence, ciprofloxacin, cross reaction, danofloxacin, detection limit, difloxacin, enrofloxacin, enzyme-linked immunosorbent assay, ethanol, fish, flumequine, horseradish, inhibitory concentration 50, levofloxacin, liquid chromatography, marbofloxacin, mass spectrometry, mutants, norfloxacin, sarafloxacin, screening, shrimp, sparfloxacin
A chemiluminescent competitive indirect enzyme-linked immunosorbent assay, based on a mutant single-chain variable fragment (scFv), was developed to detect a broad range of fluoroquinolones (FQs) in fish and shrimp matrices. In this study, the best scFvC4A9H1_mut2 was adopted, which showed 10-fold improved affinity to sarafloxacin (SAR), difloxacin (DIF), and trovafloxacin (TRO), while the affinity to other FQs was fully inherited from wild-type scFvC4A9H1. In the optimized generic test, scFvC4A9H1_mut2 in combination with norfloxacin–ovalbumin conjugate and horseradish peroxidase-labeled anti-c-myc 9E10 antibody showed 50 % binding inhibition (IC₅₀) at 0.12 μg kg⁻¹for norfloxacin in buffer. Screening for the class of FQ antibiotics is accomplished using a simple, rapid extraction carried out with ethanol/acetic acid (99:1, v/v). This common extraction was able to detect 20 FQ residues such as s ciprofloxacin (CIP), danofloxacin, DIF, enoxacin, enrofloxacin (ENR), fleroxacin, amifloxacin, flumequine, levofloxacin, lomefloxacin hydrochloride, marbofloxacin, norfloxacin (NOR), ofloxacin, orbifloxacin, pazufloxacin, pefloxacin-d5 (PEF), prulifloxacin, SAR, sparfloxacin, and TRO in fish and shrimp. The limit of detection (LOD) for NOR was 0.2 μg kg⁻¹and the LODs for CIP and ENR were all <0.2 μg kg⁻¹. Values of LODs inferred from the cross-reactivity data will range from approximately 0.23 μg kg⁻¹for PEF to 2.1 μg kg⁻¹for TRO. Field fish and shrimp samples were analyzed and compared to the results obtained from liquid chromatography tandem mass spectrometric method. All five instances (from 0.25 to 15.6 μg kg⁻¹) in which FQs were present at concentrations near or above the assay LOD were identified as positive by the newly developed assay, demonstrating the usefulness of this assay as a screening tool.