title: Chemiluminescence competitive indirect enzyme immunoassay for 20 fluoroquinolone residues in fish and shrimp based on a single-chain variable fragment.
abstract: 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(-1) for 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(-1) and 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(-1) for PEF to 2.1 μg kg(-1) for 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.
authors.affiliation: Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Beijing Key Laboratory of Detection Technology for Animal-Derived Food Safety, Beijing Laboratory For Food Quality and Safety, China Agricultural University, Yuan Ming Yuan West Road No. 2, Beijing, 100193, China.
authors.forename: Xiaoqi
authors.initials: X
authors.lastname: Tao
authors.forename: Min
authors.initials: M
authors.lastname: Chen
authors.forename: Haiyang
authors.initials: H
authors.lastname: Jiang
authors.forename: Jianzhong
authors.initials: J
authors.lastname: Shen
authors.forename: Zhanhui
authors.initials: Z
authors.lastname: Wang
authors.forename: Xia
authors.initials: X
authors.lastname: Wang
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Wu
authors.forename: Kai
authors.initials: K
authors.lastname: Wen
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D004785:Environmental Pollutants / Q000032:analysis* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005399:Fishes / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D024841:Fluoroquinolones / Q000032:analysis* / Q000378:metabolism; D057230:Limit of Detection; D008163:Luminescent Measurements; D033561:Penaeidae / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D057127:Single-Chain Antibodies / Q000737:chemistry*
chemical_list: D004785:Environmental Pollutants; D024841:Fluoroquinolones; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies