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Tao X.,China Agricultural University | Chen M.,China Agricultural University | Jiang H.,China Agricultural University | Shen J.,China Agricultural University | And 4 more authors.
Analytical and Bioanalytical Chemistry | Year: 2013

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. [Figure not available: see fulltext.] © 2013 Springer-Verlag Berlin Heidelberg. Source

Tao X.,China Agricultural University | Shen J.,China Agricultural University | Cao X.,China Agricultural University | Wang Z.,China Agricultural University | And 2 more authors.
Analytical Methods | Year: 2014

Two novel chemiluminescence immunoassays using the horseradish peroxidase (HRP)-luminol chemiluminescence system and two different enzymatic systems - HRP and alkaline phosphatase (ALP) chemiluminescence systems - were established for simultaneous detection of chloramphenicol (CAP) and clenbuterol (CLE) residues in milk. Milk samples were detected utilizing the two hybrid chemiluminescence immunoassays, with limits of detection (LODs) of 0.006 μg L-1 for CAP and 0.02 μg L-1 for CLE (Scheme 1A), 0.008 μg L -1 for CAP and 0.023 μg L-1 for CLE (Scheme 1B), respectively. The recoveries for CAP and CLE in individual and co-spiked samples were found to be between 80.0% and 96.7%. The applicability of the proposed method has been validated by determining CAP and CLE in field milk samples with satisfactory results. Thereafter, the developed hybrid chemiluminescence immunoassays can be used as reliable, rapid, and cost effective screening techniques for the simultaneous determination of CAP and CLE residues in milk. © 2014 The Royal Society of Chemistry. Source

Jiang W.,China Agricultural University | Luo P.,China National Institute for Nutrition and Food Safety | Wang X.,China Institute of Veterinary Drug Control | Chen X.,China Agricultural University | And 5 more authors.
Food Control | Year: 2012

A sensitive and specific enzyme-linked immunosorbent assay (ELISA) for the detection of 1-amino-hydantoin (AHD) is described. AHD, the marker residue for nitrofurantoin, is a tissue bound toxic metabolite. To monitor the illegal use of nitrofurantoin, a monoclonal antibody-based ELISA method was developed to detect AHD residue in animal tissues. The highly specific antibody against AHD was prepared by monoclonal antibody technology. The antibody exhibited negligible cross reactivity with other nitrofurans, their metabolites and derivatives, and 50% inhibitory concentration was 0.68 μg/L. The limit of detection in four kinds of animal tissues were all below 0.2 μg/kg and recoveries ranged from 75% to 116.7% for fortified samples at levels of 0.2-5 μg/kg with coefficient of variation values below 15%. Analysis of natural contaminated samples by the ELISA method gave similar results to those of the liquid chromatography-tandem mass spectrometry method. These results indicate the ELISA method is suitable for the detection of AHD residue in animal tissues. © 2011 Elsevier Ltd. Source

Tao X.,China Agricultural University | Yu X.,China Agricultural University | Zhang D.,China Agricultural University | Shi W.,China Agricultural University | And 7 more authors.
Journal of the Science of Food and Agriculture | Year: 2014

BACKGROUND: A rapid one-step chemiluminescent competitive indirect enzyme-linked immunosorbent assay (CL-ciELISA) for florfenicol (FF) and its major metabolite florfenicol amine (FFA) residues in animal meat products has been developed. RESULTS: The 50% binding inhibition (IC50) values of the method were 0.195 μg kg-1 for FFA and 0.24 μg kg-1 for FF under optimum conditions. The cross-reactive rates for FF and FFA were 100.0% and 81.2%, respectively. FF and FFA were easily extracted from animal meat product with an FF/FFA extraction buffer, obtaining recoveries of 81.8-92.0% (FF) and 77.2-100% (FFA). The whole one-step CL-ciELISA test can be accomplished within 40 min in theory. The detection limits (LODs) of the assay were 0.98 μg kg-1 for FF and 0.80 μg kg-1 for FFA in animal meat samples. Finally, field animal meat samples were analyzed with the CL-ciELISA method, and the results correlated well with those obtained using traditional ELISA and a previously reported liquid chromatographic-tandem mass spectrometric method. CONCLUSION: The combined results confirmed the utility of this faster one-step CL-ciELISA for simultaneous trace analysis of FF and FFA. To date, this is the most rapid developed ELISA and CL-ELISA method for detection of FF and FFA. © 2013 Society of Chemical Industry. Source

Tao X.,China Agricultural University | Jiang H.,China Agricultural University | Yu X.,China Agricultural University | Zhu J.,China Agricultural University | And 5 more authors.
Drug Testing and Analysis | Year: 2013

A competitive, direct, chemiluminescent immunoassay based on a magnetic beads (MBs) separation and gold nanoparticles (AuNPs) labelling technique to detect chloramphenicol (CAP) has been developed. Horseradish peroxidase (HRP)-labelled anti-CAP monoclonal antibody conjugated with AuNPs and antigen-immobilized MBs were prepared. After optimization parameters of immunocomplex MBs, the IC50 values of chemiluminescence magnetic nanoparticles immunoassay (CL-MBs-nano-immunoassay) were 0.017 μg L-1 for extract method I and 0.17 μg L-1 for extract method II. The immunoassay with two extract methods was applied to detect CAP in milk. Comparison of these two extract methods showed that extract method I was advantageous in better sensitivity, in which the sensitivity was 10 times compared to that of extract method II, while extract method II was superior in simple operation, suitable for high throughout screen. The recoveries were 86.7-98.0% (extract method I) and 80.0-103.0% (extract method II), and the coefficients of variation (CVs) were all <15%. The satisfactory recovery with both extract methods and high correlation with traditional ELISA kit in milk system confirmed that the immunomagnetic assay based on AuNPs exhibited promising potential in rapid field screening for trace CAP analysis. © 2013 John Wiley & Sons, Ltd. Source

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