[Show abstract][Hide abstract] 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.
Analytical and Bioanalytical Chemistry 07/2013; · 3.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUD: 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 in optimum condition. 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 a 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 chromatography-tandem mass spectrometry (LC-MS/MS). 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.
Journal of the Science of Food and Agriculture 06/2013; · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A pretreatment method was developed for the determination of four nitrofuran metabolites and chloramphenicol in pork, chicken, fish, and shrimp. Homogenized samples were hydrolyzed and derivatized with 2-nitrobenzaldehyde. Extraction was performed using ethyl acetate followed by purification of the extract by hexane. Lastly, the ethyl acetate was dried under nitrogen and the residue was redissolved for analysis. The performance of the method was satisfactory for all drugs tested at contamination levels close to or below the relevant European Union maximum levels permitted. The limits of detection (LODs) of the method were 0.025–0.13 ng/g. Recoveries higher than 72.0% were obtained for all drugs tested, and the coefficient of variation was less than 15%. Results from analysis of unknown samples by the developed ELISA were similar to those obtained by a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method
[Show abstract][Hide abstract] ABSTRACT: A novel chemiluminescent immunoassay utilising two types of primary antibodies (murine monoclonal antibody and rabbit polyclonal antibody) and two types of horseradish peroxidase-labelled secondary antibodies was established for simultaneously detecting multiple amphenicol residues in ham sausage. After combining the extract procedure of the target amphenicol into one simplified method, this hybrid chemiluminescent immunoassay could screen chloramphenicol (CAP), florfenicol (FF) and its metabolite florfenicol amine (FFA) at the same time by adding the corresponding secondary antibody. Ham sausage samples were analysed by using this hybrid immunoassay, with LODs of CAP being 0.01 μg kg(-1), of FF being 2.8 μg kg(-1) and of FFA being 3.0 μg kg(-1). The applicability of the proposed method has been validated by determining CAP, FF and FFA in ham sausage samples with satisfactory results. Good recoveries and high correlation with traditional enzyme-linked immunosorbent assay and LC-MS/MS results illustrated that the developed hybrid chemiluminescent immunoassay could screen high-throughput ultra-trace amphenicol residues effectively at one time.
Food Additives and Contaminants - Part A Chemistry, Analysis, Control, Exposure and Risk Assessment 04/2013;
[Show abstract][Hide abstract] ABSTRACT: A competitive, direct, chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for chloramphenicol (CAP) residues in milk, milk powder, honey, eggs and chicken muscle has been developed. The method gave a detection limit of 0.7 ng L−1 and a linear range of 2.1–92.4 ng L−1, with the IC50 of 13.6 ng L−1 under optimal conditions, dramatically better than any previously reported ELISA method for CAP detection. Spiked at levels of 5–60 ng L−1 in different food samples, recoveries were in the range of 72.1–116.0%, with coefficient of variations of 4.2–20.2%. In a study of incurred residues, the chicken muscle samples diluted 5-, 10- and 20-fold, results obtained by CL-ELISA correlated well with those obtained by gas chromatography with microcell electron capture detector and traditional ELISA. The developed CL-ELISA method is, therefore, suitable for rapid screening trace CAP residues in food samples
Food and Agricultural Immunology 02/2013; · 0.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A competitive indirect chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for florfenicol (FF) and its major metabolite florfenicol amine (FFA) residues in chicken muscle has been developed and validated according to Commission Decision 2002/657/EC criteria. The IC50 value of the method was 0.153 μg kg−1 for FFA with a cross-reactivity of 74.3% for FF under optimum conditions, in which FFA–F–BSA (FFA–formaldehyde–BSA) and FF–G–OVA (FF–glutaric anhydride–OVA) were used as an immunogen and a coating antigen, respectively. FFA and FF were easily extracted from chicken muscle with a 40:1 ethyl acetate–ammonia mixture, obtaining recoveries of 70.3–100% (FFA) and 71.8–102.0% (FF). Accuracy, precision, selectivity, robustness, limit of detection (LOD), limit of quantification (LOQ) and detection capability (CCβ) of the assay have been assessed during the validation process. LOD values in chicken muscle were 0.353 μg kg−1 for FFA and 0.526 μg kg−1 for FF (10-fold dilution) and 0.453 μg kg−1 for FFA and 0.657 μg kg−1 for FF (100-fold dilution). Furthermore, the CL-ELISA method gave CCβ values of 1.0 μg kg−1 for FFA and FF. Finally, real chicken muscle samples were analyzed with the CL-ELISA method, traditional ELISA and a previously reported gas chromatography-negative chemical ionization mass spectrometry (GC-MS), and results confirmed the utility of this new CL-ELISA for trace determination of FF and FFA, simultaneously.
[Show abstract][Hide abstract] ABSTRACT: A competitive indirect chemiluminescent enzyme-liked immunoassay (CL-ELISA) for chloramphenicol (CAP) residues in milk and chicken muscle has been developed. Due to the unique characteristic of the polyclonal antibody, special reaction system and modified extract method, then after optimization (concentration of Tween-20, concentration of PB and pH, incubation time and temperature), the method gave a detection limit of 0.92 ng/L and a detection range of 3.16-3035 ng/L, with the IC50 of 17.29 ng/L in optimum condition and real sample matrix. When CAP was spiked in milk and chicken muscle at levels of 5-100 ng/L, recoveries ranged from 104.9%-114.8% and 101.0%-118.8%, with coefficients of variation of 3.0%-14.6% and 9.5%-14.4%, respectively. In an actual chicken muscle residue study, although the extract of samples diluted 10-fold, or even 100-fold, which represents extremely lower concentration of CAP, the results obtained by CL-ELISA correlated well with those obtained by gas chromatography with microcell electron capture detector. The developed method is therefore suitable for screening of ultratrace CAP residues in milk and chicken muscle samples.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: A simple, rapid and sensitive immunogold chromatographic strip test based on a monoclonal antibody was developed for the detection of melamine (MEL) residues in raw milk, milk products and animal feed. The limit of detection was estimated to be 0.05 μg/mL in raw milk, since the detection test line on the strip test completely disappeared at this concentration. The limit of detection was 2 μg/mL (or 2 μg/g) for milk drinks, yogurt, condensed milk, cheese, and animal feed and 1 μg/g for milk powder. Sample pretreatment was simple and rapid, and the results can be obtained within 3-10 min. A parallel analysis of MEL in 52 blind raw milk samples conducted by gas chromatography-mass spectrometry showed comparable results to those obtained from the strip test. The results demonstrate that the developed method is suitable for the onsite determination of MEL residues in a large number of samples.
Journal of Agricultural and Food Chemistry 05/2011; 59(11):6064-70. · 3.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A rapid and sensitive colloidal gold immunochromatography test strip based on one monoclonal antibody with broad-specificity, which can detect 12 fluoroquinolones (FQs), was developed. Antigen and goat anti-mouse IgG were respectively drawn on NC membrane as test line and control line. Gold-labeled antibody was added on a pad and put on one end of the membrane. Fluoroquinolones in sample solution compete with antigen combined on NC membrane for the gold-labeled antibody. When enough fluoroquinolone exists, the test line vanishes as there are no spare gold-labeled antibodies that can bind with antigen on the membrane. The control line always exists when the antibody is activated. The lowest detection limits of the FQs in spiked chicken muscle and chicken liver samples were 25 ng mL(-1) for norfloxacin and pefloxacin. The lowest detection limit for the other 10 FQs (enrofloxacin, ciprofloxacin, norfloxacin, flumequine, pefloxacin, ofloxacin, lomefloxacin, enoxacin, danofloxacin, amifloxacin, oxolinic acid, and marbofloxacin) was 50 ng mL(-1). The whole process involving sample preparation and detection can be finished in <10 min. The results demonstrate that the developed method can be potentially used as a screening tool for the determination of 12 FQ residues in a large amount of samples on site.
Journal of Agricultural and Food Chemistry 07/2008; 56(14):5469-74. · 3.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We developed an enzyme-linked immunoassay that provides rapid and sensitive detection of gentamicin in swine tissues. Rabbit was immunized with gentamicin-BSA conjugate and antiserum was collected after the fifth immunization. After optimizing the concentration of immunoreagents, competitive indirect ELISA (ciELISA) gave an IC50 value of 0.98 ng/ml, while competitive direct ELISA (cdELISA) exhibited lower IC50 value of 0.92 ng/ml, thus cdELISA was further optimized under various pH values and ionic strengths of assay buffer, different coating methods and incubation time. The optimized ELISA can be completed within 45 min and it showed negligible cross-reactivity with other aminoglycosides. The recoveries of gentamicin from spiked swine tissues at levels of 25–200 μg/kg ranged from 64.7% to 101.2% with CVs of 4.5–12.1%, and the detection limits were 6.2 μg/kg in muscle, 3.6 μg/kg in liver and 2.7 μg/kg in kidney, respectively.