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Yu X.,China Agricultural University | Tao X.,Southwest University | Shen J.,China Agricultural University | Shen J.,National Reference Laboratory for Veterinary Drug Residues | And 7 more authors.
Analytical Methods | Year: 2015

A one-step generic chemiluminescence competitive direct enzyme immunoassay (CL-cdELISA) based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein (CL-cdELISAscFv-AP) was developed. It was capable of detecting 20 targeted fluoroquinolones (FQs) in fish and shrimp matrices within 40 min below the maximum residue levels (MRLs). In the optimized generic assay, the scFv-AP fusion protein in combination with a norfloxacin-ovalbumin conjugate (NOR-OVA) showed 50% binding inhibition (IC50) at 0.15 ± 0.01 μg kg-1 for NOR in 0.01 M phosphate-buffered saline (PBS), indicating that it is seven times as sensitive as the corresponding competitive direct enzyme immunoassay (cdELISAscFv-AP), and the linear response range of the assay was extended from 0.04 to 1.08 μg kg-1. The limits of detection (LODs) of the assay for NOR were 0.017 μg kg-1 in shrimp and 0.018 μg kg-1 in fish, and the LODs inferred from the cross reactivity (CR) ranged from 0.013 μg kg-1 for ciprofloxacin (CIP) to 4.19 μg kg-1 for trovafloxacin (TRO); the recoveries of the three representative antibiotics norfloxacin, flumequine (FLU) and sarafloxacin (SAR) from spiked fish and shrimp samples varied from 72.50 to 118.50% and the mean coefficients of variation for the inter-assay and intra-assay were 6.4% and 9.2%, respectively. Further validation of CL-cdELISAscFv-AP with high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmed that the assay was a reliable screening tool for the detection of FQs in fish and shrimp. © 2015 The Royal Society of Chemistry. Source


Zhang X.,China Agricultural University | Wen K.,China Agricultural University | Wen K.,Beijing Key Laboratory of Detection Technology for Animal Derived Food Safety | Wang Z.,China Agricultural University | And 7 more authors.
Food Control | Year: 2016

A rapid lateral flow fluorescent microsphere immunochromatographic test strip (FM-ICTS) assay has been developed for the detection of aflatoxin M1 (AFM1) residues in milk. For this purpose, an ultra-sensitive anti-AFM1 monoclonal antibody (MAb) 1D3 was prepared and identified. The IC50 value of the MAb 1D3 for AFM1 was 23 ng/L, and the cross-reactivity (CR) of the MAb with aflatoxin M2, aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatoxin G2 was 18%, 9%, <0.15%, 43% and <0.3%, respectively. The MAb was covalently conjugated with carboxylate-modified FMs, and the FMs were used as the label in a competitive immunochromatography assay. Using a moderate hapten-to-protein coupling ratio (~2) in the coating antigen resulted in improved immunoassay sensitivity. Under optimal conditions, the IC50 value of the assay was 36.3 ng/L with the limit of detection (LOD) of 4.4 ng/L in milk samples using a fluorescence reader, and the recoveries of AFM1 in spiked milk samples ranged from 76.6 to 110.8% with coefficient of variation (CV) of 4-14.7%. The whole procedure could be completed within 30 min. The results demonstrated that the FM-ICTS assay is easy to conduct, rapid, highly sensitive and specific for the detection of AFM1 residues in milk. © 2015 Elsevier Ltd. Source


Li C.,China Agricultural University | Wen K.,China Agricultural University | Mi T.,Northwest University, China | Zhang X.,China Agricultural University | And 6 more authors.
Biosensors and Bioelectronics | Year: 2016

Multi-analyte immunoassays have attracted increasing attention due to their short assay times, low sample consumption, and reduced detection costs per assay. In this work, we describe a homologous and high-throughput multi-wavelength fluorescence polarization immunoassay (MWFPIA) for the multiplexed detection of mycotoxins. Three typical Fusarium mycotoxins, deoxynivalenol (DON), T-2 toxin and fumonisin B1 (FB1), were labeled with different dyes. Tracers and specific monoclonal antibodies (mAbs) were employed in the MWFPIA to simultaneously detect the three mycotoxins. Under optimal conditions, the limits of detection using MWFPIA were 242.0μgkg-1 for DON, 17.8 μg kg-1 for T-2 toxin and 331.5μg kg-1 for FB1, providing sufficient sensitivity to meet the action levels of these three contaminants in maize as set by the European Union. The use of a methanol/water (2:3, v/v) mixture for sample pretreatment allowed recoveries ranging from 76.5-106.3%, with coefficients of variation less than 21.7%. The total time of analysis, including sample preparation, was less than 30 min. Twenty naturally contaminated maize samples were tested using MWFPIA and HPLC-MS/MS, with correlation coefficients (R2) of 0.97 for DON and 0.99 for FB1. By changing the targets of interest, homologous MWFPIA, a method with high sensitivity, a simple procedure and a short analysis time, can easily be extended to other chemical contaminants. Thus, MWFPIA represents a versatile strategy for food safety analysis. © 2015 Elsevier B.V. Source


Li C.,China Agricultural University | Mi T.,China Agricultural University | Mi T.,Northwest University, China | Oliveri Conti G.,University of Catania | And 7 more authors.
Journal of Agricultural and Food Chemistry | Year: 2015

This paper reports the development of a screening fluorescence polarization immunoassay (FPIA) for the simultaneous detection of fumonisins B1 (FB1) and B2 (FB2) in maize. Three FB1 tracers including FB1-fluorescein isothiocyanate isomer I (FB1-FITC), FB1-5-([4,6-dichlorotriazine-2-yl]amino)-fluorescein (FB1-5-DTAF), and FB1-Texas Red-X succinimidyl ester (FB1-TRX) were synthesized and studied to select appropriate tracer-antibody pairs using seven previously produced monoclonal antibodies (mAbs). An FPIA employing the pair of FB1-FITC and mAb 4B9 showing 98.9% cross-reactivity (CR) toward FB2 was used to simultaneously detect FB1 and FB2. Maize flour samples were extracted with methanol/water (2:3, v/v). After optimization, the FPIA revealed a limit of detection (LOD) of 157.4 μg/kg for FB1 and an LOD of 290.6 μg/kg for FB2, respectively. Recoveries were measured for spiked samples of FB1 or FB2 separately, ranging from 84.7 to 93.6%, with a coefficient of variation (CV) of <9.9%. Total time needed for FPIA including sample pretreatment was <30 min. The FPIA was used to screen naturally contaminated maize samples. Results detected by FPIA showed good agreement with that of HPLC-MS/MS with a fit of R2 = 0.99 for the simultaneous detection of FB1 and FB2. The established method offered a rapid, simple, sensitive, and high-throughput screening tool for the detection of fumonisins in maize. © 2015 American Chemical Society. Source


Li C.,China Agricultural University | Li J.,China Agricultural University | Jiang W.,China Agricultural University | Jiang W.,Shenzhen University | And 6 more authors.
Journal of Agricultural and Food Chemistry | Year: 2015

Salbutamol (SAL) and ractopamine (RAC) have been illegally used to promote protein synthesis and to increase the feed conversion rate in livestock. However, the residues of SAL and RAC could cause potential hazards for human health. The Ministry of Agriculture of China banned the use of SAL and RAC as growth promoters. In this paper, we provide detailed information on developing a rapid and sensitive gel-based immunoassay for on-site screening of SAL and RAC residues in pork. The detection time was shortened to 20 min. The limits of detection were 0.5 μg/kg for both SAL and RAC by visual detection, whereas the quantitative gel-based immunoassay enabled the detection of SAL (0.051 μg/kg) and RAC (0.020 μg/kg) in spiked pork samples. The gel-based immunoassay showed promise as a multiplexed immunoassay for on-site surveilling of SAL and RAC residues in pork. © 2015 American Chemical Society. Source

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