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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


Wang Z.,China Agricultural University | Wang Z.,Beijing Laboratory for Food Quality and Safety | Liang Q.,China Agricultural University | Wen K.,China Agricultural University | And 5 more authors.
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2014

The application of antibodies to small molecules in the field of bioanalytics requires antibodies with stable biological activity and high purity; thus, there is a growing interest in developing rapid, inexpensive and effective procedures to obtain such antibodies. In this work, a ractopamine (RAC) derivative, N-4-aminobutyl ractopamine (ABR), was synthesized for preparing new specific affinity chromatography to purify a murine monoclonal antibody (mAb) against RAC from ascites. The performance of the new specific chromatography was compared with four other purification methods in terms of recovery, purity and biological activity of mAb. These four purification methods were prepared by using specific ligands (RAC and RAC-ovalbumin) and commercial ligands (protein G and protein A), respectively. The results showed that the highest recovery (88.1%) was achieved using the new chromatography; in comparison, the recoveries from the other methods were all below 70%. The purity of the mAbs from the new chromatography was 88.3%, while, the highest purity of 97.6% was from protein G chromatography and the lowest purity of 84.7% was from protein A chromatography. The biological activity of the purified mAb from all of the chromatography methods was comparable in enzyme-linked immunosorbent immunoassay (ELISA). © 2014 Elsevier B.V. Source


Wang Z.,China Agricultural University | Wang Z.,Beijing Laboratory for Food Quality and Safety | Zhang H.,China Agricultural University | Ni H.,China Agricultural University | And 4 more authors.
Analytica Chimica Acta | Year: 2014

In the paper, an enzyme-linked immunosorbent immunoassay (ELISA) for detection of enrofloxacin was described using one new derivative of enrofloxacin as coating hapten, resulting in surprisingly high sensitivity and specificity. Incorporation of aminobutyric acid (AA) in the new derivative of enrofloxacin had decreased the IC50 of the ELISA for enrofloxacin from 1.3μgL-1 to as low as 0.07μgL-1. The assay showed neglect cross-reactivity for other fluoroquinolones but ofloxacin (8.23%), marbofloxacin (8.97%) and pefloxacin (7.29%). Analysis of enrofloxacin fortified chicken muscle showed average recoveries from 81 to 115%. The high sensitivity and specificity of the assay makes it a suitable screening method for the determination of low levels of enrofloxacin in chicken muscle without clean-up step. © 2014 Elsevier B.V. Source


Yang H.,China Agricultural University | Dai R.,Beijing Laboratory for Food Quality and Safety | Zhang H.,Beijing Laboratory for Food Quality and Safety | Li C.,Beijing Key Laboratory of Detection Technology for Animal Derived Food Safety | And 4 more authors.
Analytical and Bioanalytical Chemistry | Year: 2016

Microcystins (MCs) and nodularin (NOD) are cyanobacterial hepatotoxins that can greatly harm human health. Multi-analyte immunoassays provide efficient and cheap methods of screening these toxins. To develop a multi-analyte immunoassay, an antibody with both broad specificity and high affinity for structurally similar algal toxins is urgently needed. In this study, microcystin–leucine–arginine (MC-LR) and NOD were conjugated to carrier proteins using a one-step active ester (AE) method and multistep thiol-ene click chemistry and glutaraldehyde method, respectively. The immunogens obtained from these two conjugation methods were evaluated for their effectiveness in producing antibodies. The results demonstrated that the antisera derived from AE immunogens showed better performance in terms of affinity and titer. Using this simple AE method, we prepared a new immunogen for NOD and successfully produced a monoclonal antibody (mAb), 2G5, which could recognize not only NOD but also all eight of the tested MCs (MC-LR, MC-RR, MC-YR, MC-WR, MC-LA, MC-LF, MC-LY, and MC-LW) with high sensitivity and improved uniform affinities (0.23 ≤ IC50 ≤ 0.68 ng mL−1) compared with previously described mAbs. Under optimal conditions, one indirect competitive enzyme-linked immunosorbent assay was developed based on mAb2G5 for the detection of MC-LR and NOD, with limits of detection of 0.16 and 0.10 μg L−1, respectively, and a recovery of 62–86 % with a coefficient of variation below 12.6 % in water samples. © 2016 Springer-Verlag Berlin Heidelberg Source

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