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Xu F.,China Agricultural University | Jiang W.,China Agricultural University | Zhou J.,China Agricultural University | Wen K.,China Agricultural University | And 4 more authors.
Journal of Agricultural and Food Chemistry | Year: 2014

Apramycin (APR) residue in food of animal origin can cause harmful effects on human health. In this study, a monoclonal antibody (mAb) was successfully produced using APR-BSA as immunogen, which was prepared by using the glutaraldehyde method. mAb 2A2 showed low cross-reactivity (<0.1%) with other aminoglycoside antibiotics, and its IC50 value was 0.35 ng/mL. On the basis of this mAb, a novel immunoassay in the format of an immunoaffinity test column (IATC) was developed. An immunoaffinity column filled with anti-APR antibody-Sepharose 4B gel was used as solid phase. APR in sample and HRP-APR conjugate compete with each other for the limited antibody on the column. The approach was able to give a naked-eye color signal for the detection of analyte. A blue color appears for negative results and no color for positive. The method was then successfully applied to the detection of APR in animal-origin food. To further evaluate the assay, direct competitive ELISA (dcELISA) based on the same antibody was developed for comparison in different aspects. Compared to the dcELISA, the detection time of IATC is shortened to 20 min, whereas a similar sensitivity for various samples was observed. The limits of detection (LOD) for raw milk, muscles, and livers are 3 ng/mL, 3 μg/kg, and 10 μg/kg, respectively. © 2014 American Chemical Society.

Mauro D.,National Reference Laboratory for Residues of Veterinary Drugs | Ciardullo S.,National Reference Laboratory for Residues of Veterinary Drugs | Civitareale C.,National Reference Laboratory for Residues of Veterinary Drugs | Fiori M.,National Reference Laboratory for Residues of Veterinary Drugs | And 3 more authors.
Microchemical Journal | Year: 2014

Ultra performance liquid chromatography (UPLC) hyphenated to tandem mass spectrometry (MS/MS) was used for the development of an analytical method capable of simultaneous identification and quantification of 18 β-agonist compounds namely, brombuterol, chlorbrombuterol, cimaterol, cimbuterol, clenbuterol, clencyclohexerol, clenisopenterol, clenpenterol, clenproperol, hydroxymetylclenbuterol, isoxsuprine, mabuterol, mapenterol (clenbuterol-like compounds), ractopamine, ritodrine, salbutamol, salmeterol (salbutamol-like compounds) and zilpaterol in bovine urine. In compliance with the Commission Decision 2002/657/EC (CD 2002/657/EC), the method was validated applying a matrix-comprehensive in-house validation approach based on a fractional factorial design. Six experimental factors varied on two levels were selected for this purpose. The relevant validation parameters such as decision limit CCα (range, 0.24-0.51μgL-1) and detection capability CCβ (range, 0.27-0.71μgL-1), within-laboratory reproducibility (<20%) as well as recovery (range, 92-109%) were in agreement with the performance criteria set in the CD 2002/657/EC. Overall, the proposed method enabled both screening and confirmatory detection of the β-agonist compounds in the framework of official monitoring plans. © 2014 Elsevier B.V.

Zhu K.,National Reference Laboratory for Residues of Veterinary Drugs | Zhu K.,China Agricultural University | Li J.,National Reference Laboratory for Residues of Veterinary Drugs | Li J.,China Agricultural University | And 11 more authors.
Biosensors and Bioelectronics | Year: 2011

In this study, a novel immunoassay using 2 types of sensors (QDs and an enzyme) were simultaneously used for detecting multiple structurally different molecules in milk. The method integrates the fluorescence-linked immunosorbent assay (FLISA) using QD605 and QD655 as probes and an enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase (HRP) labeled secondary antibody. The FLISA was produced by anti-sulfonamide and anti-quinolone broad-specificity monoclonal antibodies (MAbs) for simultaneously detecting 6 sulfonamides and 11 quinolones. Combined with the FLISA, an ELISA was utilized for detecting melamine from the same milk samples. The cross-reactivity of the MAbs was retained while binding the QDs by using avidin and a secondary antibody as bridges. Milk samples were detected using this hybrid immunoassay, with limits of detection (LOD) of the quinolones (0.18ngmL-1), sulfonamides (0.17ngmL-1) and melamine (7.5ngmL-1), respectively. The results demonstrated that the detection limits of the integrated methods were better than required and simplified the sample pretreatment process. The developed immunoassay is suitable for high-throughput screening of low-molecular weight contaminants. © 2010 Elsevier B.V.

Wu J.,National Reference Laboratory for Residues of Veterinary Drugs | Wu J.,China Agricultural University | Xu F.,National Reference Laboratory for Residues of Veterinary Drugs | Xu F.,China Agricultural University | And 8 more authors.
Analytical Letters | Year: 2012

A rapid and sensitive indirect competitive fluorescence-linked immunosorbent assay (icFLISA) method was developed for detection of melamine in milk based on secondary antibody (Ab2)-conjugated quantum dots (QDs). The sensitivity of icFLISA with a limit of detection (LOD) of melamine (3.88 ng mL-1) and 50% inhibition value (IC50) (31.58 ng mL-1), was much higher than that of icELISA. As a proof of principle, recovery tests involving four fortification levels (150, 80, 40, and 20 ngmL-1) in blank samples were also performed. The recoveries ranged from 80.85 to 110.54% with coefficient of variations (CVs) of 2.82-8.82%. When challenged with authentic samples, these results of icFLISA were consistent with that of icELISA, immuno-chromatographic assay and gas chromatography-single quadruple mass spectrometry (GC-MS)method, indicating that icFLISA can be considered as a new screening method for detection of melamine in milk. © 2013 Copyright Taylor and Francis Group, LLC.

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