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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.

Wang Z.,China Agricultural University | Wang Z.,National Reference Laboratories for Veterinary Drug Residue | Luo P.,China National Institute for Nutrition and Food Safety | Cheng L.,China Agricultural University | And 5 more authors.
Journal of Molecular Recognition | Year: 2011

The molecular recognition of hapten-antibody is a fundamental event in competitive immunoassay, which guarantees the sensitivity and specificity of immunoassay for the detection of haptens. The aim of this study is to investigate the correlation between binding ability of one monoclonal antibody, 1H9B4, recognizing and the molecular aspects of α-zearalanol analogs. The mouse-derived monoclonal antibody was produced by using α-zearalanol conjugated to bovine serum albumin as an immunogen. The antibody recognition abilities, expressed as IC 50 values, were determined by a competitive ELISA. All of the hapten molecules were optimized by Density Function Theory (DFT) at B3LYP/ 6-31G * level and the conformation and electrostatic molecular isosurface were employed to explain the molecular recognition between α-zearalanol analogs and antibody 1H9B4. Pearson Correlation analysis between molecular descriptors and IC 50 values was qualitatively undertaken and the results showed that one molecular descriptor, surface of the hapten molecule, clearly demonstrated linear relationship with antibody recognition ability, where the relationship coefficient was 0.88 and the correlation was significant at p<0.05 level. The study shows that computational chemistry and Pearson Correlation analysis can be used as tool to help the immunochemistries better understand the processing of antibody recognition of hapten molecules in competitive immunoassay. © 2011 John Wiley &Sons, Ltd.

Zhang J.,China Agricultural University | Zhang J.,National Reference Laboratories for Veterinary Drug Residue | Wang Z.,China Agricultural University | Wang Z.,National Reference Laboratories for Veterinary Drug Residue | And 6 more authors.
Analytical Biochemistry | Year: 2013

The soluble form of penicillin-binding protein 3 (sPBP3*) from Streptococcus pneumoniae was expressed in Escherichia coli as a six-histidine fusion protein. The protein was purified and used to develop a microplate assay in direct competitive format for the detection of penicillins and cephalosporins in milk. The assay was based on competitive inhibition of the binding of horseradish peroxidase-labeled ampicillin (HRP-Amp) to the sPBP3* by free β-lactam antibiotics in milk. Under optimized conditions, most of the β-lactam antibiotics (11 penicillins and 16 cephalosporins) could be detected at concentrations corresponding to the maximum residue limits (MRLs) set by the European Union. Analysis of spiked milk samples showed that acceptable recoveries ranged from 74.06 to 106.31% in skimmed milk and from 63.97 to 107.26% in whole milk, with coefficients of variation (CVs) less than 16%. With the high sensitivity and wide-range affinities to penicillins and cephalosporins, the developed assay based on sPBP3* exhibited the potential to be a screening assay for fast detection of β-lactam antibiotics in milk.© 2013 Elsevier Inc. All rights reserved.

Zhang J.,China Agricultural University | Zhang J.,National Reference Laboratories for Veterinary Drug Residue | Wang Z.,China Agricultural University | Wang Z.,National Reference Laboratories for Veterinary Drug Residue | And 6 more authors.
Food Analytical Methods | Year: 2014

A new monoclonal antibody (mAb) against cephalexin (CEX), named 1F3, was produced by immunizing mice with immunogen CEX-keyhole limpet hemocyanin. Two fluorescein-labeled tracers (CEX-fluorescein isothiocyanate (FITC) and cefadroxil (CDX)-FITC) were synthesized and purified by thin-layer chromatography. A homogeneous fluorescence polarization immunoassay (FPIA) based on the new mAb and the selected tracer for the simultaneous determination of CEX and CDX was developed and optimized. The optimized FPIA provided a limit of detection of 1.8 and 2.3 ng ml-1 for CEX and CDX and exhibited cross-reactivities of 100 and 63.16 % for CEX and CDX, respectively, and <1 % for other 11 cephalosporins. Recoveries from fortified milk were ranged from 82 to 125 % with coefficient of variations of 3.2-14.2 %. The results indicated that the homogeneous FPIA we developed was enough sensitive to rapidly monitor CEX and CDX in milk. © 2013 Springer Science+Business Media New York.

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