Monoclonal Antibody-Based ELISA and Colloidal Gold Immunoassay for Detecting 19-Nortestosterone Residue in Animal Tissues
Monoclonal Antibody-Based ELISA and Colloidal Gold Immunoassay for Detecting 19-Nortestosterone Residue in Animal Tissues
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DOI：
10.1021/jf2012437
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发表时间：
2011-09-28
影响因子：
6.1
通讯作者：
Wang, Sanhu
中科院分区：
文献类型：
--
作者：
Jiang, Jinqing;Wang, Ziliang;Wang, Sanhu
本文介绍了通过细胞融合技术产生高特异性抗19-去甲睾酮（NT）的单克隆抗体（mab），并建立了基于单克隆抗体的间接竞争性ELISA （icELISA）方法和基于胶体金的免疫色谱法检测牛肉和猪肉样品中NT残留的方法。采用改良的碳二亚胺法合成人工抗原，用BALB/c小鼠制备抗nt单克隆抗体。在棋盘格滴定法的基础上，建立了间接竞争ELISA标准曲线。该方法灵敏度高，在磷酸盐缓冲盐水（PBS）中线性范围为0.03 ~ 38 ng/mL， IC50和LOD值分别为0.52 ng/mL和0.01 ng/mL。在所有竞争性类似物中，所制备的单抗对17 α -去甲睾酮（NT在动物组织中的主要代谢物）具有较高的交叉反应性（83.6%）。除了与trenbolone（22.6%）和-boldenone（13.8%）有中度交叉反应外，其他干扰可以忽略不计(
This article presents the generation of monoclonal antibodies (mAbs) with high specificity against 19-nortestosterone (NT) through cell fusion techniques and the development of a mAb-based indirect competitive ELISA (icELISA) method and colloidal gold-based immuno-chromatographic assay to detect NT residues in beef and pork samples. A modified carbodiimide method was employed to synthesize the artificial antigen, and BALB/c mice were used to produce anti-NT mAbs. On the basis of the checkerboard titration, an indirect competitive ELISA standard curve was established. This assay was sensitive and had a linear range from 0.03 to 38 ng/mL in phosphate buffered saline (PBS), with IC50 and LOD values of 0.52 ng/mL and 0.01 ng/mL, respectively. Of all the competitive analogues, the produced mAb exhibited a high cross-reactivity to 17 alpha-nortestosterone (83.6%), the main metabolite of NT in animal tissues. Except for moderate cross-reactivities with trenbolone (22.6%) and beta-boldenone (13.8%), the other interference to the assay was negligible (