Establishment of a sensitive time-resolved fluoroimmunoassay for detection of Bacillus thuringiensis Cry1Ie toxin based nanobody from a phage display library

文献类型： 外文期刊

第一作者： Xu, Chongxin

作者： Xu, Chongxin;Zhang, Cunzheng;Zhang, Xiao;Zhong, Jianfeng;Liu, Yuan;Hu, Xiaodan;Lin, Manman;Liu, Xianjin;Liu, Xiaoqin

作者机构：

关键词： Cry1Ie toxin;Phage display library;Nanobody;Time-resolved fluoroimmunoassay (TRFIA)

期刊名称：ANALYTICAL BIOCHEMISTRY （ 影响因子：3.365； 五年影响因子：3.049 ）

ISSN： 0003-2697

年卷期： 2017 年 518 卷

页码：

收录情况： SCI

摘要： Cry1Ie toxin was an insect-resistant protein used in genetically modified crops (GMC). In this study, a large human VH gene nanobodies phage displayed library was employed to select anti-Cry1Ie toxin antibody by affinity panning. After 5 rounds of panning, total 12 positive monoclonal phage particles were obtained. One of the identified positive phage nanobody was expressed in E.coli BL21 and the purified protein was indicated as a molecular mass of approximately 20 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then a sensitive indirect competitive time-resolved fluoroimmunoassay (IC-TRFIA) was established for detection of Cry1Ie toxin by the purified protein. The working range of detection for Cry1Ie toxin standards in the IC-TRFIA were 0.08-6.44 ng mL(-1) and the medium inhibition of control (IC50) was 0.73 ng mL(-1). It showed a weak cross-reactivity with Cry1Ab toxin (at 5.6%), but did not recognize Cry1B, Cry1C, Cry1F, and Cry2A toxins (were <0.1%). The average recoveries of Cry1Ie toxin from respectively spiked in rice, corn and soil samples were in the range of 83.5%-96.6% and with a coefficient of variation (CV) among 2.0%-8.6%. These results showed the IC-TRFIA was promising for detection of Cry1Ie toxin in agricultural and environmental samples. (C) 2016 Elsevier Inc. All rights reserved.

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