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

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• 作者：Chongxin Xu<sup>asup> ; Xiaoqin Liu<sup>bsup> ; Cunzheng Zhang<sup>asup> ; Xiao Zhang<sup>asup> ; Jianfeng Zhong<sup>asup> ; Yuan Liu<sup>asup> ; Xiaodan Hu<sup>asup> ; Manman Lin<sup>asup> ; Xianjin Liu<sup>asup><sup> ; sup><sup>hhxyxcx@163.comsup>
• 关键词：Cry1Ie toxin ; Phage display library ; Nanobody ; Time-resolved fluoroimmunoassay (TRFIA)
• 刊名：Analytical Biochemistry
• 出版年：2017
• 出版时间：1 February 2017
• 年：2017
• 卷：518
• 期：Complete
• 页码：53-59
• 全文大小：547 K
• 卷排序：518

文摘

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.