Shen Cheng, Meng Meng, Jin Jiafeng, Hu Xiaodan, Lin Manman, Gao Meijing, Zhang Xiao, Zhu Qing, Xu Chongxin, Xie Yajing, Chen Chengyu, Zhang Zhiyong, Che Guojing, Pooe Ofentse Jacob, Liu Yuan, Liu Xianjin
Key Laboratory of Food Quality and Safety of Jiangsu Province, Nanjing 210014, China; College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China.
Key Laboratory of Food Quality and Safety of Jiangsu Province, Nanjing 210014, China; School of Life Sciences, Jiangsu University, Zhenjiang 212013, China.
Int J Biol Macromol. 2024 Jan;254(Pt 3):128034. doi: 10.1016/j.ijbiomac.2023.128034. Epub 2023 Nov 14.
Bacillus thuringiensis (Bt) Cry toxins have been widely used in the development of genetically modified organisms (GMOs) for pest control. This work aimed to establish more cost effective and broader detection methods for commonly used Cry toxins. Using ligand blot and bio-layer interferometry, we confirmed that a recombinant toxin-binding fragments derived from Helicoverpa armigera cadherin-like protein (HaCad-TBR) could broadly bind Cry1Ab, Cry1Ac, Cry2Aa, and Cry2Ab with the affinity of 0.149, 0.402, 120, and 4.12 nM, respectively. Based on the affinity results, a novel receptor-antibody sandwich assay broadly detecting Cry1A and Cry2 toxins was developed by using HaCad-TBR as capture molecules, and anti-Cry1A/Cry2A polyclonal antibodies (pAbs) as the detection antibodies. The detection limit (LOD) for Cry1Ab, Cry1Ab, Cry2Aa, and Cry2Ab were 5.30, 5.75, 30.83 and 13.70 ng/mL. To distinguish Cry1A and Cry2A toxins in a singular test, anti-Cry1A pAbs and anti-Cry2A pAbs were labelled with different quantum dots (QDs). The LOD for the four toxins by receptor-QDs-pAbs sandwich assay were calculated to be 1.36, 4.71, 17.48, and 7.54 ng/mL, respectively. The two developed methods were validated by spiked rice and corn samples, suggesting they may potentially be used in monitoring and quantifying Cry toxins in food and environment.
苏云金芽孢杆菌（Bt）Cry毒素已广泛应用于转基因生物（GMO）的害虫防治研发中。这项工作旨在建立更具成本效益且适用范围更广的常用Cry毒素检测方法。利用配体印迹法和生物层干涉术，我们证实了源自棉铃虫类钙黏蛋白（HaCad-TBR）的重组毒素结合片段能够广泛结合Cry1Ab、Cry1Ac、Cry2Aa和Cry2Ab，其亲和力分别为0.149、0.402、120和4.12 nM。基于亲和力结果，开发了一种以HaCad-TBR作为捕获分子，抗Cry1A/Cry2A多克隆抗体（pAbs）作为检测抗体的新型受体-抗体夹心检测法，可广泛检测Cry1A和Cry2毒素。Cry1Ab、Cry1Ac、Cry2Aa和Cry2Ab的检测限（LOD）分别为5.30、5.75、30.83和13.70 ng/mL。为了在单一检测中区分Cry1A和Cry2A毒素，将抗Cry1A pAbs和抗Cry2A pAbs用不同的量子点（QDs）标记。受体-QDs-pAbs夹心检测法对这四种毒素的LOD分别计算为1.36、4.71、17.48和7.54 ng/mL。这两种开发的方法通过添加到水稻和玉米样品中进行了验证，表明它们可能潜在地用于监测和定量食品和环境中的Cry毒素。
