Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of Ministry of Science and Technology, Institute of Food Safety and Nutrition, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China; School of Food and Biological Engineering, Jiangsu University, Zhenjiang, 212013, China; College of Plant Protection, Nanjing Agricultural University, Nanjing, 210023, China.
Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of Ministry of Science and Technology, Institute of Food Safety and Nutrition, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China.
Anal Biochem. 2024 Oct;693:115584. doi: 10.1016/j.ab.2024.115584. Epub 2024 Jun 4.
Using the amino acid sequences and analysis of selected known structures of Bt Cry toxins, Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C and Cry1F we specifically designed immunogens. After antibodies selection, broad-spectrum polyclonal antibodies (pAbs) and monoclonal antibody (namely 1A0-mAb) were obtained from rabbit and mouse, respectively. The produced pAbs displayed broad spectrum activity by recognizing Cry1 toxin, Cry2Aa, Cry2Ab and Cry3Aa with half maximal inhibitory concentration (IC) values of 0.12-9.86 μg/mL. Similarly, 1A0-mAb showed broad spectrum activity, recognizing all of the above Cry protein (IC values of 4.66-20.46 μg/mL) with the exception of Cry2Aa. Using optimizations studies, 1A10-mAb was used as a capture antibody and pAbs as detection antibody. Double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) were established for Cry1 toxin, Cry2Ab and Cry3Aa with the limit of detection (LOD) values of 2.36-36.37 ng/mL, respectively. The present DAS-ELISAs had good accuracy and precisions for the determination of Cry toxin spiked tap water, corn, rice, soybeans and soil samples. In conclusion, the present study has successfully obtained broad-spectrum pAbs and mAb. Furthermore, the generated pAbs- and mAb-based DAS-ELISAs protocol can potentially be used for the broad-spectrum monitoring of eight common subtypes of Bt Cry toxins residues in food and environmental samples.
利用氨基酸序列和对选定的已知 Bt Cry 毒素结构的分析，我们专门设计了 Cry1Ab、Cry1Ac、Cry1Ah、Cry1B、Cry1C 和 Cry1F 的免疫原。经过抗体选择，从兔子和老鼠中分别获得了广谱多克隆抗体（pAbs）和单克隆抗体（即 1A0-mAb）。所产生的 pAbs 通过识别 Cry1 毒素、Cry2Aa、Cry2Ab 和 Cry3Aa，显示出广谱活性，半数最大抑制浓度（IC）值为 0.12-9.86μg/mL。同样，1A0-mAb 显示出广谱活性，识别所有上述 Cry 蛋白（IC 值为 4.66-20.46μg/mL），除了 Cry2Aa。通过优化研究，1A10-mAb 被用作捕获抗体，pAbs 用作检测抗体。建立了用于 Cry1 毒素、Cry2Ab 和 Cry3Aa 的双抗体夹心酶联免疫吸附测定（DAS-ELISA），检测限（LOD）值分别为 2.36-36.37ng/mL。本研究建立的 DAS-ELISA 对添加自来水中的 Cry 毒素、玉米、水稻、大豆和土壤样品具有良好的准确性和精密度。总之，本研究成功获得了广谱 pAbs 和 mAb。此外，生成的基于 pAbs 和 mAb 的 DAS-ELISA 方案可用于广泛监测食品和环境样品中八种常见 Bt Cry 毒素残留。
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