Tianjin Key Laboratory of Food Science and Health, School of Medicine, Nankai University, Tianjin, 300071, China; State Key Laboratory of Food Nutrition and Safety, Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Tianjin University of Science and Technology, Tianjin, 300457, China; ARC Centre of Excellence in Nanoscale Biophotonics (CNBP), Macquarie University, North Ryde, 2109, Australia.
State Key Laboratory of Food Nutrition and Safety, Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Tianjin University of Science and Technology, Tianjin, 300457, China.
Anal Chim Acta. 2019 Jan 24;1047:139-149. doi: 10.1016/j.aca.2018.09.058. Epub 2018 Sep 26.
In order to develop a sensitive immunoassay for Aflatoxin B1 (AFB1) monitoring, a hybridoma secreting anti-AFB1 monoclonal antibody with high binding affinity was screened. A new type of CdTe/CdS/ZnS quantum dot was synthesized and conjugated with an artificial antigen for use as a fluorescent probe in a simple one-step fluorescence immunoassay (FLISA). The developed FLISA allowed a sensitive determination of AFB1 in cereal samples in a wide linear range, from 0.08 to 1.97 ng mL, with a detection limit of 0.01 ng mL in cereal samples. The corresponding immunoglobulin genes of the Fab fragment were cloned and sequenced, and expression of Fab was successfully verified in HEK293 cells, with a K value of 1.09 × 10 mol L for AFB1. To investigate the interactions between the antibody and AFB1, molecular docking, molecular dynamic simulation, and quantum-chemical computation were performed on AFB1 and a homology model of the Fab fragment. Our results showed that residues Ser32, Trp93, and Trp98 played the most important roles in the binding through hydrogen bonds formation, Pi-Pi stacked/Pi-alkyl interactions, and van der Waals interactions. In addition, the electrostatic potential study of AFB1 demonstrated that electrostatic interactions also played an important role in the recognition process. Results from theoretical studies provide guidance for hapten design and antibody improvement through genetic engineering.
为了开发用于黄曲霉毒素 B1 (AFB1) 监测的灵敏免疫分析方法，筛选出了一种分泌高亲和力抗 AFB1 单克隆抗体的杂交瘤细胞。合成了一种新型的 CdTe/CdS/ZnS 量子点，并将其与人工抗原偶联，用作简单一步荧光免疫分析（FLISA）中的荧光探针。开发的 FLISA 允许在谷物样品中在 0.08 至 1.97 ng mL 的宽线性范围内灵敏地测定 AFB1，在谷物样品中的检测限为 0.01 ng mL。克隆并测序了 Fab 片段的相应免疫球蛋白基因，并在 HEK293 细胞中成功验证了 Fab 的表达，AFB1 的 K 值为 1.09 × 10 mol L。为了研究抗体与 AFB1 之间的相互作用，对 AFB1 和 Fab 片段的同源模型进行了分子对接、分子动力学模拟和量子化学计算。我们的结果表明，通过氢键形成、Pi-Pi 堆积/Pi-烷基相互作用和范德华相互作用，残基 Ser32、Trp93 和 Trp98 在结合中起最重要的作用。此外，AFB1 的静电势能研究表明，静电相互作用在识别过程中也起着重要作用。理论研究结果为半抗原设计和通过遗传工程改进抗体提供了指导。
