Zhao Jing, Li Peipei, Abd El-Aty A M, Xu Lingyuan, Lei Xingmei, Gao Song, Li Jia, Zhao Yun, She Yongxin, Jin Fen, Wang Jing, Hammock Bruce D, Jin Maojun
Institute of Quality Standard and Testing Technology for Agro-Products, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Department of Pharmacology, Faculty of Veterinary Medicine, Cairo University, Giza 12211, Egypt.
Chem Eng J. 2024 Jul 1;491. doi: 10.1016/j.cej.2024.152039. Epub 2024 May 7.
Immunoassays have been widely used to determine small-molecule compounds in food and the environment, meeting the challenge of obtaining false positive or negative results because of the variance in the batches of antibodies and antigens. To resolve this problem, atrazine (ATR) was used as a target, and anti-idiotypic nanobodies for ATR (AI-Nbs) and a recombinant full-length antibody against ATR (ATR-rAb) were prepared for the development of a sustainable enzyme-linked immunosorbent assay (ELISA). AI-Nb-7, AI-Nb-58, and AI-Nb-66 were selected from an immune phage display library. ATR-rAb was produced in mammalian HEK293 (F) cells. Among the four detection methods explored, the assay using AI-Nb-66 as a coating antigen and ATR-rAb as a detection reagent yielded a half maximal inhibitory concentration (IC) of 1.66 ng mL for ATR and a linear range of 0.35-8.73 ng mL. The cross-reactivity of the assay to ametryn was 64.24%, whereas that to terbutylazine was 38.20%. Surface plasmon resonance (SPR) analysis illustrated that these cross-reactive triazine compounds can bind to ATR-rAb to varying degrees at high concentrations; however, the binding/dissociation kinetic curves and the response values at the same concentration are different, which results in differences in cross-reactivity. Homology modeling and molecular docking revealed that the triazine ring is vital in recognizing triazine compounds. The proposed immunoassay exhibited acceptable recoveries of 84.40-105.36% for detecting fruit, vegetables, and black tea. In conclusion, this study highlights a new strategy for developing sustainable immunoassays for detecting trace pesticide contaminants.
免疫测定法已广泛用于测定食品和环境中的小分子化合物，解决了因抗体和抗原批次差异而导致假阳性或假阴性结果的难题。为解决这一问题，以莠去津（ATR）为靶点，制备了针对ATR的抗独特型纳米抗体（AI-Nbs）和抗ATR重组全长抗体（ATR-rAb），用于开发可持续的酶联免疫吸附测定法（ELISA）。从免疫噬菌体展示文库中筛选出AI-Nb-7、AI-Nb-58和AI-Nb-66。ATR-rAb在哺乳动物HEK293（F）细胞中产生。在所探索的四种检测方法中，以AI-Nb-66为包被抗原、ATR-rAb为检测试剂的测定法对ATR的半数抑制浓度（IC）为1.66 ng/mL，线性范围为0.35 - 8.73 ng/mL。该测定法对莠灭净的交叉反应率为64.24%，对特丁津的交叉反应率为38.20%。表面等离子体共振（SPR）分析表明，这些具有交叉反应性的三嗪化合物在高浓度下能不同程度地与ATR-rAb结合；然而，相同浓度下的结合/解离动力学曲线和响应值不同，这导致了交叉反应率的差异。同源建模和分子对接显示，三嗪环对于识别三嗪化合物至关重要。所提出的免疫测定法在检测水果、蔬菜和红茶时的回收率为84.40 - 105.36%，结果可接受。总之，本研究突出了一种开发用于检测痕量农药污染物的可持续免疫测定法的新策略。
