数字农科院2.0

Generation of a highly specific recombinant full-length antibody for detecting ethirimol in fruit and environmental water

文献类型： 外文期刊

作者： Xingmei Lei;Peipei Li;A. M. Abd El-Aty;Jing Zhao;Lingyuan Xu;Song Gao;Jia Li;Yun Zhao;Yongxin She;Fen Jin;Jing Wang;Lufei Zheng;Bruce D. Hammock;Maojun Jin

作者机构：

关键词： Ethirimol;Ic-ELISA;Monoclonal antibody;Recognition mechanism;Recombinant full-length antibody

期刊名称： Journal of Hazardous Materials

ISSN： 0304-3894

年卷期： 2024 年 469 卷

页码：

收录情况： SCIE（2024版） ; ; EI（2024版）

摘要： High-performance antibodies are core reagents for highly sensitive immunoassays. Herein, based on a novel hapten, a hybridoma secreting the high-affinity anti-ethirimol monoclonal antibody (mAb-14G5F6) was isolated with an IC50 value of 1.35 μg/L and cross-reactivity below 0.20% for 13 analogs. To further address the challenge of hybridoma preservation and antibody immortalization, a recombinant full-length antibody (rAb-14G5F6) was expressed using the HEK293(F) expression system based on the mAb-14G5F6 gene. The affinity, specificity, and tolerance of rAb-14G5F6, as characterized by indirect competitive enzyme-linked immunosorbent assay and noncompetitive surface plasmon resonance, exhibited high concordance with those of mAb-14G5F6. Further immunoassays based on rAb-14G5F6 were developed for irrigation water and strawberry fruit with limits of detection of 0.0066 and 0.036 mg/kg, respectively, recoveries of 80–100%, and coefficients of variation below 10%. Furthermore, homology simulation and molecular docking revealed that GLU(L40), GLY(L107), GLY(H108), and ASP(H114) play important roles in forming hydrogen bonds and pi-anion ionic bonds between rAb-14G5F6 and ethirimol, resulting in the high specificity and affinity of rAb-14G5F6 for ethirimol, with a KD of 5.71 × 10-10 mol/L. Overall, a rAb specific for ethirimol was expressed successfully in this study, laying the groundwork for rAb-based immunoassays for monitoring fungicide residues in agricultural products and the environment.

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