College of Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi 030801, PR China.
Beijing Key Laboratory of Biodiversity and Organic Farming, College of Resources and Environmental Sciences, China Agricultural University, Beijing, 100193, China.
Anal Methods. 2024 Mar 22;16(12):1756-1762. doi: 10.1039/d3ay01422c.
Single-chain variable fragments (scFvs) are valuable in the development of immunoassays for pesticide detection. In this study, scFvs specific to thiamethoxam (Thi) were successfully isolated from a library generated by chicken immunization through heterologous coating selection. These scFvs were subsequently expressed with fusion with an Avi tag and alkaline phosphatase. After combination and optimization, a scFv-biotin based enzyme linked immunosorbent assay (ELISA) was developed for the detection of Thi, demonstrating an impressive half-maximum signal inhibition concentration (IC) of 30 ng mL and a limit of detection (LOD) of 1.8 ng mL. The immunoassay exhibited minimal cross-reactivity with other neonicotinoid insecticides, except for 7.5% for imidacloprid and 6.7% for imidaclothiz. The accuracy of the assay was confirmed by testing spiked samples of apple, pear, cabbage, and cucumber, which resulted in average recoveries ranging between 82% and 119%, closely aligning with the results obtained through high-performance liquid chromatography. Therefore, the chicken scFv-biotin based assay showed promise as a high-throughput screening tool for Thi in agricultural samples.
单链可变片段 (scFvs) 在开发用于农药检测的免疫分析中具有重要价值。在这项研究中，通过异源包被选择从鸡免疫产生的文库中成功分离出针对噻虫嗪 (Thi) 的 scFvs。这些 scFvs 随后与 Avi 标签和碱性磷酸酶融合表达。经过组合和优化，开发了基于 scFv-生物素的酶联免疫吸附测定 (ELISA) 来检测 Thi，显示出令人印象深刻的半最大信号抑制浓度 (IC) 为 30 ng mL 和检测限 (LOD) 为 1.8 ng mL。该免疫分析与其他新烟碱类杀虫剂的交叉反应性极小，除了与吡虫啉的 7.5%和与噻虫啉的 6.7%有交叉反应。通过测试苹果、梨、白菜和黄瓜的加标样品，验证了该测定方法的准确性，结果表明平均回收率在 82%至 119%之间，与高效液相色谱法的结果非常接近。因此，鸡 scFv-生物素基测定法有望成为农业样品中噻虫嗪的高通量筛选工具。
