Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Resources and Environmental Sciences, China Agricultural University, Beijing, 100193, China.
Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, 1955 East-West Road, Honolulu, Hawaii, 96822, USA.
Anal Biochem. 2019 May 1;572:9-15. doi: 10.1016/j.ab.2019.02.022. Epub 2019 Mar 1.
Immunoassays provide a high-throughput method for monitoring pesticides in foods and the environment. Due to easy generation and capable of being manipulated, chicken single-chain variable fragment (scFv) is attractive in the development of immunoassays for pesticides. Two scFvs (X1 and X2) against the insecticide carbaryl were generated from a chicken immunized with hapten C1 conjugated to keyhole limpet hemocyanin and fused with alkaline phosphatase (AP) to develop a rapid one-step enzyme-linked immunosorbent assay for this pesticide. X2-AP showed higher binding affinity to carbaryl than X1-AP. The X2-AP-based ELISA had a half-maximum signal inhibition concentration of 15 ng mL and a limit of detection of 1.6 ng mL. This assay showed negligible cross-reactivity with other carbamate pesticides (<0.1%) and low cross-reactivity with 1-naphthol (5%). The average recoveries of carbaryl spiked in soil, apple and pear samples by the one-step assay ranged from 90% to 114% and agreed well with those of high-performance liquid chromatography. The chicken scFv-based assay showed promise as a high-throughput screening tool for carbaryl in environmental and food matrices.
免疫分析为监测食品和环境中的农药提供了高通量的方法。由于易于产生和可操作性，鸡单链可变片段（scFv）在开发用于农药的免疫分析方面具有吸引力。用半抗原 C1 与血蓝蛋白偶联物免疫的鸡产生了两种针对杀虫剂carbaryl 的 scFv（X1 和 X2），并与碱性磷酸酶（AP）融合，开发了一种用于该农药的快速一步酶联免疫吸附测定法。X2-AP 对 carbaryl 的结合亲和力高于 X1-AP。基于 X2-AP 的 ELISA 的半最大信号抑制浓度为 15ng/mL，检测限为 1.6ng/mL。该测定法与其他氨基甲酸酯类农药的交叉反应性可忽略不计（<0.1%），与 1-萘酚的交叉反应性较低（5%）。一步法测定土壤、苹果和梨样品中carbaryl 的平均回收率在 90%至 114%之间，与高效液相色谱法的结果吻合良好。基于鸡 scFv 的测定法有望成为环境和食品基质中 carbaryl 的高通量筛选工具。
