Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Resources and Environmental Sciences, China Agricultural University, No. 2 Yuanmingyuan West Road, Haidian District, Beijing, 100193, China.
Department of Entomology and UCD Comprehensive Cancer Center, University of California, 96 Briggs Hall, Davis, CA, 95616, USA.
Anal Bioanal Chem. 2019 Feb;411(6):1287-1295. doi: 10.1007/s00216-018-01563-7. Epub 2019 Feb 1.
Triazophos is mainly used in Asian and African countries for the control of insects in agricultural production. Camelid variable domains of heavy-chain antibodies (VHHs) show great promise in monitoring environmental chemicals such as pesticides. To improve the rate of success in the generation of VHHs against triazophos, genes specifically encoding VHH fragments from the unique allotype IgG3a of an immunized Camelus bactrianus were amplified by using a pair of novel primers and introduced to construct a diverse VHH library. Five out of seven isolated positive clones, including the VHH T1 with the highest affinity to triazophos, were derived from the allotype IgG3a. A one-step enzyme-linked immunosorbent assay (ELISA) using VHH T1 genetically fused with alkaline phosphatase (AP) had a half-maximum inhibition concentration of 6.6 ng/mL for triazophos. This assay showed negligible cross-reactivity with a list of important organophosphate pesticides (< 0.1%). The average recoveries of triazophos from water, soil, and apple samples determined by the one-step ELISA ranged from 83 to 108%, having a good correlation with those by a gas chromatography mass spectrometry (R = 0.99). The VHH-AP fusion protein shows potential for the analysis of triazophos in various matrices.
三唑磷主要在亚洲和非洲国家用于农业生产中防治虫害。骆驼重链抗体的可变区（VHH）在监测环境化学物质（如农药）方面显示出巨大的潜力。为了提高针对三唑磷的 VHH 生成成功率，使用一对新型引物扩增了编码免疫的双峰驼独特同种型 IgG3a 的 VHH 片段的基因，并引入构建了多样化的 VHH 文库。从同种型 IgG3a 中分离出的七个阳性克隆中有五个，包括与三唑磷亲和力最高的 VHH T1。VHH T1 与碱性磷酸酶（AP）基因融合的一步酶联免疫吸附测定（ELISA）对三唑磷的半抑制浓度为 6.6ng/mL。该测定法与一系列重要的有机磷农药的交叉反应性可忽略不计（<0.1%）。通过一步 ELISA 从水样、土壤和苹果样品中测定的三唑磷的平均回收率为 83%至 108%，与气相色谱质谱法（R=0.99）的测定结果具有良好的相关性。VHH-AP 融合蛋白在各种基质中分析三唑磷具有潜力。
