Yin Wei, Hua Xiude, Liu Xiaofeng, Shi Haiyan, Gee Shirley J, Wang Minghua, Hammock Bruce D
College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China; State and Local Joint Engineering Research Center of Green Pesticide Invention and Application, Nanjing 210095, China.
Department of Entomology and UCD Cancer Center, University of California, Davis, Davis, CA 95616, USA.
Anal Biochem. 2015 Jul 15;481:27-32. doi: 10.1016/j.ab.2015.04.015. Epub 2015 Apr 20.
A monoclonal antibody (3A5) that can recognize thiacloprid was produced, and a linear 8-residue peptide phage library was constructed. Six phage-displayed peptides were isolated from the linear 8-residue peptide phage library and a cyclic 8-residue peptide phage library. A phage enzyme-linked immunosorbent assay (ELISA) was developed to detect thiacloprid using a phage-displayed peptide. Under the optimal conditions, the half-maximal inhibition concentration (IC50) and the limit of detection (IC10) of the developed phage ELISA were 8.3 and 0.7 μg/L, respectively. Compared with the conventional ELISA, the sensitivity was improved more than 3-fold. The cross-reactivity (CR) was less than 0.08% for the tested structural analogues and was regarded as negligible. The recoveries of thiacloprid ranged from 80.3% to 116.3% in environmental and agricultural samples, which conformed to the requirements for residue detection. The amount of thiacloprid detected by phage ELISA in the samples was significantly correlated with that detected by high-performance liquid chromatography. The current study indicates that isolating phage-displayed peptides from phage display libraries is an alternative method for the development of a sensitive immunoassay and that the developed assay is a potentially useful tool for detecting thiacloprid in environmental and agricultural samples.
制备了一种能够识别噻虫啉的单克隆抗体（3A5），并构建了一个线性8肽噬菌体文库。从线性8肽噬菌体文库和环状8肽噬菌体文库中筛选出6种噬菌体展示肽。建立了一种利用噬菌体展示肽检测噻虫啉的噬菌体酶联免疫吸附测定法（ELISA）。在最佳条件下，所建立的噬菌体ELISA的半数抑制浓度（IC50）和检测限（IC10）分别为8.3和0.7μg/L。与传统ELISA相比，灵敏度提高了3倍以上。所测结构类似物的交叉反应率（CR）小于0.08%，可忽略不计。在环境和农业样品中，噻虫啉的回收率在80.3%至116.3%之间，符合残留检测要求。噬菌体ELISA法检测样品中噻虫啉的含量与高效液相色谱法检测结果显著相关。当前研究表明，从噬菌体展示文库中筛选噬菌体展示肽是开发灵敏免疫测定法的一种替代方法，所建立的测定法是检测环境和农业样品中噻虫啉的一种潜在有用工具。
