College of Plant Protection, Nanjing Agricultural University , Nanjing 210095, China.
State & Local Joint Engineering Research Center of Green Pesticide Invention and Application , Nanjing 210095, China.
Anal Chem. 2018 Feb 6;90(3):2230-2237. doi: 10.1021/acs.analchem.7b04601. Epub 2018 Jan 16.
Phage borne peptides isolated from phage libraries have proven to be valuable reagents for the development of small-molecule immunoassays. However, the large size, low diffusion rate, and biological nature of the phage particles create some limitations to their use and require secondary reagents for its detection. In this work, we explore the use of the Nano luciferase (NanoLuc) as a fusion partner to generate recombinant tracers for immunoassay development. The imidaclothiz peptidomimetic C2-15 that specifically binds to the anti-imidaclothiz monoclonal antibody (mAb) 1E was fused to NanoLuc, both at the N terminus (C2-15-NanoLuc) and C terminus (NanoLuc-C2-15). NanoLuc-C2-15 showed better performance than C2-15-NanoLuc and was adopted to develop a bioluminescent enzyme immunoassay (BLEIA) and a bioluminescence lateral flow immunoassay (BLLFIA) for imidaclothiz. The luminescence signal of NanoLuc-C2-15 rapidly reaches high intensity with slow attenuation, which enabled one to capture the BLLFIA readout by using a smartphone without an external light source. The IC of the BLEIA and BLLFIA were 3.3 ± 0.2 and 6.4 ± 0.4 ng mL, respectively. Both immunoassays exhibited good accuracy for the detection of imidaclothiz in environmental and agricultural samples.
噬菌体衍生肽从噬菌体文库中分离出来，已被证明是开发小分子免疫分析的有价值的试剂。然而，噬菌体颗粒的体积大、扩散率低和生物性质对其使用造成了一些限制，需要使用辅助试剂进行检测。在这项工作中，我们探索了使用纳米荧光素酶（NanoLuc）作为融合伴侣，生成用于免疫分析开发的重组示踪剂。特异性结合抗噻虫啉单克隆抗体（mAb）1E 的咪唑烷脒肽模拟物 C2-15 被融合到 NanoLuc 上，分别位于 N 端（C2-15-NanoLuc）和 C 端（NanoLuc-C2-15）。NanoLuc-C2-15 的性能优于 C2-15-NanoLuc，并被采用来开发用于噻虫啉的生物发光酶免疫分析（BLEIA）和生物发光侧向流动免疫分析（BLLFIA）。NanoLuc-C2-15 的发光信号迅速达到高强度，衰减缓慢，这使得可以在没有外部光源的情况下使用智能手机捕获 BLLFIA 的读数。BLEIA 和 BLLFIA 的 IC 分别为 3.3±0.2ng mL 和 6.4±0.4ng mL。两种免疫分析方法在检测环境和农业样品中的噻虫啉时均具有良好的准确性。
