Laboratory for Chemistry and Life Science, Institute of Innovative Research , Tokyo Institute of Technology , Yokohama , Kanagawa 226-8503 , Japan.
Anal Chem. 2018 Mar 6;90(5):3001-3004. doi: 10.1021/acs.analchem.7b05140. Epub 2018 Feb 15.
The homogeneous immunological detection of small molecules at high sensitivity is still a daunting task. Here, we tried sensitive noncompetitive detection of small peptides based on the open-sandwich immunoassay principle, which was combined with a bioluminescent protein-fragment complementation assay (PCA) in vitro. Since the detection of antigen-induced approximation of the two antibody variable region fragments V and V by the standard Nanoluc-based PCA utilizing larger (LgBiT) and shorter (SmBiT) fragments was not successful, we decided to further split LgBiT into two, yielding smaller N-terminal derivative (LnBiT) and two C-terminal, 11 residue peptides (LcBiT and SmBiT) corresponding to consecutive beta strands, to which V and V were each fused and expressed in Escherichia coli cells. Through the optimization of reaction conditions and peptide sequence, the antigen osteocalcin peptide can be noncompetitively detected with a low background signal and limit of detection, yielding a high light emission of 88% compared to that of the wild-type enzyme. Since the luminescence of this open sandwich bioluminescent immunoassay (OS-BLIA) can be observed with the naked eye, it could become the foundation of many point-of-care detection systems.
在高灵敏度下实现小分子的均相免疫检测仍然是一项艰巨的任务。在这里，我们尝试基于开放式三明治免疫测定原理，结合体外生物发光蛋白片段互补测定（PCA），进行敏感的非竞争性小分子检测。由于基于标准 Nanoluc 的 PCA 检测抗原诱导的两个抗体可变区片段 V 和 V 的接近，利用较大的（LgBiT）和较短的（SmBiT）片段并不成功，因此我们决定进一步将 LgBiT 分割为两个，得到较小的 N 端衍生物（LnBiT）和两个 C 端的、由 11 个残基组成的肽（LcBiT 和 SmBiT），它们分别对应于连续的β链，其中 V 和 V 分别融合并在大肠杆菌细胞中表达。通过优化反应条件和肽序列，可以非竞争地检测骨钙素肽抗原，具有低背景信号和检测限，与野生型酶相比，发光率高达 88%。由于这种开放式三明治生物发光免疫分析（OS-BLIA）的发光可以用肉眼观察到，因此它可能成为许多即时检测系统的基础。
