Homogeneous Noncompetitive Luminescent Immunodetection of Small Molecules by Ternary Protein Fragment Complementation
Homogeneous Noncompetitive Luminescent Immunodetection of Small Molecules by Ternary Protein Fragment Complementation
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DOI：
10.1021/acs.analchem.7b05140
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发表时间：
2018-03-06
影响因子：
7.4
通讯作者：
Ueda, Hiroshi
中科院分区：
文献类型：
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作者：
Ohmuro-Matsuyama, Yuki;Ueda, Hiroshi
高灵敏度的小分子同质免疫检测仍然是一项艰巨的任务。在此，我们尝试了基于开放夹心免疫分析原理的小肽的非竞争性检测，并结合生物发光蛋白片段互补法（PCA）在体外进行检测。由于使用较大（LgBiT）和较短（SmBiT）片段的标准Nanoluc-based PCA检测抗原诱导的两个抗体可变区片段V-H和V-L的近似检测不成功，我们决定进一步将LgBiT分裂为两个，产生较小的n端衍生物（LnBiT）和两个c端，11个残基肽（LcBiT和SmBiT）对应于连续的β链，其中V-H和V-L分别融合并在大肠杆菌细胞中表达。通过对反应条件和多肽序列的优化，抗原骨钙素肽具有低背景信号和检测限的非竞争性检测，与野生型酶相比，其发光率高达88%。由于这种开放式三明治生物发光免疫分析法（OS-BLIA）的发光可以用肉眼观察到，它可以成为许多即时检测系统的基础。
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-H and V-L 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-H and V-L 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.