Detection of a large antigen through the masking and exposure of a fragment of split luciferase.

IF 1.8 4区 化学 Q3 CHEMISTRY, ANALYTICAL
Cheng Qian, Ayumu Ninomiya, Natsuki Shibukawa, Hiroshi Ueda, Takanobu Yasuda, Bo Zhu, Tetsuya Kitaguchi
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引用次数: 0

Abstract

We developed PMBiT, an antibody-binding Protein M (PM)-based bioluminescent probe that detects large antigens via luciferase reconstitution by exposing a luciferase fragment. Detection is achieved by exploiting the principle that the antibody, large antigen, and PM cannot form a complex simultaneously. PMBiT was prepared by conjugating PM with a HiBiT-based peptide from split NanoLuc luciferase through an Azide-DBCO click reaction. It retained its binding activity to the antibody and showed bioluminescence upon reconstitution of the luciferase with LgBiT, the other fragment of the split NanoLuc. Mixing PMBiT with various IgG antibodies resulted in decreased bioluminescence. In contrast, when PMBiT was mixed with IgG bound to its large antigen, such as human C-reactive protein, a dose-dependent increase in bioluminescence was obtained. Molecular dynamics simulations of PM showed that two regions in the C-terminus contribute to steric clashes with antigens owing to their relatively rigid structures. Furthermore, in silico analysis of the structure suggested that the antigen size was the primary factor blocking the binding of PMBiT to IgG for antigen detection. An immunoassay utilizing PMBiT does not require genetic manipulation of antibodies, allowing for seamless and scalable antibody replacement, and will advance the future of on-site detection and rapid diagnostics.

通过掩蔽和暴露一个分裂的荧光素酶片段来检测一个大抗原。
我们开发了PMBiT,一种基于抗体结合蛋白M (PM)的生物发光探针,通过暴露荧光素酶片段,通过荧光素酶重组来检测大抗原。检测是通过利用抗体、大抗原和PM不能同时形成复合物的原理实现的。PMBiT是通过叠氮化物- dbco点击反应将PM与分离的NanoLuc荧光素酶的hibit基肽偶联而成的。它保留了与抗体的结合活性,并在与分裂的NanoLuc的另一个片段LgBiT重组荧光素酶后显示出生物发光。PMBiT与多种IgG抗体混合导致生物发光减弱。相反,当PMBiT与结合其大抗原(如人c反应蛋白)的IgG混合时,获得了剂量依赖性的生物发光增加。PM的分子动力学模拟表明,由于其相对刚性的结构,c端上的两个区域有助于与抗原的空间冲突。此外,对结构的计算机分析表明,抗原大小是阻碍PMBiT与IgG结合进行抗原检测的主要因素。利用PMBiT的免疫分析不需要对抗体进行遗传操作,允许无缝和可扩展的抗体替代,并将推进现场检测和快速诊断的未来。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Analytical Sciences
Analytical Sciences 化学-分析化学
CiteScore
2.90
自引率
18.80%
发文量
232
审稿时长
1 months
期刊介绍: Analytical Sciences is an international journal published monthly by The Japan Society for Analytical Chemistry. The journal publishes papers on all aspects of the theory and practice of analytical sciences, including fundamental and applied, inorganic and organic, wet chemical and instrumental methods. This publication is supported in part by the Grant-in-Aid for Publication of Scientific Research Result of the Japanese Ministry of Education, Culture, Sports, Science and Technology.
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