利用酪氨酸酶介导的ha标签标记生成q -小体的新方法的发展。

IF 3.8 2区 化学 Q1 BIOCHEMICAL RESEARCH METHODS
Hui-Seon Yun, Hanool Yun, Hee-Jin Jeong
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引用次数: 0

摘要

猝灭体(Q-bodies)是通过荧光猝灭和去猝灭机制对抗原结合作出反应的荧光抗体。为了增强生成方法的通用性并扩大颜色范围,我们开发了一种新的q -小体生成方法,使用酪氨酸酶介导的位点特异性偶联到富含酪氨酸的血凝素(HA)标签。利用n端ha标签设计了一种抗程序性细胞死亡配体1 (PDL1)的单链可变片段(scFv),并在大肠杆菌中高产、高纯度地表达。利用重组酪氨酸酶实现了4种不同发射波长的酰肼功能化染料的位点特异性偶联。所得到的q -小体通过荧光联免疫吸附试验进行了评估,所有这些小体都显示出抗原浓度依赖的荧光增强。四甲基罗丹明(TAMRA)标记的q -小体具有最高的信本比,检出限为0.51±0.01 μg/mL。这种ha标签介导的q -小体生成策略为生产具有广泛染料兼容性的q -小体提供了一种强大而通用的工具,实现了敏感和多路荧光免疫分析。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Development of a novel method for generating Q-bodies using tyrosinase-mediated HA-tag labeling.

Quenchbodies (Q-bodies) are fluorescent antibodies that respond to antigen binding via a fluorescence quenching and de-quenching mechanism. To enhance the versatility of the generation method and expand the color range, we developed a novel Q-body generation approach using tyrosinase-mediated site-specific conjugation to a tyrosine-rich hemagglutinin (HA)-tag. A single-chain variable fragment (scFv) against programmed cell death-ligand 1 (PDL1) was engineered with an N-terminal HA-tag and expressed in Escherichia coli with high yield and purity. Site-specific conjugation of four hydrazide-functionalized dyes with different emission wavelengths was achieved using recombinant tyrosinase. The resulting Q-bodies were evaluated via fluorescence-linked immunosorbent assay, all of which demonstrated antigen concentration-dependent fluorescence enhancement. Notably, the tetramethylrhodamine (TAMRA)-labeled Q-body showed the highest signal-to-background ratio, limit of detection of 0.51 ± 0.01 μg/mL. This HA-tag-mediated Q-body generation strategy offers a robust and versatile tool for the production of Q-bodies with broad dye compatibility, enabling sensitive and multiplexed fluorescent immunoassays.

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来源期刊
CiteScore
8.00
自引率
4.70%
发文量
638
审稿时长
2.1 months
期刊介绍: Analytical and Bioanalytical Chemistry’s mission is the rapid publication of excellent and high-impact research articles on fundamental and applied topics of analytical and bioanalytical measurement science. Its scope is broad, and ranges from novel measurement platforms and their characterization to multidisciplinary approaches that effectively address important scientific problems. The Editors encourage submissions presenting innovative analytical research in concept, instrumentation, methods, and/or applications, including: mass spectrometry, spectroscopy, and electroanalysis; advanced separations; analytical strategies in “-omics” and imaging, bioanalysis, and sampling; miniaturized devices, medical diagnostics, sensors; analytical characterization of nano- and biomaterials; chemometrics and advanced data analysis.
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