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用于免疫荧光的高性能近红外荧光二级抗体。

High-Performance Near-Infrared Fluorescent Secondary Antibodies for Immunofluorescence.

机构信息

Department of Chemistry and Biochemistry, University of Notre Dame, 251 Nieuwland Science Hall, Notre Dame, Indiana 46556, United States.

出版信息

Anal Chem. 2021 Feb 23;93(7):3643-3651. doi: 10.1021/acs.analchem.1c00276. Epub 2021 Feb 10.

Abstract

A broad array of imaging and diagnostic technologies employs fluorophore-labeled antibodies for biomarker visualization, an experimental technique known as immunofluorescence. Significant performance advantages, such as higher signal-to-noise ratio, are gained if the appended fluorophore emits near-infrared (NIR) light with a wavelength >700 nm. However, the currently available NIR fluorophore antibody conjugates are known to exhibit significant limitations, including low chemical stability and photostability, weakened target specificity, and low fluorescence brightness. These fluorophore limitations are resolved by employing a NIR heptamethine cyanine dye named s775z whose chemical structure is very stable, charge-balanced, and sterically shielded. Using indirect immunofluorescence for imaging and visualization, a secondary IgG antibody labeled with s775z outperformed IgG analogues labeled with the commercially available NIR fluorophores, IRDye 800CW and DyLight800. Comparison experiments include three common techniques: immunocytochemistry, immunohistochemistry, and western blotting. Specifically, the secondary IgG labeled with s775z was 3-8 times brighter, 3-6 times more photostable, and still retained excellent target specificity when the degree of antibody labeling was high. The results demonstrate that antibodies labeled with s775z can emit total photon counts that are 1-2 orders of magnitude higher than those currently possible, and thus enable unsurpassed performance for NIR fluorescence imaging and diagnostics. They are especially well suited for analytical applications that require sensitive NIR fluorescence detection or use modern photon-intense methods that require high photostability.

摘要

一系列广泛的成像和诊断技术采用荧光标记抗体进行生物标志物可视化,这是一种称为免疫荧光的实验技术。如果附加的荧光团发射波长> 700nm 的近红外(NIR)光,则可以获得显著的性能优势,例如更高的信噪比。然而,目前可用的 NIR 荧光团抗体缀合物已知具有显著的局限性,包括低化学稳定性和光稳定性、减弱的靶特异性和低荧光亮度。通过使用一种名为 s775z 的 NIR 七甲川花菁染料来解决这些荧光团限制,其化学结构非常稳定、电荷平衡且空间位阻屏蔽。通过间接免疫荧光进行成像和可视化,用 s775z 标记的二级 IgG 抗体的性能优于用市售 NIR 荧光团 IRDye 800CW 和 DyLight800 标记的 IgG 类似物。比较实验包括三种常见技术:免疫细胞化学、免疫组织化学和免疫印迹。具体来说,当抗体标记程度较高时,用 s775z 标记的二级 IgG 抗体的亮度提高了 3-8 倍,光稳定性提高了 3-6 倍,并且仍然保持出色的靶特异性。结果表明,用 s775z 标记的抗体可以发射比目前可能的高出 1-2 个数量级的总光子数,从而为 NIR 荧光成像和诊断提供无与伦比的性能。它们特别适合需要灵敏的 NIR 荧光检测的分析应用,或需要高光稳定性的现代光子密集型方法。

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