Detection of low-abundance membrane markers by immunofluorescence - a comparison of alternative high-sensitivity methods and reagents
Detection of low-abundance membrane markers by immunofluorescence - a comparison of alternative high-sensitivity methods and reagents
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DOI：
10.1016/j.jim.2004.04.016
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发表时间：
2004-06-01
影响因子：
2.2
通讯作者：
Zola, H
中科院分区：
文献类型：
--
作者：
Mavrangelos, C;Swart, B;Zola, H
使用免疫荧光染色和流式细胞术检测的抗体对膜分子进行分析广泛应用于研究和诊断免疫学。传统染色技术可以轻松检测每个细胞中浓度约为 2000 个分子的分子，但某些分子的表达和功能丰度要低得多。我们之前描述了一种检测每个细胞 100 个或更少分子的方法，该方法基于使用藻红蛋白作为荧光团、三层扩增过程以及仔细选择可用试剂。近年来，出现了许多新试剂、荧光团和试剂盒，其中一些用于高灵敏度应用。在本文中，许多此类试剂与已发表的方法进行了比较。虽然某些试剂在我们手中给出了不同的结果或高非特异性染色，但有几种试剂与已发表的方法相当。此外，新的荧光团可以改进同时检测两种低丰度标记物。 (C) 2004 Elsevier B.V. 保留所有权利。
The analysis of membrane molecules using antibodies detected by immunofluorescence staining and flow cytometry is used widely in research and diagnostic immunology. Conventional staining techniques readily detect molecules present at concentrations of around 2000 molecules per cell, but some molecules are expressed and function at much lower abundance. We described previously a method for the detection of molecules present at 100 molecules per cell or less based on the use of phycoerythrin as the fluorophore, a three-layer amplification process, and careful selection of available reagents. In recent years, a number of new reagents, fluorophores and kits, have become available, some of them intended for high-sensitivity applications. In this paper, a number of these reagents have been compared with the published method. While some of the reagents gave variable results or high nonspecific staining in our hands, several reagents were comparable with the published method. Furthermore, the new fluorophores allow improved simultaneous detection of two low-abundance markers. (C) 2004 Elsevier B.V. All rights reserved.