Detection of low-abundance membrane markers by immunofluorescence--a comparison of alternative high-sensitivity methods and reagents.

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.