Single epitope multiple staining to detect ultralow frequency B cells

Here we describe a method for detecting ultralow frequency target cells from within a high background of irrelevant cells by a novel method, single epitope multiple staining (SEMS). Samples of murine splenocytes were seeded with a low number of splenocytes from mice transgenic for a hen eggwhite lysozyme (HEL)-specific immunoglobulin (Ig). These samples were stained with two reagents specific for the same epitope expressed by the transgenic B cells, which had been conjugated to two different detectable labels (FITC and biotin). This dual staining of a single epitope allowed us to reduce the background due both to non-specific binding of reagents and to probabilistic distribution of the cells. We also were able to detect the cells based on knowing only one thing about them, namely, their antigen specificity. The SEMS method allowed us to reproducibly detect transgenic cells at frequencies below one cell in one million cells. SEMS could be used to increase the sensitivity of numerous fluorescence-based applications in addition to the detection and isolation of antigen-specific lymphocytes, including the detection and highly specific isolation of genetically modified cells, transformed cells, stem cells, fetal cells, or infectious organisms.