Single epitope multiple staining to detect ultralow frequency B cells

Here we describe a method for detecting ultralow frequency target cells fro m within a high background of irrelevant cells by a novel method, single ep itope multiple staining (SEMS). Samples of murine splenocytes were seeded w ith a low number of splenocytes from mice transgenic for a hen eggwhite lys ozyme (HEL)-specific immunoglobulin (Ig). These samples were stained with t wo reagents specific for the same epitope expressed by the transgenic B cel ls, which had been conjugated to two different detectable labels (FITC and biotin). This dual staining of a single epitope allowed us to reduce the ba ckground 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 s nowing only one thing about them, namely, their antigen specificity. The SE MS method allowed us to reproducibly detect transgenic cells at frequencies below one cell in one million cells. SEMS could be used to increase the se nsitivity of numerous fluorescence-based applications in addition to the de tection and isolation of antigen-specific lymphocytes, including the detect ion and highly specific isolation of genetically modified cells, transforme d cells, stem cells, fetal cells, or infectious organisms. (C) 2001 Elsevie r Science B.V. All rights reserved.