BIOTINYLATION OF INTERLEUKIN-2 (IL-2) FOR FLOW CYTOMETRIC ANALYSIS OFIL-2 RECEPTOR EXPRESSION - COMPARISON OF DIFFERENT METHODS

The main prerequisites for the use of biotinylated ligands to study th e expression of growth factor receptors on heterogeneous cell populati ons, such as peripheral blood or bone marrow, by flow cytometric metho ds, are that the biotinylated ligand retains its binding ability and t hat binding of the biotinylated ligand to the receptor does not inhibi t the subsequent interaction of biotin with fluorescently tagged avidi n or streptavidin. Using interleukin-2 (IL-2), we compared the usefuln ess of various biotinylation reagents, NHS-biotin, S-NHS-biotin, S-NHS -LC-biotin, DBB and photobiotin, and developed optimal biotinylation c onditions for the preparation of biologically active biotin-labeled IL -2 and the detection of IL-2 receptor expressing cells by flow cytomet ry. As determined by spot blot analysis, biotinylation of IL-2 was mos t efficient at the highest biotin-to-protein (B:P) ratio used. At a B: P ratio of 100, most of the biological activity of IL-2 was retained w hen S-NHS-LC-biotin was used. In contrast, most of the biological acti vity of 1L-2 samples that were labeled with NHS-biotin or photobiotin was lost under these conditions. Biotin-labeled IL-2 preparations were tested in order to detect IL-2 receptors on IL-2 dependent CTLL-2 cel ls by flow cytometry after sequential staining with the biotinylated I L-2 and fluorescence tagged streptavidin. A high B:P ratio generally r esulted in a high specific fluorescence intensity of the cells, partic ularly when S-NHS-LC-biotin was used as the biotinylation reagent. Bio tin-IL-2 could also be used to detect IL-2 receptors expressed by lymp hocytes in peripheral blood and bone marrow. Comparison of staining of lymphocytes with biotinylated IL-2 and an antibody against the IL-2 r eceptor alpha chain demonstrated that only a subset of the cells that showed a strong fluorescence signal after staining with biotinylated I L-2 expressed high numbers of the IL-2 receptor oc chain. This is in a greement with the expression of functional IL-2 receptors on resting T cells and NK cells which do not express the alpha chain.