Development of a time-resolved fluorescence resonance energy transfer assay (cell TR-FRET) for protein detection on intact cells

An assay named Cell TR-FRET based on time-resolved fluorescence resonance e nergy transfer, here utilized for detection of receptor proteins on intact cells, is described. In this assay, intact membrane-biotinylated Sf9 cells expressing human interleukin-2R alpha due to infection with a recombinant b aculovirus were prelabeled with a streptavidin-europium (Eu3+) chelate, the donor. These prelabeled cells were used in a homogeneous assay by addition of a fluorochrome-labeled anti-hIL-2R alpha -specific antibody, 7G7B6-Cy5, the acceptor. Binding of 7G7B6-Cy5 to hIL-2R alpha expressed on the cell s urface and europium-labeled streptavidin to surface biotin esters brings th e donor and the acceptor in close proximity, allowing transfer of energy fr om the excited state donor to the acceptor. This energy transfer was specif ically inhibited by unlabeled antibody and by free biotin. The described as say constitutes a general method since no specific component of the cell me mbrane is labeled, thereby allowing a number of binding studies on the cell membrane, including receptor density determinations, to be performed. In a ddition, due to the rapid fashion in which the Cell TR-FRET assay is accomp lished, it can be a valuable method not only for identifying novel membrane -associated proteins, but also for drug screening of large samples in high- throughput format. (C) 2001 Elsevier Science.