HOMOGENEOUS TIME-RESOLVED IL-2-IL-2R-ALPHA ASSAY USING FLUORESCENCE RESONANCE ENERGY-TRANSFER

A homogeneous receptor-ligand assay based on fluorescence resonance en ergy transfer is described. Tn the assay, recombinant human interleuki n 2 (IL-2) and a monoclonal antibody against the human IL-2 receptor a chain were labelled with a highly fluorescent europium chelate and Cy 5, respectively, As a result of a successful receptor-ligand complex f ormation, these labels are brought into close proximity, which will th ereby allow an energy transfer to occur from the donor (europium) to t he acceptor (Cy5), upon excitation of the donor. Utilization of specif ic non-neutralizing antibodies made it possible to use crude hIL-2R al pha membranes prepared from recombinant baculovirus-infected Sf9 insec t cells. The specific energy transfer was measured at the emission wav elength of Cy5 using a time-resolved fluorometer. The data presented, demonstrate that this assay design can be utilized for saturation as w ell as competitive binding experiments in addition to regular receptor titrations. As a rapid simple homogeneous assay it is particularily s uitable for high throughput screening analyses. (C) 1998 Academic Pres s.