Solid phase IL-2-IL-2R alpha assay with time resolved fluorometry

A time-resolved fluorometric, solid phase, receptor ligand interaction assay is described. The assay consists of wells coated with anti-human IL-2 receptor alpha (hIL-2R alpha) monoclonal antibodies (mAb), europium labelled hIL2 (Eu-IL-2) and human recombinant IL-2 receptor alpha subunits expressed in the baculovirus expression vector system (BEVS). In the assay hIL-2R alpha-Eu-IL-2 complexes bind to the solid phase mAb. Receptor bound Eu is dissociated into an enhancer solution where it forms highly fluorescent complexes. The fluorescence is measured in a time-resolved fluorometer. The Kd value calculated from the saturation curve is in good agreement with previously reported values for the low affinity type of IL-2R, making the described assay a simple and nonradioactive alternative for measurement of soluble hIL-2R alpha in biological systems. Furthermore this assay format provides convenient separation of bound ligand from unbound and is therefore suitable for high throughput screenings.