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.
Stenroos
K
K
Abo Akademi University, Department of Biochemistry and Pharmacy, Turku, Finland.
Hurskainen
P
P
Blomberg
K
K
Lindqvist
C
C
D005063:Europium; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D007163:Immunosorbent Techniques; D007376:Interleukin-2 / Q000378:metabolism*; D008024:Ligands; D011485:Protein Binding; D015375:Receptors, Interleukin-2 / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D005456:Fluorescent Dyes; D007376:Interleukin-2; D008024:Ligands; D015375:Receptors, Interleukin-2; D011994:Recombinant Proteins; D005063:Europium