Homogeneous time-resolved IL-2-IL-2R alpha assay using fluorescence resonance energy transfer.
A homogeneous receptor-ligand assay based on fluorescence resonance energy transfer is described. In the assay, recombinant human interleukin 2 (IL-2) and a monoclonal antibody against the human IL-2 receptor alpha chain were labelled with a highly fluorescent europium chelate and Cy5, respectively. As a result of a successful receptor-ligand complex formation, these labels are brought into close proximity, which will thereby allow an energy transfer to occur from the donor (europium) to the acceptor (Cy5), upon excitation of the donor. Utilization of specific non-neutralizing antibodies made it possible to use crude hIL-2R alpha membranes prepared from recombinant baculovirus-infected Sf9 insect cells. The specific energy transfer was measured at the emission wavelength of Cy5 using a time-resolved fluorometer. The data presented, demonstrate that this assay design can be utilized for saturation as well as competitive binding experiments in addition to regular receptor titrations. As a rapid simple homogeneous assay it is particularily suitable for high throughput screening analyses.
Department of Biology, Abo Akademi University, Turku, Finland.
K
K
Stenroos
P
P
Hurskainen
S
S
Eriksson
I
I
Hemmilä
K
K
Blomberg
C
C
Lindqvist
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001667:Binding, Competitive; D002232:Carbocyanines / Q000378:metabolism; D002478:Cells, Cultured; D005063:Europium / Q000378:metabolism; D019085:Fluorescent Antibody Technique, Direct; D015200:Fluoroimmunoassay / Q000379:methods; D006801:Humans; D007376:Interleukin-2 / Q000378:metabolism*; D008024:Ligands; D015375:Receptors, Interleukin-2 / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D018411:Spodoptera
D000911:Antibodies, Monoclonal; D002232:Carbocyanines; D007376:Interleukin-2; D008024:Ligands; D015375:Receptors, Interleukin-2; C085321:cyanine dye 5; D005063:Europium