Biochemical and functional analysis of soluble human interleukin-2 receptor produced in rodent cells. Solid-phase reconstitution of a receptor-ligand binding reaction.
The binding of interleukin-2 (IL-2) to the IL-2 receptor (IL-2R) on human T-cells is a key regulatory event which is absolutely required for T-cell-mediated immune responses. To understand further this binding event, we modified the human IL-2R gene to encode a secreted form of IL-2R. Secreted IL-2R was then expressed at very high levels (approximately 11 micrograms/10(6) cells/48 h) in rodent cells using gene-linked co-amplification. The soluble forms of IL-2R were shown to retain IL-2 affinity shown by cell-surface IL-2R (Kd approximately 18 nM) and were purified to homogeneity using IL-2 affinity chromatography. Purified, recombinant IL-2R and biotinylated IL-2 were used to establish a solid-phase receptor binding assay. Binding of IL-2-biotin was demonstrated to be dose-dependent at concentrations ranging from 10 to 1000 ng/ml, and the specificity of receptor-ligand binding was demonstrated by competition with non-biotinylated IL-2 and with anti-receptor antibodies known to block IL-2 binding in vivo. This immunosorbent receptor assay offers a simple and rapid method for studying the binding of IL-2 to human IL-2R.
D000818:Animals; D001667:Binding, Competitive; D001710:Biotin; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007163:Immunosorbent Techniques; D007376:Interleukin-2 / Q000378:metabolism*; D007700:Kinetics; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism*; D015375:Receptors, Interleukin-2; D014162:Transfection
D007376:Interleukin-2; D011971:Receptors, Immunologic; D015375:Receptors, Interleukin-2; D001710:Biotin