Detection of low and high affinity binding sites with fluoresceinated human recombinant interleukin-2.
In this study, we describe a new methodology to detect and quantify lymphokine receptors, using interleukin-2 as a prototype. Human recombinant interleukin-2 (IL-2) was conjugated to fluorescein isothiocyanate. Binding of fluoresceinated IL-2 to different cell types was assessed by flow cytometry analysis, on a FACS 440 calibrated using fluoresceinated Sephadex G-25 beads. This calibration procedure allowed us to quantify the actual number of binding sites for IL-2. Fluoresceinated IL-2 did not bind to normal resting T cells, whereas a highly significant binding was observed on PHA-activated human T cells. The binding was inhibited by an excess of unlabeled IL-2 and by an excess of anti-IL-2 receptor p55 antibodies (anti-TAC). Dose curves of IL-2 showed a two plateau saturation, the first plateau corresponding to the saturation of high affinity binding sites, as assessed by correlation with the biological activity on IL-2-dependent T cells. Among the cell types tested, fluoresceinated IL-2 bound to IL-2-dependent mouse T cells (the binding in that case was not inhibited by anti-IL-2 receptor p55 antibodies), and to different p70 expressing cell lines or normal cells (MLA 144, normal large granular lymphocytes). Taken together, these results indicate that fluoresceinated IL-2 can be used to detect high as well as low affinity IL-2 binding sites.
Harel-Bellan
A
A
Laboratory of Molecular Immunoregulation, National Cancer Institute, Frederick, MD 21701.
Mishal
Z
Z
Willette-Brown
J
J
Farrar
W L
WL
D000818:Animals; D002460:Cell Line; D004306:Dose-Response Relationship, Immunologic; D005347:Fibroblasts / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D006801:Humans; D006922:Hylobates; D007376:Interleukin-2 / Q000378:metabolism / Q000502:physiology; D008213:Lymphocyte Activation; D051379:Mice; D010835:Phytohemagglutinins; D015375:Receptors, Interleukin-2 / Q000032:analysis* / Q000276:immunology; D011994:Recombinant Proteins / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D013861:Thiocyanates
D005452:Fluoresceins; D007376:Interleukin-2; D010835:Phytohemagglutinins; D015375:Receptors, Interleukin-2; D011994:Recombinant Proteins; D013861:Thiocyanates; D016650:Fluorescein-5-isothiocyanate