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.

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@article{HarelBellan1989DetectionOL, title={Detection of low and high affinity binding sites with fluoresceinated human recombinant interleukin-2.}, author={Annick Harel-Bellan and Zohar Mishal and Jami Willette-Brown and William L. Farrar}, journal={Journal of immunological methods}, year={1989}, volume={119 1}, pages={127-33} }