Journal of Immunological Methods, 114 (1988) 95-99 95 Elsevier JIM04922 A simple and sensitive bioassay for the detection of IL-2 activity Torbjorn Leivestad, Gustav Gaudernack, Ragnhild Halvorsen and Erik Thorsby Institute of Transplantation Immunology, The National Hospital, Rikshospitalet, Oslo, Norway (Received 25 March 1988, revised received 29 April 1988, accepted 20 May 1988) A simple, one-step quantitative assay for the detection of biologically active interleukin-2 (IL-2) is described. It is based on culture of pure CD3 ÷ T cells which have been positively selected from blood mononuclear cells by particle (M450)-bound anti-CD3 monoclonal antibody (mAb). During culture, activation of the T cells via CD3 will occur, leading to expression of IL-2 receptors but not IL-2 production. By adding IL-2 a proliferative response is evoked, giving a linear dose-response curve for IL-2 concentrations between 0.01-20 U/ml. The cells were unresponsive to IL-1, IL-4, tumor necrosis factor a (TNF-a), interferon-a (IFN-a), IFN-'t, phytohemagglutinin and concanavalin A. The responsiveness to IL-2 was enhanced by TNF-a and inhibited by IFN-a, while the other tested lymphokines and mitogens did not influence the proliferative response. Antibodies to IL-2 and to IL-2 receptor suppressed the IL-2 response in a dose-dependent manner. The method is both simple and specific, and obviates the necessity for keeping assay cells in long term culture. Key words: Irnmunomagnetic isolation; Interleukin-2; Lymphokine bioassay Introduction Following activation of T cells various lympho- kines are produced. Included among these is inter- leukin-2 (IL-2), which is a prerequisite for a pro- liferative response of T cells (reviewed in Farrar et al., 1986). In studies of T cell responses there is Correspondence to: T. Leivestad, Institute of Transplan- tation Immunology, The National Hospital, 0027 Oslo 1, Nor- way. Abbreviations: AC, accessory cells; CM, culture medium; ConA, concanavalin A; FCS, fetal calf serum; IL-1 (-2, -4), interleukin-1 (-2, -4); rlL-2, recombinant interleukin-2; IL-2R, interleukin-2 receptor; IFN-a (-y), interferon-a (-~'); HSP, human serum pool; LPS, lipopolysaccharide; mAb, mono- clonal antibody; MLC, mixed lymphocyte culture; PBM, pe- ripheral blood mononudear cells; PHA, phytohemagglutinin; TNF, tumor necrosis factor; TPA, tetradecanoyl phorbol acetate. often a need for measurement of biologically ac- tive IL-2 in supernatants. The commonly used bioassays are based on measuring the proliferative response of IL-2-dependent T cell lines (reviewed in Hamblin and O'Garra, 1987). Activation of purified T cells with particle bound monoclonal antibodies (mAbs) reactive with the T cell receptor (Ti)/CD3 complex in the ab- sence of accessory cells results in the expression of IL-2 receptors (IL-2R), but no IL-2 production (Umetsu et al., 1987). If exogeneous IL-2 is also supplied, DNA synthesis and T cell proliferation will occur, and this IL-2 response is dose-depen- dent (Tsoukas et al., 1986; Umetsu et al., 1987). By culturing T cells positively selected with anti: CD3-coated particles, the T cells are at the same time activated to become IL-2 responsive. Using this principle we have developed a simple one-step method to detect low concentrations of IL-2. 0022-1759/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)