Optimization of the cytokine secretion assay for human IL-2 in single and combination assays.
The cytokine secretion assay identifies live cytokine-secreting cells by capturing the secreted cytokine on a surface-bound capture antibody in dilute suspension culture, followed by detection with a fluorescent anti-cytokine antibody. However, examining the kinetics of cytokine detection revealed that IL-2 staining reached a maximum at early times and then declined, whereas staining for other cytokines including interferon (IFNγ) increased for up to 90 min. The decline in IL-2 staining could have been due to rapid cessation of cytokine synthesis, coupled with internalization of cytokine/antibody complexes from the cell surface. Consistent with this model, addition of the anti-IL-2 detection antibody during the cytokine secretion step resulted in higher and more sustained staining. This modified method enhanced staining of IL-2 and IL-4, but not IFNγ, tumor necrosis factor alpha (TNFα), or IL-5. However, the longer secretion times possible in the modified assay also improved detection of other cytokines in multi-cytokine combinations.
David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, Rochester, New York, 14642.
Nan
N
Deng
David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, Rochester, New York, 14642.
Tim R
TR
Mosmann
D000906:Antibodies / Q000276:immunology; D001681:Biological Assay / Q000379:methods; D002478:Cells, Cultured; D016207:Cytokines / Q000276:immunology* / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007376:Interleukin-2 / Q000276:immunology* / Q000378:metabolism*; D007700:Kinetics
D000906:Antibodies; D016207:Cytokines; D007376:Interleukin-2
IL-2; T cells; cytokine secretion assay; human