Cytokine Assays

Abstract

Cytokines are small molecular weight proteins or glycoproteins produced by various cells. They mediate cellular interactions and play a key role in health and disease. They bind to high‐affinity receptors located on various target cells and deliver signals responsible for a variety of biological effects. Once induced by external or internal stimuli, cytokines work as cascades. They regulate physiological interactions between cells, but they also have a potential for inducing tissue damage. While cytokines are local mediators meant to transiently regulate cells within microenvironments, in disease, they may trigger powerful and long‐lasting systemic toxicities. Cytokines are mediators of inflammation, and their qualitative and quantitative ‘profiles’ change as acute states become chronic. Today, cytokine expression by individual cells, their levels in body fluids and localisation in tissues can be readily measured and serially monitored to follow disease‐associated alterations. Measurements of ‘cytokine profiles’ are emerging as an important and informative parameter in diagnosis and prognosis of many human diseases.

Key Concepts

  • Assessments of cytokines underscore their physiological importance in health and disease.
  • A broad spectrum of assays for cytokine measurements is available.
  • Measuring several cytokines, establishing a ‘cytokine profile’, is most informative.
  • Cytokines are induced mediators and their biological effects are transient.
  • Cytokines mediate inflammation and thus impact on many clinical symptoms.
  • Profiling of cytokines may be diagnostically and prognostically useful.
  • Serial monitoring of cytokine profiles in disease emerges as a clinically helpful strategy.
  • The role of cytokines as biomarkers of disease progression and outcome is still to be validated.

Keywords: cytokines; chemokines; bioassays; immunoassays; multiplex assays; single‐cell assays

Figure 1. A format for an indirect or sandwich ELISA. In the first step, the capture Ab is bound to the plate. A sample containing the cytokine is added and is captured by the Ab. Next, an enzyme (E)‐conjugated detection Ab specific for the cytokine is added to the well. After the plate is washed, substrate solution is added and the colour is developed. Colour intensity is then read in a colorimeter and related to the concentration of cytokine present in the sample based on the standard curve generated with the data obtained from the cytokine control wells.
Figure 2. Example of an IFNγ ELISPOT assay, which is testing responses of a cancer patient's peripheral blood T cells to a tumour antigen. Columns 7–9 show patients CD4+ T‐cell responses to nonspecific and tumour‐specific stimuli. Columns 10–12 show assay controls setup with healthy donor PBMC. Samples are run in triplicates, and spots are counted with an automated ELISPOT counter (CTL Technologies). Note that this cancer patient's CD4+ T cells respond well to PMA/ionomycin and to a viral antigen but only marginally to a tumour antigen.
Figure 3. Cytokine expression by human CD4+ T cells cocultured with autologous CD19+ B cells. T‐ and B‐cell subsets were isolated from PBMC using AutoMAX and coincubated for 24 h before cell permeabilisation and staining with labelled anticytokine Abs and anti‐CD4 Abs. The gate is set on CD4+ T cells. Cytokine expression in CD4+ T cells was increased following the addition of B cells. The data were obtained by Drs. Zenichiro Saze and Patrick J. Schuler.
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Butterfield, Lisa H, and Whiteside, Theresa L(Jan 2016) Cytokine Assays. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0001124.pub2]