Cytokine Assays

Fionula M Brennan, Charing Cross Sunley Research Centre, Hammersmith, London, UK Catherine Haworth, Department of Haematology, Leicester Royal Infirmary, Leicester, UK

Cytokines may be assayed by biological properties, immunological recognition (ELISA or radioimmunoassay), competitive binding to the receptor molecule and by inference from the transcription of the mRNA. The advantages and disadvantages of each technique are summarized in Table 1. In general, bioassays are very sensitive and by implication verify biological activity of the cytokine; they are not however always very reproducible or specific. In contrast immunoassays, although reproducible and specific, are not nearly as sensitive as bioassays, although more recently the sensitivity of immunoassays has improved with the use of amplification techniques.

Immunoassays

Immunoassays detect cytokines using a combination of polyclonal and monoclonal antibodies directed against the cytokine or two different complementary monoclonals. In either case the antibodies should be raised against purified cytokines, preferably recombinant products or peptide derivatives to ensure specificity. There are essentially two main types of immunoassay; the enzyme-linked immunoabsorbent assay (ELISA) and the immunoradiometric assay (IRMA). In both cases the cytokine is detected by antibody capture.

In the ELISA the first antibody (polyclonal or

Table 1 Comparison of methods of cytokine detection

Evidence of Sensitivity Technical skill Specificity Effect of bioactive protein inhibitors

mRNA - Variable depending on technique ++ ++

monoclonal) is coated to plastic, followed by incubation with the sample or cytokine standard. The cytokine is then detected using the second antibody (monoclonal) conjugated to an enzyme and visualized using the appropriate substrate (Figure 1). Further layers on this sandwich technique can be applied to increase the sensitivity. For example unconjugated second monoclonal is detected using an anti-mouse immunoglobulin G (IgG) enzyme conjugate followed by the substrate. Alternatively the second monoclonal is conjugated with biotin followed by streptavidin enzyme complex.

GoeiI well with cytokine-specilic antibody brxibrd

Block unoccupied sdes with protein

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