Detecting and measuring cytokines

Cytokines, such as interleukins, growth factors, interferons and tumour necrosis factors are soluble messengers that communicate locally between many different cells of the immune system. Often promoting multiple overlapping functions, from the maturation of B cells to the instigation of blood vessel growth, the physiological effects of cytokines depend on the relative concentrations of several different cytokines, illustrating that these chemical signalling molecules affect physiology via signalling networks.

interleukin 2 receptor, alpha chain
Interleukin 2 receptor, alpha chain

Cytokines function as indicators of inflammation and disease progression and are often used as a method to manipulate cellular responses both in vivo and in vitro. Due to their increasing importance as disease biomarkers, the ability to accurately detect and quantitate the array of cytokines present in samples is a prerequisite for both researchers and clinicians, to understand and predict disease progression and monitor the effects of treatment.

Within biological samples the concentration of different cytokines are often low, in the pg ml-1 range, thus accurate measurement of these signalling molecules is often obscured by the presence of high abundance proteins, such as albumin and IgG. For example, the dynamic range of plasma proteins is over ten orders of magnitude, with the top nine proteins making up 90% of the total plasma protein. Indeed on the primary issues in the development of new cytokine measurement platforms if the presence of many potential interferrents from biological samples.

To overcome this and ensure that the proteins are within range of the capture assay many remove the high abundance proteins from samples. However, the jury is still out on whether this additional time-consuming step is required for accurate measurement of cytokine concentrations, with one study showing that the removal of the high abundance proteins from samples also resulted in the loss of some of the cytokines present in the sample, except for GM-CSF, which showed improved measurement, so it seems this may well be protein specific, assay specific and lab specific, and ultimately tough to regulate for.

Of the currently available methods for determining cytokine concentrations within a sample the major immunoassay techniques in use are:

ELISA- Sandwich ELISAs are preferable over competitive ELISAs as they result in less variability in their results. Affimer technology has shown excellent results in ELISA applications, as they are highly specific and show no loss of function when immobilised on solid surfaces. With the increased understanding that cytokines function as part of an overall signalling network there has been much interest in assays that are multiplexed in their analyte measurement capabilities. ELISAs can be squeezed to measure more cytokines through the binding of more protein capture reagents within each well.

Cytokine bead arrays and microarrays- Both of these techniques allow multiple cytokines to be analysed at once, saving on precious samples. Concerns over these technologies include quality control and low precision between arrays, with a common observation being a difference in absolute cytokine concentrations when multiplex kits were compared to standard ELISA. Affimer binders can easily be adapted to bead arrays, which utilise their high affinity binding and rapid reactions kinetics in solution to yield a more rapid, specific and reproducible assay with improved sensitivity compared to traditional ELISAs.

Flow cytometry- This has been utilised to quantify many bead arrays and measure internal cytokines in permeabilised cells. ELISPOT assays also utilise a cell sorter to measure the cytokine production from single cells in an ELISA-based technique. Affimer reagents are well-suited to in vivo labelling applications as their small size, robust biochemical characteristics and 3D epitope recognition mean they can easily bind epitopes that are often hidden to larger affinity reagents and they are not affected by the assay environment.

IHC- This remains the preferred method for localising cytokines within sectioned tissue. The small size and high affinity and specificity of Affimer technology allows better tissue penetration and improved signal-to-noise in samples, compared to other affinity reagents.

An increased understanding of the cytokine orchestra and its regulation abnormalities could ultimately lead to promising and specific treatments for inflammatory diseases. New technologies are continuing to emerge to improve the speed and accuracy of cytokine measurements while increasing the array of cytokines measured with reduced sample sizes.