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Cytokines and their Detection

Cytokines are small (5-20kDa size), soluble, cell-impermeable proteins secreted into the extracellular environment that play key roles in cellular signaling. Many biological processes are coordinated through cytokine signaling, including apoptosis, inflammation, angiogenesis, immune responses, and cellular migration, to name but a few.

Signaling through cytokines relies on their release into the extracellular environment by the signaling cell, and subsequent binding to cell surface receptors on the receiving cell, which will lead to the activation of downstream intracellular signaling cascades. Signaling can occur in autocrine fashion, with the cytokine acting on the very same cell that secreted it; in paracrine manner, acting on other cells in the vicinity; or can even occur endocrine, affecting distant cell and organ targets by circulating through the blood stream (e.g. hormones). Cytokines commonly act pleotropic, and the same cytokine may be produced by a variety of cell types, and additionally act on several different cell types. But not necessarily to the same effect. Depending on the cell type and its activity status, a certain cytokine might act as a stimulus or inhibitor of a given cellular function. Furthermore, cytokines often act redundantly, with similar functions being fulfilled by several cytokines. And as if that wouldn’t be complex enough, cytokines often act in signaling cascaded, in which stimulation of a cell by a certain cytokine leads to the release of additional cytokines by the target cell.
The Cytokine Network in Lymphocytes

Figure 1. The Cytokine Network in Lymphocytes

The Different Types of Cytokines

The plethora of cytokine functions, their widespread redundancy, and their pleiotropy has mostly rendered the historically emerged nomenclature of cytokines useless to understand a single cytokine’s range of effects, but understating the background behind the names still provides some insight into a cytokine’s prominent function(s). The names lymphokines and monokines predate the term cytokines, and describe signaling molecules produced by lymphocytes or monocytes, respectively. Chemokines mediate chemotaxis between cells, attracting and recruiting other cells to their source location. The term interleukin was initially coined to describe cytokines involved in the interplay of leukocytes, but its widespread use to denominate newly discovered cytokines despite limited knowledge of their function has uncoupled nomenclature and function. Nonetheless, the vast majority of interleukins are produced by T-helper cells. Interferons are produced by host cells – amongst other functions – in response to viral infections and mediate viral interference, hence the name. Finally, Colony stimulating factors induce the growth of cells in semisolid media leading to the emergence of cell colonies.

Classes of Cytokines and their Function

In addition to nomenclature, cytokines are often classified due to their phylogeny, based on their genetic relation; or their structural homogeneity. However, a more useful and practical, albeit imperfect classification relies on grouping by function. Pro-inflammatory cytokines, such as Interleukin (IL)-1α/β, Tumor Necrosis Factor (TNF) α/β, or Interferon (IFN)-γ, are regulators of active immune responses and drivers of inflammatory reactions. Anti-inflammatory cytokines, such as Transforming Growth Factor (TGF)-β, or IL-10, serve to dampen immune responses. These inflammatory regulators can be further divided into those regulating a cellular immune response (e.g. TNFα, INF-γ), and those influencing the humoral immune response (e.g. TGF-β, IL-4, IL-10). Other classifications by function include chemokines, which regulate chemoattraction of leukocytes, or growth factors stimulating proliferation and differentiation. Finally, one particular functional class of cytokines encompasses the death receptor ligands (e.g. TNF- α, FasL, TRAIL). These cytokines bind to death-domain receptors on target cells and induce apoptotic cell death.

Cytokine Detection and Quantification

The complex biology of cytokines in health and disease, from viral and bacterial infection to the immune system combating cancer cells, makes them a prime focus of research and diagnostic analyses. However, detecting cytokines poses a variety of challenges. Their impact and function is directly linked to their extracellular expression levels, which often drastically vary with spatial location. Furthermore, their baseline levels are often very low and in the picomolar range. Finally, cytokines are dissolved in the extracellular fluids, which drastically limits the applicable detection methods.

Detection by ELISA

Due to the required sensitivity, range, and suitability for fluid biologic samples, Enzyme-linked Immunosorbent Assay (ELISA) is generally the method of choice to detect and quantify cytokines. Quantification of analytes by ELISA relies on immunodectection, i.e. the ability of antibodies to specifically bind a certain molecular target. ELISA is uniquely suited to analyze fluid biologic samples, including blood serum and plasma, lymph, as well as more tissue-specific fluids, such as synovial or cerebrospinal fluids, but also cell culture supernatants, and cell and tissue lysates. Additionally, sensitivities in the low picomolar ranges can easily be obtained.

With cytokines being small protein targets, the method of choice is often sandwich ELISA. Here, one immobilized antibody binds the target cytokine, and a second, enzyme-coupled detection antibody that also specifically recognizes the respective cytokine is used to quantify the amount of immobilized analyte in the assay (for more background info, please have a look at our tech notes on the different types of ELISA and the necessary controls). As this type of ELISA relies on the independent recognition by two target-specific antibodies, the resulting assays are typically of high sensitivity and specificity.

The Principle of Sandwich ELISAs

Figure 2. The Principle of Sandwich ELISAs

A plethora of cytokine ELISA kits is available on the market and researchers have a very good chance to find an ELISA suited to their target analyte and biological sample type. Additionally, ELISA analysis requires little specialized laboratory equipment and manual operation can be successfully performed utilizing standard fluid handling equipment and a microplate reader capable of measuring optical densities. The level of automation of ELISA analysis is freely scalable, from the help of plate washers and multichannel pipettes, to full automation with the aid of pipetting robots to achieve high throughput analysis.

However, ELISA assays require comparably large volumes of often precious samples, and comparably high reagent costs. Additionally, next generation methods typically offer much higher throughput rates.

ELISpot Assays

The ELISpot technique can be seen as a special form of ELISA. In contrast to ELISA, ELISpot does not serve to analyze liquid biological samples for their cytokine content, but is used to quantify the number of cells which produce a certain cytokine. Like in an ELISA, multiwell plates coated with target-specific antibodies are being used, but instead of a fluid sample, cells in suspension are being added (often in a viscous medium). Antibodies in the cells’ vicinity will bind any cytokine these cells will produce. The second difference is the use of an enzyme substrate that will generate a precipitating color product, instead of the soluble product used in ELISA. Positive detection of bound cytokines with the enzyme-coupled second antibody will result in the formation of a visible color precipitate in the direct vicinity of the antibody. Thus, colored spots will appear wherever a cytokine-producing cell was located, allowing to count the cells which produced the target cytokine in a sample.

The Principle of ELISpot

Figure 3. The Principle of ELISpot

Multiplex ELISA and Antibody Arrays

Cytokines commonly act through complex cross talk and fulfil various functions depending on the levels of other cytokines. Therefore, the examination of multiple cytokine targets in a given sample in parallel is often desirable. The simplest form of multiplex ELISA utilizes different antibodies in different wells of a microplate. For example, each row of 8 wells of a 96-well plate contains an antibody against a certain cytokine, allowing the simultaneous, qualitative analysis of 12 cytokines in up to 6 samples. However, while allowing for a convenient workflow, this type of multiplex array requires high sample volumes. A variant of this type of ELISA relies on sequential detection. A mix of capture antibodies is spotted into each well, but only one target-specific conjugated detection antibody is added at a time, analyzed, and then the next target-specific detection antibody is added. Often, enzyme-based luminescent detection with intermediate enzyme quenching or removal is used in this type of assay, but fluorescence-coupled detection antibodies can be an alternative. As only one well is needed per sample, this type of assay provides a solution to reduce sample volumes.

Spotted Multiplex ELISA

Figure 4. Spotted Multiplex ELISA
Another solution are antibody arrays. Here, antibodies are spotted with the help of microfluidics onto a carrier surface at specific locations. To allow location-specific detection, analyte binding is typically detected with a fluorescence-coupled second antibody (Read all about Fluorescence in our Technote). Read-out is performed with the help of microscopes, microplate readers, or special, often proprietary array scanners. In this type of antibody array, the signal’s location within the array provides the information on the analyte’s identity, and the signal strength provides the concentration information.

Antibody arrays exist as so-called multiplex ELISAs, where typically 9-16 different antibodies are spotted into each single well of a microplate. These plates can be either read sequentially, similar to non-array antibody mixes, or with microplate readers that can identify multiple signals in one well, or other imaging systems are utilized for read-out.

Higher-complexity antibody microarrays rely on large arrays of antibodies spotted onto glass or membrane carriers, and can analyze in the hundreds of cytokines simultaneously with a high degree of automation and very low volume sample requirements. As these technologies rely on complex, proprietary equipment, the initial investment can be substantial, despite the often moderate costs per reaction.

Bead-based Flow Assays

A clever multiplexing adaptation of the Sandwich ELISA’s principle utilizes fluorescent beads coupled to a cytokine-specific capture antibody. Detection of successful analyte binding is mediated by a fluorescence-coupled, second analyte-specific antibody. The fluorescence signals are read-out by flow cytometers or similarly operating instruments. By combining the information about the two fluorescent signals stemming from the beads or the antibodies, respectively, and eventually combining them with information on the size of the used beads, hundreds on cytokines can be analyzed in parallel. Some of these bead-based cytokine panels are compatible with widely used flow cytometers; others use proprietary instrumentation, which might even utilize additional technology, such as magnetic beads to aid with stabilization of the signal to boost detection sensitivity.

The Future of Cytokine Detection

Recent publications have suggested an interesting way forward for cytokine detection, taking advantage of the ever-advancing field of biosensors. By combining molecular target-specific sensors that bind the respective analyte, and detection of successful binding through electric signals, prototype chips were developed that allow live and continuous detection of cytokines. These biochips not only allow for fully automated detection of dozens to hundreds of cytokines in parallel, but also could potentially be integrated into implantable sensors, thus allowing to monitor cytokine levels live in patients without the need to obtain any type of sample.

Enzo’s Solutions

Enzo Life Sciences offers an extensive range of ready-to-use ELISA kits for a variety of research fields. Our kits are highly published and well renowned, and Enzo Life Sciences has decades of experience developing and optimizing commercial immunoassays to meet the ever-changing needs of our scientific community. Our cytokine ELISA kits are used in a wide variety of research fields, including inflammation and cancer research. For more information on ELISAs, please download our ELISA E-book or contact our Technical Support Team for further assistance.

Do you have more questions on ELISA and its principles? Have a look at our technotes on what are the differences between ELISA assay types, which controls to use in ELISA assays, how to QC your ELISA results, validation of sample dilutions and achieving linearity, variation in ELISA and how to reduce it, lot-to-lot reliability, and our 10 tips for successful ELISAs.

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