Cytokines are intercellular messages that regulate important functions in the human body. A greater understanding of how, when, where, and why these messages travel through the body is helping researchers and clinicians learn about cytokines’ roles in various diseases. In order to do this effectively, scientists need reliable methods for detection cytokines, and measuring their levels in different conditions.
“One of the most interesting advances in recent years is that the sensitivity of cytokine detection has dramatically increased, therefore allowing the analysis of even very rare cell populations or low cytokine producers in the context of immuno-oncology and infectious diseases,” says Marcello Stein, product manager at Miltenyi Biotec. This article looks at tools for common methods of cytokine detection, and how cytokine detection intersects with a wide variety of disease studies.
The importance of high-quality antibodies
No matter how many methods are available for cytokine analysis, detection still comes down to having good antibodies. Because cytokines disperse as soon as they are secreted into the extracellular milieu, detection relies on high-affinity antibodies. With state-of-the-art antibodies, most cytokine detection today is performed using ELISAs, bead-based assays, and flow cytometry. Cell Sciences, for example, supplies high-quality cytokine antibodies in multiple forms, including matched monoclonal antibody pairs for detection in ELISA assays; monoclonal antibodies labeled with FITC or PE for detection by immunohistochemistry; and monoclonal antibodies for staining intracellular cytokines for detection by flow cytometry. “Newer methods [such as multiplex and bead based assays] still depend on high affinity, highly specific, validated and stable antibodies,” says Anne Sloan, technical support specialist at Cell Sciences.
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Miltenyi Biotec’s recombinant REAfinity™ Antibodies for cytokine detection include a mutated FcR binding site. “This eliminates the need for FcR blocking, and results in a lower background and higher specificity when analyzing mouse and human cytokines in, for example, intracellular cytokine stainings,” says Stein.
Intracellular flow cytometry
Cytokine detection can be accomplished by flow cytometry after labeling cells intracellularly using high-quality antibodies that are tagged with fluorescent reporters. “The advantage of intracellular flow cytometry is the ability to identify the single cell that is expressing the cytokine, and multiple cytokines can be measured in each cell simultaneously,” says Stephanie Widmann, manager of MSA applications immunology at BD Biosciences.
Miltenyi Biotec’s Rapid Cytokine Inspector Kits also stain surface markers and intracellular cytokines in activated human T cells for subsequent analysis by flow cytometry. These kits allow “rapid identification and enumeration of cytokine-producing, activated, antigen-specific CD4+ and CD8+ T cells upon in vitro stimulation with the respective antigen or upon polyclonal restimulation, and are compatible with the MACSQuant® Analyzer 10’s Express Modes,” adds Stein.
ELISAs and bead-based assays
Traditional plate-based ELISAs are trustworthy tools for cytokine detection, and are ideal for routine use when speed or throughput is not crucial. This format has evolved in recent years into multiplexed and bead-based ELISAs that can be highly engineered to detect many cytokines simultaneously. The basic principle of an antigen of interest sandwiched between two specific antibodies remains—rendering this type of assay the “gold standard” for detection. Many suppliers offer both traditional and bead-based formats depending on researchers’ needs.
Bead-based assays for detecting cytokines allow researchers to maximize their output from single samples—particularly valuable for cytokines at lower concentrations. Luminex’s xMAP platform allows users to develop their own assays for cytokine detection and quantitation. “Leveraging multiplexing allows preservation of sample volume, drives down assay costs, resolves reagent availability issues, delivers improved assay performance, and exponentially increases the amount of data and statistical power of results from a single sample,” says Heather Darby, field applications scientist at Luminex.
Quanterix offers magnetic bead-based ELISA kits for up to four analytes per sample at the highest sensitivity, and more traditional plate-based ELISA kits. High sensitivity multiplexed assays, such as Quanterix’s Human 10-Plex Cytokine Panel A, “allow the simultaneous monitoring of pro-inflammatory and anti-inflammatory cytokines, which can give a more complete picture of an individual's immune system status,” says Kevin Hrusovsky, CEO and chairman of Quanterix. “[Further applications include] monitoring general immune responses, monitoring responses to specific immuno-oncology therapies, and detecting cytokine release syndrome in response to CAR-T therapy.”
Quanterix’s bead-based ELISAs offer higher sensitivity for difficult-to detect cytokines. “With these assays, very low-abundance cytokines such as IL-5 and IL12p70 may be measured with precision even in healthy individuals, or when levels decrease with disease,” says Hrusovsky. Monitoring low abundance cytokines is especially tricky because baseline levels must be measurable to detect subtle changes in cytokine levels with disease. “This allows researchers to follow cytokine levels as indicators of treatment efficacy with far more precision than typical endpoint measures of disease progression,” says Hrusovsky. Such methods are used in developing drugs for autoimmune diseases such as psoriasis, lupus, and rheumatoid arthritis.
Miltenyi Biotec’s MACSplex Cytokine Kits use their MACSplex Capture Beads technology for multiplexed analysis of soluble cytokines. Results can be read on any flow cytometer, but their MACSQuant® Analyzer 10’s fully automated Express Modes are fastest. Stein says these kits are currently used in “functional analysis of in vitro stimulated immune cells and CAR T cells, in the context of killing assays, and functional immunophenotyping by cytokine secretion analysis.”
BD Biosciences offers the BD® Cytometric Bead Array (CBA) line for multiplexing cytokine detection in three formats: CBA Kits, CBA Flex Sets, and CBA Enhanced Sensitivity Flex Sets. Kits are optimized for assaying a predetermined combination of cytokines, such as the BD® CBA Human Th1/Th2 Cytokine Kit, which measures Interleukin-2 (IL-2), Interleukin-4 (IL-4), Interleukin-5 (IL-5), Interleukin-10 (IL-10), Tumor Necrosis Factor (TNF), and Interferon-γ (IFN-γ) protein levels in a single sample. Flex Sets offer the flexibility for researchers to select analytes according to their needs. The Enhanced Sensitivity Flex Sets detect down to the low fg/mL level with a standard range of 274 to 200,000 fg/mL. “This low level of detection allows for earlier quantification in a time course, or the ability to detect cytokine expression from fewer cells,” says Widmann.
Cytokine detection in disease studies
With greater sensitivity and specificity, the ability to detect low abundance cytokines offers new targets for clinical studies. Hrusovsky says that cytokine analysis with Quanterix technology is frequently used to develop biomarkers for specific disease states. “The most common application we see is drug efficacy monitoring for translational research and early clinical trials,” he says. “Blood-based biomarkers are key to increasing the likelihood of successfully bringing a new drug to market, as they allow frequent testing for treatment response.” This makes it easier to identify minimal effective doses to reduce the likelihood of toxicity. In addition, understanding how cytokine levels change over time in healthy people can enable early detection of health abnormalities.
Cytokine detection is also an important part of immuno-oncology research today. “Understanding how immune systems can be encouraged to eliminate cancer cells is the ultimate goal,” says Widmann. “Cytokines are a key communication pathway for cells, as well as having effector functions such as granzyme and perforin for killing infected or tumor cells. The ability to detect these cytokines is critical to understand this communication and functionality.” With the enhanced assay sensitivity and multiplexing opportunities available today for detecting cytokines, researchers and clinicians are rapidly gaining tools for finding and preventing diseases.