Cytokine Detection with Multiplex Immunoassays

Cytokines are essential cell signaling molecules that can serve as biomarkers for disease. Here, we look at how cytokine detection with multiplex immunoassays is being harnessed for drug discovery and development and suggest factors to consider for platform selection.

Cytokine screening and small molecule drug discovery

For decades, researchers have measured the expression of individual cytokines in sample types such as cell culture supernatant, serum, and urine to investigate how different drug treatments impact the biological landscape. However, while single analyte immunoassays remain popular, it is increasingly common for cytokine detection to be multiplexed.

“Due to the extreme complexity of the cytokine network and its close relationship to many diseases, cytokine detection is often multiplexed to provide a more comprehensive understanding of drug effects and potential interactions,” reports Wen-Rong Lie, Principal Scientist at MilliporeSigma (the U.S. and Canada Life Science business of Merck KGaA, Darmstadt, Germany). “Platforms for simultaneously quantifying multiple cytokines are now used across the entire drug discovery and development pipeline—from monitoring of small molecule activity in preclinical lead compound screening, performing in vitro cell-based assays, and establishing critical pharmacokinetic/pharmacodynamic (PK/PD) properties of potential drug candidates. They may also play a role for in vivo modeling, preclinical toxicity studies, and the provision of supportive data during clinical research.”

Importantly, multiplexed cytokine detection may lead to more targeted therapies. “There are several groups evaluating cytokines to drive a personalized medicine type approach to treatment,” says Jarad Wilson, Ph.D., Associate Director for Business Development at RayBiotech. “As well as helping to determine the most appropriate treatment option for a particular patient, multiplexed cytokine detection may be useful for early clinical assessments of drug efficacy and characterizing negative side effect cytokine profiles.”

For a closer look at how multiplexed cytokine detection might be applied in a specific disease setting, Dominic Andrada, Scientific Applications Senior Marketing Manager at Luminex Corporation, recommends a 2017 Journal of Autoimmunity review. This covers the development of small molecule therapies targeting the Janus kinases (JAKs), a family of enzymes known to regulate the start of cytokine signaling cascades. “Approved JAK inhibitors include ruxolitinib, which selectively targets JAK1 and JAK2 to block intracellular signaling by cytokines including IL-6, IL-12, IL-23 and IFN-γ, and tofacitinib, which targets JAK1, JAK2, and JAK3 to inhibit signaling by a different array of cytokine molecules,” he says. “These drugs are successful due in part to their ability to inhibit specific JAKs and thereby limit the activity of only selected cytokines involved in an immune-related or chronic disease.”

Applicability to biologics development

Biotechnology advancement has enabled large-scale manufacturing of biologics including monoclonal antibodies, nanobodies, antibody-drug conjugates, fusion proteins, cell therapies, gene therapies, and therapeutic synthetic vaccines—and multiplexed cytokine detection technologies have had to keep pace. In a biologics setting, multiplexed immunoassays are important not only for determining how such drugs exert their effects but also for helping to monitor for cytokine release syndrome (CRS), a systemic and potentially life-threatening inflammatory response. “CRS can occur from the immune system responding to infections, but has also been linked to the administration of biologics such as therapeutic antibodies and CAR-T therapies,” explains Lie. “As these types of drugs become more widely available, careful management is required to both maximize the therapeutic benefit and minimize the risk of CRS.”

Factors to consider for platform selection

When selecting a multiplex immunoassay platform for cytokine detection, there is no such thing as one size fits all. A key factor to consider is the phase of your drug development program. “Early studies may adopt a more kitchen sink type approach to evaluate as many targets as possible, especially if not focusing on a specific type of response to a drug,” notes Wilson. “Here, something like our L8000 array—which can provide biomarker information for 8000 proteins in a high throughput manner—would be a good fit. Later on in the process, it is more usual to whittle down the target list to a much smaller panel, which may focus on the drug effects directly, the indirect or off target effects, or a patient’s response following treatment. For example, RayBiotech offers a broad selection of smaller arrays, including focused panels for studying inflammation, cancer, and obesity."

Protein profiles of 2 different serum samples visualized by the RayBio^® Human L507 Array, a 507-target antibody panel. This array comes with a nitrocellulose (left) or glass slide (right) solid support.

During the construction of smaller panels, Andrada suggests starting with a combination of markers and sample specimens that are well understood, then adding in further analytes once pilot experiments have given the expected results. “Initial steps can sometimes reveal incompatibilities with the sample matrix, or cytokine levels that are broader than anticipated,” he explains. “One way of circumventing these issues is to use an off-the-shelf kit, such as those developed by Luminex’ partners, which are centered on xMAP^® Technology and can be searched using the xMAP^® Kit Finder Portal (Research Use Only). Should the desired combination of markers be unavailable in kit form, our LuminexPLORE Lab can help with custom assay development.”

MilliporeSigma’s MILLIPLEX^® multiplex kits feature among the ready-to-use xMAP^® Technology-based options for cytokine detection. “The MILLIPLEX^® portfolio encompasses a wide selection of products for quantitative multiplexed cytokine analysis,” comments Lie. “Each kit is verified with reported assay characteristics including cross-reactivity, assay sensitivities (minimum detectable concentrations), precision (inter-assay and intra-assay %CV), and accuracy (spike recovery). And, for multiplexed cytokine detection in serum and plasma samples, quality controls and an optimized serum matrix are included to qualify assay performance and mimic the native analyte environment.” Well-known products include the MILLIPLEX® Human Cytokine/Chemokine/Growth Factor Panel A, and a corresponding Panel B, for simultaneous quantification of 48 different cytokines in just 25 µL human biofluid or lysate samples. Again, if a ready-to-use kit does not fit, MilliporeSigma (as well as other major Luminex partners) offers custom multiplex assays for Luminex instruments.

Key takeaways

• Multiplexed cytokine detection is seeing increased use for small molecule drug discovery and development
• Multiplexing provides opportunities to develop personalized therapies
• Applying multiplexed cytokine detection within a biologics setting can monitor for cytokine release syndrome
• Detecting large numbers of analytes early on, before refining the multiplex panel to include only critical analytes, is a tried and trusted approach to developing novel drugs
• Using off-the-shelf kits for multiplexed cytokine detection can save time spent on assay optimization