Cytokine Detection Methods

Cytokines are small, soluble proteins that serve as critical mediators of immune and inflammatory responses and are widely used as biomarkers for disease. While the low abundance, short half-lives, and transient secretion of many cytokines can make their detection challenging, selecting the right method will help to generate accurate results. This article provides an overview of current cytokine detection methods and suggests factors to consider when selecting a technique.

Traditional methods for cytokine detection

ELISA and PCR are among the most commonly used methods for detecting cytokines, providing high specificity and sensitivity for almost any target. “ELISAs offer many advantages for cytokine detection,” reports Mandy Lund, Ph.D., Senior Scientist for SimpleStep ELISA^® Development at Abcam. “They are easy to perform, the data can be measured with a wide variety of plate reader options, and the analysis is simple and straightforward. Historically, these benefits were offset by a time-consuming, multi-step protocol with the potential to introduce variability. However, platforms such as Abcam’s SimpleStep ELISA^® have a streamlined workflow that allows for protein quantification in just 90 minutes.”

Of the various PCR methods available to researchers, reverse transcription PCR (RT-PCR) is most often applied to cytokine detection. “RT-PCR provides a quick and easy means of detecting cytokine mRNA for insights into gene expression,” explains Amber Miller, Ph.D., Lab. Operations Manager (Flow Cytometry, IHC, and LFA) at Bethyl Laboratories (a Fortis Life Sciences brand). “Like all PCR methods, RT-PCR involves an amplification step, which may allow for detecting cytokines that are on the lower end of detection using other mechanisms. However, researchers should note that gene expression may not always correlate to protein levels—especially secreted protein levels.”

Advantages and disadvantages of traditional ELISA and RT-PCR *

*Table based on the combined feedback of all five contributors to this editorial.

Alternative tools and technologies

For those seeking an alternative to ELISA and RT-PCR for cytokine detection, a growing number of options is available. These include the following:

• Immuno-PCR

First described by Sano et al. in 1992, immuno-PCR is a technique that uses oligonucleotide-labeled antibodies for protein detection via a method analogous to ELISA.¹ Briefly, analyte-specific antibodies are immobilized on microplate wells and incubated with the sample, then oligonucleotide-labeled antibodies are added and PCR performed to amplify the signal. “Advantages of immuno-PCR are that it enables ultra-sensitive detection of low-abundance cytokines and can incorporate barcoding for multiplexed analysis,” explains Dan Lazar, Sr. Research Scientist at Promega. “However, the protocol is complex and the method is susceptible to DNA contamination. Additionally, there are not many commercially available immuno-PCR kits, and they tend to be more expensive than ELISA.”

• Bio-Plex^® Multiplex Immunoassays

Based on Luminex xMAP^® Technology, which uses color-coded beads that are pre-coated with antibodies for capturing analytes in solution, Bio-Rad’s Bio-Plex^® Multiplex Immunoassays have a wide (4 log) dynamic range that allows for quantifying both low- and high-abundance biomolecules in the same assay. “The Bio-Plex Pro™ Human Cytokine Screening Panel is a ready-to-use option that can detect 48 different cytokines in just 12.5 µL of sample,” says Vanitha Margan, Global Product Manager, Immunoassays at Bio-Rad. “This approach is faster and more efficient than traditional ELISA, saving time and sample volume, and is easily scalable to support high-throughput studies. Importantly, multiplexing provides a broader view of cytokine interactions and their roles in disease processes, improving the ability to analyze complex biological systems.”

• Flow cytometry

In recent years, flow cytometry has seen increased use for cytokine detection. “A major advantage of flow cytometry is that it identifies which cells are producing the cytokines of interest,” notes Miller. “Flow cytometry also lets you examine other factors alongside cytokines, including activation markers and additional functional markers, for deeper insights into your samples.” When performing these types of studies, it is essential to inhibit cytokine secretion (e.g., using monensin or Brefeldin A). Staining optimization is also critically important. “While fixation and permeabilization are required for assessing intracellular proteins, these steps can affect antibody staining and fluorophore intensity,” says Miller. “Staining optimization should be performed to assess whether surface staining should occur before or after fixation and permeabilization.”

• CyTOF technology

Standard BioTools™ CyTOF^® technology uses metal-labeled antibodies to assess multiple (>50) surface and functional markers in single cells, delivering insights into both phenotype and function. “Compared to flow cytometry, CyTOF technology can measure a higher number of parameters simultaneously without signal overlap, autofluorescence, or stability challenges,” reports Jennifer Ellis, MS, Director of Scientific Content at Standard BioTools. “It therefore captures immune complexity in an unconstrained manner, providing a much-needed layer of information to uncover critical biomarkers of predictive response, mechanism of action, and patient stratification in translational and clinical research.”

Importantly, the lack of signal overlap between adjacent metal channels in CyTOF makes panel design more straightforward than for fluorescence flow cytometry—and pre-validated CyTOF products can further simplify the process. “The dry-format, 30-antibody Maxpar® Direct™ Immune Profiling Assay™ can serve as a backbone to which ready-to-use panel additions measuring activation status and cytokine production can be easily added,” says Ellis. “Ultimately, the higher signal resolution afforded by CyTOF technology can help researchers to identify novel disease drivers, detect more functionally diverse cell populations, and design better therapies, all while using a single tube of limited sample.”

• Cyclical and Sequential Immunofluorescence (seqIF™)

Spatial biology techniques allow for visualizing where a cytokine is located, including which cells are producing it and what the neighboring cells are. A popular approach, known as tyramide signal amplification (TSA), involves incubating the sample with unlabeled primary antibodies and HRP-conjugated secondary antibodies before adding a tyramide substrate, resulting in the deposition of a tyramide-fluorophore at the target site. The antibodies are then removed with heat-induced epitope retrieval and the process repeated with a different tyramide substrate.

Another method, sequential immunofluorescence (seqIF™), combines unlabeled primary antibodies with fluorescently labeled secondary antibodies for target detection. It is based on repeated sequences of staining, imaging, and gentle, buffer-based elution of antibody complexes and has been fully automated on the COMET™, for 40-plex detection in less than 24 hours.² “Because TSA and seqIF use off-the-shelf antibodies, no upstream conjugations are needed,” says Miller. “However, careful optimization is required, including the order in which the antibodies are added during TSA and the length of the incubation times when performing seqIF on the COMET.”

• Imaging Mass Cytometry™

Imaging Mass Cytometry™ (IMC™), a technique based on CyTOF technology, is also widely used for studying spatial cytokine expression patterns. Notably, the high sensitivity and multiplexing capacity of IMC can efficiently reveal key phenotypic differences between samples. For example, a 2019 Cell publication reports the use of IMC to evaluate the expression of 73 proteins in 144 human breast tumor samples and 50 non-tumor tissue samples, leading to the identification of tumor and immune ecosystem characteristics related to prognosis.³

• Lumit^® Immunoassays

Developed as fast, homogeneous alternatives to ELISA, Lumit Immunoassays have a simple “add-mix-read” protocol, with no wash steps, that enables direct detection of cytokines and other analytes in cell culture wells without the need for sample transfer to a separate assay plate. “By minimizing sample handling, the direct protocol reduces variability and the potential for operator error,” says Lazar. Other noteworthy features of Lumit Immunoassays include a total assay time of less than 2 hours, compared with up to 5 hours or more for a traditional ELISA, and the use of luminescence detection, which provides high sensitivity and a broader linear range than colorimetric detection. “In combination, the homogenous workflow, assay compatibility with miniaturization, and the long signal half-life make Lumit Immunoassays ideal for automation and high-throughput screening applications,” adds Lazar.

• Develop your own assay with validated antibody pairs and conjugation kits

With 67 human cytokines reported in the literature, an off-the-shelf product may not always be readily available for detecting the analyte(s) of interest.⁴ In this situation, researchers may find themselves developing an assay from scratch, either through partnership with a technology provider or by using existing antibody reagents. To facilitate the latter, Abcam offers validated in-house antibody pairs in a ready-to-conjugate format (BSA and azide-free) and user-friendly Lightning Link^® conjugation kits.

“Our antibody pairs have been validated in multiplex assays and across various platforms to give customers confidence in the specificity,” says Lund. “Lightning-Link conjugation kits take as little as 30 seconds of hands-on time to label antibodies with tags including enzymes, fluorophores, oligos, and metals. These tools give customers the versatility to flow between ELISAs and their own in-house screening platform, while reducing the variation that is often observed when comparing data from different techniques.”

Key questions to ask when selecting a cytokine detection method

• Is the method compatible with your sample type?
• What sample volume is required?
• Do you want to detect proteins or mRNA?
• How straightforward is the protocol?
• How many cytokines do you wish to detect?
• Is the dynamic range of the assay suitable for detecting the cytokine of interest?
• Are matrix effects likely to cause problems? Can these be addressed with optimized buffer systems?
• Is there a risk of cross-reactivity with other targets, such as related cytokines that share conserved domains?
• Do you need single-cell data?
• Are specialized reagents or equipment necessary?
• Is the assay amenable to high-throughput automation?
• How reproducible is the method?
• Are there tools available to simplify data analysis?
• What level of throughput is possible—sample numbers, hands-on time, total assay time?
• Is the assay commercially available and how much does it cost?

References

1. Sano T, Smith CL, Cantor CR. Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates. Science. 1992;258(5079):120-122.

2. Rivest F, Eroglu D, Pelz B, et al. Fully automated sequential immunofluorescence (seqIF) for hyperplex spatial proteomics. Sci Rep. 2023;13(1):16994.

3. Wagner J, Rapsomaniki MA, Chevrier S, et al. A Single-Cell Atlas of the Tumor and Immune Ecosystem of Human Breast Cancer. Cell. 2019;177(5):1330-1345.e18.

4. Jiang P, Zhang Y, Ru B, et al. Systematic investigation of cytokine signaling activity at the tissue and single-cell levels. Nat Methods. 2021;18(10):1181-1191.