by Caitlin Smith
Cytokines are vital intercellular communication molecules. These proteins carry signals or messages when they are released from one cell and are subsequently sensed by another cell. Releasing information into the extracellular milieu means dilution of the signal, however. “Sensitivity is always an issue in cytokine biology,” says Valerie Jones, Marketing and Technical Support Manager at Raybiotech. “Although some cytokines are produced at ng/ml concentrations in body fluids, most are expressed at pg/ml levels. This is the main reason that traditional methods of proteomics, such as 2D gel electrophoresis and mass spectrometry, are not suitable for cytokine detection. Immunoassays are capable of detecting most of these low-abundance proteins. In the case of inflammatory cytokines, their concentrations in the serum or plasma of normal individuals [are] at or below 1 pg/ml. At this point, you begin to approach the technical limits of all immunodetection assays using current technology.”
There are several methods of detecting and analyzing cytokines in a sample: traditional ELISA assays, enzyme-linked immunosorbent spot (ELIspot) assays, antibody array assays and bead-based assays. There is no one best method, because choosing a method depends upon the particular situation and application. Rather, each has its own merits and limitations, which are discussed here to help you understand applications and which method might be best for you.
ELISA assays
Traditional ELISA assays are reliable, tried-and-true solutions that many researchers depend on. A successful ELISA system depends on having good antibodies and detection reagents for the assay. In the drawback category, ELISA assays are not fast, and they usually require larger sample volumes than the other assay types.
However, for many researchers who aren’t performing high-throughput research on tight deadlines, ELISA is still the reliable way to go. “While next-generation techniques such as bead-based assays or antibody arrays offer the possibility of much higher throughput, traditional ELISAs still provide a sufficient level of throughput for most researchers at a much lower cost per assay,” says Matt Baker, director of business development for protein biology at Thermo Fisher Scientific. “Besides the reagent costs, the instrumentation to perform ELISAs is much less expensive than that used by the newer technologies. In addition, colorimetric or chemiluminescent-based ELISAs require less time to set up and optimize than bead-based assays or antibody arrays, while providing better assay-to-assay consistency.”
The Thermo Scientific Pierce Cytokine ELISAs and antibodies offer a range of cytokine products, including the Thermo Scientific SuperSignal ELISA Pico Chemiluminescent Substrate, a detection reagent. “The biggest challenge facing researchers is the availability of high-quality antibodies and ELISAs to measure specific analytes,” says Baker. “The number of targets is growing rapidly, and researchers will look to those companies that offer an ever-expanding line of validated antibodies to keep up.” Reagents for ELISA assays also are offered by other companies, including BD Biosciences, BioLegend, eBioscience and Raybiotech. Quansys offers an ELISA-based array that allow the researcher to simultaneously assay for 16 markers.
A modified form of the ELISA assay is the ELIspot assay. The ELIspot is useful in determining the number and type of cytokine-producing cells in a sample, at the level of the single cell. The assay is sensitive enough to reveal (with developing spots) which individual cells are producing which cytokine, allowing the researcher to count cells individually. This is possible because the assay reagents rapidly “capture” the secreted cytokine as it is released from the plated cell, before it is diluted or degraded. Standard detection systems (such as biotin-avidin-HRP) are used to detect the spots of cytokine release, which correspond to the location of a single cytokine-releasing cell. Spot counting also can be automated, for greater throughput and more objective readings.
Antibody arrays
Antibody arrays, spotted onto chips or glass slides, have particular advantages that ELISA lacks. “Because cytokines commonly have complex cross-talk and overlapping functions with many other cytokines, examination of only one or a few molecules (by Western blot or ELISA, for instance) can give an incomplete picture of their roles in an experimental model,” says Jones. “Antibody arrays measure the levels of dozens or even hundreds of proteins simultaneously; this approach not only gives researchers a broader perspective but unveils other relevant molecules they may not have previously considered. Compared to the traditional approaches of ELISA or Western blotting, antibody arrays are also highly cost-effective and consume far less sample.” Raybiotech’s arrays come in multiple formats, including semi-quantitative and fully quantitative arrays and membrane- or glass-chip solid supports.
An advantage of antibody arrays is their high density. “Sandwich-based arrays can detect up to 100 targets in a single array, while label-based arrays (in which sample proteins are labeled with a reporter) can be constructed to detect 1,000 proteins or more,” says Jones. “But compared to ELISA, it is more difficult to optimize the antibody array to detect single targets. In antibody arrays, chemiluminescent signals can be detected using multiple exposures and fluorescent signals on the glass chip, using multiple laser power and/or signal gain settings. These multiple images can be obtained using a single finished array, with no requirement to repeat the experiment with a new array and sample.”
Affymetrix also offers antibody arrays (in addition to the bead-based arrays discussed below). The company’s Angiogenesis Antibody Arrays are made to detect multiple human or mouse cytokines from cell lysates, conditioned media, sera, plasma and/or urine. “Cytokine Antibody Arrays have several advantages over traditional methods like ELISA,” says George Bers, executive vice president of commercial operations at Affymetrix. “The high sensitivity is a key benefit. For example, the Cytokine Antibody Arrays can detect soluble cytokine proteins at concentrations in the pg/ml range. More importantly, the Cytokine Antibody Arrays enable the simultaneous detection of multiple cytokines in one assay.”
Bead-based assays
Bead-based assays confer savings in cost, time and money because they allow multiplexing, i.e. the simultaneous detection of multiple targets. Multiplexing allows for smaller sample volumes and high-throughput automation. A drawbacks include interfering interactions between antibodies that may lead to false positives—something that does not happen, in antibody arrays. “When the antibodies are printed in fixed positions on a planar surface, there is no possibility of interactions between the capture antibodies,” explains Jones..
Luminex offer a new bead-based assay system, MagPix, for its xMAP technology platform. MagPix uses magnetic polystyrene beads that, when subjected to a magnetic force, remain still long enough for fluorescence imaging with light-emitting diodes and a CCD camera. This is a simpler and more affordable system than others that involve injecting beads single-file through a cuvette and imaging with lasers. As such, according to Christoph Cordes, group marketing manager at Luminex, MagPix is an ideal entry-level multiplexing bead-based assay tool. “We’re using a technology that closely resembles ELISA plate readers [as opposed to flow cytometry], so it’s more familiar to people who have not moved to multiplexing yet,” he says. “From a workflow perspective, we have a similar workflow as an ELISA in terms of assay preparation. The reduction in complexity, the accessibility, the price, as well as compatibility with existing methods, really allow us to target people who are switching from things like ELISA and Western blotting.” Cordes also notes the increasing importance of studying pathways in biology and says that bead-based assays allow you to easily add one more type of bead to your experiment. To do this with an antibody array, you’d need to re-spot the entire chip or slide just to add one item to your panel.
Sherry Dunbar, director of scientific marketing at Luminex, notes one of the limitations of multiplexing with bead-based assays: antibodies. Currently, labs have reported going up to 70-plex for proteins (higher for DNA). “We could go up to 500-plex, but we’re held back by the lack of available antibodies that don’t cross-react or interfere with each other,” says Dunbar. “Being able to do truly huge multiplex assays is dependent upon that next type of biological reagent that may mitigate these issues—something that will take the place of the antibody in detection. It’s more difficult with proteins, because you’re looking at [not just a sequence] but also specificity.” She suggests potential candidates, such as oligos or aptamers, which have shape and structure, yet aren’t generated by mice or rabbits.
Affymetrix’s bead-based Procarta assay uses Luminex’s xMAP technology to quantify the cytokines in multiple samples simultaneously. “Our Procarta multiplex assays have been used for biomarker discovery and validation of actual cytokine expression following RNA analysis to better characterize host immune response in vaccine developments,” says Bers, “and to reflect physiological and pathological state of organs, tissue or cells in the context of immune surveillance and disease diagnosis, development and progression.”
Bio-Rad offers two systems for detecting cytokines using bead-based assays, both based on the Luminex platform. Their Bio-Plex 200 reads 96-well plates, and their higher-throughput instrument, the Bio-Plex 3D, reads 96- and 384-well plates. Bio-Rad also offers various cytokine detection panels for several species. According to Jessica Dines, senior product manager at Bio-Rad, the Bio-Plex only needs about 12.5 ul sample volume per well to measure over 25 analytes simultaneously, as compared to 100 ul per well to measure one target for an ELISA. “Bio-Rad optimizes their assays and software for flexibility and performance, enabling sensitive, accurate and precise measurements across a broad dynamic range. Our analysis tools help the researcher digest all of that data. The reliability and ease of use has made Bio-Plex appealing across research segments from the discovery phase to clinical trials.”
Dines says the limitations of bead-based arrays depend in part on the research area in which they’re being applied. “For example, for diabetes, the core group of biomarkers is pretty well defined,” says Dines. “In contrast, in cancer, it’s just daunting how many markers are currently used, depending on whether you want to look at angiogenesis or metastatic markers, for example. Multiplexing is a huge advantage in either case. Either we have a discrete panel, and we’re monitoring changes in levels in certain disease or drug-treatment conditions. Or, in the case of cancer, it depends on which cancer and which tissue you’re looking at—those markers are still being defined.”
There is no doubt that the story of cytokines has only just begun. “The interconnected, extracellular signaling networks of cytokines [are] at least as complex as intracellular signaling pathways, such as those involved in phosphorylation signal cascades,” says Jones. “As a result, detection of multiple cytokines is necessary to get a more global view of these signals. The less that is known about a particular physiologic or pathologic mechanism directed by cytokine signaling, the greater the need to use multiplexed cytokine detection.” Using the assay system best suited to your needs will help you to advance the story.
The image at the top of the page is Luminex's MagPix.