Cytokines are small, secreted proteins that play a key role in cell signaling, with multiple classes of cytokines having regulatory functions throughout a network of different cellular pathways. Identifying and characterizing the interplay and interactions between cytokines and other target proteins—both to understand the healthy state and to identify biomarkers of disease—is a complex study that mandates more than merely detecting the presence of individual species. Rather, it is increasingly important to document even subtle changes in multiple cytokines at the same time.
In addition, especially when sample volume is limited and many cytokines need to be measured, “then it’s best to do it in a multiplex-type of setting,” notes Philip Wong, head of the Immune Monitoring Core Facility at Memorial Sloan Kettering Cancer Center, whose customers are often physicians involved in setting up clinical trials.
Although the traditional ELISA and its kin remain the gold standard of quantifying single cytokine species, this type of assay cannot simply be scaled. Here we look at several platforms that scientists are using to simultaneously and quantitatively interrogate multiple cytokines.
Double down
A traditional sandwich ELISA starts with an antibody bound to a surface. The antibody captures its target antigen, which is then bound by a second (“detection”) antibody at a different epitope. The detection antibody itself is reported in one of several ways, directly or indirectly, so that eventually an enzyme catalyzes a reaction that changes the color of the solution. The more antigen captured, the more enzyme present, the more substrate that is converted, the darker the solution and the higher its optical density as read by an ELISA (absorbance) reader. Similar assays increasingly are being performed using a fluorogenic or luminescent substrate in place of colorimetric readouts. Detecting the color of solution in a well is very difficult to multiplex, to say the least. Even multiplexing fluorescent immunoassays in solution, by using fluors with distinct excitation and emission wavelengths, becomes exponentially more difficulty because of inherent cross-talk as the number of analytes being detected increases.
Thus, most multiplex immunoassays need a solid phase to which the antigen can attach as well as a means of detecting its specific location, notes Robert Matson, founder and president of QuantiScientifics. That location can be a spot on a planar surface such as a glass slide, membrane or 96-well plate, for example, or on a uniquely identifiable polystyrene or magnetic bead.
This often means that assays are performed by directly or indirectly (through a biotin/streptavidin intermediary, for example) attaching a fluorophore or luminescent label to the detection antibody. Although, “if you don’t use an enzyme, formally speaking it’s not an ELISA,” Matson says. The assay can then be read with an instrument that maps the location (or identity of the bead) and intensity to the antigen being bound.
Luminex
Of the players in the field, “Luminex® is the overwhelming 800-pound gorilla,” says Per Hellsund, founder and CEO of CyVek (acquired last year by Bio-Techne), which developed the Simple Plex multiplex-assay technology now sold under the ProteinSimple© brand.
The Luminex website maintains a database of more than 23,000 peer-reviewed publications related to the company’s xMAP® technology. With this technology, microspheres are internally labeled with up to 500 combinations of fluorescent dye, allowing for the company’s highest-end reader to distinguish up to 500 distinct spectral addresses (which the company calls “regions”). “You can perform a different assay on the surface of each,” notes Luminex product marketing manager Carlos Garcia.
In the research arena, Luminex licenses the technology to companies such as Bio-Rad; EMD Millipore; Life Technologies, a Thermo Fisher Scientific company; R&D Systems; and eBioscience, to develop assays compatible with the Luminex platform. Many of these can be found as individual assays or as multiplex kits, such as R&D Systems’ 100-plex screening panel, in Luminex’s xMAP Kit Finder database.
Because Luminex is an “open platform,” customers can design their own immunoassays, as well, using their own proprietary antibodies and conjugating their antibodies to the beads using a coupling kit, Garcia says. “The workflow for these immunoassays, before you go on the instrument, is very, very similar to an ELISA,” he notes. The beads are then read either by imaging or flow cytometry, in 20 to 60 minutes for a 96-well plate, depending on the instrument.
In some cases, partners may market Luminex instrumentation “with additional accessories around that product” under their own name, says Garcia, citing the example of Millipore’s Milliplex® assay system “with Milliplex Analyst Software for running their specific assays.” But he is quick to add that “it doesn’t matter what brand is on the box, [the systems] should all be able to run any assay that is designed for the Luminex platform, regardless of who they are distributed by.”
Planar and simple
Off-the-shelf glass or membrane microarrays—essentially mini ELISAs—designed to be read by a standard laser scanner, can be purchased from a variety of manufacturers. At least one, RayBiotech’s Human L1000 Array, can simultaneously detect up to 1,000 target proteins, “including cytokines, chemokines, adipokines, growth factors, proteases, soluble receptors, adhesion molecules, and other proteins,” according to the company’s website. More modest versions—for detecting the 11 cytokines in the IL-1 family, for example—are available from the same company. But microarrays typically query one or very few samples at a time.
More diminutive microarrays also can be printed onto a microtiter plate; several companies offer these, as well. Among them, at least two platforms have emerged as major players in the field.
Meso Scale Discovery (MSD)—the system used by Wong’s core—prints up to 10 capture antibody spots onto the bottom of 96- or 384-well plates embedded with carbon electrodes. Detection antibodies are conjugated to electrochemiluminescent labels that emit light when a charge is applied to the plate, enabling highly sensitive assays with a broad dynamic range. The IL-4 assay, for example, is listed on the company’s website as being able to detect 0.02 to 158 pg/ml. “The imager is pretty fast—it takes an image of the entire plate, so you have your signals read in less than five minute,” notes Wong.
Aushon BioSystems’ multiplex immunoassays can “get down to the subpicogram to femtogram [per milliliter] levels of detection” with high levels of accuracy and reproducibility, claims CEO Susan Vogt. Assays, chosen from a menu of about 245 antibody pairs, are spotted in wells in a circular format “equidistant to the outer edge, so that they’re interrogating the sample all to the same degree,” she says. The chemiluminescent signal is read from the bottom up (to avoid any distortion from residual liquid that might be remaining in the plate), using the Cira™ platform’s high-end CCD camera. As with the MSD system, an entire plate takes less than five minutes to read and process, enabling high throughput.
In June 2015, Quanterix, known for its singleplex immunoassays, announced “commercial availability of its multiplex panels, providing users with the ability to develop their own multiplex assays using the company’s ultra-sensitive Simoa technology,” according to a press release. “The first commercially available multiplex kits are 3-plex panels of cytokines, which are very useful for measuring inflammatory response across a wide range of conditions.”
All of these systems are still confronted with the issue of multiple antibodies and other reagents needing to play well together. The Simple Plex microfluidics cartridge-based system “overcomes some of the conventional and inherent problems associated with the typical multiplexing” by separating the single sample into multiple channels, explains Hellsund. “We’re not using cocktails of reagents, we’re doing individual, single-analyte reactions/assays.” This system also boasts excellent reproducibility and “up to 4.5 logs” of dynamic range, but the real advantage is the hands-free ease of use, he says. Compared with other systems’ 25 to 50 manual steps to process the assay, “you simply add the sample and buffer to the cartridge, put the cartridge in the system, and within one hour you get results,” he says.
The local custom
These platforms offer either panels of cytokine immunoassays or menus from which to construct them—or both. Few offer the ability to customize assays beyond what’s on the menu (for example, using antibodies generated in-house). One exception is the A2® MicroArray System from QuantiScientifics, which offers arrays with pre-spotted linkers, designed to attach to capture antibodies conjugated to the complementary linker and thus self-assemble. This, says Matson, gives a more uniform density across the array and, he speculates, gives the tethered antibodies a greater range of motion, which would then lead to improved binding efficiency. Another exception is MSD’s new U-PLEX platform, which lets customers similarly construct their own arrays, in a fashion.
The good news is that there are now many choices to query multiple cytokines beyond running singleplex ELISAs. Multiplex assays provide a wide range of assay and performance features and formats that potentially benefit researchers by increasing throughput, sensitivity and time to results. Identify what is most important, or the hardest challenge you are facing, and it is likely that there is a system out there that can get the job done!