Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
For all the fancy technology today’s biomedical researchers have at their disposal, when it comes to quantifying specific proteins, one of the most popular choices is decidedly old-school: ELISA.
In an enzyme-linked immunosorbent assay (ELISA) the wells of a flat-bottomed, 96-well plate are coated with antibodies recognizing the protein of interest—interleukin-2, say. After incubating the plate with the sample, a second labeled antibody, which also recognizes the target protein, is added. The assay is then read by quantifying that second antibody’s abundance, for instance by adding a chromogenic substrate, and comparing the signal to a standard curve.
ELISAs are easy enough to do and relatively fast for one or two analytes, but sample and time can quickly become prohibitive as the analytes scale up. That’s where multiplex immunoassays come in. Multiplexed immunoassay systems enable researchers essentially to run multiple ELISAs in a single tube, simplifying workflow, reducing reagent requirements and conserving sample.
“You can get so much more information from one experiment [by multiplexing],” says Jane Schmidt, director of new technologies at R&D Systems. One 11-plex Luminex assay from R&D Systems requires just 125 μl of sample and 4.5 hours, for instance; getting the same data via ELISA would take more than a week, she estimates, and consume 3.5 ml of sample. “Getting that [much sample] for a research study is really difficult.”
Bead-based multiplexing
Multiplex immunoassays are available in basically two flavors. One approach, exemplified by Luminex Corp., is solution-based (as is BD Biosciences’ alternative, the BD™ Cytometric Bead Array (CBA)).
Luminex’s so-called xMAP® method uses libraries of microbeads, each internally labeled with differing amounts of two (or sometimes three) fluorescent dyes, to create spectrally distinct populations, each of which is assigned to a particular analyte. One set of beads, for instance, might contain 5% dye A and 95% dye B and be coupled to an anti-IL-2 antibody; another might be 20% A, 80% B and target IL-4.
To run the assay, beads representing each analyte are pooled into a single well, mixed with sample and incubated. After washing, secondary antibodies are added, followed by a fluorescent label. The assay is then read bead by bead, first identifying a bead by its fluorescent signature and then quantifying the associated analyte by the secondary antibody’s fluorescence. The result, says Cindy Fry, product manager at EMD Millipore, which offers Luminex assays under the MILLIPLEX® MAP brand name, is an “ELISA-on-a-bead.”
Luminex has developed three dedicated instruments to read these assays. The Luminex® 100/200 (which can distinguish up to 100 different bead signatures) and the FLEXMAP 3D, a higher-throughput instrument that can distinguish up to 500 signatures, are both flow-based, reading the beads as they pass a series of lasers as in a flow cytometer. Luminex’s newest (and least-expensive) offering, the MAGPIX, loads beads onto a magnetic plate and images them with a CCD instead. (Despite this level of theoretical multiplexing, few assays approach even 50-plex. Millipore’s human cytokine panel, its largest, is 42-plex, and most are much smaller. According to documentation, BD CBAs, which are read on actual flow cytometers, can multiplex up to 30 analytes.)
Though Luminex produces the beads and the instrumentation, the xMAP immunoassays run on those machines are available through corporate partners, 20 of whom are Affymetrix, listed on the company’s web site and include Bio-Rad Laboratories, EMD Millipore, Life Technologies and R&D Systems. Each company offers a different menu of assays, in both singleplex and multiplex configurations, and the menus do overlap, especially for common targets like cytokines.
There are differences, though. For instance, Bio-Rad Laboratories, with partner Myriad RBM, is developing panels specifically for use in drug-development testing. The two companies recently released kidney toxicity panels for human, rat and dog, and intrinsic apoptosis panels are anticipated by the end of the year, says Chris Linnevers, senior product manager for Bio-Rad’s Bio-Plex system.
Life Technologies offers a panel specifically for autoimmune-disease research, says Thao Sebata, senior product manager for immunoassays at Life Technologies, which offers Luminex assays under the Novex® brand name. Rather than coupling beads to antibodies, the ProtoPlex™ Immune Response Assay couples them to proteins, Sebata explains. “We conjugate the bead to the antigen and probe for antibodies instead.”
Luminex has an xMAP Kit Finder web app, and even an iPhone/iPad app, to help users find the assays they need.
Going magnetic
Initially, Luminex assays used only nonmagnetic polystyrene beads. More recently, the company has made magnetic beads available, as well. Both bead formats perform equally well, Fry says, but magnetic beads simplify and improve washing steps and automation. (Bio-Rad, with partner Tecan, offers the automated Bio-Plex Pro™ Wash Station for magnetic-bead assays; the Bio-Plex Pro II, for both magnetic and polystyrene beads, is also available.)
All three Luminex platforms can use magnetic beads, but the MAGPIX instrument actually requires them. But, not all assays are necessarily available in both formats, and different companies have different plans regarding the transition. Bio-Rad is aggressively phasing out its nonmagnetic offerings, says Linnevers. So, too, is Millipore: “As of 2014, we will no longer carry the nonmagnetic beads,” says Fry.
Life Technologies will support both formats for the foreseeable future, if for no other reason than there’s a large installed base of flow-based instruments on the market, and many users have validated their assays on that platform, says Sebata. Nevertheless, Sebata recommends new users adopt magnetic beads, as she sees “the polystyrene eventually being phased out.” But that won’t happen in the near term, she adds. “As long as [Luminex] continue[s] to provide [polystyrene] and customers buy it, we’ll still continue providing it.”
Planar microarrays
A second multiplexing strategy multiplexes the ELISA using a small, planar antibody microarray on the bottom of each well of a 96-well plate. The assay is run just like a standard ELISA, but to read the results, the plate is imaged to measure signal intensity at each spot.
R&D’s Proteome Profiler™ 96 microplate-based antibody arrays can detect up to 16 analytes per well, either using chemiluminescent or infrared-fluorescence detection. The company offers four fixed-content arrays for detecting phosphorylated human receptor tyrosine kinases and soluble kinases. (R&D also offers membrane-based Proteome Profiler protein microarrays for up to 119 proteins.)
Meso Scale Discovery (MSD) uses an electrochemiluminescence approach to quantify up to 10 spots per round-sided well (96-well format) or 25 per square-sided well. The assays are read in a dedicated instrument called a SECTOR® Imager. Application of electrical current to electrodes on the bottom of the wells causes a SULFO-TAG™ affixed to the secondary antibody to luminesce. According to Jonathan Klein-Evans, vice president and general counsel at MSD, imagers range from $25,000 to $200,000.
Customization
Luminex assays are relatively easily customized. All it takes is buying individual beads and mixing them together (so long as the beads’ spectral signatures don’t overlap). The hardest part, says Fry, is making sure the conjugated beads work well together. “That’s where you can get background issues,” she says.
Bespoke antibodies can be coupled to Luminex beads using Luminex’s assay-development kits.
R&D Systems’ Proteome Profiler 96 assays also are customizable; users can select up to eight antibodies per well from its catalog of 40-odd human phospho-RTK and 18 phospho-kinase antibodies.
MSD offers some 300 premade assays, according to Klein-Evans, and custom-development options are available. Alternatively, MSD offers a STREPTAVIDIN GOLD® kit, which enables researchers to deposit custom antibodies on MSD plates in a singleplex format by conjugating the antibodies to biotin and capturing them on a streptavidin-coated plate.
Before you buy
One variable to consider when making a purchase decision is sensitivity. On the face of it, that variable favors MSD. Electrochemiluminescence “is widely recognized as the most sensitive of the immunoassay detection methods, while also maintaining the widest dynamic range,” says Klein-Evans.
But sensitivity actually depends on multiple variables, Sebata notes, including antibody quality and the buffers used, so your mileage may vary. If sensitivity is likely to be a concern, you might consider testing each platform to see how it performs with your real-world samples.
Vanderbilt University Medical Center assistant professor of medicine Julie Bastarache, who uses the MSD system in her work on acute respiratory distress syndrome, says time and sample requirements drove her and her colleague Lorraine Ware to explore different multiplex platforms. In particular, she says, it’s difficult to get more than “a couple hundred microliters” of plasma from a mouse, [so] to do multiple singleplex ELISAs would be prohibitive.”
In 2011, Bastarache and Ware detailed their difficulties with one particular platform, the SearchLight® planar array then sold by Thermo Fisher Scientific [1]. (In 2009 SearchLight was acquired by Aushon BioSystems, but the assays are no longer available -- at least they are not listed on the company's web site. The company now offers chemiluminescent, planar array-based Ciraplex™ assays instead.)
As a result of her experience, Bastarache recommends users do some basic quality-control (QC) assessment before diving into any multiplex platform. Make sure the assay works as expected with your particular sample matrix (plasma, sera, cell extracts, for instance); ensure the protein survives (and can be detected) after multiple freeze-thaw cycles; check reproducibility from day to day and plate to plate; and see how the assay fares in terms of “spike-in recovery.”
“If the assay doesn’t pass those QC measures, we look for another assay,” she says.
Reference
[1] Bastarache, JA, et al., “Accuracy and reproducibility of a multiple immunoassay platform: A validation study,” J Immunol Methods, 367:33-9, 2011.
The image at top illustrates the Luminex xMAP assay principle, from Affymetrix.