Multiplex Platform Review and Selection Guide

When choosing a multiplex platform, the number of available options can be overwhelming. But, provided you keep the end goal in sight, platform selection shouldn’t be a formidable task. Here, we share tips for identifying a multiplex platform that best meets your needs.

Multiplexing offers a bigger picture

According to Marissa Beck, Senior Product Manager for Ella™ at Bio-Techne^®, multiplexed assays are becoming increasingly critical in understanding disease. “With the rise in proteomics analysis for not only research, but also disease detection, researchers need methods to quickly assess multiple targets impacting disease outcome,” she says. “Being able to see the bigger picture is important for identifying biomarkers, narrowing down drug candidates in preclinical studies, and evaluating therapeutic responses during clinical trials. In addition, for transplants and cell and gene therapy, profiling the immune response through multiple biomarker analysis may help better predict rejection of treatment.”

Many different options are available

Commercial multiplex platforms span technologies for detecting just a handful of analytes to those capable of detecting hundreds of targets simultaneously. Rather than attempting to list every possible option here, this section provides an overview of four very different approaches being employed.

Proximity extension assay

Proximity extension assay (PEA) uses matched pairs of antibodies, each labeled with a different oligonucleotide, to detect target analytes in solution. When an antibody pair is bound to the same protein, hybridization and subsequent extension with a DNA polymerase create an amplicon that can then be measured by quantitative real-time PCR or NGS. “Advantages of PEA include its exceptional specificity and sensitivity, which can reach fg/mL levels, and low sample consumption, which spans just 1 µL to 6 µL of a variety of biofluids,” reports Dale Yuzuki, M.A., M.Ed., Director of Marketing at Olink Proteomics. “Our technology currently scales from a mix-and-match set of 15 to 21 biomarker Olink^® Flex Panels, where users can choose from 200 predefined markers, through 15 standard 96-plex Olink^® Target 96 panels, all the way up to the 3,000-plex Olink^® Explore 3072 panel. These are designed to cover as many biological pathways and functions as possible.”

Paramagnetic bead-based array

Paramagnetic beads are attracted by the poles of a magnet, but are not magnetic in their own right, meaning they are easily separated in suspension and unlikely to clump. They form the basis of Quanterix’ Simoa^® technology, where they are coupled to antibodies and used for target pulldown from samples including plasma, cell lysate, and urine. “Following analyte capture on the bead surface and the addition of detection antibodies, the sample is loaded into a disc containing over 200,000 microwells, each capable of holding a single bead,” explains David H. Wilson, Ph.D. VP for Clinical Strategy at Quanterix. “At low concentrations, each bead will have either one bound protein, or none, meaning it is possible to count single protein molecules with enzyme-based signal amplification.” Quantification is performed using the semi-automated SR-X™ Biomarker Detection System, which can multiplex up to 4 targets, or the fully automated HD-X™ Automated Immunoassay Analyzer for measuring up to 6 targets.

Flow-based analysis with color-coded beads

Luminex’s xMAP^® Technology uses color-coded, antibody-coated beads for analyte capture. In addition, a combination of biotinylated target-specific antibodies and PE-conjugated streptavidin is used for detection with a dual-laser flow-based instrument. “By using one laser to identify the beads, and another to measure the PE-derived signal, Luminex technology allows researchers to measure as many as 500 analytes at once,” reports Dominic Andrada, Scientific Applications Senior Marketing Manager at Luminex Corporation. “Through partnership with companies including Bio-Techne, Bio-Rad, MilliporeSigma, and Thermo Fisher Scientific, we are able to offer a broad selection of kits for detecting many different combinations of analytes, as well as support custom panel development.” Recently, xMAP^® Technology was adapted to incorporate a second detection channel (xMAP INTELLIFLEX^®), effectively doubling the number of readouts per sample.

Fully automated, cartridge-based ELISA

While multiplexed ELISAs based on spatial separation remain popular for scientific research, a limitation of these methods is that they often involve numerous manual steps, which take time and can introduce user variability. Bio-Techne’s Ella™ platform was developed as an alternative to manual, plate-based ELISAs and other types of multiplexing assays, and requires less user intervention while providing faster time to results. Specifically, Ella uses customizable microfluidic cartridges containing R&D Systems reagents for accurate and consistent detection of up to 8 analytes in less than 90 minutes. “Unlike other platforms, Ella comes with factory-generated standard curves and pre-loaded cartridges,” explains Beck. “So, researchers simply add samples and buffer, insert the cartridge into the instrument, and walk away. Importantly, because each analyte is detected in a separate channel, the risk of unwanted cross-reactivity is eliminated.”

Schematic representation of Ella technology microfluidic circuit. Image provided by Bio-Techne.

Factors to consider for multiplex platform selection

So, what should you be thinking about when deciding on a multiplex platform? Wilson notes that understanding the expected concentration range of the different analytes being investigated is key. “If the concentration of one analyte being measured with plex is very high and requires dilution of the sample, reliably measuring a less abundant analyte in the diluted sample may be difficult due to a lack of sensitivity,” he says. One way of addressing this is to divide neat and diluted sample material across several, lower plex panels according to target expression.

It is also important to have flexibility in panel design. “Each disease requires different configurations of analytes,” says Beck. “An ideal multiplex platform will have a broad menu of fully validated assays to customize specific panels.” Andrada agrees, commenting that being able to build your own panels can also help mitigate costs. “Customers I have worked with are sometimes surprised at how expensive multiplex kits can be,” he reports. “While price typically increases with analyte number, high costs can also be due to a lack of alternative products. Building your own panel is often a good cost saving alternative.”

Other considerations include how easy the multiplex platform is to set up and use, and the level of ongoing technical support available. In addition, researchers should plan to ensure the chosen platform aligns with upcoming requirements. “When selecting a multiplex platform, it is essential to consider what analytes are of interest now, and what applications will be relevant later,” says Andrada. “By establishing what is most important for everyone involved—from laboratory leaders to the bench scientists who will be running the experiments—and speaking with providers, you should be able to identify a multiplex platform to match your specific needs for both today and the near future.”