The advantages of multiplexing are well known, and it has become common practice for researchers to analyze many different target analytes in parallel. But with such a broad range of multiplexing technologies available, why should you consider using them and what should you be looking for when deciding on a platform that meets your needs? Here, we highlight some of the different multiplexing technologies available.
Recommended eBook
Download this ebook now to get answers to questions like "why multiplex?" and "which multiplexing platform should I use?" as well as find out what you need to do to incorporate multiplexing in to your experimental workflow.
Multiplexing provides many advantages
Ask any researcher why they choose to multiplex, and you’ll likely receive the same response: lower sample volume requirements, more results per sample, greater throughput, and lower cost per data point compared to traditional singleplex assays such as ELISA. However, another important benefit of multiplexing is that it improves data consistency by enabling many different target analytes to be investigated within the same assay environment. This not only eliminates confounding variables like different buffer systems, time points, or operators, but it also provides a more complete picture of the sample that is under investigation. Additionally, with most multiplexing workflows designed to be compatible with automation, researchers can now achieve unprecedented levels of productivity.
Multiplex immunoassays comprise various formats
Multiplex immunoassays can broadly be categorized according to how the antibody reagents are used to capture and detect target analytes. Platforms where the antibodies are spatially separated on a solid surface include Meso Scale Discovery’s multi-array technology and the Ella™ system from ProteinSimple®, whereas Quanterix’ Simoa® platform and Luminex’ xMAP® technology both employ a bead-based approach. Olink instead uses antibodies that have been labeled with DNA oligonucleotides to bind target analytes in solution.
Multi-array technology from Meso Scale Discovery
Meso Scale Discovery’s multi-array technology is similar to an ELISA but provides greater sensitivity and a broader dynamic range. Using 96- and 384-well plates where up to 10 different capture antibodies are immobilized as distinct spots, the reaction is completed with detection antibodies bound to electrochemiluminescent labels. Plates are read on MSD’s flagship instrument, the MESO SECTOR S 600, or using the smaller but lower throughput MESO QuickPlex SQ 120.
Ella™ from ProteinSimple®, a Bio-Techne brand
ProteinSimple’s Ella is an automated microfluidic cartridge-based system that allows researchers to measure 1–8 biomarkers in just 25 µL sample. It relies on Simple Plex™ microfluidic technology to achieve a fluorescent readout, with multianalyte cartridges allowing for parallel quantitation of up to four biomarkers in discrete channels. “Ella’s multianalyte approach provides both high data quality and a high degree of panel flexibility,” explains Colin Merrifield, Product Manager. “The platform also delivers incredibly fast immunoassay results with minimal setup time—researchers simply add sample and buffer to the microfluidic cartridge and insert this into the instrument; fully analyzed results are produced in an hour for up to 32 samples in the multianalyte or multiplex formats.”
Quanterix’ Simoa® platform
Quanterix’ Simoa platform employs digital ELISA technology to provide approximately 1,000 times greater sensitivity compared to conventional immunoassays. Antibody-coupled paramagnetic beads are presented as an array within a specialized disc containing >200,000 wells, each capable of holding a single bead, where they are used to capture target analytes from a solution. This allows for the measurement of up to 6 analytes in a single sample, with readouts generated using the fully automated Simoa HD-1 or HD-X analyzers, or the more compact SR-X.
Proximity extension assay technology from Olink
While the three platforms previously described all require dedicated instrumentation, readout from Olink’s proximity extension assay (PEA) technology is achieved via qPCR or NGS systems that have been widely used in genomics studies, with Olink recommending the Biomark™ HD from Fluidigm. “During a typical Olink immunoassay, pairs of oligonucleotide-labeled antibodies bind as many as 384 different target proteins using only 1 µL of solution,” notes Jon Heimer, Olink CEO. “When the two probes are in close proximity, a new DNA amplicon is formed that can subsequently be detected and quantified using qPCR or NGS. Olink’s sensitive technology covers a 10-log dynamic range, from fg/mL to µg/mL. Because unique DNA oligo sequences report only matched DNA-pairs, the risk of cross-reactive events is eliminated.” Olink recently launched Olink® Explore, a platform capable of detecting 1536 analytes from ~3 µL of sample; this can generate >1.3 million protein measurements per week from one Illumina® NovaSeq NGS instrument.
Increasing the power of Luminex xMAP® technology
Luminex’ xMAP® technology has been on the market for over 20 years, making it one of the most widely used multiplexing platforms around. Using a mixture of color-coded beads, pre-coated with capture antibodies, it is possible to bind up to 500 different analyte targets in solution, with detection simply requiring the addition of biotinylated target-specific antibodies and PE-conjugated streptavidin.
Upon completion of an xMAP® immunoassay, beads are analyzed with a dedicated dual-laser flow-based detection instrument (Luminex 200™ or FLEXMAP® 3D), where one laser (635 nm) classifies the bead and the other (532 nm) determines the magnitude of the PE-derived signal. As an alternative to flow-based detection, magnetic MagPlex® beads can be read using the Luminex MAGPIX® Analyzer.
The xMAP® INTELLIFLEX is a new addition to the xMAP® family, designed to take Luminex’ technology to new levels. Featuring a second reporter laser (405 nm) alongside the traditional reporter channel, it enables the detection of two parameters on a single bead. This equates to decreased sample volumes and the potential for an even greater number of readouts than before, as well as enhancing the scope of biological assays that are currently performed using existing xMAP® technologies.
For example, on a single bead, researchers can now compare total and phosphorylated forms of a protein or monitor two distinct post-translational modifications. It is also possible to use the same bead for measuring two antigen-specific antibody isotypes, an approach that has received significant interest from researchers wishing to perform serology assays to study the immune response to COVID-19. Further uses for the xMAP® INTELLIFLEX include free versus bound drug testing, detection of protein cleavage and splice variants, and genomic applications such as SNP genotyping and evaluation of gene expression.
With an enhanced dynamic range of up to 5.5 logs and a compact footprint enabled by an integrated computer with touchscreen to reduce bench space, the xMAP® INTELLIFLEX complements current multiplexing technologies, giving researchers even greater choice when it comes to selecting a platform that best fits their requirements. Surely now the question isn’t ‘why multiplex?’ but rather ‘which multiplexing platform should I use?’