Types of Multiplex Immunoassays

When asked to suggest some different types of multiplex immunoassays, many researchers include Luminex’ xMAP^® Technology in their answer. This isn’t surprising. Over the past few decades, collaboration between Luminex and its partners has led to the development of over 1,300 research use only (RUO) kits and more than 58,000 peer-reviewed publications. In this article, we review the various xMAP platforms and gather expert insight from four leading providers of xMAP products and services.

xMAP instrumentation

xMAP technology is centered around the covalent coupling of capture molecules to color-coded beads. The beads are identified and bound analytes detected with a dedicated instrument, of which there are currently four flexible options to choose from. “The Luminex^® 200™ System is the most widely used xMAP instrument,” says Dominic Andrada, Scientific Applications Senior Marketing Manager at Luminex. “It is based on a specialized flow-cell where the beads are interrogated individually, and can measure up to 100 different analytes per well of a 96-well plate.”

Another model, the xMAP INTELLIFLEX^®, builds on this by enabling multiplexing of up to 500 analytes in each well of a 96- or 384-well plate. It also incorporates a second detection channel to measure two parameters per bound analyte. For example, using the xMAP INTELLIFLEX platform, it is possible to measure two antibody isotypes targeting the same antigen or detect both a target of interest and a post-translationally modified isoform. “Additionally, the xMAP INTELLIFLEX platform offers greater compatibility with automation,” notes Andrada.

Other xMAP instruments are the FLEXMAP 3D^® System, which is also designed for high-throughput applications and can rapidly measure up to 500 analytes per sample, and the MAGPIX^® System, which is the easiest to use, most portable, and most affordable instrument. “The MAGPIX System can perform up to 50 different tests in a single reaction,” explains Andrada. “Rather than being based on a flow-cell, these instruments feature an innovative technology to analyze the beads.”

The power of partnership

While Luminex manufactures these instruments, it does not offer off-the-shelf RUO biomarker kits. Instead, it relies on a network of partners to develop and commercialize immunoassays and genomic tests based on xMAP Technology. These can be viewed using Luminex’ Kit Finder application.

If you cannot find an off-the-shelf product that meets your needs, both Luminex and its partners offer training and services for custom assay development. It should be noted that the xMAP INTELLIFLEX platform is for research use only, not for use in diagnostic procedures.

The remainder of this editorial features commentary from Bio-Rad, Bio-Techne, MilliporeSigma (the U.S. and Canada Life Science business of Merck KGaA, Darmstadt, Germany), and Thermo Fisher Scientific, who each outline their own xMAP portfolio.

Bio-Rad

Bio-Rad markets its xMAP products as Bio-Plex and offers the Luminex 200 and FLEXMAP 3D instruments branded as the Bio-Plex 200 and Bio-Plex 3D, respectively. “We provide installation and technical service for these systems and have our own Bio-Plex Software Manager for data acquisition,” explains Vanitha Margan, Bio-Rad’s Global Product Manager.

The Bio-Plex instrumentation and software are complemented by a suite of xMAP based immunoassays, named Bio-Plex Pro Assays. “Our Bio-Plex Pro Assays include products for studying cytokines, chemokines, and cell signaling, as well as assays for isotyping, detecting immunotherapy biomarkers, and measuring SARS-CoV-2 neutralizing antibodies, to name but a few,” says Margan. “Researchers can select from fixed multi-target kits, singleplex sets, or custom kits with their preferred targets.”

In addition, Bio-Rad offers Luminex raw beads as Bio-Plex Pro Magnetic Beads and Bio-Plex Pro Non-Magnetic Beads that can be used in combination with its Bio-Plex Amine Coupling Kit for in-house xMAP multiplex assay development.

Bio-Techne

R&D Systems, a Bio-Techne brand, offers a diverse portfolio of products utilizing xMAP Technology. “These include a large menu of assays, instruments, and analysis software,” reports Megan Larson, Senior Product Manager. “We make use of our extensive in-house collection of antibodies, proteins, and specialized diluents to develop our xMAP assays, all of which go through stringent quality control testing to ensure consistent, reproducible results. Importantly, the use of internal reagents allows for scale up and bulk shipments.”

Larson highlights the company’s mix-and-match analyte menu of over 450 analytes, as well as its pre-configured customizable High Performance Assays, which are optimized with custom diluents and correlated to Quantikine^® ELISA Kits. She also notes that the new Quantist Data analysis software, compatible with all xMAP instruments, simplifies data manipulation. “With Quantist, users can easily make inter-assay comparisons of multiple targets, assess quality standards and controls, and better evaluate the long-term consistency of their xMAP data.” 

MilliporeSigma

MilliporeSigma distributes the entire portfolio of xMAP instruments, along with ancillary equipment for streamlining multiplex workflows (e.g., BioTek^® Plate Washers, handheld magnetic separation blocks, and MultiScreen^®_HTS vacuum manifolds). In addition, the company offers a broad selection of xMAP immunoassays under the MILLIPLEX^® brand. “Our MILLIPLEX assays span multiple research areas and many different species, including human as well as research, companion, and agricultural animals,” says Brooke Gilliam, Global Product Manager. Each kit includes the following:

• Selectable capture antibody-immobilized magnetic beads in solution
• A premixed standard cocktail allowing you to easily rehydrate and prepare your serial dilutions
• Two premixed quality control cocktails to qualify assay performance
• A premixed detection antibody cocktail
• A prediluted optimized concentration of Streptavidin Phycoerythrin
• An optimized serum matrix mimicking the native sample environment (included for kits verified for serum or plasma samples)
• Bead diluent containing proprietary blocking reagents to reduce non-specific binding to the antibodies in the assay
• 10X wash buffer and ready-to-use assay buffer optimized specifically for the kit
• 96- or 384-well plate with two plate sealers

“Researchers can choose from our premixed panels or select any combination of analytes to design an assay that meets their needs,” adds Gilliam. “We also have a dedicated Custom Assay Development & Innovation (CADI) team, who can develop MILLIPLEX assays for detecting specific proteins of interest.” It is worth noting that MILLIPLEX cell signaling and autoantibody kits have different assay formats.

Thermo Fisher Scientific

Thermo Fisher Scientific distributes Luminex instruments and offers an extensive menu of targets and panels for both protein and gene expression applications. Invitrogen ProcartaPlex assays are marketed for detection and quantification of proteins, and Invitrogen QuantiGene Plex assays for gene expression.

“ProcartaPlex assays can be purchased as Simplex kits for single analytes, which can be combined to create a larger panel,” reports Craig Tin, Senior Manager for Immunoassays. “Preconfigured and customized panels for measuring up to 80 analytes in a single well are also available. For users processing large numbers of samples, 384-well plate assays are provided in two formats: with liquid bead formulation and with lyophilized beads already in the wells, which saves time aliquoting and for the overall preparation.”

When matched with corresponding ProcartaPlex panels, the QuantiGene Plex assays enable researchers to study proteins and RNA on the same Luminex instrument. “The QuantiGene platform provides higher plex and throughput compared to traditional techniques, in part by eliminating the need for RNA purification and adding flexibility to run in 384- and 96-well microplate formats,” says Tin.

Whichever type of multiplex immunoassay you decide to use, manufacturers and their partners are on hand to offer guidance.