Luminex® Multiplex Assay Overview

As your research uncovers the significant role of more and more biomarkers, you need a reliable partner for quality assays and consistent performance. Our MILLIPLEX® multiplex immunoassays are the largest portfolio of multiplex biomarker assays, based on Luminex® xMAP® multiplex assay technology, offering you consistent, high-quality results, so that you can do your best work while saving time, labor, and cost. Discover the benefits of these assays and see how they are being used to advance research for immunology, cancer, veterinary medicine, metabolism/endocrinology, and more.

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What Is a Multiplex Assay?

A multiplex assay is an assay that combines tests for various analytes in a single panel. These assays have gained popularity for their advantages of saving sample volume and time by improving workflows when compared to assays that measure a single analyte, such as enzyme-linked immunosorbent assays (ELISAs). 

What Are MILLIPLEX® Multiplex Assays and How Do They Work?

MILLIPLEX® multiplex assays use the proprietary Luminex® xMAP® bead-based multiplex assay platform. This is how these multiplex assays work:

  • Each magnetic MagPlex® microsphere bead is fluorescently coded with one of 500 specific ratios of two fluorophores (each spectrally distinct set is known as a bead region, fluorescent at ex 635 nm).
  • Analyte-specific capture antibodies are bound to beads of a specific region, then unbound sites of the bead are blocked.
  • Beads are incubated with sample, washed, and then incubated with a biotinylated secondary antibody.
  • The unbound secondary antibody is washed away and the added streptavidin-phycoerythrin binds to the biotin of the “sandwich” immunoassay.
  • The Luminex® instrument detects individual beads by region plus the streptavidin-conjugated R-Phycoerythrin (SAPE) signal, ex 525 nm, indicating the analyte is present.

Figure 1 shows a general illustration of how these assays work.

Diagram showing the MILLIPLEX® multiplex assay format based on Luminex® technology, using Luminex® magnetic beads and phycoerythrin-labeled detection antibodies.

Figure 1.MILLIPLEX® multiplex assay format based on Luminex® technology.

How Do The Luminex® Detection Systems Work?

There are different detection systems for Luminex® multiplexing platforms, including the xMAP® INTELLIFLEX, Luminex® 200™, and FLEXMAP 3D® instruments. These Luminex® instruments use a flow cytometry-based system where beads flow in sheath fluid through a cell with a red laser (635 nm) identifying the bead region and a green laser (525 nm) quantifying the bound analyte “sandwich”. The instruments integrate key xMAP® detection components, such as lasers, optics, advanced fluidics, and high-speed digital signal processors.

Each individual microsphere is identified by its “bead signature,” (or bead region) and the result of its bioassay is quantified based on fluorescent reporter signals. We combine the power of Luminex® acquisition software with sophisticated analysis capabilities of Belysa® Immunoassay Curve Fitting software, integrating data acquisition and analysis seamlessly on all Luminex® instruments. Use of Belysa® Immunoassay Curve Fitting software affords the user advanced features not available within most data acquisition packages: auto-curve fitting functions, data hygiene rules, easy data visualization and standard curve comparison tools, with all data exportable in a variety of file formats.

The capability of adding multiplexed conjugated beads to each sample allows multiple assay results from each sample. Open architecture xMAP® technology enables the multiplexing of many types of bioassays, reducing time, labor, and costs over traditional methods.

Find the right platform for your research in our Luminex® multiplexing platform comparison article.

Why Choose MILLIPLEX® Multiplex Assays?

MILLIPLEX® multiplex assays contain all the components and reagents you need to simultaneously detect multiple analytes and offer the largest selection of kits, analytes, and species across many research areas. This includes:

  • >130 kits to study circulating soluble proteins
  • >500 unique circulating analytes (not counting different species)
  • >25 premixed multiplex and singleplex kits to study cell signaling proteins
  • >120 cell signaling analytes
  • Multiple species, including human, research animals, companion animals, and agricultural animals
  • 96- and 384-well formats

The benefits of multiplex protein detection assays are endless, including saving on time, cost, reagents, and sample. With this broad portfolio, researchers can:

  • Find assays for analytes they need
  • Achieve greater consistency by purchasing assays from one vendor
  • Retain the flexibility to meet needs now and in the future
  • Use one technology to quantify biomarkers in preclinical studies involving animal models and translational studies utilizing human samples
  • Use one technology across multiple research areas

Discover more about the benefits of MILLIPLEX® multiplex immunoassays in this short video. 

Quality in Every Kit

Most of all, the quality makes MILLIPLEX® assays stand out. From kit development and verification to manufacturing and quality control, researchers can rely on the quality we build into each kit to produce consistent, reliable results. In addition to the assay specifications listed in the protocol, we evaluate other performance criteria during our kit development and verification process: cross-reactivity, dilutional linearity, kit stability, and sample behavior (e.g., detectability and stability). We write the protocol with assay performance and your workflow in mind, making it easy to find the information you need. Learn more about the quality built into these kits in our Quality in MILLIPLEX® Multiplex Kits article.

Custom Multiplex Assay Services

We understand that some commercial multiplex kits may not provide everything needed for your research. To help address this, we also offer custom MILLIPLEX® multiplex assay services.

Our custom assay development services can help to:

  • Develop new analytes
  • Multiplex new analytes with existing analytes
  • Combine analytes from 2 or more panels into one custom panel
  • Design and verify assays on your samples
  • Bridge kits for lot-to-lot reproducibility

MILLIPLEX® Multiplex Workflow

MILLIPLEX® assays work seamlessly with Luminex® multiplex assay platforms, making them easy to run and analyze. We also offer ancillary equipment to enhance your multiplex workflows, such as the Biotek® Plate Washers.

Each MILLIPLEX® kit includes all necessary reagents to prepare the assay to read on your Luminex® instrument, just add your sample and water:

  • 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 (QCs) to qualify assay performance
  • A premixed detection antibody cocktail designed to yield consistent analyte profiles within the panel
  • A prediluted optimized concentration of Streptavidin Phycoerythrin (SAPE)
  • An optimized serum matrix is included for kits verified for serum or plasma samples, to mimic the native sample environment
  • Bead diluent containing proprietary blocking reagents to reduce non-specific binding to the antibodies in the assay
  • 10X wash buffer and assay buffer optimized specifically for the kit
  • 96- or 384-well plate (depending on assay format) with two plate sealers

Note: MILLIPLEX® cell signaling kits have a different format to support the use of cell lysate samples.

Note: MILLIPLEX® antibody detection kits have a different format to measure antibodies in serum and plasma samples.

Research Areas

Multiplex immunoassays have a vast utility in a variety of research areas because they can provide a bigger picture of disease mechanisms and processes. MILLIPLEX® multiplex assays can be used in the following research areas:

For Research Use Only. Not For Use In Diagnostic Procedures.

Tips and Tricks for Running MILLIPLEX® Assays

Have questions about running MILLIPLEX® assays? Check out these articles for helpful tips:

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Publications Using MILLIPLEX® Assays

Every year, thousands of researchers experience the benefits of MILLIPLEX® kits, publishing in scientific journals around the world. Check out the highlighted recent publications below separated by research area to see how researchers are using MILLIPLEX® assays.

For Research Use Only. Not For Use In Diagnostic Procedures.

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