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Firefly Multiplex Immunoassay Validation Data Graph

Multiplex immunoassay techniques: a review of current methods

Understand key differences between the main multiplex immunoassay technologies, and their advantages over immunoassays that only measure one analyte at a time.

Summary of common methods used to measure proteins 

TechniqueAdvantagesDisadvantagesWhen to use
Western blot
  • Gauge specificity through band position
  • No need for an antibody pair
  • Semi-quantitative
  • Difficult to automate
  • Difficult to transfer large or hydrophobic proteins
  • To detect presence of a single protein
  • Quantitative
  • Higher throughput than western blot
  • Can only readily measure one analyte at a time
  • To quantify a single protein

Antibody microarray

  • Membrane arrays use the same equipment as a chemiluminescent western blot 
  • Hard to  customize, some array types need specialized equipment
  • To screen for changes across a large number of proteins

Luminex? technology

  • Multiplex and customizable
  • Requires specialized equipment and a well-validated antibody pair
  • To quantify multiple proteins 

FirePlex? particle technology and bead-based immunoassays for flow cytometers 

  • Customizable multiplex panels
  • Runs on standard bench-top flow cytometers
  • Requires a well-validated  antibody pair
  • To quantify multiple proteins 

Popular and robust assays for single analytes

Assays to measure the level of a single protein in a homogenous liquid sample (typically ELISA or western blot) have been a mainstay of biological research for decades. Accurately quantifying protein levels is informative in research and diagnostics, whether quantifying levels of a particular protein or testing for the presence of protein markers.

Many ELISA kits using optimized antibody pairs are now available, featuring capture antibodies that pull a target of interest out of the biological milieu and a specific detector antibody. Antibody pairs in ELISA are specific, sensitive and can be read on ubiquitous plate readers.

Limitations of assays for single analytes

Although these singleplex assays (i.e. assays to measure a single analyteare robust and popular, they may not be the best format for your research as it is often more biologically informative to measure more than one target protein.

Measuring multiple targets by ELISA involves performing several assay workflows in parallel; this is time consuming and increases the risk of error. Moreover, sample volume requirements increase in proportion with the number of analytes measured; a key challenge as biological samples are often scarce and precious.

Assays can be miniaturized to compensate for the increased sample volume requirements, however this often requires expensive specialized equipment. Multiple assays can be run in parallel with minimal manual processing using microfluidics; however the complexity of the fluidics routes scales rapidly as more assays and samples are added, limiting flexibility in the number of samples and analytes that can be measured.

Multiplex immunoassays

To address workflow and sample volume problems, methods are required that combine assays for multiple target analytes. Multiplex immunoassays combine assays for many target analytes in a single reaction volume, reducing workflow and sample volume problems.

Multiplex platforms can offer distinct advantages over singleplex assays. The need for lower sample input has been alluded to already; typically 25–50 μL sample volume is required to test multiple markers compared with 100 μL per target required for ELISA.

Additionally, multiplex platforms often provide a gain in assay dynamic range. Whereas ELISA rapidly loses linearity over a few orders of magnitude, multiplex assays are reported to maintain linearity over three or even five orders of magnitude. As panels often contain targets that are present at radically different concentrations, linearity over a wide range is essential for reliable data.

A number of methods are typically used for multiplex immunoassays, and they loosely fit into two categories:

  • Immobilization on a solid surface in which the assays for each analyte are spatially separated
  • Immobilization on beads or particles in which the assays for each analyte are on a different bead/particle

Multiplex immunoassays: spatially separate assays

The first approach is spatially separate assays, these are similar to an ELISA except multiple antibody pairs share the same reaction volume. There are several variations on this theme, but a common feature is the physical isolation of capture antibodies on a shared solid surface.

For example, analytes can be interrogated by large numbers of capture antibodies spotted on microarrays, such as with semi-quantitative membrane antibody arrays, analyzed just like a chemiluminescent western blot, or quantitative glass slide-based antibody arrays. Alternatively the same can be achieved with batches of 1–25 capture antibodies immobilized in small arrays at the bottom of individual wells of 96- or 384-well plates, for examples with the Meso Scale discovery platform or the Quansys system. 

Each spot captures a specific target protein then a second, target-specific, detector antibody is used for quantification. A whole array can be read at once and spot coordinates can then be used as an address to determine what protein is being detected. 

Multiplex immunoassays: bead or particle immobilization

The bead immobilization approach for multiplex immunoassays faces the challenge of identifying which analyte is being measured on which bead. For this purpose, technologies use combinations or levels of dyes to encode analyte specific capture beads.

For example, technology from Luminex? uses combinations of red and near-infrared dyes to encode solid polystyrene microspheres, potentially generating codes for 500 different targets. These codes are used to identify the bound capture antibodies, and a sandwich assay with phycoerythrin-labeled detector antibodies is used to measure protein levels.

A limitation to this technology is the requirement for specialized equipment: beads are read using a specialized flow cytometer, or in the case of Luminex’s xMAP? technology, by capture on a flat surface, followed by imaging using LEDs and a camera. Decoding sample signals is performed after sorting by dedicated software.

Bead-based assays also exist that can be used with standard flow cytometers – these are limited in the number of codes that can be distinguished by the cytometer but do not require an expensive dedicated instrument. We have developed multiplex assays, using our FirePlexTM particle technology, that are compatible with standard flow cytometers. The FirePlex particles contain multiple regions for coding and analyte detection, which increases the number of available codes and the number of analytes that can be assayed simultaneously.


There are many good reasons to opt for multiplex immunoassays over singleplex and several platforms are available, although requirements for specialist equipment currently restrict access to the technology in some cases.

We offer a broad range of multiplex immunoassays, including membrane antibody arrays and glass-slide based quantitative antibody arrays, which can be used with standard laboratory equipment. For powerful multiplexing performance, with convenient analysis by flow cytometry, we recommend our FirePlex? particle multiplex immunoassays.

FirePlex? is a registered trade mark in the United States and is an unregistered trademark elsewhere.