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Introduction to Multiplex Immunoassays
Multiplex immunoassays allow researchers to simultaneously detect two or more analytes in the same sample. They offer several advantages over conventional 'singleplex' immunoassays, including lower sample requirements and reduced hands-on time.
What are multiplex immunoassays?
Conventional immunoassays such as Western blot and ELISA are designed to measure a single protein analyte in a complex sample. By detecting the target of interest with a primary antibody - which may either be directly labeled or require the use of a secondary antibody for indirect detection - they provide information about the presence and relative abundance of the analyte in different sample types.
Multiplex immunoassays follow similar principles. However, instead of using just one primary antibody, they use multiple primary antibodies, each recognizing a different target. Provided a different readout is assigned to each analyte, multiplex immunoassays allow two or more protein targets to be measured at the same time.
What are the advantages of multiplex immunoassays?
A major advantage of multiplex immunoassays is that they use less sample compared to conventional singleplex assays. For instance, rather than requiring that a separate 100 µL aliquot of tissue homogenate be used for each of two different ELISAs, a single 100 µL aliquot can instead be used for measuring both analytes simultaneously.
In turn, this reduces the level of sample processing involved, freeing up researchers' time to be spent on other tasks. Multiplex immunoassays also offer improved experimental consistency by eliminating inter-assay variables such as temperature, timing, and sample diluent.
What different types of multiplex immunoassays are available?
Almost any conventional immunoassay can now be run in a multiplexed format. Well-known examples include Western blot, immunocytochemistry (ICC), immunohistochemistry (IHC), and lateral flow immunoassays, which are typically configured to detect 2-3 readouts per sample; ELISA, which offers multiplexing in the region of 4-10 different targets; and flow cytometry, which can simultaneously measure up to 40 different analytes provided due care is given to panel design.
Other types of multiplex immunoassays include mass cytometry, which is currently capable of detecting around 50 targets in parallel; bead-based technologies such as Luminex®, which can measure up to 500 analytes; and various antibody microarrays that provide multiplexed detection of as many as 2,000 different targets.
Luminex' xMAP® technology centers on color-coded beads that are pre-coated with target-specific antibodies. Target capture is detected with PE-conjugated antibodies, or biotinylated target-specific antibodies and PE-conjugated streptavidin, which are read on a dual-laser flow-based instrument - while one laser identifies the bead, the other measures the PE-derived signal.
Our PE- and biotin-conjugated anti-human IgG subclass antibodies are cited frequently in this application.
What readouts do multiplex immunoassays use?
Multiplex immunoassays tend to use a fluorescent or chemiluminescent readout, although mass cytometry is an exception in that it employs time-of-flight mass spectrometry. In general, chemiluminescent detection is favored for assays based on spatially separate target detection, such as ELISA or antibody microarrays, while fluorescent detection is more commonly used for cell staining applications like ICC, flow cytometry, and IHC, as well as for Western blot and bead-based applications.
It is also possible for multiplexed detection to use a colorimetric readout, such as when a lateral flow immunoassay employs antibodies that have been conjugated to colored latex beads.
What are some factors to consider for running multiplex immunoassays?
Deciding whether to multiplex your immunoassay - and which platform to use - will mainly come down to the aims of your research project, the amount of sample you can access, and the technologies available.
For example, if you are trying to discover a novel biomarker in a limited quantity of tumor biopsy material, an antibody microarray may be a good place to start provided you have a suitable reader, while for investigating a small number of targets (e.g., several biomolecules in a well-characterized signaling pathway) in a lysate prepared from a cancer cell line, a multiplex ELISA could be a better choice.
Critically, multiplex immunoassays must avoid unwanted cross-reactivities that could lead to inaccurate results. Where secondary antibodies will be used for indirect detection, it is important to select products that have been cross-adsorbed, whereas multiplex immunoassays based on fluorescence must be carefully designed to avoid spillover from one channel into another.
Other important factors to consider include the expected expression level of each target in your chosen sample type and whether there is a need for signal amplification. You may also want to think about the pros and cons of performing assay development in-house compared to purchasing a pre-configured antibody panel or off-the-shelf immunoassay kit.
We offer an extensive selection of secondary antibodies labeled with enzymes or fluorescent dyes that have been cross-adsorbed against IgG and serum proteins from multiple species. These include secondary antibodies specific to human, mouse, rabbit, goat, and chicken conjugated to PE, biotin, and Alexa Fluor® dyes.
We also provide labeled streptavidin products including streptavidin-HRP and streptavidin-AP that can be used in combination with biotinylated secondary antibodies for signal amplification.
