Plex It up with LEGENDplex™ Multiplex Bead-Based Immunoassays!

blog / Immunology April 12 2022
biolegend legendplex

Updated in April 2022.

LEGENDplex™ from BioLegend is a bead-based multiplex immunoassay that allows you to simultaneously detect and quantify up to 14 different analytes from panels that span hundreds of protein, chemokine, and cytokine targets across the fields of cardiovascular biology, extracellular matrix biology, immunology, metabolism and endocrinology, stem cell biology and toxicity.

The LEGENDplex™ technology is built upon the same basic principles of sandwich immunoassays, whereby a soluble analyte is captured between two antibodies and quantified. However, with LEGENDplex™, you can bypass many of the challenges associated with single-plex analyte detection assays, e.g., conventional ELISAs, which are time-consuming, require relatively large sample volumes, and are not feasible to run in large numbers simultaneously.

LEGENDplex™ assays are compatible with low sample volumes and provide higher detection sensitivities and broader dynamic ranges than traditional ELISA methods. The assays have been validated for use on cell culture supernatant, serum, and plasma samples.

So How Does a LEGENDplex™ Assay Work?

  1. The LEGENDplex™ assays contain populations of beads of different sizes and varying APC intensities, allowing them to be clearly distinguishable from each other (Figure 1.1). Each bead set is conjugated with a specific antibody on the surface, and this antibody serves as the capture bead for a particular analyte. When a selected panel of capture beads are mixed together and incubated with an unknown sample containing target analytes, each analyte will be bound by its specific capture bead.
  2. After washing the sample/bead mixture, biotinylated detection antibodies are added, where each will bind to its specific analyte that is already bound by the capture beads. This leads to the formation of capture bead-analyte-detection antibody sandwiches. Streptavidin-phycoerythrin (PE) is subsequently added. This then binds to the biotinylated detection antibodies, providing fluorescent signal with intensities proportional to the amount of bound analyte (Figure 1.2).
  3. For each bead population, the PE signal fluorescence intensity is then quantified using a flow cytometer (Figure 1.3).
  4. The concentration of a particular analyte is determined based on a known standard curve using the LEGENDplex™ data analysis software (Figure 1.4).

Figure 1: The LEGENDplex™ Principle. Each LEGENDplex™ kit contains reagents for at least 100 tests. You may perform the assay in a 96-well filter plate, allowing you to run 40 samples plus 8 standard curve titrations in duplicate, or in FACS tubes, where duplicate titrations may not be necessary. Figure courtesy of BioLegend.

Full Flexibility in Choice of Target Analytes

BioLegend’s LEGENDplex™ kits provide the user with great flexibility, with a large number of panel options available that encompass the broad research fields mentioned earlier. Current panels options include:

Pre-defined panels: A range of pre-defined panels spanning the research areas listed above, for the detection of up to 14 analytes from human, mouse, rat, or non-human primate samples.

Mix and Match: Here, you can mix any subset of a pre-defined panel to suit your needs e.g., if you are only interested in 3 of the analytes in the Human B Cell panel.

Custom Panels: To combine specificities across different pre-defined panels, i.e., Human Cytokine and Human Chemokine panels. To find out more about custom panel options, click here and scroll to the custom panels tab.

You can also view all pre-defined panels here – simply scroll down, click on Pre-defined panels and select the species of interest!

COVID-19 Panels

In response to the COVID-19 pandemic and the urgent need to understand the varied immune responses to SARS-CoV-2 infection and vaccination, a number of COVID-19 panels were added to the LEGENDplex™ portfolio, including:

  • COVID-19 Cytokine Storm Panels 1 and 2
  • SARS-CoV-2 Serological IgA Panel
  • SARS-CoV-2 Serological IgG Panel
  • SARS-CoV-2 Neutralization Antibody Assay
  • SARS-CoV-2 Variants Neutralization Antibody Panel
  • SARS-CoV-2 Serological IgM Panel

You can learn more about the COVID-19/SARS-CoV-2 LEGENDplex panels here.

LEGENDplex™ Will Simplify Your Lab-Life and Help Your Budget!

Besides excellent assay performance, you can expect many additional advantages from LEGENDplex™ kits:

Compatibility with standard flow cytometers

Cost-effectiveness: Free data analysis software and dongle, and kits themselves are very competitively priced

Time-saving: You can analyse up to 14 analytes in a single experiment, thus reducing hands-on time.

Small sample volumes: You only need 50 µL to run a sample in duplicate. This is significantly less than you would need to run 13 separate ELISAs!

Free data analysis software: For analysis of LEGENDplex™ assays, BioLegend offers complementary data analysis software. You can learn more about the programme options and request account access by clicking the software tab on this page.

View the complete listing of the LEGENDplex portfolio