Today we are going to review the basic principles behind LEGENDplex™, BioLegend’s multiplexing, bead-based immunoassay for quantification of soluble analytes by flow cytometry. These kits are particularly relevant for those measuring cytokine levels, like in patients with cytokine storm syndrome.

LEGENDplex™ makes it easy to quantify multiple soluble targets in biological samples ranging from serum or plasma to culture supernatant. Unlike other bead-based assays, LEGENDplex™ is compatible with standard flow cytometers.

Currently, LEGENDplex™ kits are available for mouse, human, rat, and non-human primate targets.

LEGENDplex™ allows the researcher to take a “systems biology” approach to their work by studying multiple soluble factors at once. The biology of diseases can be initiated by inflammation and driven by complex cytokine responses.

Identifying these responses can help shed light on the underlying mechanisms of disease. In this recent publication, cytometric bead-based analysis was used to help define patterns associated with symptomatic versus asymptomatic malaria patients. Orange boxes indicate an increase in the cytokine expression, while blue boxes indicate a decrease.

The multiplexing nature of LEGENDplex™ provides a sensitive, reproducible snapshot of your system’s “cytokinome” in a convenient package.

So why use LEGENDplex™? LEGENDplex™ allows you to avoid running multiple ELISAs. This means you can generate one dataset encompassing all your targets.

LEGENDplex™ also offers greater sensitivity than traditional ELISAs. Example data, shown in the chart in the lower right, compares sensitivities of selected analytes from our LEGENDplex™ human T helper cell cytokine panel and corresponding Human ELISA kits.

One kit provides all the reagents needed for 100 tests, including 8 standards and 40 samples in duplicate. Analysis is a breeze with our free-to-use cloud-based software solution. Downloadable options are also available for both Mac and PC platforms.

Now let’s review the basic principles of how LEGENDplex™ works. LEGENDplex™ utilizes the same basic principles of sandwich immunoassays, whereby a soluble analyte is captured onto a bead between two antibodies. The biotinylated detection antibody is then labeled with a streptavidin PE molecule to allow for quantification. Individual beads are distinguishable by size and varying levels of internal APC or a suitable fluorescence detection channel.

The labeled beads are then acquired on a conventional flow cytometer. Users have the option of collecting directly from the 96 well plate, or from FACS tubes.

Data analysis is performed using our free cloud-based software. As seen in the figures on the left, the software automatically finds and gates the beads based on forward and side scatter profile, followed by internal APC fluorescence. As seen on the right, it uses a 5-parameter curve fitting algorithm to generate a standard curve for each analyte from the provided titrations. Samples are then quantified based on these curves.

Each kit includes all of the reagents listed in the left hand column. Minimal additional equipment, as seen on the right, is required to run a LEGENDplex™ assay.

An additional benefit to LEGENDplex™ is that it offers flexible ordering options to get the most out of your research dollar.

Full panels are predefined groups of target analytes related to a specific research field. These include our T helper cell Cytokine Panels, B cell Panels, and Growth Factor Panels. Full panels are typically sold as 13-plex assays.

We also offer Mix and Match options for researchers who are interested in creating an assay from a sub-group of analytes within our full panels. For example, you might be interested in only 6 of the 13 targets in the T helper cell Cytokine Panel. With Mix and Match, you can order an assay that will include beads for only the 6 targets you choose.

Custom panels are available for researchers who would like to combine a set of targets from multiple full panels into one assay. Custom panels must be assessed for feasibility before ordering.

Getting the best possible results with your LEGENDplex™ assay requires attention to a few key points.

When preparing the kit reagents, always make sure to thoroughly suspend the premixed antibody-bound beads to avoid clumping. This is achieved by placing the beads in a sonicator bath for 1 minute at room temperature. If the bath is not available, increase vortex time from 30 seconds to 1 minute.

Be sure to only use polypropylene plastics, as polystyrene or tissue culture treated plates are known to bind recombinant proteins to the surface of the wells. This causes flat standard curves at the low end, leading to errors in calculating sample concentrations.

Finally, we recommend that users run a standard curve each time they run the assay. Reusing old curves introduces error. When curves are generated independently of their current assay, they may not reflect variations in bench technique and/or cytometer setup for that particular day.

In summary, LEGENDplex™ is a powerful platform to study the soluble factor profiles of your experimental model using only standard equipment found in many laboratories. LEGENDplex™ is available in numerous off-the shelf solutions, including custom options. Combined with our easy to use software, LEGENDplex™ offers a complete system for data acquisition. Be sure to watch our video tutorial on our cloud-based software and read our blog on common LEGENDplex™ questions.

For any additional questions and feedback, please contact us here.