Multiplex Bead Assays (LEGENDplex)

Principle of the assay

BioLegend’s LEGENDplex™ is a bead-based immunoassay that utilizes the same basic principles of sandwich immunoassays, whereby a soluble analyte is captured between two antibodies.

(1) Bead populations come in two sizes and differing levels of APC fluorescence, allowing them to be clearly distinguished from each other. Each bead set is conjugated with a specific antibody on the surface and serves as the capture bead for that 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, biotinylated detection antibodies are added and each detection antibody will bind to its specific analyte bound on the capture beads, thus forming capture bead-analyte-detection antibody sandwiches. Streptavidin-phycoerythrin (SA-PE) is subsequently added, which will bind to the biotinylated detection antibodies, providing fluorescent signal with intensities in proportion to the amount of bound analyte.

(3) For each bead population, the PE signal fluorescence intensity is then quantified using a flow cytometer.

(4) The concentration of a particular analyte is determined based on a known standard curve using the LEGENDplex™ data analysis software.

For the analysis of LEGENDplex™ flow cytometry data files, download the LEGENDplex™ Data Analysis Software. Learn more about the software options that consists of an online cloud-based program as well as downloadable desktop applications for both PC and Mac computers.