Luminex Multiplex Assay: Bead-Based Immunoassays
Luminex multiplex assay is a bead-based immunoassay technique that allows multiple analytes (ex. cytokines, chemokines, metabolic biomarkers, etc.) to be quantitated simultaneously in a single biological matrix sample. Generally, the results obtained using a standardized Luminex assay are superior or equivalent to conventional ELISA, even as the differentiated xMAP technology allows high throughput biomarker testing with reduced sample volume, test preparation, and reagent costs. Luminex multiplex immunoassays can detect up to 100 compatible analytes simultaneously using a commercial or custom kit. Over the past few years, Luminex cytokine assays have been one of our most requested biomarker testing services.
Luminex Multiplex utilizes xMAP technology comprising a magnetic or non-magnetic bead-based flow cytometric platform. The Luminex system creates unique spectral addresses for the microspheres by labeling them as per the different ratios of red and far-red fluorophores. This technology can detect multiple targets in a single immunoassay system by assigning each of them with a unique address through the covalent attachment of these fluorescent antibodies to the beads. Thus, this multiplexed immunoassay system with a microplate format can simultaneously analyze up to 100 different nucleic acids, proteins, or peptides.
By using the Luminex Multiplex platform, we can detect and quantitate multiple biomarker proteins in the provided samples to evaluate the safety and efficacy of your therapeutics. Furthermore, Luminex bead-based assays can be used in bioanalytical studies utilizing cell-based assays and biomarkers for cardiac, kidney, inflammation, cancer, apoptosis, toxicity, obesity, and intracellular cell signaling (including phosphoproteins and nucleic acids). High-performance multiplex assay kits available through this platform can be a valuable asset in basic and translational research on various diseases for the simultaneous analysis of multiple proteins, including growth factors and cytokines such as interferons, interleukins, chemokines, and members of the tumor necrosis superfamily.
Luminex Multiplex Assay xMAP® Technology and Step-by-step Protocol
Luminex assays employ xMAP® technology that reliably uses colored magnetic beads/microspheres to perform multiplex immunoassays based on enzyme-linked immunosorbent assay (ELISA). An immunoassay measures the presence or concentration of a macromolecule through an antibody or immunoglobulin. We color-code each bead set with a capture molecule and later measure its specific biological antigen using Luminex’s two laser systems. Here, a green laser identifies the bead set by exciting its internal dyes, and a red laser excites the fluorescent reporting molecule to quantitate the MFI signal for each of these analytes. We continue recording the MFI signals for each Luminex assay run until 50-100 microspheres of each bead set get analyzed. The Luminex xPONENT software then provides the average result for each analyte per bead set per sample. Thus, Luminex multiplex assays can simultaneously analyze up to 100 compatible analytes in a single biological sample. Consequently, multiplex cytokine assays continue to gain popularity in life sciences as a valuable research tool.
Here’s a comprehensive protocol for Luminex Multiplex bead based assay –
- Incubate Luminex beads pre-coated with target-specific capture antibodies in a sample containing various target proteins
- Wash off the beads and incubate them again with target-specific biotinylated detection antibodies to sandwich the target proteins between the capture and detection antibodies
- Wash off the beads and incubate them with phycoerythrin (PE)-conjugated streptavidin (streptavidin-PE)
Generally, a chain is formed comprising the bead—target protein—fluorescent capture antibody—biotinylated target-specific antibody—streptavidin-PE when the samples contain a target of any given biotinylated antibody. Then, we draw a single-file of these Luminex beads into a flow cell to be excited by two lasers. This dual laser flow-based system identifies the signature of each bead and simultaneously detects the presence and intensity of the associated reporter (phycoerythrin). This Luminex Multiplex assay helps identify and quantitate targets in the sample as a high-speed digital signal processor manages the fluorescent output.