Luminex Multiplex Assay: Bead-Based Immunoassays

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.’

Step-by-step Luminex Multiplex Assay Protocol

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.

Luminex Assay: High Throughput Multiplex with Strong Sensitivity and Specificity

Luminex immunoassays can multiplex by incubating several pre-coated bead sets with different samples in a microtiter plate. The Luminex Multiplex reader identifies the address of each bead and quantitates the present target antigen. Thus, Luminex technology can analyze up to 100 target proteins in as little as 12.5 μL sample per reaction as compared to a single-plex Enzyme-linked Immunoassay (ELISA) that requires 100­-­200 μL sample per well to detect one target protein.

Moreover, high-performance Luminex multiplex assay kits are available that streamline assay time and costs during the simultaneous analysis of multiple growth factors and cytokines, etc. Also, a more extensive range of analyses is possible as patient specimens and other valuable analytes can be utilized more judiciously, given the lower sample volumes. That said, we must consider antibody cross-reactivity when designing multiplex experiments using Luminex Multiplex.

Luminex can facilitate the identification and quantification of multiple biomarker proteins to identify higher-risk patients, determine disease prognosis, or monitor treatment efficacy. A few benefits of Luminex Multiplex assay are listed below –

  • Single platform to analyze many different types of targets for various diseases and purposes (nucleic acids, phosphoproteins)
  • Multiplex, thus reduce sample volume requirement and assay cost, labor, and time with more reproducible results and a wide range of detection
  • Monitor biological interactions in real-time (such as antigen-antibody binding, enzyme/receptor-ligand)
  • Higher intra-assay precision and standard curve recovery than conventional ELISAs
  • Kits available with antibodies for human, monkey, rat, and mouse targets (up to 25­30
Basis of Bioanalysis

The Basics of Bioanalysis

Bioanalysis is an essential tool in drug discovery and development for determining the concentration of drugs and their metabolites as well as various pharmacodynamics biomarkers in biological fluids. In these analyses, scientists use developed an….

Unmatched and Affordable Luminex Multiplex Assay

At NorthEast BioLab, we perform your bioanalytical assays to analyze your tissue and fluid-based biomarkers using state-of-the-art technology, such as the Luminex Multiplex platform. We are experts in GLP and GCP biomarker assessments, including method development and validation for novel and identified biomarkers. Luminex Multiplex is highly customizable and includes user-friendly software that automatically generates standard curves of known concentrations of all analytes and displays data in graphic or tabular form. Furthermore, the integration of kits, software, calibration, and validation beads, and instrumentation makes Luminex Multiplex a reliable high-throughput multiplex assay system.