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.

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.

Generally, ELISA assays are most widely accepted for quantitating proteins and considered the “gold standard” in biomedical research and clinical laboratory testing. That said, Luminex assays are often a better option since they can multiplex several of these robust single analyte ELISA assays within a single and small-volume biological sample. The needed reaction volume is typically ~50 uL per sample for multiplex immunoassay compared to ~100+ uL per analyte for ELISA. Luminex multiplex assays also support a more extensive dynamic range given their linearity over 3-5 orders of magnitude, supporting target analyte concentrations over a broad range without multiple dilution factors. Consequently, Luminex cytokine assays are extensively popular since they can cost-effectively measure various cytokines from minimal sample volume simultaneously.

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)

Biomarker Assay List

Biomarker research, discovery, and qualification are pivotal in modern drug development from demonstrating proof-of-concept (POC) to establishing drug safety and efficacy in the clinic…

Unmatched and Affordable Luminex Multiplex Assay

At NorthEast BioLab, we focus on Luminex multiplex assay development, validation, and sample analysis. This powerful technique has been an industry standard and extensively used for detecting multiple analytes simultaneously due to its high accuracy, specificity, and ability to measure a wide variety of biological molecules such as hormones, enzymes, drugs, and clinical biomarkers. Additionally, Luminex ELISA assays are advantageous since we can run these on untreated biological samples such as blood serum, plasma, urine, and saliva.

Our scientists 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.

Related FAQs

Answers to additional Luminex Multiplex
Assay questions popular among our potential clients.

How Are Luminex Assays Validated?

What are the acceptance criteria for a robust Luminex Assay?

What types of Luminex Multiplex Assays are commercially available?

What types of biological samples can be analyzed on the Luminex Platform?

Can Luminex Multiplex Assays be used for analytes that occur in vastly different biological concentrations (e.g., ng/mL vs. pg/mL)?

What are the optimization parameters for Luminex Assays?

Are Luminex Assay samples analyzed in singlicate or duplicate?

How long does a Luminex Assay take?

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How many microspheres should be analyzed per analyte in a Luminex Assay?

What are the various formats of Luminex Assay?

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