Luminex assay is a bead-based immunoassay multiplex 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 immunoassays can multiplex 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.
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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 assays can simultaneously multiplex 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 bead based assay multiplex –
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 assay multiplex helps identify and quantitate targets in the sample as a high-speed digital signal processor manages the fluorescent output.
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…
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 assay multiplex also supports 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 assay multiplex 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 Assay.
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 assay multiplex are listed below –
At NorthEast BioLab, we focus on Luminex assay multiplex 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 platform 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.
Luminex assays are based on the principles of traditional ELISA. Thus, Luminex ELISA method validation must be fit for purpose (FFP) and follow the FDA Bioanalytical Method Validation Guidance for Industry (May, 2018). For several Luminex multiplex assay or sponsor developed custom Luminex assay, validation starts with verifying appropriate reference standards, critical reagents, calibrators, and controls. Then, each Luminex multiplex assay gets evaluated for accuracy and precision. The accuracy is assessed by treating each Luminex assay standard as an unknown and back-calculating the concentration using the standard curve. According to regulatory guidelines, the ratio (recovery) of the calculated value to the expected value should be within ± 20% (±25% LLOQ and ULOQ) of the nominal value. The precision of each standard, calibrator, and study sample is based on the calculated coefficient of variation (%CV) and should be no more than ±20% (±25% LLOQ and ULOQ). As needed, we perform sensitivity, selectivity, specificity, matrix effects (linearity and spike recovery), inter and intra-day variation, and sample stability to validate your Luminex multiplex assay further.
Although there are numerous Luminex ELISA variations, the underlying inter and intra-day accuracy and precision acceptance criteria remain the same as highlighted in the FDA Bioanalytical Method Validation Guidance for Industry (May, 2018). Additionally, we strictly follow Luminex multiplex assay protocols to prepare duplicated eight or six-point calibration curves, negative controls, and up to three positive controls using materials in every multiplex assay kit. Here, the calibration curves are evaluated using a 5-parameter logistic model, and each standard/control must have a deviation under 20% of the nominal value. Our scientists can add extra calibrators or controls at the lower concentrations for increased sensitivity from the multiplex immunoassay. Furthermore, each Luminex Assay gets rigorously assessed for the run to run variability.
Luminex assays, i.e. bead based multiplex, are readily available commercially from multiple life sciences vendors as they extend traditional ELISA benefits to multiplex immunoassay. We collaborate with numerous qualified vendors to acquire high-quality Luminex assay multiplex kits and reagents for your preclinical and clinical studies. Although Luminex cytokine assays are the most popular ones, hundreds of other singleplex and multiplex assays are available for wide-ranging drug and medical device R&D applications. These multiplex assays cover therapeutic areas such as immunology/ inflammation (i.e., cytokines/ chemokine, t-cell), cell signaling, cardiology, oncology, toxicology, and endocrinology. Multiplex immunoassays are available for various species and biological matrices such as blood serum, plasma, urine, and lysates. Our scientists can help you readily find Luminex based assay materials and perform appropriate assay development for your biomarkers. We can also develop a custom Luminex assay to suit your needs if kits are unavailable commercially. Additionally, we can optimize and validate your multiplex assay if you have already acquired a bead set of interest.
Luminex multiplex assays can analyze biomarkers in multiple biological matrices such as blood serum, plasma, urine, CSF, saliva, synovial fluid, and cell lysates. Luminex immunoassays can also be performed using tissue lysates. The preparation of the tissue lysates varies according to tissue type, and 0.1 mM DPP-IV and Aprotinin can be added to inhibit degradation by proteases. Before starting the Luminex ELISA assay protocol, the total protein in the tissue lysate must be quantified using a Bradford/BCA assay and diluted to a concentration of 12.5 ug/mL total protein per well using recommended buffers. Luminex platform is unsuitable for whole blood assays since eukaryotic cells are much larger than the xMAP microspheres used for Luminex multiplex bead assays.
One common challenge for Luminex assay multiplex tends to be finding a dilution factor that allows all analytes to be analyzed simultaneously. Some analytes at low biological concentrations require samples to be diluted as little as possible. This often causes analytes at higher biological concentrations to fall above the dynamic range of the assay. Typically, we can address these issues when developing a custom Luminex assay by altering bead concentration, using antibodies with a lower/higher binding affinity, and employing a weaker reporter to reduce the MFI signal. With commercially available Luminex assays, however, the best option is to utilize a multiplex assay where all desired analytes’ sensitivity is optimized to fall within the dynamic range of the assay. That said, the analytes of interest are sometimes too far apart in biological concentrations. In this case, the best option is to create two or more smaller multiplex immunoassay panels that allow all analytes to fall within the dynamic range of the multiplexed assays. Using these assay techniques, scientists and vendors create Luminex ELISA assays for various biomarkers, including multiplex cytokine assays.
Luminex platform utilizes multiplex immunoassays that offer several advantages over singleplex assays, but these assays also require optimizing parameters such as the dynamic range, cross-reactivity, and matrix effects for each analyte. All Luminex multiplex assay kits come standardized with bead concentrations, antigen-antibody binding, detection antibodies, incubation times, and buffers for optimal sensitivity over each analyte’s reported dynamic range. The biological matrix effect, however, may still be a concern with commercial Luminex immunoassays. Although the multiplex assays get prevalidated for most biological matrices, concentrated biological samples must be diluted to prevent matrix effects originating from material interference and bead aggregation. We recommend a pre-validation run to test dilution linearity and spike recovery if biological samples are known to have a high level of interferents. Our scientists suggest not stacking and shaking the plates at the correct speed in the dark for optimal binding, preventing bead aggregation, and preserving bead integrity with all Luminex assay protocols. We can also increase the number of bead events analyzed for each analyte if a more significant statistical analysis is required than stated in the Luminex multiplex immunoassay protocol.
We recommend that calibration standards and samples are assayed at least in duplicate to evaluate reproducibility and robustness within each Luminex assay. From duplicated samples on the Luminex platform, our scientists calculate the analyte concentration average, standard deviation, and coefficient of variation (%CV). According to GLP guidelines, each sample replicate should have a % CV ± 20%. Thus, we become confident in assay performance based on sample analysis in duplicate when the multiplex assay data is reproducibly accurate and precise. Here, our team calculates the %CV for each analyte if the Luminex multiplex assay quantitates more than one analyte.
Like traditional ELISA, most Luminex assay protocols are optimized for completing sample preparation to multiplex analysis in a typical workday. That said, incubation times between steps for each Luminex based assay can vary but are tightly controlled to retain high inter-assay accuracy and precision. Generally, the 96 well plate takes about 45 min to 1 hour for analysis once the samples are loaded onto the Luminex platform. The time it takes to evaluate the multiplex immunoassays is related to the concentration of each microsphere and the number of events recorded for each analyte. We understand that Luminex assay results are an integral part of your study and aim to share data within a couple of weeks after all study samples and Luminex assay reagents are received. Naturally, more extensive multiplex immunoassay studies may take longer due to the larger number of plate runs.
After data collection on the Luminex platform, we examine the calibration curves for every analyte of the multiplex immunoassay for accuracy and precision. If acceptable, the results from study samples get reported to the sponsor in multiple formats. Here, our summary report contains information concerning the catalog and lot number of each manufacturer’s Luminex assay kit, assay conditions, and results for each analyte in the multiplex assay. Additionally, we transparently share the spreadsheet and pdf files generated by the Luminex xPONENT software for each analyzed batch. The raw data for each Luminex ELISA assay includes the mean, standard deviation, %CV, and bead count for each calibration standard, control, and study sample. Furthermore, our scientists would be happy to share a formal audited and hyperlinked pdf report as needed. Finally, we are glad to help if you have any additional questions regarding your Luminex project results.
We recommend 50-100 microspheres per analyte in a Luminex multiplex bead assay to obtain highly accurate and precise results. To meet your unique scientific objectives, we sometimes increase the number of events even as most commercial Luminex multiplex assay protocols recommend 50 bead events per analyte per sample. The increased bead count does not affect the kinetics of the multiplex immunoassay. It only increases the time needed to acquire results per sample. Generally, each Luminex assay takes about 30 mins to 1 hour to analyze a 96 well plate on the platform. That said, we would sometimes need to spend several hours on meticulous sample preparation and incubation.
Generally, we can analyze your Luminex protein assay specimens using one of three commercially available formats. A traditional sandwich ELISA format is one of the most commonly used approaches. In a Luminex sandwich assay, the analyte binds to a bead conjugated capture antibody and detected by a secondary antibody that contains a fluorophore. In a competitive assay, another Luminex immunoassay format, the bead conjugated capture antibody gets bound reversibly to a labeled antigen. Here, analytes in the sample get detected by decreased MFI signal resulting from competition with the labeled and bound antigen. Typically, we utilize this format when the target molecule has a low molecular weight or when only one antibody is available. The third format is an indirect Luminex assay, where the antigen gets conjugated to the beads and bound by antibodies in the biological sample. Here, we use a secondary antibody labeled with a fluorophore for detection. An indirect Luminex assay format is used for serological assays to measure the concentration of antigen-specific antibodies present in a sample. Our scientists have 30+ years of experience performing singleplex and multiplex immunoassays to produce accurate and reliable results. Frequently, we perform multiplex cytokine assays using a sandwich ELISA assay.
