Luminex Multiplex Assays for Cytokine and Biomarker Screening by PhDs at 20+ Year, FDA-Inspected Lab

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 commercial or custom Luminex assay development, validation begins with verifying appropriate reference standards, critical reagents, calibrators, and controls. Each assay is evaluated for accuracy and precision. Accuracy is assessed by treating each Luminex standard as an unknown and back-calculating 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, sensitivity, selectivity, specificity, matrix effects (linearity and spike recovery), inter and intra-day variation, and sample stability are performed to further validate the multiplex assay.

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). Scientists 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. 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. Extra calibrators or controls at lower concentrations can be added for increased sensitivity from the multiplex immunoassay. 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. The laboratory collaborates with numerous qualified vendors to acquire high-quality Luminex assay multiplex kits and reagents for preclinical and clinical studies.

While Luminex cytokine assays are the most popular, hundreds of other singleplex and multiplex assays exist for wide-ranging drug and medical device R&D applications. These multiplex assays cover:

• Immunology/inflammation (cytokines/chemokine, t-cell)
• Cell signaling
• Cardiology
• Oncology
• Toxicology
• Endocrinology

Multiplex immunoassays are available for various species and biological matrices such as blood serum, plasma, urine, and lysates. Scientists can help identify commercially available Luminex-based assay materials and perform appropriate assay development for biomarkers. Custom Luminex assays can be developed if kits are unavailable commercially. Existing multiplex assays can also be optimized and validated if a bead set of interest has been acquired.

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. Tissue lysate preparation varies by 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.

Once a panel is qualified, a plate runs inside a working day, and results on a typical study land within about two weeks of samples and reagents arriving, larger studies scoped upfront rather than promised optimistically. Incubation windows are controlled tightly because inter-assay precision, not instrument time, is what protects a multiplex dataset.

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.

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.

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. The results from standardized Luminex assays generally match or exceed conventional ELISA, while the xMAP technology enables high throughput biomarker testing with reduced sample volume, test prep, and reagent costs. Up to 100 compatible analytes can be multiplexed simultaneously.

Luminex Multiplex utilizes xMAP technology comprising a magnetic or non-magnetic bead-based flow cytometric platform. The system creates unique spectral addresses by labeling microspheres with different ratios of red and far-red fluorophores. This allows multiple targets to be assigned unique addresses through fluorescent antibody attachment to beads, enabling simultaneous analysis of up to 100 different nucleic acids, proteins, or peptides in microplate format.

Through the Luminex Multiplex platform, multiple biomarker proteins can be detected and quantitated to evaluate therapeutic safety and efficacy. Applications include cardiac, kidney, inflammation, cancer, apoptosis, toxicity, obesity, and intracellular cell signaling biomarkers (including phosphoproteins and nucleic acids). High-performance multiplex assay kits available through this platform support basic and translational research on various diseases.

Luminex assays employ xMAP® technology that reliably uses colored magnetic beads/microspheres to perform multiplex immunoassays based on enzyme-linked immunosorbent assay (ELISA). Each bead set is color-coded with a capture molecule, and biological antigens are measured using two Luminex laser systems. A green laser identifies the bead set by exciting internal dyes, while a red laser excites the fluorescent reporting molecule to quantitate the MFI signal for each analyte. MFI signals are recorded until 50-100 microspheres of each bead set are analyzed. The Luminex xPONENT software provides average results for each analyte per bead set per sample, enabling simultaneous multiplex of up to 100 compatible analytes.

Comprehensive protocol:

• 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. Single-file beads are drawn into a flow cell to be excited by two lasers. This dual laser flow-based system identifies each bead signature and simultaneously detects the presence and intensity of the associated reporter (phycoerythrin). A high-speed digital signal processor manages fluorescent output to identify and quantitate targets.