PB4400_Luminex_Assays

Luminex® assays are designed to simultaneously measure multiple protein targets in a single sample. They are used  for the multiplexed quantitation and detection of cytokines and signal transduction proteins using as little as 50 µL of sample  using xMAP® (multi-analyte profiling) technology. These assays offer consistent performance and work with a broad range of sample types and species.

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Matched, pre-titered, and fully optimized capture (coating) and detection antibodies allow you to build your own immunoassay.

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Luminex® Assays

Bead-based technology enables the analysis of multiple targets in a single sample from a wide range of biological sources. Check our custom Luminex® assay panel.

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Luminex® singleplex, multiplex, and custom assays

Our Luminex® assays, available in polystyrene and magnetic bead formats, are an integral part of cytokine, pathway analysis, and biomarker discovery screening workflows.

  • Singleplex assays can be used to analyze individual protein targets—mix your own custom panels or try premixed multiplex assays
  • Premixed multiplex assays are available for the most widely-studied cytokines, adhesion proteins, and growth factors for human, primate (monkey), mouse, rat, and swine research samples
  • Custom luminex assays from a range of available protein targets, blended for you by our team to optimize for low background, high signal-to-noise ratios, and linearity of dilution

Luminex® assays are easy to set up and run, using a procedure that is very similar to an ELISA (Figure 1). If you are new to Luminex® xMAP® technology or are using Luminex® assays for the first time, we have a team of dedicated Field Application Scientists (FASs) ready to help. Our FASs come to your lab to guide your efforts running Luminex® assays and using Luminex® instruments and software. Contact LuminexFAS@thermofisher.com  to request your personal Luminex® consultation today.

Luminex® xMAP® technology is based on polystyrene and paramagnetic microspheres (beads) that are internally dyed with red and infrared fluorophores of differing intensities.  Each dyed bead is given a unique number, or bead region, allowing the differentiation of individual beads.  Next, individual bead sets are coated with a capture antibody qualified for a specific analyte. Multiple analyte-specific beads can then be combined in a single well of a 96-well microplate assay format to detect and quantify up to 100 targets simultaneously using a Luminex® instrument for analysis.

Tables 1, 2, and 3 provide examples of the results achieved with our Luminex® assays and protein standards (included in the assays). Unlike other assays, we use the maximum number of beads in each Luminex® assay, so that data are collected from at least 100 events per bead region per target. Measurement of a minimum of 100 events, compared to the 50 recommended by some manufacturers, enables lower %CVs and more precise results.

Our Luminex® assays are subject to rigorous quality control and validation prior to release; we extensively test cross-reactivity to help ensure the specificity of each assay for its intended target. In addition, the protein standards we supply are precisely calibrated to reference materials for maximum consistency.

Figure 1. Fast and easy Luminex® assay protocol. Beads of defined spectral properties are conjugated to protein-specific capture antibodies and added along with samples (including standards of known protein concentration, control samples, and test samples), into the wells of a filter-bottom microplate. Target protein binds to the capture antibodies over the course of a two hour incubation. After washing the beads, protein-specific, biotinylated detector antibodies are added and incubated with the beads for 1 hr. Next, excess biotinylated detector antibodies are removed, and streptavidin-conjugated fluorescent protein, R-Phycoerythrin (SAV-R-PE), is added and incubated for 30 min. SAV-R-PE binds to the biotinylated detector antibodies, forming a four-member, solid-phase sandwich. After washing to remove unbound SAV-RPE, the beads are analyzed with a Luminex® detection system. By monitoring the spectral properties of the beads and the amount of associated R-Phycoerythrin (R-PE) fluorescence, the concentration of one or more proteins can be determined.

Table 1. More reliable data

These data indicate that %CVs and assay precision are improved by collecting data from 100 bead events compared to collecting data from only 50 bead events.

Sample Description
%CV (MFI)
50 Bead events
(0.5X Beads/ well)
100 Bead events
(1X Beads/ well)
Average (Serum)8.712.0
Average (Plasma)14.8 7.3
Average (TCS)8.55.2
Average (All)10.56.9

Table 2. Better-calibrated standards

Our Luminex® assays are calibrated to National Institute of Biological Standards and Controls (NIBSC), a set of international reference standards, when available, or to published intracellular cell models for phosphoprotein activity to help ensure that measurements are relevant. Protein standards are tested before and after lyophilization, in single as well as multiplexed assays, for consistency in standard integrity and performance.

Novex® Multiplex Luminex® Assay Standards

  Expected
(pg/ml)
Calc.
(pg/ml)
%
Differ
GM-CSF1,9641,9881
IFN-γ1,078948-12
IL-21,4321,4894
IL-49821,08911
IL-61,0401,1026
IL-81,3571,266-7
IL-10954910-5
TNF-α2,3752,278-4

Competitor Standards

  Expected
(pg/ml)
Calc.
(pg/ml)
%
Differ
GM-CSF500271-46
IFN-γ500445-11
IL-2500484-3
IL-4500237-53
IL-6500301-40
IL-8500306-39
IL-1050076854
TNF-α5003,620624

Table 3. Greater assay specificity

We measured assay response from dozens of like-target proteins to help ensure that the antibodies selected for use in our Luminex® assays are highly specific for their desired targets. In this table, we compare results (fluorescence units shown) for similar target proteins using our Luminex® assays or assays from another manufacturer. None of the Luminex® assays showed significant signal from similar, non-target proteins, whereas a few of the assays from the other supplier showed significant cross-reactivity with non-target proteins.

Novex® Multiplex Luminex® Assay

  IL-6  IL-10  TNF-
α
IFN-
γ
IL-66,827312212
IL-10128,1512312
TNF-
α
7306,64210
IFN-
γ
835253,540
Zero1110137

Competitor Assay Fluorescence Units

  IL-6  IL-10  TNF-
α
IFN-
γ
IL-68,878153240
IL-1057,090812
TNF-
α
132159,999330
IFN-
γ
902020614,248
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