Luminex Applications: Multiplex Luminex technology allows for simultaneous analysis of a broad selection of analytes (e.g. hormones, cytokines, and adipokines) across a wide range of disease states, including metabolic disease, inflammation, neurodegenerative disease, toxicity, cancer and more.
These multiplexed assays use x-map technology via the MagPix system. The reactants in these assays are attached to the surfaces of tiny, fluorescent, microspheres, each of which carries a unique biological reagent distinguishable by internal dye ratios. Identification of an analyte is based upon specific fluorescent emission spectra of the bead associated with the analyte. Two laser beams with high-speed digital signal processors and computer algorithms distinguish which assay is being carried on each microsphere while quantifying the reaction based on fluorescent reporter signals. This instrumentation allows for the analysis of multiple analytes from a single aliquot of sample.
Multiplex kits are available from several vendors, including Millipore, BioRad, Life Sciences, ThermoFisher, and R&D Systems. Millipore has the most extensive selection but our core has used kits from other companies (the procedures are quite similar despite the vender). We have the most experience with the kits described below. Others may be available upon request.
Pending approval by the core, investigators may use the instrument for a fee to run their own plates. Please contact Susan Hajizadeh.
***A minimum of at least 27 samples must be submitted allowing us to cover the purchase of each panel. Contact the core for more information***
-
We have experience with the following assays; others may be available upon request:
Mouse Single Plex Adiponectin Panel - 7 µl - Adiponectin (total)
Mouse Cytokine/Chemokine Panel 1 - 5-60 µl, depending on dilution - Eotaxin/CCL11, G-CSF, GM-CSF, IFN-y, IL-1a, IL-1b, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A, IP-10/CXCL10, KC/CXCL1, LIF, LIX, MCP-1/CCL2, M-CSF, MIG/CXCL9, MIP-1a/CCL3, MIP-1b/CCL4, MIP-2/CCL8, RANTES/CCL5, TNFa, VEGF
Mouse/Rat IGF-1 Panel - 10 µl - IGF-1
Mouse Th17 Panel - 60 µl - GM-CSF, INFy, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12(p70), IL-13, IL-15, IL-17A, IL-17E/IL-25, IL-17F, IL-21, IL-22, IL-23, IL-27, IL-28A, IL-28B, IL-31, IL-33, MIP-3a/CCL20, TNFa, TNFb/LTA
Mouse High Sensitivity T Cell - 60ul - GM-CSF, IFNγ, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12 (p70), IL-13, IL-17A, KC/CXCL1, LIX, MCP-1, MIP-2, TNF-α
Mouse Metabolic Hormone Panel - note warning below - 30 µl - Amylin (Active)*, C-Peptide 2, Ghrelin (Active)*, GIP (Total), GLP-1 (Active)*, Glucagon*, IL-6, Insulin, Leptin, MCP-1, PP, PYY, Resistin, TNF-α. We have had poor results measuring GLP-1 and glucagon (low sensitivity) with this panel, and do not recommend it for that purpose. We have also seen poor precision measuring leptin. *Needs preservative: after collecting blood sample, invert tube several times to mix, immediately add DPPIV inhibitor (for GLP-1 measurement), protease inhibitor cocktail (for Amylin measurement), Aprotinin (for Glucagon measurement) and serine protease inhibitor (for Active Ghrelin measurement). Millipore recommends using EMD Millipore’s DPPIV Inhibitor (Cat # DPP4), Sigma’sProtease Inhibitor Cocktail, and Roche’s Pefabloc SC (AEBSF) following manufacturers’ instructions.
-
We have experience with the following assays; others may be available upon request:
Human Adipokine Panel 1 - 20 µl - Adiponectin (total), Resistin, PAI-1 (total), Lipocalin-2/NGAL, Adipsin
Human Cytokine/Chemokine Panel 1 - 60 µl - EGF, Eotaxin/CCL11, FGF-2, Flt-3 Ligand, Fractalkine/CX3CL1, G-CSF, GM-CSF, GRO, IFNa2, IFN-y, IL-1a, IL-1b, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8CXCL8, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A, IP-10, MCP-1/CCL2/CXDL10, MCP-3/CCL7, MDC/CCL22, MIP-1a/CCL3, MIP-1b/CCL4, PDGF-AA, PDGF-AB/BB, RANTES/CCL5, sCD40L, TGFa, TNFa, TNFb/LTA, VEGF
Human High Sensitivity T Cell - 60ul - Fractalkine, GM-CSF, IFNγ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p70), IL-13, IL-17A, IL-21, IL-23, ITAC, MIP-1α, MIP-1β, MIP-3α, TNF-α
Human Metabolic Hormone Panel - 60 µl - Active Amylin*, Total Amylin*, C-Peptide, Ghrelin (active)*, GIP (total), GLP-1(active)*, GLP-1 (total)*, Glucagon*, IL-6, Insulin, Leptin, MCP-1/CCL2, Pancreatic Polypeptide, PYY (total), TNFa - *Needs preservative: after collecting blood sample, invert tube several times to mix, immediately add DPPIV inhibitor (for GLP-1 measurement), protease inhibitor cocktail (for Amylin measurement), Aprotinin (for Glucagon measurement) and serine protease inhibitor (for Active Ghrelin measurement). Millipore recommends using EMD Millipore’s DPPIV Inhibitor (Cat # DPP4), Sigma’s Protease Inhibitor Cocktail, and Roche’s Pefabloc SC (AEBSF) following manufacturers’ instructions.
Human IGF Panel - 60 µl - IGF-1, IGF-2
Human Th17 Panel - 60 µl - GM-CSF, INFy, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12(p70), IL-13, IL-15, IL-17A, IL-17E/IL-25, IL-17F, IL-21, IL-22, IL-23, IL-27, IL-28A, IL-31, IL-33, MIP-3a/CCL20, TNFa, TNFb/LTA
-
There are general advantages and disadvantages of Luminex assays compared to traditional radioimmunoassays (RIAs) offered by the core.
RIA advantages:
- Sensitivity
- Accuracy
- Cost is typically lower
- High throughput
RIA disadvantages:
- More sample volume needed than multiplex analysis
- Only one analyte may be measured at a time in a single sample
Luminex advantages:
- Small sample volume is especially beneficial for mouse research
- Ability to analyze up to 100 analytes in a single assay
- Customizable (one, all, or any combination of analytes may be chosen within a given panel)
- Greater selection of analytes
Luminex disadvantages:
- Not as sensitive as RIA
- Not as accurate as RIA
- Cost is typically higher (although there is a significant reduction in price for additional analytes run within a single panel)
-
1. Do all the analytes in a panel have to be run or do I pick the ones that I am interested in?
- You pick the ones that you are interested in.
2. How does the pricing work?
- For VU/VUMC investigators, the first analyte within a panel is $66 and each additional analyte within the same panel is $10, per sample in duplicate (current pricing, including for external users, is available here and in iLab where orders are placed).
- For example, to measure three analytes in one sample from panel 1, and two analytes in another sample from panel 2, the price will be $66+$10+$10/sample for panel 1 and $66+$10/sample for panel 2.
3. Can I get a reduced price if I submit more samples?
- No. We are an NIH funded non-profit core and run assays at cost.
4. Do I need to provide the required sample volume for each analyte in the panel?
- No. The required sample volume is enough to measure all analytes within the panel simultaneously.
5. What is the required sample volume?
- This is specific to each assay, typically ranging from 5-60 µl plasma (see above).
6. How long will it be until I get my data?
- We process samples in the order that we receive them so it depends on the current demand for the assay. The wait time is typically 2 to 3 weeks once we have the kit and reagents.
***Because Luminex is a custom assay it often requires extra time to order reagents. If you are in a rush, place your order a few weeks before you submit your samples to expedite the process.***
7. What is the sensitivity, cross-reactivity, etc of the assay?
- Vendors supply those details for their individual panels. Please note that analyte concentrations may be below the limits of detection of the assay -- this is out of our control and important for you to verify ahead of time.
8. Can I purchase and run my own Luminex panel?
- Yes, for a fee Vanderbilt users may use the core's Luminex instrument.
9. Who can I contact for further information?