Immunoassays

We have standardized immunoassays for human and mouse cytokines, which can be used to assay serum, plasma, cell culture supernatants, tissue homogenates, or other fluids. We measure multiple cytokines simultaneously using either Luminex (up to 63 analytes) or MesoScale Discovery (MSD, up to 10 analytes) systems.  We also provide assays for other soluble proteins on the MSD platform (see their website at www.mesoscale.com for available kits). Download background information on the protein analytes in our standard kits here.

Standard Assays (per-sample pricing, no minimum sample number): For human cytokines, we offer a 63-plex Luminex assay custom-built by eBioscience. We have compared this assay to our previous 51-plex, for which we have extensive performance data.  For mouse cytokines, we offer a 38-plex Luminex array.  Analytes in these arrays are shown here:

Special order assays (charged as actual materials and labor):  We do not maintain our own inventory of these kits, but can usually obtain them within 1-2 weeks.  For these kits, you should plan to run at least 40 samples for cost efficiency, since approximately 40 duplicate samples can be run per plate, with standards and controls.  Please contact Yael Rosenberg Hasson (yaelhr@stanford.edu or 650-723-4984) for availability and order options.

Human 41 plex cytokines:  http://www.millipore.com/catalogue/item/HCYTMAG-60K-PX41

EGF, Eotaxin, FGF-2, Flt-3 ligand, Fractalkine, G-CSF, GM-CSF, GRO, IFN-α2, IFN-γ, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17, IL-1ra, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IP-10, MCP-1, MCP-3, MDC (CCL22), MIP-1α, MIP-1β, PDGF-AA, PDGF-AB/BB, RANTES, TGFα, TNF-α, TNF-β, VEGF, sCD40L

Human adiponectin:  http://www.millipore.com/catalogue/item/HADK1MAG-61K

Option to order 1-6 biomarkers:  Adiponectin, Adipsin, Lipocalin-2/NGAL, PAI-1 (Total), Resistin

Human Soluble Receptor 14 Plex:  http://www.millipore.com/catalogue/item/hscrmag32kpx14

sCD30, sEGFR, sIL-1RI, sIL-1RII, sIL-2Rα, sIL-4R, sIL-6R, sRAGE, sTNFRI, sTNFRII, sVEGF-R1, sVEGF-R2, sVEGF-R3, sgp130

Rat 27 plex cytokines:  http://www.millipore.com/catalogue/item/recymag65k27pmx

EGF, Eotaxin, Fractalkine, G-CSF, GM-CSF, GRO/KC, IFN-γ, IL-10, IL-12 (p70), IL-13, IL-17A, IL-18, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IP-10, LIX, Leptin, MCP-1, MIP-1α, MIP-2, RANTES, TNF-α, VEGF

Human cardiovascular disease Panel III:  http://www.millipore.com/catalogue/item/hcvd3mag-67k

Option to run 1-11 of the biomarkers: AGP, Adipsin, CRP, Fetuin A, Fibrinogen, Haptoglobin, L-Selectin, Platelet Factor-4, Serum Amyloid P, von Willebrand Factor (vWF), α2-Macroglobulin

Human immunoglobulin Isotyping Panel:  http://www.millipore.com/catalogue/item/hgammag-301k

IgA, IgG1, IgG2, IgG3, IgG4, IgM

Human TIMP’s in Cell culture:  http://www.millipore.com/catalogue/item/HTMP2MAG-54K

TIMP-1, TIMP-2, TIMP-3, TIMP-4

Human TIMP’s in Serum/Plasma:  http://www.millipore.com/catalogue/item/htmp1mag-54k

Human MMP’s:http://www.millipore.com/catalogue/item/HMMP2MAG-55K

MMP-1, MMP-10, MMP-2, MMP-7, MMP-9

MSD Kits:  These kits are essentially multiplexed ELISA assays, read out via electrochemiluminescence.

V-plex -Panels: http://www.mesoscale.com/CatalogSystemWeb/webroot/products/ProductDetail.aspx?ItemNumber=K15054D-1

Eotaxin, Eotaxin-3, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-10, IL-12 p70, IL-12/IL-23p40, IL-13, IL-15, IL-16, IL-17A, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-8 (HA), IP-10, MCP-1, MCP-4, MDC, MIP-1α, MIP-1β, TARC, TNF-α, TNF-β, VEGF

Hypoxia Markers:  http://www.mesoscale.com/CatalogSystemWeb/WebRoot/Products/ProductDetail.aspx?ItemNumber=K15122C-1

Human   EPO, IGFBP-1, VEGF

Please inquire for CRP or BDNF.

A custom 5 plate order  may be required

ELISA Assays:  A few ELISA assays are also run in HIMC.

CMV ( IgG or IgM)  by GSD

EBV ( IgG or IgM)  by GSD

User Instructions

1.  Planning your assay:  To minimize run-to-run variability, we suggest assaying all samples to be compared in a single experiment, if possible.  Since there can also be plate-to-plate variation, it is helpful to fit the experiment into a single plate (40 duplicate samples), or to randomize groups across multiple plates (i.e., don't put all controls on one plate and all test samples on another).  We will run samples in the order provided, unless instructed otherwise.  Please consult us in the planning phase if you have questions.

2.  Collecting your samples:  We need a minimum of 120 microliters per sample to run duplicate wells without dilution.  Be sure samples undergo a hard spin (10,000 G) before freezing, to remove cells and debris that can clog the Luminex instrument; see our SERUM PROCESSING or PLASMA PROCESSING protocols for details on serum or plasma collection and processing.  Process serum and plasma within two hours of blood draw if possible.  Avoid hemolysis, which interferes with cytokine detection.  Serum and plasma processing services are also available through our Blood Processing Unit.  Tissue homogenates should include a buffer with protease inhibitors, and normalization of total protein concentration may be desirable (we can measure total proteins using our Bioanalyzer platform; see our genomics page for details).  You may wish to include a control of cell-free media (for cell supernatants) or lysis buffer (for homogenates), for background.

All samples should be frozen at -80C and transferred to the HIMC on dry ice.  Multiple freeze-thaw cytcles should be avoided.  Please label all tubes clearly on the side and top, and email an excel list of your samples to yaelhr@stanford.edu (or post to Basecamp for existing projects).

3. Running samples:  An online order is required before samples can be run.  We will batch samples sets where possible, to run complete plates.  Turnaround time varies depending on the size of your project and the current workload; smaller projects can usually be completed within two weeks of order placement and sample delivery.

Plasma samples are run with a 1:3 dilution due to known issues with clogging of the instrument; dilution of other sample types is optional; it can improve quantitation of high-level cytokines, with minimal impact on the detection of most low-level cytokines.  Leftovers will be discarded unless you provide other instructions, and will not be saved for more than two weeks.

4.  Data analysis:  Data is transmitted via our Basecamp project management system.  New customers will get an email invitation to join the system  An Excel report will be posted there, containing summaries of median fluorescence intensity (MFI), calculated concentration, bead count, and coefficient of variation (CV) of replicate wells, for each cytokine in each sample, standard, and control.  QC notes are also provided in the report.  The report data is simultaneously uploaded to our online databawse, Stanford Data Miner, where it can be integrated with other HIMC data from your lab, as well as clinical/demographic data if provided.  Click the help link in the upper left corner of the Data Miner homepage for instructions on using this system.

We recommend using MFI rather than concentration values, especially for analytes that are outside of the linear range of the standards.  This is usually the case for serum and plasma samples.  Normalization (to controls or to global sample median for each plate) is recommended when comparing values across plates.

Protocols

See the Protocols page for complete HIMC protocols as well as short descriptions suitable for publications and grant applications.

Addtional information:

• Methods for collection mouse blood
• BD Vacutainer guide