Institute for Immunity, Transplantation and Infection

The Human Immune Monitoring Center (HIMC)

For more information

Contact:
Yael Rosenberg-Hasson, Immunoassay Manager
Email » [yaelhr]

Address:
Human Immune Monitoring Center
Fairchild Science Bldg, D033
299 Campus Drive
Stanford, CA 94305-5124

phone:  650-723-4984
fax:  650-498-7495

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).

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

Human 63-plex Human 51-plex (legacy) Mouse 38-plex Mouse 26-plex (legacy)
BDNF IL-21 SDF-1 ENA-78 IP-10 eotaxin LIF eotaxin
bNGF IL-22 sFas ligand eotaxin leptin

G-CSF/

CSF-3

LIX/ENA78/

CXCL5

G-CSF
CD40L IL-23 sICAM-1 FGF-basic LIF GM-CSF M-CSF GM-CSF
EGF IL-27 sVCAM-1 G-CSF MCP-1 GRO-alpha/KC MCP-1 IFN-gamma
eotaxin IL-31 TGF-alpha GM-CSF MCP-3 IFN-alpha MCP-3 IL-10
ENA-78 IL-4 TGF-beta GRO-alpha M-CSF IFN-gamma MIP-1-alpha IL-12p40
FGF-2 IL-5 TNF-alpha HGF MIG IL-10 MIP-1-beta IL-12p70
G-CSF IL-6 TNF-beta IFN-alpha MIP-1-alpha IL-12p70 MIP-2 IL-13
GM-CSF IL-7 TRAIL IFN-beta MIP-1-beta IL-13 RANTES IL-17
GRO-alpha IL-8 VEGF-A IFN-gamma NGF IL-15/IL-15R TGF-beta IL-1-alpha
HGF IL-9 VEGF-D IL-1-alpha PAI-1 IL-17A TNF-alpha IL-1-beta
IFN-alpha IP-10   IL-1-beta PDGF-BB IL-18 VEGF-A IL-2
IFN-beta leptin   IL-1RA RANTES IL-1-alpha   IL-3
IFN-gamma LIF   IL-2 resistin IL-1-beta   IL-4
IL-1-alpha M-CSF   IL-4 sCD40 ligand IL-2   IL-5
IL-10 MCP-1   IL-5 SCF IL-22   IL-6
IL-12p40 MCP-3   IL-6 sFas ligand IL-23   IL-23
IL-12p70 MIG   IL-7 sICAM-1 IL-27   IP-10
IL-13 MIP-1-alpha   IL-8 sVCAM-1 IL-28   KC
IL-15 MIP-1-beta   IL-10 TGF-alpha IL-3   MCP-1
IL-17A PAI-1   IL-12p40 TGF-beta IL-31   MCP-3
IL-17F PDGF-BB   IL-12p70 TNF-alpha IL-4   MIP-1-alpha
IL-18 PIGF-1   IL-13 TNF-beta IL-5   RANTES
IL-1RA RANTES   IL-15 TRAIL IL-6   TGF-beta
IL-1-beta resistin   IL-17A VEGF IL-9   TNF-alpha
IL-2 SCF   IL-17F   IP-10   VEGF

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

1. Short protocol descriptions, suitable for publications or grant applications:

Luminex (Polystyrene bead kits):  This assay was performed in the Human Immune Monitoring Center at Stanford University.  Human 51-plex or Mouse 26 plex kits were purchased from Affymetrix and used according to the manufacturer’s recommendations with modifications as described below. Briefly, samples were mixed with antibody-linked polystyrene beads on 96-well filter-bottom plates and incubated at room temperature for 2 h followed by overnight incubation at 4°C. Room temperature incubation steps were performed on an orbital shaker at 500-600 rpm. Plates were vacuum filtered and washed twice with wash buffer, then incubated with biotinylated detection antibody for 2 h at room temperature. Samples were then filtered and washed twice as above and resuspended in streptavidin-PE. After incubation for 40 minutes at room temperature, two additional vacuum washes were performed, and the samples resuspended in Reading Buffer. Each sample was measured in duplicate. Plates were read using a Luminex 200 instrument with a lower bound of 100 beads per sample per cytokine.

Luminex (Magnetic bead kits):  This assay was performed in the Human Immune Monitoring Center at Stanford University.  Human 42-plex kits were purchased from EMD Millipore and used according to the manufacturer’s recommendations with modifications as described below. Briefly, samples were mixed with antibody-linked magnetic beads on a 96-well plate and incubated overnight at 4°C with shaking. Plates were washed twice with wash buffer in a Biotek ELx405 washer. Following a one hour incubation at room temperature with biotinylated detection antibody, streptavidin-PE was added for 30 minutes with shaking. Plates were washed as above and PBS added to wells for reading in the Luminex 200 Instrument with a lower bound of 100 beads per sample per cytokine. Each sample was measured in duplicate.

MSD Assays:  This assay was performed in the Human Immune Monitoring Center at Stanford University.  Kits were purchased from Meso Scale Discovery and the protocol was followed as recommended. Briefly, antibody-coated 96 well plates were pre-incubated for 30 minutes with 25 uL/well of diluent 2. Sample or 7 point diluted Calibrators were added and plates were sealed and incubated at room temperature for 2.5 h with shaking. Plates were aspirated and washed three times with PBST (PBS+0.05% Tween-20). 25 uL of Sulfo-Tag secondary antibody in diluent 3 was then added and the plate incubated for 2.5 h with shaking. Plates were washed 3 times in PBST as above and 150 uL of 2X reading buffer was added. Plates were read immediately in the Sector Imager 2400. Results were analyzed with Masterplex software by MiraiBio.

For the MSD Hypoxia 3 plex kit, the following changes apply: Plates were pre-blocked with 150 uL of Blocker C for 1hour at room temperature with shaking followed by PBST washes as above. 25 uL per well of diluent 7 was added followed by either sample or calibrators. The Sulfo-Tag antibody was diluted in diluent 8 and the assay was completed as outlined above except that 1X reading buffer was used.

2.  Detailed protocols and addtional information:

Stanford Medicine Resources:

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