Human IFN-γ elisapot 說(shuō)明書(shū)

ELISpot  Human IFN-
Catalog Number EL285
For the quantitative determination of the frequency of cells
releasing human IFN-.
This package insert must be read in its entirety before using this product.
FOR RESEARCH USE ONLY.
NOT FOR USE IN DIAGNOSTIC PROCEDURES.
TABLE OF CONTENTS
Contents Page
INTRODUCTION 2
PRINCIPLE OF THE ASSAY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
ELISpot SCHEMATIC 4
LIMITATIONS OF THE PROCEDURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
PRECAUTIONS 5
MATERIALS PROVIDED. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
STORAGE 5
OTHER SUPPLIES REQUIRED . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
TECHNICAL HINTS 6
REAGENT PREPARATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
SAMPLE PREPARATION 7
ASSAY PROCEDURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
CALCULATION OF RESULTS 8
REPRODUCIBILITY DATA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
TROUBLESHOOTING GUIDE 9
REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
ASSAY RECORD TEMPLATE 11
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INTRODUCTION
Interferon gamma （IFN-, also known as Type II interferon） is an important immunoregulatory
cytokine that was originally identified through its anti-viral activity （1）. It plays key roles in host
defense by exerting anti-viral, anti-proliferative and immunoregulatory activities （2-4）. IFN-
induces the production of cytokines and upregulates the expression of various membrane
proteins including class I and II MHC antigens, Fc receptors, leukocyte adhesion molecules,
and B7 antigen. IFN- is a potent activator of macrophage effector functions. It potentiates the
secretion of immunoglobulins by B cells and affects isotype switching. IFN- also influences
T-helper cell phenotype determination by inhibiting Th2 differentiation and stabilizing Th1 cells
（2-4）.
IFN- is produced primarily by activated NK cells, activated Th1 cells and activated CD8+
cytotoxic cells （2-4）. Additional cell types that produce IFN- include macrophages （5）, mast
cells （6）, dendritic cells （7）, neutrophils （8）, and peripheral T cells （9）. The production of
IFN- is upregulated synergistically by IL-12 and IL-18 （10-12）. Human IFN- cDNA encodes a
166 amino acid （aa） residue precursor protein containing a 23 aa residue predicted signal
peptide that is cleaved to generate the 143 aa residue mature human IFN- containing a
pyroglutamate residue at the N-terminus （2-4）. Natural IFN- is heterogeneously glycosylated
and contains truncations of up to 16 aa residues at the carboxy-terminus （13-16）. In solution
human IFN- exists exclusively as a noncovalent homodimer. Human IFN- shares
approximay 40% aa sequence identity with mouse IFN- and does not have cross-species
activity （2-4）.
The functional IFN- receptor complex consists of two distinct subunits （17）. The subunit
（IFN- R1） binds IFN- with high-affinity and species-specificity. The subunit （IFN- R2, also
referred to as the accessory factor 1, AF-1） interacts with the IFN--occupied subunit in a
species-specific manner and is required for signal transduction via the JAK-STAT pathway.
Both the and the subunits are type I membrane proteins. Whereas the subunit is
expressed constitutively at low levels on many cell types, the cellular expression of the
subunit correlates with IFN- responsiveness and is tightly-regulated.
The Human IFN- ELISpot assay is designed for the detection of IFN- secreting cells at the
single cell level and can be used to quantitate the frequency of human IFN- secreting cells.
ELISpot assays are well suited for monitoring immune responses to various treatments and
therapies and have been used for the quantitation of antigen-specific CD4+ and/or CD8+ T cells
responses. Other methods for assessment of antigen-specific T cell responses, such as
chromium release assays with quantitation by limiting dilution, are tedious and require previous
in vitro expansion of T cells for several days. These assays typically are not suitable for
measuring infrequent T cell responses that occur at less than 1 in 1000. ELISpot assays are
highly reproducible and sensitive and can be used to measure responses with frequencies well
below 1 in 100,000. ELISpot assays do not require prior in vitro expansion of T cells and are
suitable for high-throughput analysis using only small volumes of primary cells. As such,
ELISpot assays are useful tools for research in vaccine development and for the monitoring of
various clinical trials.
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PRINCIPLE OF THE ASSAY
The enzyme-linked immunospot （ELISpot） assay was originally developed for the detection of
individual B cells secreting antigen-specific antibodies （18, 19）. This method has since been
adapted for the detection of individual cells secreting specific cytokines or other antigens
（20, 21）. ELISpot assays employ the quantitative sandwich enzyme-linked immunosorbent
assay （ELISA） technique. A monoclonal antibody specific for human IFN- has been
pre-coated onto a PVDF （polyvinylidene difluoride）-backed microplate. Appropriay
stimulated cells are pipetted into the wells and the microplate is placed into a humidified 37° C
CO2 incubator for a specified period of time. During this incubation period, the immobilized
antibody in the immediate vicinity of the secreting cells binds secreted IFN-. After washing
away any cells and unbound substances, a biotinylated polyclonal antibody specific for human
IFN- is added to the wells. Following a wash to remove any unbound biotinylated antibody,
alkaline-phosphatase conjugated to streptavidin is added. Unbound enzyme is subsequently
removed by washing and a substrate solution （BCIP/NBT） is added. A blue-black colored
precipitate forms and appears as spots at the sites of cytokine localization, with each individual
spot representing an individual IFN- secreting cell. The spots can be counted with an
automated ELISpot reader system or manually using a stereomicroscope.
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ELISpot SCHEMATIC
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Incubate IFN--secreting
cells in an antibody-coated
well.
Remove cells by washing.
Secreted IFN- is captured
by the immobilized antibody.
Incubate with biotinylated
anti-IFN- antibody.
Incubate with alkaline
phosphatase conjugated
streptavidin.
Add substrate and monitor
the formation of colored
spots.
LIMITATIONS OF THE PROCEDURE
· FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
· The kit should not be used beyond the expiration date on the kit label.
· Any variation in pipetting and washing techniques, incubation time or temperature, or kit age
can cause variation in density of spots, intensity of specific staining and background levels.
PRECAUTIONS
Some components of this kit contain sodium azide, which may react with lead and copper
plumbing to form explosive metallic azides. Flush with large volumes of water during disposal.
Do not use reagents from this kit with components from other R&D Systems’ ELISpot or ELISA kits
and/or components manufactured by other vendors.
Do not remove the flexible plastic underdrain on the bottom of the microplate before or during
incubation and development since it may damage the PVDF membrane filters. The underdrain
cover may be removed only after completing the incubation with BCIP/NBT chromogen.
Although the toxicity of the chromogenic substrate BCIP/NBT is not currently known, wear gloves
to avoid contact with skin. Follow local, state and federal regulations to dispose of used BCIP/NBT.
MATERIALS PROVIDED
Human IFN- Microplate （Part 890882） - One 96-well PVDF-backed microplate coated with
monoclonal antibody specific for human IFN-.
Detection Antibody Concentrate （Part 890883） - 150 L of a 120X concentrated solution of
biotinylated polyclonal antibody specific for human IFN- with preservatives.
Streptavidin-AP Concentrate A （Part 895358） - 150 L of a 120X concentrated solution of
Streptavidin conjugated to Alkaline Phosphatase with preservatives.
Dilution Buffer 1 （Part 895307） - 12 mL of a buffer for diluting Detection Antibody Concentrate
with preservatives.
Dilution Buffer 2 （Part 895354） - 12 mL of a buffer for diluting Streptavidin-AP Concentrate A with
preservatives.
10X Wash Buffer Concentrate （Part 895308） - 50 mL of a 10X concentrated solution of a
buffered surfactant with preservative.
BCIP/NBT Chromogen （Part 895867） - 12 mL of a stabilized mixture of 5-Bromo-4-Chloro-3
Indolylphosphate p-Toluidine Salt （BCIP） and Nitro Blue Tetrazolium Chloride （NBT）.
Human IFN- Positive Control （Part 890893） - 1 vial （2 ng） of recombinant human IFN-;
lyophilized.
STORAGE
Store the unopened kit at 2-8° C. Do not use beyond the kit expiration date. This kit is validated for
single use only. Results obtained with opened/reconstituted reagents at a later date may not be
reliable.
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OTHER SUPPLIES REQUIRED
Pipettes and pipette tips
Deionized or distilled water
Squirt bottle, manifold dispenser, or automated microplate washer
· 500 mL graduated cylinder
· 37° C CO2 incubator
· Sterile culture media
· Dissection microscope or an automated ELISpot reader
TECHNICAL HINTS
· To minimize edge effect, place the microplate （bottom down） onto a piece of soft aluminum foil
（about 4 x 6 inches）. Add cells, cover the microplate with the lid and shape the foil around the
edges of the microplate. The foil may be left on the microplate for the rest of the experimental
procedure and removed after the BCIP/NBT has been washed off.
· Do not touch PVDF membrane filters with pipette tips when pipetting cells and reagents to
avoid damage to the membrane.
· After completion of the experiment, do not dry the microplate at a temperature higher than
37° C since it may cause cracking of the PVDF membrane filters.
· The 96-well microplate provided in the kit is not sterile. However, due to the short incubation
period and presence of antibiotics in the culture media, microbial contamination has not been
a problem during the ELISpot procedure.
· The kit is designed for single use only. The layout of the assay should be carefully planned
to maximize the use of the plate and reagents provided.
· The controls listed are recommended for each ELISpot experiment.
Positive Control - Use recombinant human IFN-.
Unstimulated/Negative Control - Use the same number of unstimulated cells as
stimulated cells.
Background Control - Use sterile culture media.
Detection Antibody Control - Substitute phosphate buffered saline for Detection Antibody.
REAGENT PREPARATION
10X Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and
mix gently until the crystals have compley dissolved. To prepare Wash Buffer, add 50 mL of
Wash Buffer Concentrate to 450 mL of deionized water and mix well.
Human IFN- Positive Control - Reconstitute lyophilized human IFN- with 250 L of culture
medium that is used to incubate cells.
Detection Antibody - Tap or vortex the vial to release reagent collected in the cap. Transfer
100 L of Detection Antibody Concentrate into the vial labeled Dilution Buffer 1 and mix well.
For optimal performance, prepare Detection Antibody immediay before use.
Streptavidin-AP - Tap or vortex the vial to release reagent collected in the cap. Transfer
100 L of Streptavidin-AP Concentrate A into the vial labeled Dilution Buffer 2 and mix well.
For optimal performance, prepare Streptavidin-AP immediay before use.
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SAMPLE PREPARATION
The types of effector and responder cells used, method of cell separation, mode of stimulation,
and length of incubation are to be determined by each investigator. R&D Systems’ cell
selection products are suitable for the purification of effector and responder cells. For a
complete product listing of human, mouse, and rat cell selection products, see the
R&D Systems catalog or visit our website at www.RnDSystems.com.
ASSAY PROCEDURE
Bring all reagents to room temperature, except the Detection Antibody Concentrate and
Dilution Buffer 1, which should remain at 2-8° C. All samples and controls should be assayed at
least in duplicate. An Assay Record Template is provided at the back of this insert to record
controls and samples assayed.
1. Fill all wells in the microplate with 200 L of sterile culture media and incubate for approximay
20 minutes at room temperature.
2. When cells are ready to be plated, aspirate the culture media from the wells. Immediay add
100 L of the appropriate cells or controls to each well （see Technical Hints for appropriate
controls）.
3. Incubate cells in a humidified 37° C CO2 incubator. Optimal incubation time for each stimuli
should be determined by the investigator. Do not disturb the cells during the incubation
period.
4. Aspirate each well and wash, repeating the process three times for a total of four washes. Wash
by filling each well with Wash Buffer （250-300 L） using a squirt bottle, manifold dispenser, or
autowasher. Complete removal of liquid at each step is essential to good performance. After the
last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and
blot it against clean paper towels.
Note: Adjust the height of the prongs of the manifold dispenser or autowasher to prevent
damage to the membranes.
5. Add 100 L of diluted Detection Antibody into each well and incubate at 2-8° C overnight.
6. Repeat step 4.
7. Add 100 L of diluted Streptavidin-AP into each well and incubate for 2 hours at room
temperature.
8. Repeat step 4.
9. Add 100 L of BCIP/NBT Chromogen into each well and incubate for 1 hour at room
temperature. Protect from light.
10. Discard the chromogen solution from the microplate and rinse the microplate with deionized
water. Invert the microplate and tap to remove excess water. Remove the flexible plastic
underdrain from the bottom of the microplate, wipe the bottom of the plate thoroughly with paper
towels and dry compley either at room temperature （60-90 minutes） or 37° C （15-30 minutes）.
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CALCULATION OF RESULTS
The developed microplate can be analyzed by counting spots either manually using a
dissection microscope or by using a specialized automated ELISpot reader. Specific spots are
round and have a dark center with slightly fuzzy edges. Quantitation of results can be done, for
example, by calculating the number of spot forming cells （SFC） per number of cells added into
the well.
REPRODUCIBILITY DATA
Peripheral blood mononuclear cells （5 x 105 cells/mL） were stimulated with 50 ng/mL of phorbol
12-myristate-13-acetate and 0.5 g/mL calcium ionomycin overnight at 37° C in a 5% CO2
incubator. The sample was assayed in seven wells according to the procedure and analyzed
with a dissection microscope.
Well Number of Spots Counted
1 72
2 81
3 100
4 98
5 78
6 100
7 76
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TROUBLESHOOTING GUIDE
Observation Problem Corrective Action
Following the incubation with
BCIP/NBT chromogen and
rinsing the microplate with
deionized water, the dark-blue
background color of filter
membrane attenuates
visualization and quantitation of
spots.
Wet membrane
Microplates cannot be analyzed
accuray until PVDF filter
membranes are compley dry. Wait
until membrane becomes dry, usually
15-30 minutes at 37° C or
60-90 minutes at room temperature.
The number of spots in the
wells that contained the cells is
high but their contrast as well
as intensity of staining in the
Positive Control wells is low.
Underdevelopment-perhaps the
result of using Streptavidin-AP
and/or BCIP/NBT solutions that
have not been brought to room
temperature
Warm the reagents to room
temperature before adding to the
wells.
The number of spots in the
wells that contained cells is
lower than expected whereas
Positive Control wells turned
black-blue.
Cell stimulation problem
Ensure that reagents used to
stimulate the cytokine release from
the cells retained their biological
activity. One way to check is to
perform immunocytochemistry on
fixed cells after stimulation.
Too few cells added to the wells
Increase the number of cells added
per well.
Following incubation with
BCIP/NBT and drying the
microplate, the density of the
spots makes it difficult to
quantify them.
Too many cells were added to the
wells
Make dilutions of cells （i.e.,
1 x 106, 5 x 105, 1 x 105, 5 x 104,
1 x 104 cells per well） to determine the
optimal number of cells that will result
in formation of distinct spots.
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REFERENCES
1. Wheelock, E.F. （1965） Science 146:310.
2. Billiau, A. （1996） Adv. Immunol. 62:61.
3. Boehm, U. et al. （1997） Annu. Rev. Immunol. 15:749.
4. Farrar, M.A. and R.D. Schreiber （1993） Annu. Rev. Immunol. 11:571.
5. Puddu, P. et al. （1997） J. Immunol. 159:3490.
6. Gupta, A.A. et al. （1997） J. Immunol. 157:2123.
7. Sugaya, M. et al. （1999） J. Invest. Dermatol. 113:350.
8. Yeaman, G.R. et al. （1998） J. Immunol. 160:5145.
9. Battistini, L. et al. （1997） J. Immunol.. 159:3723.
10. Trinchieri, G. and F. Gerosa （1996） J. Leukoc. Biol. 59:505.
11. Lebel-Binay, S. et al. （2000） Eur. Cytokine Netw. 11:15.
12. Tominaga, K. et al. （2000） Int. Immunol. 12:151.
13. Rinderknecht, E. et al. （1984） J. Biol. Chem. 259:6790.
14. Pan, Y-C.E. et al. （1987） Eur. J. Biochem. 166:145.
15. Dobeli, H. et al. （1988） J. Biotech. 7:199.
16. Sakaguchi, M. et al. （1988） FEBS Lett. 230:201.
17. Bach, E.A. et al. （1997） Annu. Rev. Immunol. 15:563.
18. Czerkinsky, C.C. et al. （1983） J. Immunol. Methods 65:109.
19. Sedgwick, J.D. and P.G. Holt （1983） J. Immunol. Methods 57:301.
20. Czerkinsky, C.C. et al. （1984） J. Immunol. Methods 72:489.
21. Helms, T. et al. （2000） J. Immunol. 164:3723.
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ASSAY RECORD TEMPLATE
This template may be used as a record of samples and controls run in an assay.
© 2011 R&D Systems, Inc.
11.00 750532.7 7/11
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