Sanquin
PeliSPOT ä
IFN-Gamma
Sample ASSAY PROCEDURE
See each kit for batch specific information
RDI-M2533clb, new cat#: 55R-M2533clb $525.00
Sanquin Reagents1006 AD The |
Order through: RDI Division of Fitzgerald Industries Intl Phone (978) 371-6446 or (800) 370-2222 Fax (978)
371-2266 Email: antibodies@fitzgerald-fii.com Web: http://www.researchd.com |
The ELISPOT or ELISA spot technique is a
method that has increased its significance in the last few years mainly in the
field of anti-viral immunity and tumor-immunology. The ELISPOT assay for the
detection of IFN-g secreting T-cells was first described by Czerkinski (1). Compared
to the ELISA technique the substantial improvement is the increased
sensitivity. Where ELISA requires at least 400 cells per well to produce
sufficient cytokines to give detectable levels, in ELISPOT only one positive
cell in a well containing 100,000 cells can be detected (2).
Reactivitity of T-cells against peptides or proteins can be easily monitored
directly ex vivo with the
ELISPOT technique, in vitro restimulation of the cells is not required. Cytokine release
upon stimulation provides information about Th1/Th2 profiles and cytoxicity.
Different studies showed that ELISPOT is a sensitive and reproducible assay for
determining antigen reactive T-cells (3).
This PeliSPOTä kit meets the requirements of a
standardized and optimized assay protocol. This kit has been developed for
reproducible and specific quantification of the frequency of human cells
secreting the cytokine of interest. The kits are for research use only.
Cells
are stimulated in a well coated with a high affinity monoclonal antibody.
Secreted products of interest will bind locally to the antibody. Subsequently
cells are washed away and a biotinylated antibody is added. Signal amplification
is established by adding a streptavidin-conjugated enzyme. The area is
visualised as a spot by a precipitating blue/purple coloured substrate.
Enumeration of spots can be reliably performed with the A.EL.VIS, or other
automated plate reader system. The results can be expressed as the number of
spot forming cells per million cells.
The
components of the PeliSPOT kits (except TMB substrate solution) should be stored
at -18 to -32°C. The TMB substrate solution should be stored at 2 to 8 °C. The performance of the kit is
guaranteed until the expiration date shown on the box label. Kit components are
stable upto three freeze-thawing cycles, however dividing the components in
small aliquots is recommended.
The
PeliSPOTä kit contains material sufficient for 288
tests.
The
reagents provided are:
|
Kit component |
Volume |
Storage |
Cap colour |
Handling |
|
Coating antibody |
190 µl |
-18 to -32°C |
Red |
Dilute 1:100 in PBS |
|
Biotinylated antibody |
375 µl |
-18 to -32°C |
Yellow |
Dilute 1:100 in PeliSPOT
buffer |
|
Streptavidin-poly-HRP |
20 µl |
-18 to -32°C |
Brown |
Dilute 1:6,000 in
PeliSPOT buffer |
|
Positive assay control |
See vial |
-18 to -32°C |
Black |
See vial |
|
PeliSPOT buffer
stock solution |
50 ml |
-18 to -32°C |
|
Dilute 1:5 in distilled
water |
|
TMB Substrate
for PeliSPOT |
18 ml |
2 to 8 °C |
|
Ready for use |
1. The PeliSPOTä kits are intended for research purposes only, ingredients are not for in vivo use.
2. Only use the reagents supplied with the kit; do not mix reagents from different kit lots.
3. Handle all blood, tissue and cell samples with care to prevent transmission of infections.
4. Sodium azide inactivates HRP, do not use sodium azide-containing solutions, nor add sodium azide to the supplied materials.
5. Centrifuge all vials before use (1 minute at 3,000 x g).
6. The supplied reagents have only been tested and approved for use on MAIPN45 microplates from Millipore.
Materials
- 96-wells microtiter plate: preferably MiIlipore MAIPN45 (PVDF membrane) in combination with a single cell culture tray , Millipore MAMC S01
- Device for delivery of wash buffer: automated or manual
- CO2 Incubator
- A.EL.VIS or other ELISPOT analyser
- Pipetting devices for accurate delivery of 1-20 µl, 50 µl, 100 µl and 1 ml volumes
- Beakers, flasks, cylinders, tubes necessary for preparation of reagents
- Device for counting of cells
Reagents
- Cell culture medium: IMDM or RPMI-1640
- Supplemented with:
- 5% FCS
- 2 mM Glutamin
- 100 U/ ml Penicillin
- 100 mg/ ml Streptomycin
Recommended for positive secretion control, (polyclonal stimulation):
-
PMA (phorbol 12-myristate
13-acetate); Sigma, P1585
stock solution: 2 µg/ ml in ethanol, store at -18 to –32°C
-
Ionomycin (Calcium
ionophore); Sigma I0634
stock solution: 1 mg/ml in ethanol, store at -18 to –32°C
- Ethanol 96% (v/v) (70 % is also suitable)
- Distilled water
- Tween-20
- PBS, pH 7.2-7.4 (do not use PBS tablets as a component for the coating buffer)
Preparation of PBS (phosphate
buffered saline) stock solution (20x)
Dissolve in 900 ml distilled water:
38 mM 6 g NaH2PO.2H2O
18
mM 32 g Na2HPO.2H2O
280 mM 164 g NaCl
0.001% (w/v) 20 mg Merthiolate (do not use Na-azide as preservative!)
Check pH and adjust to 6.8- 6.9 with concentrated NaOH or
HCl and add distilled water to a volume of 1 L. The prepared buffer can be stored up to three months at 2 to 8°C.
Preparation of PBS pH 7.2- 7.4
50 ml PBS stock solution (20x)
950 ml Distilled water
Preparation of wash buffer
50 µl Tween-20
1 L PBS
1. Czerkinsky C.C., et al., Reverse ELISPOT assay for clonal analysis of cytokine production. I. Enumeration of gamma-interferon-secreting cells. Journal of Immunological Methods, 110 (1): 29-36 (1988)
2. Scheibenbogen C, et al., Quantitation of antigen-reactive T-cells in peripheral blood by IFNg- ELISPOT assay and chromium-release assay: a four-centre comparative trial. Journal of Immunological Methods, 244:81-89 (2000)
3. Mwau M, et al., Design and validation of an Enzyme–Linked Immunospot
assay for use in clinical trials of candidate HIV vaccines. AIDS research and
human retroviruses, 18(9): 611-618 (2002)
Microtiter plate
Millipore
MAIP N45 (PVDF membrane) is recommended. Make sure not to scrape the bottom of
the wells and do not let the membrane dry out, empty plate just before adding
reagents. Best results are obtained after activating the membrane with ethanol
before use.
A
disadvantage of using PVDF membrane plates is the chance of leakage through the
membrane. This can result in a high background or unequal staining of the well.
To prevent this, remove the bottom of the plate and use a separate culture tray
(Millipore MAMC S01). Rinsing the
underside of the membrane with distilled water after incubation with the
biotinylated antibody and streptavidin-poly-HRP will further reduce eventual
background staining.
Washing
thoroughly will result in a low background and good spot development. Washing
can be carried out manually with a squirt bottle or with an automated washing
device.
PeliSPOT buffer
The provided PeliSPOT buffer is 5-fold concentrated, dilute with distilled water to obtain a working strength buffer.The buffer can be used as a dilution buffer for the biotinylated antibody and the streptavidin-poly-HRP.
The buffer does not contain a preservative, store the buffer at -18 to -32°C after use.
Positive
assay control
Each kit contains a positive assay control. Dilute as described on the label of the vial and add 100 µl to one well in the assay plate. If the assay is performed correctly it will result in a totally blue/purple well.
Avoid repeated freeze-thawing of the control. Thaw at room temperature (18-25°C), do not use a waterbath for this purpose. Immediately store control after use at -18 to -32°C.
Working
conditions
Sterile conditions are not necessary for coating if the required cell incubation period is not longer than 40 hours and the cell culture medium is supplemented with antibiotics. When working with human specimen the use of a flow cabinet is recommended.
Freshly isolated or frozen peripheral blood mononuclear cells (PBMC) as well as cell lines or cultured cells can be used.
After being drawn, anti-coagulated blood is kept at room temperature for a maximum of 24 hours. PBMC are isolated from venous blood by density centrifugation according to the manufacturers protocol. Wash cells twice with cell culture medium.
Add a mitogen as a positive secretion control, for example PMA (1 ng/ml) and Ionomycin (0.5 µg/ml). Wells that exceed the number of 2.5×104 mitogen stimulated cells per well are generally not countable due to high cytokine production.
For antigen specific stimulation the optimal concentration of the antigen and the optimal number of cells per well have to be determined experimentally. Start with a cell concentration of e.g. 2 ×105 cells per well and an antigen concentration between 1 and 10 µg/ml.
Please read the chapter IX. IMPORTANT INFORMATION first.
For your convenience an easy reference protocol and a suggested plate plan are available on the last pages of this leaflet.
COATING
Make sure not to scrape the
membrane bottom of the wells during pipetting and do not let the membrane dry
out, empty plate just before adding reagents.
Prepare PBS as described in chapter VII of this leaflet.
Bring the coating antibody (red-capped vial) to room temperature.
Centrifuge vial before use (30 seconds 3,000 x g).
Add 60 ml of coating antibody to 6 ml PBS per microtiter plate.
Activate the PVDF microplates by adding 100 µl of 70% or 96% ethanol.
Cover the plate with the lid and incubate for 10 minutes at room temperature.
Empty the microtiter plate by flicking over a sink and completely fill the wells with distilled water, repeat this with PBS.
Empty plate and tap plate gently on absorbent paper.
Directly add 50 ml of the diluted coating antibody per well.
Cover the plate with the lid and incubate overnight at 2 to 8 °C.
Prepare wash buffer (PBS + 0.005% Tween-20) as described in CHAPTER VII.
CELLS
Please read the chapter X. PREPARATION OF CELLS for recommendations regarding the best method for cell preparation.
Optimal concentrations for cells and stimuli and the optimal incubation period have to be determined experimentally.
Prepare cell culture medium
Supplement cell culture medium, e.g. IMDM or RPMI-1640, with:
5% FCS
100 mg/ml
streptomycin
2 mM glutamin
Prepare cell suspension
Isolate, thaw or harvest cells.
Centrifuge cells and wash them twice with cell culture medium.
Resuspend the cell suspension to avoid cell clumping. Handle cells with care, do not vortex.
Determine the concentration of cells in the cell suspension.
Dilute the cell suspension to a concentration of e.g.:
4×106 cells/ml for a final concentration of 2×105 cells per well, or
2×106 cells/ml for a final concentration of 1×105 cells per well.
Prepare cell culture medium with
antigen or mitogen
The concentration of the antigen and mitogen must be twice the final concentration.
Antigen, e.g.: 20 µg/ml for a final concentration of 10 µg/ml, or
10 µg/ml for a final concentration of 5 µg/ml.
PMA/Ionomycin
Add 1 µl per ml cell culture medium from each stock solution (CHAPTER VII) to obtain a final concentration of 1 ng/ml PMA and 0,5 µg/ml Ionomycin.
Positive
assay control
Bring the positive assay control (black-capped vial) to room
temperature.
Centrifuge vial before use (30 seconds 3,000 x g).
Prepare as
indicated on the vial.
Cell incubation
Empty the microtiter plate by flicking over a sink and wash plate five times with wash buffer. Empty plate.
Pipette the prepared solutions in triplicate according to the recommended plate plan on the last pages of this leaflet:
- Add 50 µl cell culture medium with antigen, PMA / Ionomycin or without stimulus to the appropriate wells.
- Resuspend the cell suspension carefully to obtain a homogeneous cell suspension and directly add 50 µl to each well
- Add 100 µl of the diluted positive assay control to one well (do not add cells).
Cover the plate with lid and incubate as
indicated in the specification sheet at 37°C in a CO2 incubator.
During this period make sure that the plate is
completely horizontal and do not agitate or move the plate.
DAY 3
Bring the PeliSPOT buffer, the biotinylated antibody and the TMB
substrate to room temperature.
Prepare 30 ml PeliSPOT buffer per microtiter plate:
6 ml PeliSPOT buffer stock solution
24 ml Distilled water
BIOTINYLATED ANTIBODY
Centrifuge the biotinylated antibody (yellow capped vial) before use (30 seconds 3,000 x g).
Dilute 120 ml of biotinylated antibody in 12 ml PeliSPOT buffer per microtiter plate.
Empty the microtiter plate by flicking over a sink. Rinse the underside of the membrane with distilled water and wash plate five times with wash buffer as described in chapter IX.
Empty plate and tap plate gently on absorbent paper.
Directly add 100 ml diluted biotinylated antibody per well.
Cover the plate with lid and incubate 1 hour at room temperature .
STREPTAVIDIN-POLY-HRP
Keep the streptavidin-poly-HRP (brown capped vial) at -18 to -32 °C to maintain maximal stability. The contents of the vial will not freeze at this temperature.
Centrifuge vial before use (30 seconds 3,000 x g).
Dilute 2 ml of coating antibody in 12 ml PeliSPOT buffer per microtiter plate.
Do not prepare in advance of assay.
Empty the microtiter plate by flicking over a sink. Rinse the underside of the membrane with distilled water and wash plate five times with wash buffer as described in chapter IX.
Empty plate and tap plate gently on absorbent paper.
Directly add 100 ml diluted streptavidin-poly-HRP per well.
Cover the plate with lid and incubate 1 hour at room temperature .
TMB SUBSTRATE
Empty the microtiter plate by flicking over a sink. Rinse the underside of the membrane with distilled water and wash plate five times with wash buffer as described in chapter IX.
Empty plate and tap plate gently on absorbent paper.
Directly add 50 ml ready to use TMB substrate per well.
Cover the plate with lid and incubate 4 –15 minutes at room temperature.
Note: The speed of the enzymatic colour development is influenced by many factors including temperature, therefore it is advised to monitor the colour development by eye.
To stop the staining empty the microtiter plate by flicking over a sink. Rinse the underside of the membrane with distilled water and wash plate five times with distilled water. Empty plate and tap plate gently on absorbent paper.
ANALYSE THE SPOTS
Dry the plate overnight at room temperature in the dark or place the plate in an airflow in front of a fan until the membranes are completely dry.
Count the number of spots preferably using the A.EL.VIS automated spot analyser, recommended analysis settings are stated in the specification sheet.
Store the plates in the dark to prevent fading of the spots.
PROTOCOL SUMMERY AND CHECKLIST
PLEASE READ CHAPTER IX. IMPORTANT INFORMATION FIRST!
Day 1:
¡ Bring coating antibody to room temperature.
¡ Prepare PBS.
¡ Dilute the coating antibody 1:100 in PBS.
¡ Activate PVDF-bottomed-well microtiter plate with 70% or 96% ethanol for 10 minutes at room temperature.
¡ Rinse once with distilled water and wash once with PBS.
¡ Remove all residual buffer by repeated tapping on absorbent paper.
¡ Add 50 ml per well of diluted coating antibody.
¡ Cover the plate with the lid and incubate overnight at 2 to 8°C.
Day 2:
¡ Prepare wash buffer.
¡ Prepare cell culture medium.
¡ Prepare cell suspensions and solutions with antigen and mitogen in cell culture medium.
¡ Prepare positive assay control as indicated on the vial.
¡ Empty wells by flicking the plate over a sink and tapping it on absorbent paper.
¡ Wash the plate five times with wash buffer.
¡ Add 50 µl cell culture medium with antigen, PMA / Ionomycin or without stimulus to the appropriate wells according to the plate plan (see plate plan example).
¡ Add 50 ml of the cell suspension containing the appropriate number of cells
¡ Add 100 ml of the positive assay control per plate to one well.
¡ Cover the plate with the lid and incubate overnight (16 – 24 hours) at 37°C in a CO2 incubator.
During this period make sure that the plate is completely horizontal and do not agitate or move the plate.
Day 3
¡ Prepare PeliSPOT buffer.
¡
Bring all required reagents,
with the exception of streptavidin-poly-HRP, to room temperature.
¡ Dilute the biotinylated antibody 1:100 in PeliSPOT buffer.
¡ Empty wells, rinse underside of the membrane with distilled water, wash the plate five times with wash buffer and tap plate on absorbent paper.
¡
Add 100 ml of the diluted biotinylated antibody per well, cover the plate
with the lid and incubate for one hour at room temperature.
¡ Dilute the streptavidin-poly-HRP 1:6,000 in PeliSPOT buffer.
¡ Empty wells, rinse underside of the membrane with distilled water, wash the plate five times with wash buffer and tap plate on absorbent paper.
¡
Add 100 ml of the diluted streptavidin-poly-HRP per well, cover the plate
with the lid and incubate for one hour at room temperature.
¡ Empty wells, rinse underside of the membrane with distilled water, wash the plate five times with wash buffer and tap plate on absorbent paper.
¡ Add 50 ml TMB substrate per well, cover the plate with the lid and incubate for 4-15 minutes at room temperature (monitor spot intensity by eye).
¡ Rinse underside of the membrane and wash the plate with an excess of distilled water.
¡
Remove all residual water by
repeated tapping on absorbent paper
¡ Dry the plate overnight at room temperature in the dark or place the plate in an air flow in front of a fan until the membranes are completely dry.
¡ Count the number of spots preferably using the A.EL.VIS automated spot analyzer.
Suggested plate plan
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1 |
2 |
3 |
4 |
5 |
6 |
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9 |
10 |
11 |
12 |
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Sample 1 |
A |
NEG |
NEG |
NEG |
POS |
POS |
POS |
A1 |
A1 |
A1 |
A2 |
A2 |
A2 |
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B |
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PAC |
Key: NEG :
negative secretion control (cell suspension only)
POS : positive secretion control (polyclonal stimulator e.g. PMA / Ionomycin)
A1 +A2 : antigen (peptides) of interest
PAC : positive assay control (no cells)
|
AVAILABLE
PRODUCTS FOR ELISPOT |
|
Order
number |
|
Human IFN-gamma PeliSPOT kit |
288 tests |
RDI-M2533clb |
|
Human Granzyme B PeliSPOT kit |
288 tests |
RDI-M2536clb |
|
Human IL-4 PeliSPOT kit |
288 tests |
RDI-M2514clb |
|
PeliSPOT buffer (stock solution) |
50 ml |
RDI-M2540clb |
|
TMB for PeliSPOT |
18 ml |
RDI-M2521clb |
|
Streptavidin poly-HRP |
0.1 ml |
RDI-M2051clb |
|
|
1 ml |
RDI-M2032clb |
Please inquire for information
or demos of the A.EL.VIS spot analyzer systems
Order through:
RDI Division of Fitzgerald Industries Intl
Phone (978) 371-6446 or (800) 370-2222
Fax (978)
371-2266
Email: antibodies@fitzgerald-fii.com
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