I. INTRODUCTION

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).

II. INTENDED USE

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.

III. PRINCIPLE OF THE TEST

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.

IV. STORAGE AND STABILITY

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.

V. CONTENTS OF THE KIT

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

VI. PRECAUTIONS 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.

VII. ADDITIONAL MATERIALS AND REAGENTS REQUIRED

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

VIII. REFERENCES

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)

IX. IMPORTANT INFORMATION

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.

Washing

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.

X. PREPARATION OF CELLS

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 ×10⁵cells per well and an antigen concentration between 1 and 10 µg/ml.

XI. ASSAY PROCEDURE

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.

DAY 1

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.

DAY 2

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 U/ ml penicillin

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.:

4×10⁶cells/ml for a final concentration of 2×10⁵ cells per well, or

2×10⁶cells/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