Components
1.Vial 1 (blue top):Monoclonal antibody MT57 (1200 μl);Concentration: 0.5 mg/ml.
2.Vial 2 (red top):Biotinylated monoclonal antibody MT20 (100 μl);Concentration: 0.5 mg/ ml.
3.Vial 3 (yellow top):Biotinylated monoclonal antibody MT57 (100 μl);Concentration: 0.5 mg/ ml.
4.Vial 4 (white top):Streptavidin Alkaline Phosphatase (50 ul).
5.Vial 5 (black top):Polyclonal activator: R848 (100 μl), Concentration: 1 mg/ml.
6.Vial 6 (blue top):Lyophilised recombinant human IL-2(1 ug).
7.Antibodies are supplied in sterile filtered (0.2 um) PBS with 0.02% sodium azide. Streptavidin-ALP is supplied in 0.1 M Tris bufer with 0.002% Kathon CG. R848 is supplied in sterile filtered (0.2 um) PBS containing 1% DMSO. V ials have been overfilled to ensure recovery of the specified amount.
Assay Procedure
1.Dilute the coating antibodies (MT57) to 15 ug/ml in sterile PBS, pH 7.4.
2.Remove the ELISpot plate from the package and if using a PVDF plate, pre-wet the membrane by adding ethanol.
3.Wash plate 5 times with sterile water, 200 ul/well.
4. Add 100 ul/well of the antibody solution and incubate overnight at 48°C.
5.Remove excess antibody and wash plate 5 times with sterile PBS, 200 ul/well.
6. Add 200 ul/well of medium containing 10% of the same serum as used for the cell suspensions Incubate for at least 30 minutes at room temperature.
7.Remove the medium and add the stimuli followed by the cell suspension. Alternatively, cells and stimuli can be mixed before addition to the plate.
8.Put the plate in a 37°C humidified incubator with 5% CO2, and incubate for 18-48 hours.
9.Remove the cells by emptying the plate and wash 5 times with PBS, 200 ul/well.
10.Dilute the detection antibody(MT20-biotin) to 0.05 ug/ml in PBS containing 0.5% fetal calf serum (PBS-O.5%FCS). Add 100 ul/well and incubate for 2 hours at room temperature.
11.Wash plate as above.
12.Dilute the Streptavidin-ALP(1:1000) in PBS-0.5% FCS and add 100 ul/well. Incubate for 1 hour at room temperature.
13.Wash plate as above.
14.Filter the ready-to-use substrate solution(BCIP/NBT-plus) through a 0.45 um filter and add 100 ul/well. Develop until distinct spots emerge.
15.Stop color development by washing extensively in tap water. If desirable, remove the underdrain (the soft plastic under the plate) and rinse the underside of the membrane.
16.Leave the plate to dry. Inspect and count spots in an ELISpot reader or in a dissection microscope.
17. Store plate in the dark at room temperature.
Format
Capture mAb (MT57), Biotinylated detection mAb (MT20), Biotinylated detection mAb (MT57), Streptavidin-ALP, R848, Recombinant human IL-2
Precaution of Use
We recommend the use of PVDF-based membrane plates. Maximal antibody binding capacity of these platesis obtained by a brief treatment with ethanol.
Handling Advice
PBS for washing and dilution should be filtered (0.3 um) for optimal results. Although possible to use, we do not recommend the inclusion of Tween or other detergents in the washing and incubation buffers.
Storage
4°C-8°C. R848 and IL-2 shoud be stored frozen at -20°C
Storage Comment
Plates should be kept at room temperature.
Expiry Date
The expiry date indicates how long unopened products, stored according to instructions, are recommended for use.
Note
The serum should be selected to support cell culture and give low background staining. We recom-mend the use of fetal calf serum. Alternatively serum-free medium evaluated for cell culture can beused.
Restrictions
For Research Use Only. Not for use in diagnostic procedures.
Datasheet