Human IgE ELISpotBASIC kit (ALP) (CAT#: ITS-0322-P96)

4 plates

1.Vial 1 (green top):Monoclonal antibody 107 (600 μl);Concentration: 1 mg/ ml.
2.Vial 2 (purple top):Biotinylated monoclonal antibodies 107/182/101 (50 μl);Concentration: 1 mg/ml.
3.Vial 3 (white top):Streptavidin-Alkaline Phosphatase (50 μl).
4.Activators:Anti-CD40 monoclonal antibody S2C6 (80 μl);Concentration: 1 mg/ ml.
5.Lyophilized recombinant humanIL-4(1 ug).

200 µL

2h

Pre-coated

1.Dilute the coating antibodies (107) 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% CO, 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(107/182/101-biotin) to 0.05 ug/ml in PBS containing 0.5% fetal calfserum(PBS-O.5%FCS).Add 100 ul/well and incubate for 2 hours at room temperature.
11.Wash plate as above (step C1).
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 (step C1).
14.Filter the ready-to-use substrate solution(BCIP/NBT-plus) through a 0.45 um filter and add100 ul/well. Develop until distinct spots emerge.
15.Stop color development by washing extensively in tap water. If desirable, remove the underd-rain (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 micro-scope.
17. Store plate in the dark at room temperature.

human

Capture mAb (107),Biotinylated detection mAbs (107/182/101),Streptavidin-ALP,Anti-CD40 mAb,Recombinant human IL-4

We recommend the use of PVDF-based membrane plates. Maximal antibody binding capacity of these platesis obtained by a brief treatment with ethanol.

PBS for washing and dilution should be filtered (0.3 um) for optimal results.Although possible to use, we donot recommend the inclusion of Tween or other detergents in the washing and incubation buffers.

4°C-8°C. Human IL-4 shoud be stored frozen at -20°C

Plates should be kept at room temperature.

The expiry date indicates how long unopened products, stored according to instructions, are recommended for use.

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.

For Research Use Only. Not for use in diagnostic procedures.

3497

1.Antibody coating:Cytokine-specific monoclonal capture antibodies are immobilized on an ethanol-treated PVDF membrane plate.
2.Cell incubation:Cells are added to the wells in the presence or absence of activating stimuli, and then incubated to allow for cytokine secretion.
3.Cytokine capture:Secreted cytokines bind to the capture antibodies on the membrane immediately surrounding the activated cells.
4.Detection antibodies:Following removal of the cells and washing of the plate wells, biotinylated cytokine-specific detection antibodies are added to the wells.
5.Streptavidin-enzyme conjugate:To enable the formation of spots on the membrane, a streptavidin-enzyme conjugate is added to the wells.
6.Addition of substrate:Colorimetric substrate is added to the wells and will form an insoluble precipitate when catalyzed by the enzyme; a visible representation of cytokine release by a single activated cell.
7.Analysis Spots are counted in an automated ELISpot reader or under a dissection microscope, and the frequency of secreting cells is calculated.