Human IgM ELISpotBASIC kit (ALP) (CAT#: ITS-0322-P101)

4 plates

1.Vial 1 (red top):Monoclonal antibodies MT11/12(1.2 ml);Concentration: 0.5 mg/ml.
2.Vial 2 (yellow top):Biotinylated monoclonal antibody MT22 (100 ul);Concentration: 0.5 mg/ml.
3.Vial 3 (white top):Streptavidin-Alkaline Phosphatase (50 μl).
4.Vial 4 (black top):Polyclonal activator: R848 (100 μul);Concentration: 1 mg/ ml.
5.Vial 5 (blue top):Lyophilised recombinant human IL-2(1 ug).
6.Antibodies are supplied in sterile filtered (0.2 μm) 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. Vials have been overilled to ensure recovery of the specified amount.

200 µL

2h

Pre-coated

1.Dilute the coating mAb MT11/12 to 15 ug/ ml in sterile PBS, pH7.4.
2.PVDF plates need to be treated (activated) with ethanol before coating.
3.Wash plate 5 times with sterile water, 200 ul/well.
4.Add 100 pul/well of the antibody solution and incubate overnight at 4-8°C.
5.Wash plate 5 times with sterile PBS, 200 ul/well, to remove excess antibody.
6.For analysis of in vivo activated B cells, add the cell suspension to the ELISpot plate. For analysis of memory B cells, the cells can be pre-activated separately in tubes prior to addition to the plates.
7.Put the plate in a 37°C humidifed incubator with 5% CO, and incubate for 16-24 hours.
8.To remove the cells, empty the plate and wash 5 times with PBS, 200 μl/well.
9.Dilute biotinylated antigen to a suitable concentration (0.01-1 ug/ml) in PBS. Add 100 ul/well and incubate for 2 hours at room temperature.
10.Wash plate as above (step C1).
11.Dilute the Streptavidin-ALP (1:1000) in PBS and add 100 μl/well. Incubate for 1 hour at room temperature.
12.Wash plate as above (step C1).
13.Add 100 ul/well of subtrate solution (e.g. BCIP/NBT) and develop until distinct spots emerge.
14.Stop colour development by washing extensively in tap water. If desirable, remove the plate from the plate tray or the underdrain and rinse the underside of the membrane.
15.Leave the plate to dry. Inspect and count spots in an ELISpot reader or in a dissection microscope.
16.Store plate in the dark at room temperature.

human, non-human primates

Capture mAbs (MT11/12),Biotinylated detection mAb (MT22),Streptavidin-ALP,R848,Recombinant human IL-2

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 R848 and IL-2 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.

IgM

3507

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.