Bovine IgG ELISpotBASIC kit (ALP) (CAT#: ITS-0322-P90)

4 plates

1.Vial 1 (green top):Monoclonal antibody MT17.1 (800 ul);Concentration: 0.5 mg/ ml.
2.Vial 2 (yellow top):Biotinylated monoclonal antibody MT391 (40 ul);Concentration: 0.5 mg/ml.
3.Vial 3 (white top):Streptavidin-Alkaline Phosphatase (50 ul).
4.Vial 4 (black top):Polyclonal activator: R848 (100 ul);Concentration: 1 mg/ ml.

200 µL

2h

Pre-coated

1.Dilute the coating mAb MT134 to 10 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.

Cow

Capture mAb (MT134),Biotinylated detection mAb (MT391),Streptavidin-ALP,R848

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

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.

281850

Please note that antigen must be biotinylated prior to starting the experiment. IgG from all secreting B cells are captured by anti- IgG antibodies coated on the ELISpot plate. The secretion of specific IgG is then visualised by the addition of biotinylated antigen followed by Streptavidin-enzyme and substrate.