Mouse IgG2c ELISpotBASIC kit (ALP) (CAT#: ITS-0322-P37)

4 plates

1.Vial 1(green top):anti-mouse Ig mAbs MT24/JC5-1 (2x1600 ul); Concentration:0.5 mg/ml.
2.Vial 2(red top):anti-mouse IgG2c mAb MTG2c, biotinylated (50 ul);Concentration:0.5 mg/ml.
3.Vial 3 (white top):Streptavidin-Alkaline Phosphatase(ALP) (50 ul);Polyclonal activator: R848(100 ul);Concentration: 1 mg/ml;Lyophilised recombinant mouse IL-2(0.5 ug).

200 µL

2h

Pre-coated

1.Total IgG2c: Dilute the coating antibody(MT24/JC5-1)to 40 ug/ml in sterile PBS,pH 7.4.Antigen-specific IgG2c: Dilute the antigen to suitable concentration(1-50 ug/ml) in sterilePBS, pH7.4.
2.Remove the ELISpot plate from the package and if usinga PVDF plate, pre-wet the membrane by adding ethanol.PVDF-plates from Millipore Corp.,MAIPSWU, should be treated with 50ul 70%ethanol per well for 2 minutes.PVDF-plates, type MSIP, should be treated with 15 ul 35% ethanol per well for maximum 1 minute.
3.Wash plate 5 times with sterile water, 200 ul/well.
4.Add 100 ul/well of the antibody solution and incubate overnight at 4-8°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 cell suspension. The included R848 and recombinant IL-2can be used to induce IgG2c secretion.
8.Put the plate in a 37°C humidified incubator with 5% CO, and incubate for 16-24hours. Do not move the plate during this time and take measures to avoid evaporation.
9.Remove the cells by emptying the plate and wash 5 times with PBS,200 ul/well.
10.Dilute the detection antibody(MTG2c-biotin) to 0.5 ug/ml in PBS containing 0.5% fetal calf serum (PBS-0.5% FCS).Add 100 ul/well and incubate for 2 hours at room temperature.
11.Wash as above(step C1).
12.Dilute the Streptavidin-ALP(1:1000) in PBS-0.5%FCS and add 100 ul/well. Incubate for l hour at room temperature.
13.Wash as above(step C1).
14.Add 100 ul/well of substrate solution(e.g.BCIP/NBT) and develop until distinct spots emerge.
15.Stop colour development by washing extensively in tap water. If desirable, remove the plate from the tray or the underdrain 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.

Mouse

Capture mAbs (MT24/JC5-1),Biotinylated detection mAb (MTG2c),Streptavidin-ALP,R848,Recombinant mouse 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 (0.2 um) for optimal results.Although possible to use, we donot recommend the inclusion of Tween or other detergents in the washing and incubation buffers.

On arrival antibodies and Strepta-vidin-ALP should be stored refrigerated at 4-8C. R848 and IL-2should be stored at -20°C.

Antibodies are supplied in sterile filtered(0.2 um)PBS with0.02% sodium azide. Streptavidin-ALP is supplied in 0.1 M Tris bufferwith 0.002% Kathon CG. Vials have been overfilled to ensure recov-ery of stated quantity.

The expiry date indicates how long un-opened products, stored according to instructions, are recommendedfor use.

lne serum should be selected to support cell culture and gve low bacKground staning. we recommena tneuse of fetal calf serum.Alternatively serum-free medium evaluated for cell culture can be used.

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

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.