Human IgG SARS-CoV-2 RBD ELISpotPLUS (ALP) (CAT#: ITS-0322-P127)

1 plate

1.1 pre-coated plate, mAbs MT91/145.
2.For detection of RBD-specific IgG spots:Antigen: RBD-WASP, lyophilized (1 vial);anti-WASP-ALP (60 μl).
3.For detection of total IgG spots:Biotinylated monoclonal antibodies MT78/145, 0.5 mg/ml (40 μl);Streptavidin-ALP (40 μl);Standard reconstitution buffer AS (1 ml);Polyclonal activator: R848, 1 mg/ ml, (100 ul);Lyophilized recombinant human IL-2(1 μg).
4.BCIP/NBT-plus substrate (25 ml):The biotinylated mAbs contain 0.02% sodium azide. Anti-WASP-ALPand SA-ALP are supplied in PBS with 0.002% Kathon CG. Vials havebeen overfilled to ensure recovery of the specified amount.

200 µL

2h

Pre-coated

1.Remove the plate from the sealed package and wash 4 times with sterile PBS(200 ul/well).
2.Condition the plate with medium (200 ul/well) containing 10% of the same serum as used for the cell suspensions. Incubate for at least 30 minutes at room temperature.
3.Remove the medium and add the cell suspension. The included R848 and recombinant IL-2 can be used to induce IgG secretion.
4.Put the plate in a 37°C humidified incubator with 5% CO, and incubate for 18-24 hours.
5.Remove the cells by emptying the plate and wash 5 times with PBS, 200 ul/well.
6.RBD-specific IgG spots: Add 500 ul standard reconstitution bufter to the lyophilized RBD-WASP, allow it to dissolve for 5 minutes and mix thoroughly. Dilute the RBD WASP solution 1:25.Add 100 ul/well.Total IgG spots: Dilute the detection mAbs MT78/145-biotin to 1 ug/ml in PBS-0.5% FCS. Add 100 ul/well.Incubate for 2 hours at room temperature.
7.Wash plate as above (step C1).
8.Dilute the anti-WASP-ALP (1:200) in PBS-0.5% FCS and add 100 ul/well. Incubate for 1 hour at room temperature.
9.Wash plate as above (step C1).
10.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.
11.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.
12.Leave the plate to dry.Inspect and count spots in an ELISpot reader or in a dissection micro-scope.
13. Store plate in the dark at room temperature.

human

RBD-WASP,Biotinylated detection mAbs (MT78/145),anti-WASP-ALP,Streptavidin-ALP,R848,Recombinant human IL-2,Pre-coated MSIP white plate (mAbs MT91/145),Reconstitution buffer

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.

On arrival all reagents should be stored refrigerated at 4-8 °C except the RBD-WASP, R848 and IL-2 that should be stored frozen at -20°C or below.

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.

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.