Human IFN-gamma SARS-CoV-2 ELISpotPLUS kit (ALP), strips (CAT#: ITS-0322-P123)

1 plate

1 pre-coated strip plate (mAb 1-D1K) and empty plate frame.
Biotinylated detection mAb 7- B6-1, 1 mg/ml (40 μl).
Streptavidin-ALP (40 μl).
SARS- CoV-2 S2 N defined peptide pool.
SARS- CoV-2 SNMO defined peptide pool.
SARS- CoV-2 S1 scanning pool (2 vials: pool 1 and pool 2).
Co-stimulator anti-CD28 mAb CD28-A, 0.1 mg/ml (100 μl).
Positive control antiCD3 mAb CD3-2 (100 μl).
BCIP/NBT-plus substrate (25 ml)The detection antibody is supplied in sterile fltered (0.2um) PBS with0.02% sodium azide. Streptavidin-ALP is supplied in 0.1 M Tris bufferwith 0.002% Kathon CG. Anti-CD28 mAb and anti-CD3 mAb is supplied in sterile fltered (0.2 um) PBS. Vials have been overilled 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.Empty the plate and add 50 ul/well of peptides or controls, followed by 50 ul cell suspension/ well. Alternatively, mix cells and stimuli before addition of 100 yul/well. The final peptide concentration should be 2 ug/ ml of each peptide. Anti CD28 can be included at a final concentration of 0.1 ug/ml.
4.Put the plate in a 37°C humidified incubator with 5% CO, and incubate for 12-48 hours.
5.Remove the cells by emptying the plate and wash 5 times with PBS, 200 ul/well.
6.Dilute the detection antibody((7-B6-1-biotin) to 1/ml in PBS containing 0.5% fetal calfserum(PBS-O.5%FCS).Add 100 ul/well and incubate for 2 hours at room temperature.
7.Wash plate as above (step C1).
8.Dilute the Streptavidin-ALP(1:1000) 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

SARS-CoV-2 S2 N defined peptide pool,SARS-CoV-2 S1 scanning pool,SARS-CoV-2 S N M O defined peptide pool,Anti-CD28 mAb,Biotinylated detection mAb (7-B6-1),Streptavidin-ALP,anti-CD3 mAb,Substrate (BCIP/NBT-plus),Pre-coated strip clear plates (mAb 1-D1K)

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 peptide pools 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.

The SARS-CoV-2 S1 scanning pool contains 166 peptides from the human SARS-CoV-2 virus. The peptides are 15-mers overlapping with 11 amino acids, covering the S1 domain of the spike protein (amino acid 13-685). The pool is supplied in two vials: pool 1 SARS-CoV-2 S1 peptides 1-83 (83 peptides) and pool 2 SARS-CoV-2 S1 peptides 84-166 (83 peptides).
The SARS-CoV-2 S2 N defined peptide pool contains 41 synthetic peptides from the human SARS-CoV-2 virus. The peptides are derived from the spike and nucleoprotein.
The SARS-CoV-2 SNMO defined peptide pool contains 47 synthetic peptides from the human SARS-CoV-2 virus. The peptides are derived from the spike, nucleoprotein, membrane protein, ORF3a and ORF7a.

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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.