Human TNF-alpha ELISpot BASIC (ALP)

The human TNF-α ELISpot kit is ideal for users who want the flexibility of setting up their own assay.

The kit can count cells that secrete human TNF-α, and contains a pair of matched monoclonal capture and detection antibodies and Streptavidin-ALP. The human TNF-α ELISpot kit can also use streptavidin-hrp.

ELISpot

Washing of plates can be done using a multi-channel micropipette. In washing steps not requiring sterile conditions(C1-C5), a regular ELISA plate washer can also be used, provided that the washing head isadapted to the ELISpot plates. Avoid getting liquid on the underside of the membrane as this may causeleakage due to capillary drainage. If using ELISpot PVDF (ELIIP) plates, always remove the plate from the plate traybefore manually emptying the plate.

TNFA

Human

Sandwich

Sandwich

cell suspension

Recognizes natural human TNF-α

4 plates

1.Vial 1(green top):Monoclonal antibody MT25C5 (1.2 ml);Concentration: 0.5 mg/ml.
2.Vial 2(yellow top):Biotinylated monoclonal antibody MT20D9 (50 ul);Concentration: 0.5 mg/ml.
3.Vial 3(white top):Streptavidin-Alkaline Phosphatase (50 ul).

200 µL

2h

Pre-coated

1.Dilute the coating antibody (MT25C5) to 15 ug/ml in sterile PBS, pH7.4.
2.Remove the ELISpot plate from the package and if using a PVDF plate, pre-wet the membrane by adding ethanol.
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 stimuli followed by the cell suspension. Alternatively, cells and stimuli can be mixed before addition to the plate.
8.Put the plate in a 37°C humidified incubator with 5% CO2, and incubate for 12-48 hours.
9.Remove the cells by emptying the plate and wash 5 times with PBS, 200 ul/well.
10.Dilute the detection antibody (MT20D9-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.
12.Dilute the Streptavidin-ALP(1:1000) in PBS-0.5% FCS and add 100 ul/well. Incubate for 1 hour at room temperature.
13.Wash as above.
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.

Human

Capture mAb (MT25C5), Biotinylated detection mAb (MT20D9), Streptavidin-ALP

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 do not recommend the inclusion of Tween or other detergents in the washing and incubation buffers.

4°C-8°C

Antibodies are supplied in sterile filtered (0.2 um) PBS with 0.02% sodium azide. Streptavidin-ALP is supplied in 0.1 M Tris buffer with 0.002% Kathon CG. Vials have been overfilled to ensure recovery of stated quantity.

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.

Tumor necrosis factor ligand superfamily member 2; DIF; Cachectin; ICD2; ICD1; N-terminal fragment; TNF-a; TNFA; TNFSF2; TNF-alpha; Tumor necrosis factor; NTF

TNF-a

This gene encodes a multifunctional proinflammatory cytokine that belongs to the tumor necrosis factor (TNF) superfamily. This cytokine is mainly secreted by macrophages. It can bind to, and thus functions through its receptors TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. This cytokine is involved in the regulation of a wide spectrum of biological processes including cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation. This cytokine has been implicated in a variety of diseases, including autoimmune diseases, insulin resistance, and cancer. Knockout studies in mice also suggested the neuroprotective function of this cytokine.

7124

P01375

Among its related pathways are Allograft rejection and TNF signaling.

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.