Human IFNg / IL-5 Dual ELISpot Set

The purpose of ELISPOT analysis is to count cytokine producing cells in a single cell suspension. The advantage of this method is that it requires minimal in vitro manipulations, making the analysis of cytokine production as close to in vivo conditions as possible, and in a highly specific manner.

The Elispot test is an ideal tool for TH1/TH2 response, vaccine development, viral infection monitoring and treatment, oncology, infectious diseases, autoimmune diseases and transplantation. Elispot detection is based on sandwich immunoenzyme technology. Cytokines or soluble molecules secreted by cells are captured by coating antibodies to avoid diffusion in the supernatant, protease degradation or binding to soluble membrane receptors. After the cells are removed, the captured cytokines are displayed by the tracer antibody and the appropriate conjugate.

ELISpot (ES)

Small volumes of IFNg / IL-5 ELISpot set vial(s) may occasionally become entrapped in the seal of the product vial during shipment and storage. If necessary, briefly centrifuge the vial on a tabletop centrifuge to dislodge any liquid in the container's cap. Certain products may require to ship with dry ice and additional dry ice fee may apply.

After cell stimulation, locally produced cytokines are captured by IFNg and IL-4 specific monoclonal antibodies. After cell lysis, trapped cytokine molecules are revealed by a secondary anti-IFNg FITC conjugated antibody and a biotinylated anti-IL-4 antibody. Those are in turn recognised by anti-FITC HRP and streptavidin-AP conjugates. PVDF-bottomed-well plates are then incubated first with AEC substrate buffer, washed and subsequently incubated with BCIP/NBT. Coloured red/brownish spots indicate IFNg production while IL-4 is revealed by blue/purple spots.

IFN-g / IL-5

Human

Sandwich

Sandwich

cell suspension

Recognizes both natural and recombinant human IFN-g and IL-5

Cross-reactive with simian IFN-g.

1x96 Wells

1.Capture antibody for IFNγ (0.50 mL). Supplied sterile.
2.Capture antibody for IL-5 (0.50 mL). Supplied sterile.
3.FITC conjugated detection antibody for IFNγ (lyophilised, resuspend in 0.55mL).
4.Biotinylated detection antibody for IL-5 (lyophilised, resuspend in 0.55mL).
5.Anti-FITC antibody HRP conjugate (100 µL).
6.Streptavidin Alkaline Phosphatase conjugate (50 µL).
7.Bovine Serum albumin
8.Dry skimmed milk:non sterile Elispot / Liquid sterile milk: sterile Elispot.
9.50 x concentrate AEC substrate buffer (1mL).
10.10 x concentrate buffer for the preparation of AEC buffer (5mL).
11.Ready-to-use BCIP/NBT substrate buffer (50mL).
12.96 PVDF-bottomed-well plates (5 if ordered).

1.96 PVDF-bottomed-well plates.
2.Cell culture media.
3.CO2 incubator.
4.70% ethanol.
5.Tween 20.
6.Phosphate buffered saline.
7.ELISPOT reading system.

100 µL

1.5h

Sterile-plate

1.Detection antibody:Reconstitute the lyophilised antibody with 0.55mL of distilled water. Gently mix the solution and wait until all the lyophilised material is back into solution.
2.Streptavidin alkaline phosphatase:Dilute 1/1000 in PBS 1% BSA.
3.Phosphate buffered saline (10X Concentrate solution):For 1 liter weight : 80g NaCl ; 2g KH2PO4 ; 14.4g Na2HPO4 2H2O. Add distilled water to 1 liter. Check that pH is comprised between 7.4 +/- 0.1. Dilute the solution to 1X before use.
4.Skimmed milk in PBS:For one non-sterile plate dissolve 0.2g of powder in 10mL of 1X PBS;For one sterile plate dilute 5ml of liquid milk in 5ml of 1X PBS.
5.1% BSA in PBS:For one plate dissolve 0.2 g of BSA in 20 mL of 1X diluted PBS.
6.0.05% Tween in PBS:For one plate dissolve 50µl of Tween 20 in 100 ml of 1X diluted PBS.
7.35% ethanol in water:For one plate mix 3.5 ml of ethanol with 6.5 ml of distilled water.
8.AEC buffer:For one plate mix 1 ml of AEC buffer A with 9 ml of distilled water. Then add 200µl of AEC buffer B.

1.Incubate PVDF-bottomed-well plates with 25µl / well of 35% ethanol for 30 sec at room temperature.
2.Empty wells and wash three times with 100µl / well of PBS.
3.Pipette 100µl of IFNγ capture antibody and 100µl of IL-5 capture antibody in 10 mL of plain PBS. Mix and dispense 100 µl into each well, cover the plate and incubate overnight at +4°C.
4.Empty wells and wash once with 100 µl of PBS.
5.Dispense 100 µl of skimmed milk in PBS into wells, cover and incubate for 2 hours at room temperature.
6.Empty wells by flicking the plate over a sink and tapping it on absorbent paper.
7.Wash plate once with PBS.
8.Dispense into wells 100 µl of cell suspension containing the appropriate number of cells and appropriate concentration of stimulator. Cells may have been previously in-vitro stimulated (Indirect ELISPOT). Cover the plate with a standard 96-well plate plastic lid and incubate cells at 37°C in a CO2 incubator for an appropriate length of time (15-20 hours).
9.Empty wells by flicking the plate over a sink and gently tapping it on absorbent paper.
10.Dispense 100µl of PBS-0.05% tween 20 into wells and incubate for 10 min at +4°C.
11.Wash wells three times with PBS-0.05% tween 20.
12.For 1 plate dilute 100µl of reconstituted IFNγ detection antibody and 100µl of reconstituted IL-5 detection antibody into 10 mL of PBS containing 1% BSA. Dispense 100µl into wells, cover the plate and incubate 1 hour 30 min at 37°C.
13.Empty wells and wash three times with PBS-0.05% tween 20.
14.For 1 plate dilute 20µl of anti-FITC HRP and 10 µl of streptavidin-Alkaline phosphatase conjugates into 10 mL of PBS-1% BSA.Dispense 100µl of the dilution into wells. Seal the plate and incubate for 1 hour at 37°C.
15.Empty wells and wash three times with PBS-0.05% tween 20.
16.Peel off the plate bottom; wash three times both sides of the membrane under running distilled water. Remove all residual buffer by repeated tapping on absorbent paper.
17.Prepare AEC buffer (see reagents preparation). Dispense 100µl of solution in wells.
18.Let the colour reaction proceed for 5-20 min at room temperature. When the spots have developed empty the buffer into an appropriate tray.
19.Wash three times both sides of the membrane with distilled water. Remove residual water by tapping the plate on absorbent paper. 20.Dispense 100µl of ready-to-use BCIP/NBT buffer into wells.
21.Let the colour reaction proceed for about 5-20 min at room temperature. When spots have developed, empty the buffer into an appropriate tray.
22.Wash thoroughly both sides of the membrane with distilled water
23.Dry the membrane by repeatedly tapping the plate on absorbent paper. Store the plate upside down so no remaining liquid will go back on the membrane. Read spots once the membrane is dried. Note that spots may become sharper after one night at +4°C.

Human

Liquid

4°C/-20°C

If not used within a short period of time, reconstituted detection antibody should be aliquoted and stored at -20°C.

at least one year at -20°C

Select online data sheet information is drawn from bioinformatics databases, occasionally resulting in ambiguous or non-relevant product information. It is the responsibility of the customer to review, verify, and evaluate the information to make sure it matches their requirements before purchasing the kit. Our ELISA Kit assays are dynamic research tools and sometimes they may be updated and improved.

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

IFN-g / IL-5

1.96 PVDF-bottomed-well plates are first treated with 35% ethanol and then coated with anti-IFNγ and anti-IL-5 capture antibodies.
2.Cells are incubated in the presence of the antigen. Upon stimulation they release cytokines molecules which bind to the capture antibodies.
3.Cells are lysed. Anti-IFNγ-FITC and anti-IL-5 biotin detection antibodies are added and bind to the captured cytokines.
4.Detection antibodies are in turn bound by anti-FITC-HRP for IFNγ and biotin-AP for IL-5.
5.Finally coloured spots are developed by separate incubations with first AEC and then BCIP/NBT substrate buffers. Cells producing IFNγ give red/brownish spots while those producing IL-5 give blue/purple spots.