Components
1.Capture antibody for r IL-17A (0.50 mL). Supplied sterile.
2.Capture antibody for IL-4 (0.50 mL). Supplied sterile.
3.FITC conjugated detection antibody for r IL-17A (lyophilised, resuspend in 0.55mL).
4.Biotinylated detection antibody for IL-4 (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).
Material not included
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.
Reagent Preparation
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.Anti FITC-green fluorescence conjugate / Steptavidin-phycoerythrin:Dilute each reagent with the volume indicated on each vial in 10 ml of PBS 1% BSA. For 1 plate, prepare 10 ml of anti FITC-green fluorescence conjugate / Streptavidin-phycoerythrin solution.
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.
8.5% Fluorescence buffer (optional use):5% Fluorescence buffer (optional use).
Assay Procedure
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 IL-17A capture antibody and 100µl of IL-4 capture antibody in 10 mL of 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/well of RPMI 10% FCS 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/well 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 IL-17A detection antibody and 100µl of reconstituted IL-4 detection antibody into 10 mL of PBS containing 1% BSA. Dispense 100µl into wells, cover the plate and incubate 1 hour 30 min at room temperature.
13.Empty wells and wash three times with PBS-0.05% Tween 20.
14.Distribute 100 µl of anti FITC-green fluorescence conjugate / Streptavidin-phycoerythrin solution (see reagent preparation) in each well. Seal the plate and incubate for 1 hour at room temperature.
15.Empty wells and wash three times with PBS-0.05% Tween 20.
16.Peel off the plate bottom and wash three times both sides of the membrane under running distilled water. Remove all residual buffer by repeated tapping on absorbent paper.
17.Dry wells away from light.
18.Read spots on an Eli-spot reader under a UV light source.
19.Store the plate at +4°C away from light.
Storage Comment
If not used within a short period of time, reconstituted detection antibody should be aliquoted and stored at -20C°.
Expiry Date
at least one year at -20°C
Note
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.
Restrictions
For Research Use Only. Not for use in diagnostic procedures.