Components
1.Capture antibody (0.50 mL). Supplied sterile.
2.Biotinylated detection antibody (lyophilised, resuspend in 0.55mL).
3.Streptavidin - Alkaline Phosphatase conjugated (50 µL).
4.Bovine Serum albumin.
5.Ready-to-use substrate buffer (50mL).
6.96 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.
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.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.
4.1% BSA in PBS:For one plate dissolve 0.2 g of BSA in 20 mL of 1X diluted PBS.
5.0.05% Tween in PBS:For one plate dissolve 50µl of Tween 20 in 100 ml of 1X diluted PBS.
6.35% ethanol in water:For one plate mix 3.5 ml of ethanol with 6.5 ml of distilled water.
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 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 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 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 (10-15 hours).
9.Empty wells by flicking the plate over a sink and gently tapping it on absorbent paper.
10.Distribute 100µl of PBS-0.0.5% tween 20 in wells and let sit 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 detection antibody into 10 mL of PBS containing 1% BSA. Distribute 100µl in 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.For 1 plate dilute 10µl of streptavidin-Alkaline phosphatase conjugate into 10 mL of PBS-1% BSA. Distribute 100µl of the dilution in wells. 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.Distribute 100µl of ready-to-use BCIP/NBT buffer in wells.
18.Let the reaction go for about 5-20 min at room temperature. Monitor spot formation visually.
19.Rinse three times both side of the membrane under running distilled water.
20.Dry wells. Read spots. Note that spots may become sharper after one night at +4°C.
Accession Number
NP_004122.2
Storage Comment
If not used within a short period of time, reconstituted detection antibody should be aliquoted and stored at -20°C.
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.
Datasheet
