DUAL ELISPOT PROTOCOL

staining provides the opportunity to detect two target proteins at the same time in the ELISPOT cell assay. An advantage of this is that it enables the researcher to detect a marker protein to determine which cell type is secreting the protein, as well as the secreted protein of interest.
The following dual ELISPOT procedure describes a protocol using an FITC-conjugated primary antibody and a biotinylated primary antibody. Those are in turn recognized 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. The colored spots will either be red/brownish blue/purple so the two target proteins can be distinguished.

Materials and Reagents:
1.Detection antibody
2.Streptavidin alkaline phosphatase conjugate
3. Phosphate buffered saline (10 x Concentrate solution).
For 1 liter weight : 80g NaCl ; 2g KH2PO4; 14.4g Na2HPO42H2O. Add distilled water to 1 liter. Check that pH is 7.4+/- 0.1. Dilute the solution to 1X before use.
4. 2% dry skimmed milk in PBS
For one plate dissolve 0.2 g of powder in 10 mL of 1X diluted PBS.
5. 1% BSA in PBS
For one plate dissolve 0.2 g of BSA in 20 mL of 1X diluted PBS.
6. 0.1% Tween in PBS
For one plate dissolve 100􀀁l of Tween 20 in 100 ml of 1X diluted PBS.
7. 70% ethanol in water
For one plate mix 7 ml of ethanol with 3 ml of distilled water.
8. AEC Buffer
For one plate mix 1 ml of ACE Buffer A with 9ml of distilled water. Then add 200􀀁l of ACE Buffer B.
4. ELISPOT 96 well plates
(provided with Abcam ELISPOT kits)