Elisa protocol

Elisa Testing Protocols

Materials Needed:

1 X PBS
200 mM Bicarbonate pH 8.3
Microtiter Plates
1:10 Blocking BSA
1:15 Dilution BSA
Goat Anti-Rabbit HRP
ABTS Substrate

Storage of Peptide:

For ELISA, peptide should be stored in a 1:1 ratio of peptide to 1 X PBS.

Preparation of the Plate:

1. Prepare a working solution/coating concentration 1:100 ratio of stored peptide to Bicarbonate 10µg/ml - Example: 30 µl to 3ml.
2. Pipette 50 µl of working solution/coating concentration into each well. Leave at room temperature for >2 hours or overnight at 4oC.
3. Empty plate. Add 200 µl of Blocking BSA into each well. Leave for 30 minutes to 1 hour at room temperature and empty plate.
4. Add 50 µl of Dilution BSA into each well.
5. Prepare working solution of antibody by adding 10 µl of serum to 1 ml of Dilution BSA.
6. Add 50 µl of working solution into the first row of wells only.
7. Perform serial dilutions by taking 50 µl from the first row and transferring it to the second, mix, then take 50 µl from the second row and transfer to the third row, mix, etc., taking 50µl out of the last wells.
8. Let this incubate for 1 hour at room temperature.
9. Wash 4 times with 1 X PBS.
10. Add second antibody: HRP conjugate/Goat anti-rabbit 1:1000 dilution with dilution BSA. Add 50µl per well.
11. Let this incubate for 1 hour at room temperature.
12. Wash 4 times with 1 X PBS.
13. Add 50 µl of ABTS substrate.
14. Let this sit for 2 minutes.
15. Add 50 µl of Stopping solution to all wells (1% SDS).
16. Run ELISA machine twice and read results.