Sean Lauber:Isotype ELISA against Adenovirus

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Want to determine what antibody isotypes are directed against a specific antigen (in my case it was adenovirus).

 Wash buffer (10X):
 800 ml dH2O
 24.23 g Tris
 87.66 g NaCl
 3.73 g KCl
 5.0 ml Tween 20
 Adjust to pH 7.4 with concentrated HCl. Store at 4°C
 Reagent diluent (1% BSA):
 100 ml Wash buffer
 1 g BSA
 Make fresh and store at 4°C

1. Coat ELISA plate with 50 μg/mL Ad lysate (50 μL, diluted with PBS) overnight at 4°C

2. Wash 3x with wash buffer. Blot against paper towels.

3. Block with 200 μL reagent diluent for 1 h at RT.

4. Wash 3x and blot.

5. Load samples (100 μL, diluted with reagent diluent) for 2 h at RT.

6. Wash 3x and blot.

7. Apply Ab-biotin (100 μL, diluted with reagent diluent) for 2 h at RT.

8. Wash 3x and blot.

9. Add working concentration of strep-HRP (cat# DY998, 100 μL, diluted with reagent diluent) for 20 min in the dark at RT.

10. Wash 3x and blot.

11. Add working substrate solution (cat# DY999, 100 μL final volume; or BD Bioscience 555214) in the dark at RT.

12. Stop the reaction by adding 50 μL 2M H2SO4. Stop it when color development is sufficient, usually after 20 min of adding substrate solution.

13. Read at 450 nm.