Peptide Inhibition ELISA Protocol

1. PBS
2.  PBS:0.1% BSA:0.05% Tween 20 (PBT)
3. PBS:2% BSA
4. 10X Substrate Buffer, pH 6.0
   36.6 g      Citric Acid, monohydrate
   113.5 g      Potassium dibasic phosphate
   Dissolve in 900 ml di-H₂O.  Check pH and adjust to 6.0 if necessary.  Qs. to 1 liter.
5. 30% H₂O₂
6. OPD Stock, 4.0%
   4 g OPD in 100 ml di-H₂O.  Aliquot and store at -20^oC.  Protect from light.
7. 4.5N H₂SO₄
   12.0 ml      Concentrated Sulfuric Acid
   88.0 ml      di-H₂O

 Procedure

1. Titer MAb supes at serial 5-fold dilutions to determine the concentration at which the titer begins to drop.
2.  Dilute the MAb to one-half the above dilution in PBT
3. Dilute specific and non-specific peptides to 200 mg/ml in PBT.
4. In a microtiter plate, mix equal volumes of the peptide dilutions and the MAb supe.  Add an equal volume of PBT and MAb as the control.
5.  Incubate 1 hour at 37^oC.
6. Transfer 50 ml to an antigen coated plates, which has been blocked with PBS:2% BSA for 1 hour at 37^oC.
7.  Incubate for 1 hour at 37^oC.
8. Wash 3 times with di-H₂O.
9. Add 50 ml/well anti-mouse IgG:HRP diluted in PBS:0.1% BSA:0.05% Tween 20.
10. Incubate for 1 hr at 37^oC.
11. Wash 5 times with di-H₂O.
12. Add 50 ml/well working substrate solution
    0.5 ml      4.0% OPD
    50 ml      30% H₂O₂
    1.0 ml      10X Substrate buffer
    8.5 ml      di-H₂O.
13. Incubate for 20 minutes at room temperature.
14. Add 25 ml/well 4.5N Sulfuric Acid
15. Read A₄₉₀