Peptide Inhibition ELISA Protocol

Peptide Inhibition ELISA Protocol

2. PBS
4. PBS:0.1% BSA:0.05% Tween 20 (PBT)
6. PBS:2% BSA
8. 10X Substrate Buffer, pH 6.0
   36.6 g Citric Acid, monohydrate
   113.5 g Potassium dibasic phosphate
   Dissolve in 900 ml di-H₂ O. Check pH and adjust to 6.0 if necessary. Qs. to 1 liter.
10. 30% H₂ O₂
12. OPD Stock, 4.0%
    4 g OPD in 100 ml di-H₂ O. Aliquot and store at -20^o C. Protect from light.
14. 4.5N H₂ SO₄
    12.0 ml Concentrated Sulfuric Acid
    88.0 ml di-H₂ O

Procedure

2. Titer MAb supes at serial 5-fold dilutions to determine the concentration at which the titer begins to drop.
4. Dilute the MAb to one-half the above dilution in PBT
6. Dilute specific and non-specific peptides to 200 mg/ml in PBT.
8. 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.
10. Incubate 1 hour at 37^o C.
12. Transfer 50 ml to an antigen coated plates, which has been blocked with PBS:2% BSA for 1 hour at 37^o C.
14. Incubate for 1 hour at 37^o C.
16. Wash 3 times with di-H₂ O.
18. Add 50 ml/well anti-mouse IgG:HRP diluted in PBS:0.1% BSA:0.05% Tween 20.
20. Incubate for 1 hr at 37^o C.
22. Wash 5 times with di-H₂ O.
24. Add 50 ml/well working substrate solution
    0.5 ml 4.0% OPD
    50 ml 30% H₂ O₂
    1.0 ml 10X Substrate buffer
    8.5 ml di-H₂ O.
26. Incubate for 20 minutes at room temperature.
28. Add 25 ml/well 4.5N Sulfuric Acid
30. Read A₄₉₀