Peptide Inhibition ELISA Protocol

A. Reagents

PBS

 PBS:0.1% BSA:0.05% Tween 20 (PBT)

PBS:2% BSA

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.

30% H₂O₂

OPD Stock, 4.0%
4 g OPD in 100 ml di-H₂O. Aliquot and store at -20^oC. Protect from light.

4.5N H₂SO₄
12.0 ml      Concentrated Sulfuric Acid
88.0 ml      di-H₂O

B. Procedure

Titer MAb supes at serial 5-fold dilutions to determine the concentration at which the titer begins to drop.

 Dilute the MAb to one-half the above dilution in PBT

Dilute specific and non-specific peptides to 200 mg/ml in PBT.

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.

 Incubate 1 hour at 37^oC.

Transfer 50 ml to an antigen coated plates, which has been blocked with PBS:2% BSA for 1 hour at 37^oC.

 Incubate for 1 hour at 37^oC.

Wash 3 times with di-H₂O.

Add 50 ml/well anti-mouse IgG:HRP diluted in PBS:0.1% BSA:0.05% Tween 20.

Incubate for 1 hr at 37^oC.

Wash 5 times with di-H₂O.

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.

Incubate for 20 minutes at room temperature.

Add 25 ml/well 4.5N Sulfuric Acid

Read A₄₉₀