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Peptide Inhibition ELISA Protocol

Author: Nanci Donacki
Source: Contributed by Nanci Donacki
Date Added: Tue May 14 2002
Date Modified: Wed Apr 28 2004

Reagents
  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 -20oC.  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 37oC.
  6. Transfer 50 ml to an antigen coated plates, which has been blocked with PBS:2% BSA for 1 hour at 37oC.
  7.  Incubate for 1 hour at 37oC.
  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 37oC.
  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
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