Reagents

1.  DMEM:10% FBS
2. DMEM:10% FBS:1% HEPES (DFH)
3. 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.
4. 30% H₂O₂
5. OPD Stock, 4.0%
   4 g OPD in 100 ml di-H₂O.  Aliquot and store at -20^oC.  Protect from light.
6. 4.5N H₂SO₄
   12.0 ml      Concentrated Sulfuric Acid
   88.0 ml      di-H₂O
7.  Hanks Balanced Salt Solution (HBSS)

Procedure

1. Wash ELISA plates once with 150 ml/well DFH by centrifugation.
2. Add 50 ml/well supe, ascites, or controls diluted in DFH.
3. Incubate for 2 hr at 37^oC.
4. Wash 4X with DFH
5.  Add 50 ml/well anti-mouse IgG:HRP diluted in DFH
6.  Incubate for 1 hr at 37^oC.
7.  Wash 3X with DFH and 2X with HBSS.
8. 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.
9.  Incubate for 20 minutes at room temperature.
10. Add 25 ml/well 4.5N Sulfuric Acid
11. Read A₄₉₀

Notes

1.  Test all supernatants at 1:5 dilution.
2. Test ascites at 1:100