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-H2 O. Check pH and adjust to 6.0 if necessary. Qs. to 1 liter.

4. 30% H2 O2

5. OPD Stock, 4.0% 4 g OPD in 100 ml di-H2 O. Aliquot and store at -20o C. Protect from light.

6. 4.5N H2 SO4 12.0 ml Concentrated Sulfuric Acid 88.0 ml di-H2 O

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 37o C.

4. Wash 4X with DFH

5. Add 50 ml/well anti-mouse IgG:HRP diluted in DFH

6. Incubate for 1 hr at 37o C.

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% H2 O2 1.0 ml 10X Substrate buffer 8.5 ml di-H2 O.

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