Live Cell ELISA Protocol

A. 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 -20oC.  Protect from light.

6. 4.5N H2SO4

12.0 ml Concentrated Sulfuric Acid

88.0 ml di-H2O

7.  Hanks Balanced Salt Solution (HBSS)

B. 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 37oC.

4. Wash 4X with DFH

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

6.  Incubate for 1 hr at 37oC.

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

C. Notes

1.  Test all supernatants at 1:5 dilution.

2. Test ascites at 1:100