Live Cell ELISA Protocol

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 -20^o 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 37^o C.

4. Wash 4X with DFH

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

6. Incubate for 1 hr at 37^o 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