Live Cell ELISA Protocol

Live Cell ELISA Protocol

Reagents

2. DMEM:10% FBS
4. DMEM:10% FBS:1% HEPES (DFH)
6. 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.
8. 30% H₂ O₂
10. OPD Stock, 4.0%
    4 g OPD in 100 ml di-H₂ O. Aliquot and store at -20^o C. Protect from light.
12. 4.5N H₂ SO₄
    12.0 ml Concentrated Sulfuric Acid
    88.0 ml di-H₂ O
14. Hanks Balanced Salt Solution (HBSS)

Procedure

2. Wash ELISA plates once with 150 ml/well DFH by centrifugation.
4. Add 50 ml/well supe, ascites, or controls diluted in DFH.
6. Incubate for 2 hr at 37^o C.
8. Wash 4X with DFH
10. Add 50 ml/well anti-mouse IgG:HRP diluted in DFH
12. Incubate for 1 hr at 37^o C.
14. Wash 3X with DFH and 2X with HBSS.
16. 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.
18. Incubate for 20 minutes at room temperature.
20. Add 25 ml/well 4.5N Sulfuric Acid
22. Read A₄₉₀

Notes

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