Cellular ELISA Protocol

1.Trypsinize confluent flasks
2. Pool and count cells
3.Centrifuge at 1500 rpm for 10 minutes
4.Resuspend to the appropriate concentration in complete medium
4 x 10⁵ cells/ml for epithelial cells
2 x 10⁵ cells/ml for fibroblast cells
5.Add 100 ml/cell to 96 well culture plates.
6. Incubate overnight at 37^oC.
7.Wash plates twice with PBS
8.Add 125 ml/well 10% Buffered Formalin
9.Fix for 15 minutes at room temperature
10. Wash three times with di-H₂O.
11. Blot dry.
12. Store at 2-8^oC.

B. Reagents
1.PBS:1% BSA
2.PBS:2% BSA
3.Carbonate Buffer
1.59 g Na₂CO₃
2.93 g NaHCO₃
Dissolve in 900 ml di-H₂O. Check pH and adjust to 9.6 necessary. Qs. to 1 liter.
4.10X 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.
5.0.3% H₂O₂
Dilute 30% stock Peroxide 1:100 in di-H₂O.
6.OPD Stock, 4.0%
4 g OPD in 100 ml di-H₂O. Aliquot and store at -20^oC. Protect from light.
7.4.5N H₂SO₄
12.0 ml Concentrated Sulfuric Acid
88.0 ml di-H₂O

B. Procedure
1.Wash ELISA plates once with di-H₂O.
2.Add 250 ml/well PBS:2% BSA.
3.Incubate 1 hour at 37^oC.
4.Wash 3 times with di-H₂O.
5.Add 50 ml/well supe, ascites, or controls diluted in PBS:1%BSA.
6.Incubate for 2 hr at 37^oC.
7.Wash 5 times with di-H₂O.
8.Add 50 ml/well anti-mouse IgG:HRP diluted in PBS:1% BSA.
9.Incubate for 1 hr at 37^oC.
10. Wash 5 times with di-H₂O. Wash once with carbonate buffer.
11. Add 50 ml/well working substrate solution
0.5 ml 4.0% OPD
5 ml 30% H₂O₂
1.0 ml 10X Substrate buffer
8.5 ml di-H₂O.
12. Incubate for 20 minutes at room temperature.
13. Add 25 ml/well 4.5N Sulfuric Acid
14. Read A₄₉₀

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