Cellular ELISA Protocol

A. Formalin Fixed Cell Plates

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

C. 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

D. Notes

1.     Test all supernatants at 1:5 dilution.

2.     Test ascites at 1:100