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 medium4 x 105 cells/ml for epithelial cells2 x 105 cells/ml for fibroblast cells

5.     Add 100 ml/cell to 96 well culture plates.

6.     Incubate overnight at 37oC.

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 2-8oC.

B.  Reagents

1.     PBS:1% BSA

2.     PBS:2% BSA

3.     Carbonate Buffer1.59 g      Na2CO32.93 g      NaHCO3Dissolve in 900 ml di-H2O.  Check pH and adjust to 9.6 necessary.  Qs. to 1 liter.

4.     10X Substrate Buffer, pH 6.036.6 g      Citric Acid, monohydrate113.5 g      Potassium dibasic phosphateDissolve in 900 ml di-H2O.  Check pH and adjust to 6.0 if necessary.  Qs. to 1 liter.

5.     0.3% H2O2Dilute 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 -20oC.  Protect from light.

7.     4.5N H2SO412.0 ml      Concentrated Sulfuric Acid88.0 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 37oC.

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 37oC.

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 37oC.

10. Wash 5 times with di-H2O.  Wash once with carbonate buffer.

11. Add 50 ml/well working substrate solution0.5 ml      4.0% OPD5 ml      30% H2O21.0 ml      10X Substrate buffer8.5 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