Cellular ELISA Protocol
A. Formalin Fixed CellPlates 1. Trypsinizeconfluent flasks
2. Pool andcount cells
3. Centrifugeat 1500 rpm for 10 minutes
4. Resuspendto the appropriate concentration in complete medium
4 x 105 cells/ml for epithelial cells
2 x 105 cells/ml for fibroblast cells
5. Add 100ml/cell to 96 well cultureplates.
6. Incubateovernight at 37oC.
7. Wash platestwice with PBS
8. Add 125ml/well 10% BufferedFormalin
9. Fix for 15minutes 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. CarbonateBuffer
1.59 g Na2CO3
2.93 g NaHCO3
Dissolve in 900 ml di-H2O. Check pH and adjust to 9.6necessary. Qs. to 1liter.
4. 10XSubstrate Buffer, pH 6.0
36.6 g CitricAcid, monohydrate
113.5 g Potassiumdibasic phosphate
Dissolve in 900 ml di-H2O. Check pH and adjust to 6.0 ifnecessary. Qs. to 1liter.
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-20oC. Protect from light.
7. 4.5NH2SO4
12.0 ml Concentrated Sulfuric Acid
88.0 ml di-H2OC.Procedure
1. Wash ELISAplates once with di-H2O.
2. Add 250ml/well PBS:2% BSA.
3. Incubate 1hour at 37oC.
4. Wash 3times with di-H2O.
5. Add 50ml/well supe, ascites, orcontrols diluted in PBS:1%BSA.
6. Incubatefor 2 hr at 37oC.
7. Wash 5times with di-H2O.
8. Add 50ml/well anti-mouse IgG:HRPdiluted in PBS:1% BSA.
9. Incubatefor 1 hr at 37oC.
10. Wash 5 times with di-H2O. Wash once with carbonate buffer.
11. Add 50 ml/well workingsubstrate solution
0.5 ml 4.0%OPD
5 ml 30%H2O2
1.0 ml 10XSubstrate buffer
8.5 ml di-H2O.
12. Incubate for 20 minutes at room temperature.
13. Add 25 ml/well 4.5NSulfuric Acid
14. Read A490D.Notes
1. Test allsupernatants at 1:5 dilution.
2. Testascites at 1:100