Reagents

1. DMEM:10%FBS
2. DMEM:10%FBS:1%HEPES(DFH)
3. 10XSubstrateBuffer,pH6.036.6gCitricAcid,monohydrate113.5gPotassiumdibasicphosphateDissolvein900mldi-H₂O.CheckpHandadjustto6.0ifnecessary.Qs.to1liter.
4. 30%H₂O₂
5. OPDStock,4.0%4gOPDin100mldi-H₂O.Aliquotandstoreat-20^oC.Protectfromlight.
6. 4.5NH₂SO₄12.0mlConcentratedSulfuricAcid88.0mldi-H₂O
7. HanksBalancedSaltSolution(HBSS)

Procedure

1. WashELISAplatesoncewith150ml/wellDFHbycentrifugation.
2. Add50ml/wellsupe,ascites,orcontrolsdilutedinDFH.
3. Incubatefor2hrat37^oC.
4. Wash4XwithDFH
5. Add50ml/wellanti-mouseIgG:HRPdilutedinDFH
6. Incubatefor1hrat37^oC.
7. Wash3XwithDFHand2XwithHBSS.
8. Add50ml/wellworkingsubstratesolution0.5ml4.0%OPD50ml30%H₂O₂1.0ml10XSubstratebuffer8.5mldi-H₂O.
9. Incubatefor20minutesatroomtemperature.
10. Add25ml/well4.5NSulfuricAcid
11. ReadA₄₉₀

Notes

1. Testallsupernatantsat1:5dilution.
2. Testascitesat1:100