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-20oC.Protectfromlight. 6.4.5NH2SO412.0mlConcentratedSulfuricAcid88.0mldi-H2O 7.HanksBalancedSaltSolution(HBSS) Procedure 1.WashELISAplatesoncewith150ml/wellDFHbycentrifugation. 2.Add50ml/wellsupe,ascites,orcontrolsdilutedinDFH. 3.Incubatefor2hrat37oC. 4.Wash4XwithDFH 5.Add50ml/wellanti-mouseIgG:HRPdilutedinDFH 6.Incubatefor1hrat37oC. 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
