Overview | TMB,(3,3",5,5"-tetramethybenzidine)hasbeenshowntobeasafe-sensitivesubstratefortheassayofHRP.Initially,inthepresenceofHRPandhydrogenperoxide,aone-electronoxidationproductisformed.Thiscompound,acationfreeradical,isblueincolorwithanadsorptionmaximumof653nm.FurtherreactionwithHRP/H2O2oracidificationoftheradicalwithacidyieldsthediimineterminaloxidationproductadsorbinglightat450nm.Theextinctioncoefficientoftheradical(E653nm=3.9x104mol-1cm-1)anddiimine(E450nm=5.9x104mol-1cm-1)providearemarkablysensitivesystemfortheassayofHRPandHRPlabeledprobes.ES022isasinglecomponentreagentstableatroomtemperatureandnotsensitivetonormallaboratorylight.ItisoptimizedwithrespecttoTMBandhydrogenperoxideconcentrationsandyieldsalinearresponsewiththeconcentrationsofHRPusuallyemployedinimmunologicalassays. |