Description:ProductName:UrinaryCortisol#ofSamples:1x96AssaysIntendedUse:CompetitiveimmunoenzymaticcolorimetricmethodforquantitativedeterminationofCortisolinurine.Introduction:CortisolisasteroidhormonereleasedfromtheadrenalcortexinresponsetoanhormonecalledACTH(producedbythepituitarygland),itisinvolvedintheresponsetostress;itincreasesbloodpressure,bloodsugarlevels,maycauseinfertilityinwomen,andsuppressestheimmunesystem.Cortisolactsthroughspecificintracellularreceptorsandhaseffectsinnumerousphysiologicsystems,includingimmunefunction,glucose-counterregulation,vasculartone,substrateutilizationandbonemetabolism.Cortisolisexcretedprimarilyinurineinanunbound(free)form.Cortisolisbound,inplasma,fromcorticosteroid-bindingglobulin(CBG,transcotin),withhighaffinity,andfromalbumin.Onlyfreecortisolisavailabletomostreceptors.Thesenormalendogenousfunctionsarethebasisforthephysiologicalconsequencesofchronicstress-prolongedcortisolsecretioncausesmusclewastage,hyperglycaemia,andsuppressesimmune/inflammatoryresponses.Thesameconsequencesarisefromlong-termuseofglucocorticoiddrugs.Thefreecortisolfractionrepresentsthemetabolicallyactivecortisol.Innormalconditions,lessthen1%itcomesexcreteinurines.Inpathologicalconditions(syndromeofCushing)thelevelsoffreeurinarycortisoloareelevate,becausetheCBGdon’tboundtheplasmaticcortisolinexcessanditwasremovewithurines.Duringpregnancyorestro-progestogentreatmentanincreaseofplasmaticcortisolcausedbyanincrementoftheproductionofthetransportprotein,butthelevelsoffreeurinarycortisolresultsnormaltoindicatecorrectsuprarenalfunctionality.Thistestisveryusefultoestimatetherealsuprarenalfunction,becauseisdosethefreecortisol,itisthemetabolicallyactiveform.MoreoverthemeasurementoffreeurinarycortisoloisthebetterparameterforthediagnosisoftheCushing’ssyndrome.Principlesoftheassay:Microtiterstripwellsareprecoatedwithanti-Cortisolantibodies(solid-phase).CortisolinthesamplecompeteswithaddedhorserADIshperoxidaselabelledCortisol(enzyme-labelledantigen)forantibodybinding.Afterincubationabound/freeseparationisperformedbysolid-phasewashing.Theimmunecomplexformedbyenzyme-labelledantigenisvisualizedbyaddingTetramethylbenzidine(TMB)substratewhichgivesabluereactionproduct.TheintensityofthisproductisinverselyproportionaltotheamountofCortisolinthesample.Sulphuricacidisaddedtostopthereaction.Thisproducesayellowendpointcolour.Absorptionat450nmisreadusinganELISAmicrowellplatereader.StorageandStABIlity:Thereagentsarestableuptotheexpirydatestatedonthelabelwhenstoredat+2...+8°Cinthedark.LimitationsoftheTest:Sample(s),whicharecontaminatedmicroBIOLOGically,shouldnotbeusedintheassay.Highlylipemeicorhaemolysedspecimen(s)shouldsimilarlynotbeused.ItisimportantthatthetimeofreactionineachwellisheldconstantforreproducIBLeresults.Pipettingofsamplesshouldnotextendbeyondtenminutestoavoidassaydrift.Ifmorethanoneplateisused,itisrecommendedtorepeatthedoseresponsecurve.Additionofthesubstratesolutioninitiatesakineticreaction,whichisterminatedbytheadditionofthestopsolution.Therefore,theadditionofthesubstrateandthestoppingsolutionshouldbeaddedinthesamesequencetoeliminateanytimedeviationduringreaction.Platereadersmeasurevertically.Donottouchthebottomofthewells.Failuretoremoveadheringsolutionadequatelyintheaspirationordecantationwashstep(s)mayresultinpoorreplicationandspuriousresults.Ifcomputercontrolleddatareductionisusedtocalculatetheresultsofthetest,itisimperativethatthepredictedvaluesforthecalibratorsfallwithin10%oftheassignedconcentrations.ReferencesFoster,L.B.andDunn,R.T.(1974)Clin.Chem20(3),365.DeLacerda,L.,Kowarski,A.,andMigeon,C.J.(1973)J.Clin.Endocr.andMetab36,227.Rolleri,E.,Zannino,M.,Orlandini,S.andMalvano,R.(1976)ClinchimActa66,319.Kobayashi,Y.etal.(1978)Steroids32(1),137–44.Arakawa,H.,Maeda,M.,Tsuji,A.(1979)Anal.Biochem.97,248.AdditionalInformation:NameUrinaryCortisolRelatedProductNamesUrinaryCortisolStorageThereagentsarestableuptotheexpirydatestatedonthelabelwhenstoredat+2...+8°Cinthedark.IntendedUseResearchUseOnly