Description:ProductName:TotalT4#ofSamples:1x96AssaysIntendedUse:CompetitiveimmunoenzymaticcolorimetricmethodforquantitativedeterminationoftotalT4(Thyroxine)inserumorplasma.Introduction:Thethyroidhormone,thyroxine(T4)isproducedbythethyroidgland.Animportantcomponentinthesynthesisisiodine.Themajorformofthyroidhormoneinthebloodisthyroxine(T4).ThyroxineisconvertedtotheactiveT3(threetofourtimesmorepotentthanT4)withincellsbydeiodinases(5"-iodinase).Thyroxine-bindingglobulin(TGB)isthemajorcarrierproteinforcirculatingthyroidhormone.Onlyaverysmallfractionofthecirculatinghormoneisfree(unbound)-T40.03%.Thethyroninesactonthebodytoincreasethebasalmetabolicrate,affectproteinsynthesisandincreasethebody"ssensitivitytocatecholamines(suchasadrenaline)bypermissiveness.Thethyroidhormonesareessentialtoproperdevelopmentanddifferentiationofallcellsofthehumanbody.Thesehormonesalsoregulateprotein,fat,andcarbohydratemetabolism,affectinghowhumancellsuseenergeticcompounds.Numerousphysiologicalandpathologicalstimuliinfluencethyroidhormonesynthesis.Thyrotoxicosisorhyperthyroidismistheclinicalsyndromecausedbyanexcessofcirculatingfreethyroxine,freetriiodothyronine,orboth.BothT3andT4areusedtotreatthyroidhormonedeficiency(hypothyroidism).Principlesoftheassay:Microtiterstripwellsareprecoatedwithanti-T4antibodies(solid-phase).T4inthesamplecompeteswithaddedhorserADIshperoxidaselabelledT4(enzyme-labelledantigen)forantibodybinding.Afterincubationabound/freeseparationisperformedbysolid-phasewashing.Theimmunecomplexformedbyenzyme-labelledantigenisvisualizedbyaddingTetramethylbenzidine(TMB)substratewhichgivesabluereactionproduct.TheintensityofthisproductisinverselyproportionaltotheamountofT4inthesample.Sulphuricacidisaddedtostopthereaction.Thisproducesayellowendpointcolour.Absorptionat450nmisreadusinganELISAmicrowellplatereader.StorageandStABIlity:Thereagentsarestableuptotheexpirydatestatedonthelabelwhenstoredat2...8°Cinthedark.Openedreagentsarestablefor60dayswhenstoredat2?8°C.LimitationsoftheTest:ItisimportantthatthetimeofreactionineachwellisheldconstantforreproducIBLeresults.Pipettingofsamplesshouldnotextendbeyondtenminutestoavoidassaydrift.Ifmorethanoneplateisused,itisrecommendedtorepeatthedoseresponsecurve.Additionofthesubstratesolutioninitiatesakineticreaction,whichisterminatedbytheadditionofthestopsolution.Therefore,theadditionofthesubstrateandthestoppingsolutionshouldbeaddedinthesamesequencetoeliminateanytimedeviationduringreaction.Platereadersmeasurevertically.Donottouchthebottomofthewells.Failuretoremoveadheringsolutionadequatelyintheaspirationordecantationwashstep(s)mayresultinpoorreplicationandspuriousresults.ReferencesBarker,S.B.;J.BIOLOGicalChemistry(1948)173,175Chopra,I.J.,Solomon,D.H.andHo,R.S.;J.ClinicalEndocrinol.(1971)33,865Young,D.S.,Pestaner,L.C.andGilberman,U.;ClinicalChemistry(1975)21,3660Sterling,L.;DiagnosisandTreatmentofThyroidDisease,ClevelandCRCPress(1975)p.19-51AdditionalInformation:NameTotalT4RelatedProductNamesTotalT4MolecularWeight0.5StorageThereagentsarestableuptotheexpirydatestatedonthelabelwhenstoredat2...8°Cinthedark.Openedreagentsarestablefor60dayswhenstoredat2?8°C.IntendedUseResearchUseOnly