Description:

ProductName:FreeT4

#ofSamples:1x96Assays

IntendedUse:CompetitiveimmunoenzymaticcolorimetricmethodforquantitativedeterminationoffreeT4(Thyroxine)inserumorplasma.

Introduction:Thethyroidhormone,thyroxine(T4)isproducedbythethyroidgland.Animportantcomponentinthesynthesisisiodine.Themajorformofthyroidhormoneinthebloodisthyroxine(T4).ThyroxineisconvertedtotheactiveT3(threetofourtimesmorepotentthanT4)withincellsbydeiodinases(5"-iodinase).
Thyroxine-bindingglobulin(TGB)isthemajorcarrierproteinforcirculatingthyroidhormone.Onlyaverysmallfractionofthecirculatinghormoneisfree(unbound)-T40.03%.Whenthyroidhormoneisbound,itisnotactive,sotheamountoffreeT3/T4iswhatisimportant.Forthisreason,measuringtotalthyroxineinthebloodcanbemisleADIng.
TheconcentrationoffreethyroidhormonesinthebloodisregulatedbyanegativefeedbackmechanisminvolvingTSH.ThebindingofT4byTBGplaysakeyroleinthisfeedbackmechanismandthemostsignificantchangesthatoccurinT4bindingcapacityaretheresultofalterationsinTBG.ChangesinthecirculatinglevelsofTBGwillresultinaproportionalincreaseordecreaseintheconcentrationoftotalT4.However,measurementofserumfreeT4isunaffectedbychangesinT4proteinbindinglevelsandthereforecorrelateswellwiththefunctionalthyroidstateinmostindividuals.FactorsresponsIBLefordiscrepanciesbetweenserumtotalT4levelsandtruethyroidstatesincludeTBGconcentration,estrogenichormones(pregnancy,oralcontraceptivesandestrogen)anddrugsthatbindtoTBGpreventingitsbindingtofreeT4.
Thethyroninesactonthebodytoincreasethebasalmetabolicrate,affectproteinsynthesisandincreasethebody"ssensitivitytocatecholamines(suchasadrenaline)bypermissiveness.Thethyroidhormonesareessentialtoproperdevelopmentanddifferentiationofallcellsofthehumanbody.Thesehormonesalsoregulateprotein,fat,andcarbohydratemetabolism,affectinghowhumancellsuseenergeticcompounds.Numerousphysiologicalandpathologicalstimuliinfluencethyroidhormonesynthesis.Thyrotoxicosisorhyperthyroidismistheclinicalsyndromecausedbyanexcessofcirculatingfreethyroxine,freetriiodothyronine,orboth.BothT3andT4areusedtotreatthyroidhormonedeficiency(hypothyroidism).

Principlesoftheassay:Microtiterstripwellsareprecoatedwithanti-T4antibodies(solid-phase).FreeT4inthesamplecompeteswithaddedhorseradishperoxidaselabelledT4(enzyme-labelledantigen)forantibodybinding.Afterincubationabound/freeseparationisperformedbysolid-phasewashing.Theimmunecomplexformedbyenzyme-labelledantigenisvisualizedbyaddingTetramethylbenzidine(TMB)substratewhichgivesabluereactionproduct.TheintensityofthisproductisinverselyproportionaltotheamountoffreeT4inthesample.Sulphuricacidisaddedtostopthereaction.Thisproducesayellowendpointcolour.Absorptionat450nmisreadusinganELISAmicrowellplatereader.

StorageandStABIlity:Thereagentsarestableuptotheexpirydatestatedonthelabelwhenstoredat2...8°Cinthedark.Openedreagentsarestableforanothertwomonthsifstoredat2…8°C.

LimitationsoftheTest:Bacterialcontaminationorrepeatedfreeze-thawcyclesofthespecimenmayaffecttheabsorbancevalues.
Totalserumthyroxineconcentrationisdependentuponamultiplicityoffactors:thyroidglandfunctionanditsregulation,thyroxine¬bindingglobulin(TBG)concentration,andthebindingofthyroxinetoTBG.Thus,totalthyroxineconcentrationaloneisnotsufficienttoassessclinicalstatus.TotalserumthyroxinevaluesmaybeelevatedunderconditionssuchaspregnancyoradmiNISTrationoforalcontraceptives.AT3uptaketestmaybeperformedtoestimatetherelativeTBGconcentrationinordertodetermineiftheelevatedT4iscausedbyTBGvariation.
Adecreaseintotalthyroxinevaluesisfoundwithproteinwastingdiseases,certainliverdiseasesandadministrationoftestosterone,diphenylhydantoinorsalicylates.Atableofinterferingdrugsandconditions,whichaffecttotalthyroxinevalues,hasbeencompiledbytheJournaloftheAmericanAssociationofClinicalChemists.
Notintendedfornewbornscreening.

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.19-51

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Name	FreeT4
RelatedProductNames	FreeT4
MolecularWeight	0.5
Storage	Thereagentsarestableuptotheexpirydatestatedonthelabelwhenstoredat2...8°Cinthedark.Openedreagentsarestableforanothertwomonthsifstoredat2 8°C.
IntendedUse	ResearchUseOnly