Humantransthyretin,TTRELISAKit货号为：HRR-H0001FORRESEARCHUSEONLY;NOTFORTHERAPEUTICORDIAGNOSTICAPPLICATI***!PLEASEREADTHROUGHENTIREPROCEDUREBEFOREBEGINNING!IntendeduseThisimmunoassaykitallowsfortheinvitroquantitativedeterminationofhumantransthyretin,TTRconcentrati***incellculturesupernates,serum,pla***aandotherbiologicalfluids.IntroductionTransthyretin(TTR)isaserumandcerebrospinalfluidcarrierofthethyroidhormonethyroxine(T4).TTRwasoriginallycalledprealbuminbecauseitranfasterthanalbuminsonelectrophoresisgels.Itfuncti***inconcertwithtwootherthyroidhormonebindingproteins:thyroxine-bindingglobulin(TBG)andalbumins.TTRalsoactsasacarrierofretinol(vitaminA)throughanassociationwithretinolbindingprotein(RBP).Numerousother***allmoleculesareknowntobindinthethyroxinebindingsites,inTTRdingmanynaturalproducts(suchasresveratrol),drugs(diflunisal,flufenamicacid),andtoxinsPCB.SinceTTRbindspromiscuouslytomanyaromaticcompounds,andgenerallydoesnotbindT4inserum,thereisspeculationthatTTR's"truefunction"istogenerallysweepuptoxicandforeigncompoundsinthebloodstream.TTRisa55kDahomotetramerwithadimerofdimersconfigurationthatissynthesizedintheliver,choroidplexusandretinalpigmentepithelium.Eachmonomerisa127residuepolypeptiderichinbetasheetstructure.Associationoftwomonomersformsanextendedbetasandwich.Furtherassociationofanotheridenticalsetofmonomersproducesthehomotetramericstructure.Thetwothyroxinebindingsitespertetramersitattheinterfacebetweenthelattersetofdimers.TestprincipleThemicrotiterplateprovidedinthiskithasbeenpre-coatedwithanantibodyspecifictoTTR.Standa***orsamplesarethenaddedtotheappropriatemicrotiterplatewellswithabiotin-conjugatedpolyclonalantibodypreparationspecificforTTRand***idinconjugatedtoHorseradishPeroxidase(HRP)isaddedtoeachmicroplatewellandincubated.ThenaTMBsubstratesolutionisaddedtoeachwell.OnlythosewellsthatcontainTTR,biotin-conjugatedantibodyandenzyme-conjugated***idinwillexhibitachangeincolor.Theenzyme-substratereactionisterminatedbytheadditionofasulphuricacidsolutionandthecolorchangeismeasuredspectrophotometricallyataw***elengthof450nm&plu***n;2nm.TheconcentrationofTTRinthesamplesisthendeterminedbycomparingtheO.D.ofthesamplestothestandardcurve.MaterialsandcomponentsReagentQuantityAssayplate1×20mlStandard2SampleDiluent1×20mlAssayDiluentA1×10mlAssayDiluentB1×10mlDetectionReagentA1×120μlDetectionReagentB1×120μlWashBuffer(25xconcentrate)1×30mlSubstrate1×10mlStopSolution1×10mlPlatesealerfor96wells5Instruction1OthersuppliesrequiredLuminometer.Pipettesandpipettetips.EPtubeDeionizedordistilledwater.SamplecollectionandstorageSerum-Useaserumseparatortube(SST)andallowsamplestoclotfor30minutesbeforecentrifugationfor15minutesatapproximately1000×g.Removeserumandassayimmediatelyoraliquotandstoresamplesat-20℃or-80℃.Pla***a-Collectpla***ausingEDTAorheparinasananticoagulant.Centrifugesamplesfor15minutesat1000×gat2-8℃within30minutesofcollection.Storesamplesat-20℃or-80℃.***oidrepeatedfreeze-thawcycles.Cellculturesupernatesandotherbiologicalfluids-Removeparticulatesbycentrifugationandassayimmediatelyoraliquotandstoresamplesat-20℃or-80℃.***oidrepeatedfreeze-thawcycles.Note:Serum,pla***a,andcellculturesupernatantsamplestobeusedwithin7daysmaybestoredat2-8℃,otherwisesamplesmuststoredat-20℃(≤1months)or-80℃(≤2months)to***oidlossofbioactivityandcontamination.***oidfreeze-thawcycles.Whenperformingtheassayslowlybringsamplestoroomtemperature.DONOTUSEHEAT-TREATEDSPECIMENS.SamplepreparationSerum/pla***asamplesrequirea20folddilution.Asuggested20-folddilutionis50uLsample+950uLSampleDiluent.Limitati***oftheprocedure1.Thekitshouldnotbeusedbeyondtheexpirationdateonthekitlabel.2.Donotmixorsubstitutereagentswiththosefromotherlotsorsources.3.Ifsamplesgeneratevalueshigherthanthehigheststandard,furtherdilutethesampleswiththeAssayDiluentandrepeattheassay.Anyvariationinstandarddiluent,operator,pipettingtechnique,washingtechnique,incubationtimeortemperature,andkitagecancausevariationinbinding.4.Thisassayisdesignedtoeliminateinterferencebysolublereceptors,ligands,bindingproteins,andotherfactorspresentinbiologicalsamples.Untilallfactorsh***ebeentestedintheQuantikineImmunoassay,thepossibilityofinterferencecannotbeexTTRded.ReagentpreparationBringallreagentstoroomtemperaturebeforeuse.WashBuffer-Ifcrystalsh***eformedintheconcentrate,warmtoroomtemperatureandmixgentlyuntilthecrystalsh***ecompletelydissolved.Dilute30mLofWashBufferConcentrateintodeionizedordistilledwatertoprepare750mLofWashBuffer.Standard-Rec***titutetheStandardwith1.0mLofSampleDiluent.Thisrec***titutionproducesastocksolutionof30ng/ml.Allowthestandardtositforaminimumof15minuteswithgentleagitationpriortomakingserialdiluti***(Makingserialdilutioninthewellsdirectlyisnotpermitted).Theundilutedstandardservesasthehighstandard(30ng/ml).TheSampleDiluentservesasthezerostandard(0ng/ml).ng/mL30157.53.751.880.940.470DetectionReagentAandB-DilutetotheworkingconcentrationusingAssayDiluentAandB(1:100),respectively.AssayprocedureAllowallreagentstoreachroomtemperature(Pleasedonotdissolvethereagentsat37℃directly.).Allthereagentsshouldbemixedthoroughlybygentlyswirlingbeforepipetting.***oidfoaming.Keepappropriatenumbersofstripsfor1experimentandremoveextrastripsfrommicrotiterplate.Removedstripsshouldberesealedandstoredat4℃untilthekitsexpirydate.Prepareallreagents,workingstanda***andsamplesasdirectedintheprevioussecti***.Pleasepredicttheconcentrationbeforeassaying.Ifvaluesforthesearenotwithintherangeofthestandardcurve,usersmustdeterminetheoptimalsamplediluti***fortheirparticularexperiments.1.Add100μlofStandard,Blank,orSampleperwell.CoverwiththePlatesealer.Incubatefor2hoursat37℃.2.Removetheliquidofeachwell,don’twash.3.Add100μlofDetectionReagentAworkingsolutiontoeachwell.CoverwiththePlatesealer.Incubatefor1hourat37℃.DetectionReagentAworkingsolutionmayappearcloudy.Warmtoroomtemperatureandmixgentlyuntilsolutionappearsuniform.4.Aspirateeachwellandwash,repeatingtheprocessthreetimesforatotalofthreewashes.WashbyfillingeachwellwithWashBuffer(approximately400μl)usingasquirtbottle,multi-channelpipette,manifolddispenserorautowasher.Completeremovalofliquidateachstepisessentialtogoodperformance.Afterthelastwash,removeanyremainingWashBufferbyaspiratingordecanting.Inverttheplateandblotitagainstcleanpapertowels.5.Add100μlofDetectionReagentBworkingsolutiontoeachwell.CoverwithanewPlatesealer.Incubatefor1hoursat37℃.6.Repeattheaspiration/washasinstep4.7.Add90μlofSubstrateSolutiontoeachwell.CoverwithanewPlatesealer.Incubatewithin30minutesat37℃.Protectfromlight.8.Add50μlofStopSolutiontoeachwell.Ifcolorchangedoesnotappearuniform,gentlytaptheplatetoensurethoroughmixing.9.Determinetheopticaldensityofeachwellatonce,usingamicroplatereadersetto450nm.ImportantNote:1.Absorbanceisafunctionoftheincubationtime.Therefore,priortostartingtheassayitisrecommendedthatallreagentsshouldbefreshlypreparedpriortouseandallrequiredstrip-wellsaresecuredinthemicrotiterframe.Thiswillensureequalelapsedtimeforeachpipettingstep,withoutinterruption.2.Pleasecarefullyrec***tituteStanda***orworkingDetectionReagentAandBaccordingtotheinstruction,and***oidfoamingandmixgentlyuntilthecrystalsh***ecompletelydissolved.Therec***titutedStanda***canbeusedonlyonce.Thisassayrequirespipettingof***allvolumes.Tominimizeimprecisioncausedbypipetting,ensurethatpipettorsarecalibrated.Itisrecommendedtosuckmorethan10μlforoncepipetting.3.Toensureaccurateresults,properadhesionofplatesealersduringincubationstepsisnecessary.Donotallowwellstosituncoveredforextendedperiodsbetweenincubationsteps.Oncereagentsh***ebeenaddedtothewellstrips,DONOTletthestripsDRYatanytimeduringtheassay.4.Foreachstepintheprocedure,totaldispensingtimeforadditionofreagentstotheassayplateshouldnotexceed10minutes.5.To***oidcross-contamination,changepipettetipsbetweenadditi***ofeachstandardlevel,betweensampleadditi***,andbetweenreagentadditi***.Also,useseparatereservoirsforeachreagent.6.Thewashprocedureiscritical.Insufficientwashingwillresultinpoorprecisionandfalselyelevatedabsorbancereadings.7.Duplicationofallstanda***andspecimens,althoughnotrequired,isrecommended.8.SubstrateSolutioniseasilycontaminated.Pleaseprotectitfromlight.SpecificityThisassayrecognizesrecombinantandnaturalhumanTTR.Nosignificantcross-reactivityorinterferencewasobserved.SensitivityTheminimumdetectabledoseofhumanTTRistypicallylessthan0.24ng/mL.Thesensitivityofthisassay,orLowerLimitofDetection(LLD)wasdefinedasthelowestproteinconcentrationthatcouldbedifferentiatedfromzero.DetectionRange0.47-30ng/mL.Thestandardcurveconcentrati***usedfortheELISA’swere30ng/mL,15ng/mL,7.5ng/mL,3.75ng/mL,1.88ng/mL,0.94ng/mL,0.47ng/mL.Calculationofresults***eragetheduplicatereadingsforeachstandard,control,andsampleandsubtractthe***eragezerostandardopticaldensity.Createastandardcurvebyreducingthedatausingcomputersoftwarecapableofgeneratingafourparameterlogistic(4-PL)curve-fit.Asanalternative,c***tructastandardcurvebyplottingthemeanabsorbanceforeachstandardonthex-axisagainsttheconcentrationonthey-axisanddrawabestfitcurvethroughthepointsonthegraph.ThedatamaybelinearizedbyplottingthelogoftheTTRconcentrati***versusthelogoftheO.D.andthebestfitlinecanbedeterminedbyregressionanalysis.Itisrecommendedtousesomerelatedsoftwaretodothiscalculation,suchascurveexpert13.0.Thisprocedurewillproduceanadequatebutlessprecisefitofthedata.Ifsamplesh***ebeendiluted,theconcentrationreadfromthestandardcurvemustbemultipliedbythedilutionfactor.Storageoftestkitsandinstrumentation1.Unopenedtestkitsshouldbestoredreferringtothepackagelabelforfrequentuse,andstoredat-20℃forlongtimestorage.Theunusedstripsshouldbekeptinasealedbagandstoredat2-8℃intheirpouchwiththedesiccantprovidedtominimizeexposuretodampair.Thetestkitmaybeusedthroughouttheexpirationdateofthekit(sixmonthsfromthedateofmanufacture).Openedtestkitswillremainstableuntiltheexpiringdateshown,provideditisstoredasprescribedabove.2.Theremaybesomefoggysubstanceinthewellswhentheplateisopenedatthefirsttime.Itwillnoth***eanyeffectonthefinalassayresults.3.Donotremovemicrotiterplatefromthestoragebaguntilneeded.4.Amicrotiterplatereaderwithabandwidthof10nmorlessandanopticaldensityrangeof0-3ODorgreaterat450nmw***elengthisacceptableforuseinabsorbancemeasurement.5.Usefreshdisposablepipettetipsforeachtransferto***oidcontamination.6.Donotsubstitutereagentsfromonekitlottoanother.Useonlythereagentssuppliedbymanufacturer.7.Validperiod:sixmonths.PrecautionTheStopSolutionsuggestedforusewiththiskitisanacidsolution.Weareye,hand,face,andclothingprotectionwhenusingthismaterial.)