Humantrypsinogenactivationpeptide,TAPELISAKit货号为：HRR-H0015IntendeduseThisimmunoassaykitallowsforthespecificmeasurementofhumantrypsinogenactivationpeptide,TAPconcentrati***incellculturesupernates,serum,andpla***a.TestprincipleThisassayemploysthequantitativesandwichenzymeimmunoassaytechnique.AmonoclonalantibodyspecificforTAPhasbeenpre-coatedontoamicroplate.Standa***andsamplesarepipettedintothewellsandanyTAPpresentisboundbytheimmobilizedantibody.Anenzyme-linkedpolyclonalantibodyspecificforTAPisaddedtothewells.Followingawashtoremoveanyunboundantibody-enzymereagent,asubstratesolutionisaddedtothewellsandcolordevelopsinproportiontotheamountofTAPboundintheinitialstep.Thecolordevelopmentisstoppedandtheintensityofthecolorismeasured.MaterialsandcomponentsReagentQuantityAssayplate1Standard2SampleDiluent1x20mlAssayDiluentA1x10mlAssayDiluentB1x10mlDetectionReagentA1×120ulDetectionReagentB1×120ulWashBuffer1x30ml(25xconcentrate)Substrate1x10mlStopSolution1x10mlSamplecollectionandstorageCellculturesupernates-Removeparticulatesbycentrifugationandassayimmediatelyoraliquotandstoresamplesat≤-20°C.***oidrepeatedfreeze-thawcycles.Serum-Useaserumseparatortube(SST)andallowsamplestoclotfor30minutesbeforecentrifugationfor15minutesatapproximately1000xg.Removeserumandassayimmediatelyoraliquotandstoresamplesat-20°C.Pla***a-Collectpla***ausingEDTAorheparinasananticoagulant.Centrifugesamplesfor15minutesat1000xgat2-8°Cwithin30minutesofcollection.Storesamplesat≤-20°C.***oidrepeatedfreeze-thawcycles.Note:Citratepla***ahasnotbeenvalidatedforuseinthisassay.Limitati***oftheprocedureFORRESEARCHUSEONLY.NOTFORUSEINDIAGNOSTICPROCEDURES.1.Thekitshouldnotbeusedbeyondtheexpirationdateonthekitlabel.2.Donotmixorsubstitutereagentswiththosefromotherlotsorsources.3.Ifsamplesgeneratevalueshigherthanthehigheststandard,furtherdilutethesampleswiththeAssayDiluentandrepeattheassay.Anyvariationinstandarddiluent,operator,pipettingtechnique,washingtechnique,incubationtimeortemperature,andkitagecancausevariationinbinding.4.Thisassayisdesignedtoeliminateinterferencebysolublereceptors,ligands,bindingproteins,andotherfactorspresentinbiologicalsamples.Untilallfactorsh***ebeentestedintheQuantikineImmunoassay,thepossibilityofinterferencecannotbeexcluded.ReagentpreparationBringallreagentstoroomtemperaturebeforeuse.WashBuffer-Ifcrystalsh***eformedintheconcentrate,warmtoroomtemperatureandmixgentlyuntilthecrystalsh***ecompletelydissolved.Dilute20mLofWashBufferConcentrateintodeionizedordistilledwatertoprepare500mLofWashBuffer.Standard-Rec***titutetheStandardwith1.0mLofSampleDiluent.Thisrec***titutionproducesastocksolutionof50nmol/mL.Allowthestandardtositforaminimumof15minuteswithgentleagitationpriortomakingserialdiluti***.Theundilutedstandardservesasthehighstandard(50nmol/mL).TheSampleDiluentservesasthezerostandard(0nmol/mL).DetectionReagentAandB-DilutetotheworkingconcentrationspecifiedontheviallabelusingAssayDiluentAandB(1:100),respectively.AssayprocedureAllowallreagentstoreachroomtemperature.Arrangeandlabelrequirednumberofstrips.1.Prepareallreagents,workingstanda***andsamplesasdirectedintheprevioussecti***.2.Add100uLofStandard,Control,orsample*perwell.Coverwiththeadhesivestrip.Incubatefor2hoursat37°C.3.Removetheliquidofeachwell,don’twash.4.Add100uLofDetectionReagentAtoeachwell.Incubatefor1hourat37°C.DetectionReagentAmayappearcloudy.Warmtoroomtemperatureandmixgentlyuntilsolutionappearsuniform.5.Aspirateeachwellandwash,repeatingtheprocessthreetimesforatotalofthreewashes.WashbyfillingeachwellwithWashBuffer(350uL)usingasquirtbottle,multi-channelpipette,manifolddispenserorautowasher.Completeremovalofliquidateachstepisessentialtogoodperformance.Afterthelastwash,removeanyremainingWashBufferbyaspiratingordecanting.Inverttheplateandblotitagainstcleanpapertowels.6.Add100uLofDetectionReagentBtoeachwell.Coverwithanewadhesivecubatefor1hoursat37°C.7.Repeattheaspiration/washasinstep5.8.Add90uLofSubstrateSolutiontoeachwell.Incubatefor30minutesatroomtemperature.Protectfromlight.9.Add50uLofStopSolutiontoeachwell.Ifcolorchangedoesnotappearuniform,gentlytaptheplatetoensurethoroughmixing.10.Determinetheopticaldensityofeachwellwithin30minutes,usingamicroplatereadersetto450nm.SpecificityThisassayrecognizesrecombinantandnaturalhumanTAP.Nosignificantcross-reactivityorinterferencewasobserved.ImportantNote:1.Thewashprocedureiscritical.Insufficientwashingwillresultinpoorprecisionandfalselyelevatedabsorbancereadings.2.Itisrecommendedthatnomorethan32wellsbeusedforeachassayrunifmanualpipettingisusedsincepipettingofallstanda***,specimensandcontrolsshouldbecompletedwithin5minutes.Afullplateof96wellsmaybeusedifautomatedpipettingis***ailable.3.Duplicationofallstanda***andspecimens,althoughnotrequired,isrecommended.4.Whenmixingorrec***titutingproteinsoluti***,always***oidfoaming.5.To***oidcross-contamination,changepipettetipsbetweenadditi***ofeachstandardlevel,betweensampleadditi***,andbetweenreagentadditi***.Also,useseparatereservoirsforeachreagent.6.Toensureaccurateresults,properadhesionofplatesealersduringincubationstepsisnecessary.Calculationofresults***eragetheduplicatereadingsforeachstandard,control,andsampleandsubtractthe***eragezerostandardopticaldensity.Createastandardcurvebyreducingthedatausingcomputersoftwarecapableofgeneratingafourparameterlogistic(4-PL)curve-fit.Asanalternative,c***tructastandardcurvebyplottingthemeanabsorbanceforeachstandardonthey-axisagainsttheconcentrationonthex-axisanddrawabestfitcurvethroughthepointsonthegraph.ThedatamaybelinearizedbyplottingthelogoftheTAPconcentrati***versusthelogoftheO.D.andthebestfitlinecanbedeterminedbyregressionanalysis.Thisprocedurewillproduceanadequatebutlessprecisefitofthedata.Ifsamplesh***ebeendiluted,theconcentrationreadfromthestandardcurvemustbemultipliedbythedilutionfactor.Storageoftestkitsandinstrumentation1.Unopenedtestkitsshouldbestoredat2-8°Cuponreceiptandthemicrotiterplateshouldbekeptinasealedbagwithdesiccantstominimizeexposuretodampair.Thetestkitmaybeusedthroughouttheexpirationdateofthekit(sixmonthsfromthedateofmanufacture).Refertothepackagelabelfortheexpirationdate.2.Openedtestkitswillremainstableuntiltheexpiringdateshown,provideditisstoredasprescribedabove.3.Amicrotiterplatereaderwithabandwidthof10nmorlessandanopticaldensityrangeof0-3ODorgreaterat450nmw***elengthisacceptableforuseinabsorbancemeasurement.PrecautionTheStopSolutionsuggestedforusewiththiskitisanacidsolution.Weareye,hand,face,andclothingprotectionwhenusingthismaterial.)