人尿激酶型纤溶酶原激活物受体（PLAUR/uPAR）Elisa试剂盒说明书

文档简介FORINVITROUSEANDRESEARCHUSEONLYNOTFORUSEINDIAGNOSTICORTHERAPEUTICPROCEDURES7thEdition（RevisedinNovember,2011）[INTENDEDUSE]ThekitisasandwichenzymeimmunoassayforinvitroquantitativemeasurementofuPARinhumanserum,plasmaandotherbiologicalfluids.[REAGENTSANDMATERIALSPROVIDED]ReagentsQuantityReagentsQuantityPre-coated,readytouse96-wellstripplate1Platesealerfor96wells4Standard（lyophilized）2StandardDiluent1×20mLDetectionReagentA（green）1×120μLAssayDiluentA（2×concentrate）1×6mLDetectionReagentB（red）1×120μLAssayDiluentB（2×concentrate）1×6mLTMBSubstrate1×9mLStopSolution1×6mLWashBuffer（30×concentrate）1×20mLInstructionmanual1[MATERIALSREQUIREDBUTNOTSUPPLIED]1.Microplatereaderwith450±10nmfilter.2.Precisionsingleormulti-channelpipettesanddisposabletips.3.EppendorfTubesfordilutingsamples.4.Deionizedordistilledwater.5.Absorbentpaperforblottingthemicrotiterplate.6.ContainerforWashSolution[STORAGEOFTHEKITS]1.Forunopenedkit:Allthereagents首ldbekeptaccordingtothelabelsonvials.TheStandard,DetectionReagentA,DetectionReagentBandthe96-wellstripplate首ldbestoredat-20oCuponreceiptwhiletheothers首ldbeat4oC.2.Foropenedkit:Whenthekitisopened,theremainingreagentsstillneedtobestoredaccordingtotheabovestoragecondition.Besides,pleasereturntheunusedwellstothefoilpouchcontainingthedesiccantpack,andresealalongentireedgeofzip-seal.Note:Itishighlyrecommendedtousetheremainingreagentswithin1monthprovidedthisiswithintheexpirationdateofthekit.[SAMPLECOLLECTIONANDSTORAGE]Serum-Allowsamplestoclotfortwohoursatroomtemperatureorovernightat4oCbeforecentrifugationfor20minutesatapproximately1000×g.Assayimmediatelyorstoresamplesinaliquotat-20oCor-80oC.Avoidrepeatedfreeze/thawcycles.Plasma-CollectplasmausingEDTAorheparinasananticoagulant.Centrifugesamplesfor15minutesat1000×gwithin30minutesofcollection.Removeplasmaandassayimmediatelyorstoresamplesinaliquotat-20oCor-80oC.Avoidrepeatedfreeze/thawcycles.Otherbiologicalfluids-Centrifugesamplesfor20minutesat1000×g.Removeparticulatesandassayimmediatelyorstoresamplesinaliquotat-20oCor-80oC.Avoidrepeatedfreeze/thawcycles.Note:1.Samplestobeusedwithin5daysmaybestoredat4oC,otherwisesamplesmustbestoredat-20oC（1month）or-80oC（2months）toavoidlossofbioactivityandcontamination.2.Samplehemolysiswillinfluencetheresult,sohemolyticspecimencannotbedetected.3.Whenperformingtheassay,bringsamplestoroomtemperature.[REAGENTPREPARATION]1.Bringallkitcomponentsandsamplestoroomtemperature（18-25oC）beforeuse.2.Standard-ReconstitutetheStandardwith1.0mLofStandardDiluent,keptfor10minutesatroomtemperature,shakegently（nottofoam）.Theconcentrationofthestandardinthestocksolutionis200ng/mL.Pleasefirstlydilutethestocksolutionto20ng/mLandthedilutedstandardservesasthehigheststandard（20ng/mL）.Thenprepare7tubescontaining0.5mLStandardDiluentandusethedilutedstandardtoproduceadoubledilutionseriesaccordingtothepictureshownbelow.Mixeachtubethoroughlybeforethenexttransfer.Setup7pointsofdilutedstandardsuchas20ng/mL,10ng/mL,5ng/mL,2.5ng/mL,1.25ng/mL,0.625ng/mL,0.312ng/mL,andthelastEPtubeswithStandardDiluentistheblankas0ng/mL.3.AssayDiluentAandAssayDiluentB-Dilute6mLofAssayDiluentAorBConcentrate（2×）with6mLofdeionizedordistilledwatertoprepare12mLofAssayDiluentAorB.（Infact,morethan6mLAssayDiluentAandAssayDiluentBarecontainedinthebottles.Therefore,ineverytest,pleasepreciselypipetterequiredamountofDiluentandmakedoubledilutioninanewcontainer.Thepreparedworkingdilutioncan'tbefrozen.）Tube123456789ng/mL200201052.51.250.6250.31204.DetectionReagentAandDetectionReagentB-BrieflyspinorcentrifugethestockDetectionAandDetectionBbeforeuse.DilutetotheworkingconcentrationwithworkingAssayDiluentAorB,respectively（1:100）.5.WashSolution-Dilute20mLofWashSolutionconcentrate（30×）with580mLofdeionizedordistilledwatertoprepare600mLofWashSolution（1×）.6.TMBsubstrate-Aspiratetheneededdosageofthesolutionwithsterilizedtipsanddonotdumptheresidualsolutionintothevialagain.Note:1.Makingserialdilutioninthewellsdirectlyisnotpermitted.2.Preparestandardwithin15minutesbeforeassay.Pleasedonotdissolvethereagentsat37oCdirectly.3.PleasecarefullyreconstituteStandardsorworkingDetectionReagentAandBaccordingtotheinstruction,andavoidfoamingandmixgentlyuntilthecrystalsarecompletelydissolved.Tominimizeimprecisioncausedbypipetting,usesmallvolumesandensurethatpipettorsarecalibrated.Itisrecommendedtosuckmorethan10μLforoncepipetting.4.ThereconstitutedStandards,DetectionReagentAandDetectionReagentBcanbeusedonlyonce.5.IfcrystalshaveformedintheWashSolutionconcentrate（30×）,warmtoroomtemperatureandmixgentlyuntilthecrystalsarecompletelydissolved.6.Contaminatedwaterorcontainerforreagentpreparationwillinfluencethedetectionresult.[SAMPLEPREPARATION]1.Uscn,Inc.isonlyresponsibleforthekititself,butnotforthesamplesconsumedduringtheassay.Theuser首ldcalculatethepossibleamountofthesamplesusedinthewholetest.Pleasereservesufficientsamplesinadvance.2.Pleasepredicttheconcentrationbeforeassaying.Ifvaluesforthesearenotwithintherangeofthestandardcurve,usersmustdeterminetheoptimalsampledilutionsfortheirparticularexperiments.3.Ifthesamplesarenotindicatedinthemanual,apreliminaryexperimenttodeterminethevalidityofthekitisnecessary.4.TissueorcellextractionsamplespreparedbychemicallysisbuffermaycauseunexpectedELISAresultsduetotheimpactsfromcertainchemicals.5.Duetothepossibilityofmismatchingbetweenantigenfromotheroriginandantibodyusedinourkits（e.g.,antibodytargetsconformationalepitoperatherthanlinearepitope）,somenativeorrecombinantproteinsfromothermanufacturersmaynotberecognizedbyourproducts.6.Influencedbythefactorsincludingcellviability,cellnumberorsamp领time,samplesfromcellculturesupernatantmaynotbedetectedbythekit.7.Freshsampleswithoutlongtimestorageisrecommendedforthetest.Otherwise,proteindegradationanddenaturalizationmayoccurinthosesamplesandfinallyleadtowrongresults.[ASSAYPROCEDURE]1.Determinewellsfordilutedstandard,blankandsample.Prepare7wellsforstandard,1wellforblank.Add100μLeachofdilutionsofstandard（readReagentPreparation）,blankandsamplesintotheappropriatewells.CoverwiththePlatesealer.Incubatefor2hoursat37oC.2.Removetheliquidofeachwell,don’twash.3.Add100μLofDetectionReagentAworkingsolutiontoeachwell.Incubatefor1hourat37oCaftercoveringitwiththePlatesealer.4.Aspiratethesolutionandwashwith350μLof1×WashSolutiontoeachwellusingasquirtbottle,multi-channelpipette,manifolddispenserorautowasher,andletitsitfor1~2minutes.Removetheremainingliquidfromallwellscompletelybysnappingtheplateontoabsorbentpaper.Totallywash3times.Afterthelastwash,removeanyremainingWashBufferbyaspiratingordecanting.Inverttheplateandblotitagainstabsorbentpaper.5.Add100μLofDetectionReagentBworkingsolutiontoeachwell.Incubatefor30minutesat37oCaftercoveringitwiththePlatesealer.6.Repeattheaspiration/washprocessfortotal5timesasconductedinstep4.7.Add90μLofSubstrateSolutiontoeachwell.CoverwithanewPlatesealer.Incubatefor15-25minutesat37oC（Don'texceed30minutes）.Protectfromlight.TheliquidwillturnbluebytheadditionofSubstrateSolution.8.Add50μLofStopSolutiontoeachwell.TheliquidwillturnyellowbytheadditionofStopsolution.Mixtheliquidbytappingthesideoftheplate.Ifcolorchangedoesnotappearuniform,gentlytaptheplatetoensurethoroughmixing.9.Removeanydropofwaterandfingerprintonthebottomoftheplateandconfirmthereisnobubbleonthesurfaceoftheliquid.Then,runthemicroplatereaderandconductmeasurementat450nmimmediately.Note:1.Assaypreparation:Keepappropriatenumbersofwellsfor1experimentandremoveextrawellsfrommicroplate.Restwells首ldberesealedandstoredat-20oC.2.SamplesorreagentsadditionPleaseusethefreshlypreparedStandard.Pleasecarefullyaddsamplestowellsandmixgentlytoavoidfoaming.Donottouchthewellwall.Foreachstepintheprocedure,totaldispensingtimeforadditionofreagentsorsamplestotheassayplate首ldnotexceed10minutes.Thiswillensureequalelapsedtimeforeachpipettingstep,withoutinterruption.Duplicationofallstandardsandspecimens,althoughnotrequired,isrecommended.Toavoidcross-contamination,changepipettetipsbetweenadditionsofstandards,samples,andreagents.Also,useseparatedreservoirsforeachreagent.3.Incubation:Toensureaccurateresults,properadhesionofplatesealersduringincubationstepsisnecessary.Donotallowwellstosituncoveredforextendedperiodsbetweenincubationsteps.Oncereagentsareaddedtothewellstrips,DONOTletthestripsDRYatanytimeduringtheassay.Incubationtimeandtemperaturemustbecontrolled.4.Washing:Thewashprocedureiscritical.Completeremovalofliquidateachstepisessentialforgoodperformance.Afterthelastwash,removeanyremainingWashSolutionbyaspiratingordecantingandremoveanydropofwaterandfingerprintonthebottomoftheplate.Insufficientwashingwillresultinpoorprecisionandfalseelevatedabsorbancereading.5.Control领ofreactiontime:ObservethechangeofcolorafteraddingTMBSubstrate（e.g.observationonceevery10minutes）,ifthecoloristoodeep,addStopSolutioninadvancetoavoidexcessivelystrongreactionwhichwillresultininaccurateabsorbancereading.6.TMBSubstrateiseasilycontaminated.Pleaseprotectitfromlight.7.Theenvironmenthumiditywhichislessthan60%mighthavesomeeffectsonthefinalperformance,therefore,ahumidifierisrecommendedtobeusedatthatcondition.[TESTPRINCIPLE]Themicrotiterplateprovidedinthiskithasbeenpre-coatedwithanantibodyspecifictouPAR.Standardsorsamplesarethenaddedtotheappropriatemicrotiterplatewellswithabiotin-conjugatedantibodypreparationspecificforuPAR.Next,AvidinconjugatedtoHorseradishPeroxidase（HRP）isaddedtoeachmicroplatewellandincubated.AfterTMBsubstratesolutionisadded,onlythosewellsthatcontainuPAR,biotin-conjugatedantibodyandenzyme-conjugatedAvidinwillexhibitachangeincolor.Theenzyme-substratereactionisterminatedbytheadditionofsulphuricacidsolutionandthecolorchangeismeasuredspectrophotometricallyatawavelengthof450nm±10nm.TheconcentrationofuPARinthesamplesisthendeterminedbycomparingtheO.D.ofthesamplestothestandardcurve.[CALCULATIONOFRESULTS]Averagetheduplicatereadingsforeachstandard,control,andsamplesandsubtracttheaveragezerostandardopticaldensity.Createastandardcurveonlog-loggraphpaper,withuPARconcentrationonthey-axisandabsorbanceonthex-axis.Drawthebestfitstraightlinethroughthestandardpointsanditcanbedeterminedbyregressionanalysis.Usingsomeplotsoftware,forinstance,curveexpert1.30,isalsorecommended.Ifsampleshavebeendiluted,theconcentrationreadfromthestandardcurvemustbemultipliedbythedilutionfactor.[TYPICALDATA]Inordertomakethecalculationeasier,weplottheO.D.valueofthestandard（X-axis）againsttheknownconcentrationofthestandard（Y-axis）,althoughconcentrationistheindependentvariableandO.D.valueisthedependentvariable.However,theO.D.valuesofthestandardcurvemayvaryaccordingtotheconditionsofassayperformance（e.g.operator,pipettingtechnique,washingtechniqueortemperatureeffects）,plottinglogofthedatatoestablishstandardcurveforeachtestisrecommended.Typicalstandardcurvebelowisprovidedforreferenceonly.TypicalStandardCurveforHumanuPARELISA.[DETECTIONRANGE]0.312-20ng/mL.ThestandardcurveconcentrationsusedfortheELISA’swere20ng/mL,10ng/mL,5ng/mL,2.5ng/mL,1.25ng/mL,0.625ng/mL,0.312ng/mL.[SENSITIVITY]TheminimumdetectabledoseofhumanuPARistypicallylessthan0.123ng/mL.Thesensitivityofthisassay,orLowerLimitofDetection（LLD）wasdefinedasthelowestproteinconcentrationthatcouldbedifferentiatedfromzero.Itwasdeterminedbyaddingtwostandarddeviationstothemeanopticaldensityvalueoftwentyzerostandardreplicatesandcalculatingthecorrespondingconcentration.[SPECIFICITY]ThisassayhashighsensitivityandexcellentspecificityfordetectionofhumanuPAR.Nosignificantcross-reactivityorinterferencebetweenhumanuPARandanalogueswasobserved.Note:Limitedbycurrentskillsandknowledge,itisimpossibleforustocompletethecross-reactivitydetectionbetweenhumanuPARandalltheanalogues,therefore,crossreactionmaystillexist.[RECOVERY]MatriceslistedbelowwerespikedwithcertainlevelofrecombinanthumanuPARandtherecoveryrateswerecalculatedbycomparingthemeasuredvaluetotheexpectedamountofuPARinsamples.MatrixRecoveryrange（%）Average（%）humanserum（n=5）91-10597humanEDTAplasma（n=5）95-10498humanheparinplasma（n=5）80-9688[LINEARITY]ThelinearityofthekitwasassayedbytestingsamplesspikedwithappropriateconcentrationofhumanuPARandtheirserialdilutions.Theresultsweredemonstratedbythepercentageofcalculatedconcentrationtotheexpected.Sample121418116humanserum（n=5）95-109%80-91%91-107%81-95%humanEDTAplasma（n=5）93-101%76-99%90-99%83-93%humanheparinplasma（n=5）78-96%94-107%101-106%89-98%[PRECISION]Intra-assayPrecision（Precisionwithinanassay）:3sampleswithlow,middleandhighlevelhumanuPARweretested20timesononeplate,respectively.Inter-assayPrecision（Precisionbetweenassays）:3sampleswithlow,middleandhighlevelhumanuPARweretestedon3differentplates,8replicatesineachplate.CV（%）=SD/meanX100Intra-Assay:CV<10%Inter-Assay:CV<12%[STABILITY]ThestabilityofELISAkitisdeterminedbythelossrateofactivity.Thelossrateofthiskitislessthan5%withintheexpirationdateunderappropriatestoragecondition.Thelossratewasdeterminedbyacceleratedthermaldegradationtest.Keepthekitat37oCfor3days,andcompareO.D.valuesofthekitkeptat37oCwiththatofatrecommendedtemperature.（referringfromChinaBiologicalProductsStandard,whichwascalculatedbytheArrheniusequation.ForELISAkit,1daystorageat37oCcanbeconsideredas2monthsat4oC,whichmeans3daysat37oCequa领6monthsat4oC）.Note:Tominimizeextrainfluenceontheperformance,operationproceduresandlabconditions,especiallyroomtemperature,airhumidity,incubatortemperature首ldbestrictlycontrolled.Itisalsostronglysuggestedthatthewholeassayisperformedbythesameoperatorfromthebeginningtotheend.[ASSAYPROCEDURESUMMARY]1.Prepareallreagents,samplesandstandards;2.Add100μLstandardorsampletoeachwell.Incubate2hoursat37oC;3.Add100μLpreparedDetectionReagentA.Incubate1hourat37oC;4.Aspirateandwash3times;5.Add100μLpreparedDetectionReagentB.Incubate30minutesat37oC;6.Aspirateandwash5times;7.Add90μLSubstrateSolution.Incubate15-25minutesat37oC;8.Add50μLStopSolution.Readat450nmimmediately.[IMPORTANTNOTE]1.Limitedbythecurrentconditionandscientifictechnology,wecan'tcompletelyconductthecomprehensiveidentificationandanalysisontherawmaterialprovidedbysuppliers.Sotheremightbesomequalitativeandtechnicalriskstousethekit.2.Thefinalexperimentalresultswillbecloselyrelatedtovalidityoftheproducts,operationskillsoftheendusersandtheexperimentalenvironments.Pleasemakesurethatsufficientsamplesareavailable.3.Kitsfromdifferentbatchesmaybealittledifferentindetectionrange,sensitivityandcolordevelopingtime.Pleaseperformtheexperimentexactlyaccordingtotheinstructionattachedinkitwhileelectroniconesfromourwebsite（www.uscnk.us;www.uscnk.cn;www.uscnk.com）isonlyforinformation.4.Donotmixorsubstitutereagentsfromonekitlottoanother.Useonlythereagentssuppliedbymanufacturer.5.Protectallreagentsfromstronglightduringstorageandincubation.Allthebottlecapsofreagents首ldbecoveredtightlytopreventtheevaporationandcontaminationofmicroorganism.6.Theremaybesomefoggysubstanceinthewellswhentheplateisopenedatthefirsttime.Itwillnothaveanyeffectonthefinalassayresults.Donotremovemicrotiterplatefromthestoragebaguntilneeded.7.Wrongoperationsduringthereagentspreparationandloading,aswellasincorrectparametersettingfortheplatereadermayleadtoincorrectresults.Amicroplateplatereaderwithabandwidthof10nmorlessandanopticaldensityrangeof0-3O.D.orgreaterat450±10nmwavelengthisacceptableforuseinabsorbancemeasurement.Pleasereadtheinstructioncarefullyandadjusttheinstrumentpriortotheexperiment.Formoreinformation,pleaserefertotheoperationvideo（http://www.uscnk.com/homepage/operate-elisa.htm）.8.Eventhesameoperatormightgetdifferentresultsintwoseparateexperiments.Inordertogetbetterreproducibleresults,theoperationofeverystepintheassay首ldbecontrolled.Furthermore,apreliminaryexperimentbeforeassayforeachbatchisrecommended.9.EachkithasbeenstrictlypassedQ.Ctest.However,resultsfromendusersmightbeinconsistentwithourin-housedataduetosomeunexpectedtransportationconditionsordifferentlabequipments.Intra-assayvarianceamongkitsfromdifferentbatchesmightarisefromabovefactors,too.10.Kitsfromdifferentmanufacturerswiththesameitemmightproducedifferentresults,sincewehaven’tcomparedourproductswithothermanufacturers.11.Validperiod:sixmonths.12.Theinstructionmanualalsosuitsforthekitof48T,butallreagentsof48Tkitarereducedbyhalf.[PRECAUTION]TheStopSolutionsuggestedforusewiththiskitisanacidsolution.Weareye,hand,face,andclothingprotectionwhenusingthismaterial.