FORRESEARCHUSEONLY;NOTFORTHERAPEUTICORDIAGNOSTICAPPLICATI***!PLEASEREADTHROUGHENTIREPROCEDUREBEFOREBEGINNING!IntendeduseThisimmunoassaykitallowsfortheinvitroquantitativedeterminationofmouseNeuregulin1,NRG-1concentrati***intissuehomogenatesandotherbiologicalfluids.IntroductionNeuregulin1orNRG1isoneofthefourproteinsoftheneuregulinfamilywhichactonEGFRfamilyofreceptors.Neuregulin1isproducedinnumerousisoformsbyalternativesplicing,andthisallowsittoperformawidevarietyoffuncti***.Neuregulin1-ErbB4interacti***arethoughttoplayaroleinthepathologicalmechani***ofschizophrenia.Theproteinalsohasaputativeabilitytoprotectthebrainfromdamageinducedbystroke.TestprincipleThemicrotiterplateprovidedinthiskithasbeenpre-coatedwithanantibodyspecifictoNRG-1.Standa***orsamplesarethenaddedtotheappropriatemicrotiterplatewellswithabiotin-conjugatedpolyclonalantibodypreparationspecificforNRG-1and***idinconjugatedtoHorseradishPeroxidase(HRP)isaddedtoeachmicroplatewellandincubated.ThenaTMBsubstratesolutionisaddedtoeachwell.OnlythosewellsthatcontainNRG-1,biotin-conjugatedantibodyandenzyme-conjugated***idinwillexhibitachangeincolor.Theenzyme-substratereactionisterminatedbytheadditionofasulphuricacidsolutionandthecolorchangeismeasuredspectrophotometricallyataw***elengthof450nm&plu***n;2nm.TheconcentrationofNRG-1inthesamplesisthendeterminedbycomparingtheO.D.ofthesamplestothestandardcurve.MaterialsandcomponentsReagentQuantityAssayplate1×20mlStandard2SampleDiluent1×20mlAssayDiluentA1×10mlAssayDiluentB1×10mlDetectionReagentA1×120μlDetectionReagentB1×120μlWashBuffer(25xconcentrate)1×30mlSubstrate1×10mlStopSolution1×10mlPlatesealerfor96wells5Instruction1OthersuppliesrequiredLuminometer.Pipettesandpipettetips.EPtubeDeionizedordistilledwater.SamplecollectionandstorageTissuehomogenates-Thepreparationoftissuehomogenateswillvarydependingupontissuetype.Forthisassay,tissuewasrinsedwith1XPBStoremoveexcessblood,homogenizedin20mLof1XPBSandstoredovernightat≤-20°C.Aftertwofreeze-thawcycleswereperformedtobreakthecellmembranes,thehomogenateswerecentrifugedfor5minutesat5000xg.Removethesupernateandassayimmediatelyoraliquotandstoreat≤-20°C.Otherbiologicalfluids-Removeparticulatesbycentrifugationandassayimmediatelyoraliquotandstoresamplesat-20℃or-80℃.***oidrepeatedfreeze-thawcycles.Note:Tissuehomogenatestobeusedwithin7daysmaybestoredat2-8℃,otherwisesamplesmuststoredat-20℃(≤1months)or-80℃(≤2months)to***oidlossofbioactivityandcontamination.***oidfreeze-thawcycles.Whenperformingtheassayslowlybringsamplestoroomtemperature.DONOTUSEHEAT-TREATEDSPECIMENS.Limitati***oftheprocedure1.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.Dilute30mLofWashBufferConcentrateintodeionizedordistilledwatertoprepare750mLofWashBuffer.Standard-Rec***titutetheStandardwith1.0mLofSampleDiluent.Thisrec***titutionproducesastocksolutionof250ng/mL.Allowthestandardtositforaminimumof15minuteswithgentleagitationpriortomakingserialdiluti***(Makingserialdilutioninthewellsdirectlyisnotpermitted).Pleasefirstlydilutethestocksolutionto50ng/mLandthedilutedstandardservesasthehighstandard(50ng/mL).TheSampleDiluentservesasthezerostandard(0ng/mL).ng/mL250502512.56.253.121.560.780DetectionReagentAandB-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°C.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°C.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.SpecificityThisassayrecognizesrecombinantandnaturalmouseNRG-1.Nosignificantcross-reactivityorinterferencewasobserved.SensitivityTheminimumdetectabledoseofmouseNRG-1istypicallylessthan0.39ng/ml.Thesensitivityofthisassay,orLowerLimitofDetection(LLD)wasdefinedasthelowestproteinconcentrationthatcouldbedifferentiatedfromzero.DetectionRange0.78-50ng/mL.Thestandardcurveconcentrati***usedfortheELISA’swere50ng/mL,25ng/mL,12.5ng/mL,6.25ng/mL,3.12ng/mL,1.56ng/mL,0.78ng/mL.Calculationofresults***eragetheduplicatereadingsforeachstandard,control,andsampleandsubtractthe***eragezerostandardopticaldensity.Createastandardcurvebyreducingthedatausingcomputersoftwarecapableofgeneratingafourparameterlogistic(4-PL)curve-fit.Asanalternative,c***tructastandardcurvebyplottingthemeanabsorbanceforeachstandardonthex-axisagainsttheconcentrationonthey-axisanddrawabestfitcurvethroughthepointsonthegraph.ThedatamaybelinearizedbyplottingthelogoftheNRG-1concentrati***versusthelogoftheO.D.andthebestfitlinecanbedeterminedbyregressionanalysis.Itisrecommendedtousesomerelatedsoftwaretodothiscalculation,suchascurveexpert13.0.Thisprocedurewillproduceanadequatebutlessprecisefitofthedata.Ifsamplesh***ebeendiluted,theconcentrationreadfromthestandardcurvemustbemultipliedbythedilutionfactor.Storageoftestkitsandinstrumentation1.Unopenedtestkitsshouldbestoredreferringtothepackagelabelforfrequentuse,andstoredat-20°Cforlongtimestorage.Theunusedstripsshouldbekeptinasealedbagandstoredat2-8°Cintheirpouchwiththedesiccantprovidedtominimizeexposuretodampair.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.中文说明书Email:info@索取;13801829628定货；HR-M0292)