人血管紧张素转化酶(ACE)ELISA试剂盒
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HumanAngiotensinconverting
enzyme,ACEELISAKit
CatalogNo.CSB-E04489h
(96T)
Thisimmunoassaykitallowsfortheinvitroquantitativedeterminationofhuman
ACEconcentrationsinserum,plasmaandotherbiologicalfluids.
Expirationdatesixmonthsfromthedateofmanufacture
FORRESEARCHUSEONLY.NOTFORUSEINDIAGNOSTICPROCEDURES.
PRINCIPLEOFTHEASSAY
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Themicrotiterplateprovidedinthiskithasbeenpre-coatedwith
anantibodyspecifictoACE.Standardsorsamplesarethen
addedtotheappropriatemicrotiterplatewellswitha
biotin-conjugatedantibodypreparationspecificforACEand
AvidinconjugatedtoHorseradishPeroxidase(HRP)isaddedto
eachmicroplatewellandincubated.ThenaTMB(3,3',5,5'
tetramethyl-benzidine)substratesolutionisaddedtoeachwell.
OnlythosewellsthatcontainACE,biotin-conjugatedantibody
andenzyme-conjugatedAvidinwillexhibitachangeincolor.The
enzyme-substratereactionisterminatedbytheadditionofa
sulphuricacidsolutionandthecolorchangeismeasured
spectrophotometricallyatawavelengthof450nm±2nm.The
concentrationofACEinthesamplesisthendeterminedby
comparingtheO.D.ofthesamplestothestandardcurve.
DETECTIONRANGE
3.12U/ml-200U/ml.Thestandardcurveconcentrationsusedfor
theELISA’swere200U/ml,100U/ml,50U/ml,25U/ml,12.5
U/ml,6.25U/ml,3.12U/ml.
SPECIFICITY
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ThisassayrecognizesrecombinantandnaturalhumanACE.No
significantcross-reactivityorinterferencewasobserved.
SENSITIVITY
TheminimumdetectabledoseofhumanACEistypicallyless
than0.78U/ml.
Thesensitivityofthisassay,orLowerLimitofDetection(LLD)
wasdefinedasthelowestproteinconcentrationthatcouldbe
differentiatedfromzero.
MATERIALSPROVIDED
ReagentQuantity
Assayplate1
Standard2
SampleDiluent1x20ml
Biotin-antibodyDiluent1x10ml
HRP-avidinDiluent1x10ml
Biotin-antibody1x120μl
HRP-avidin1x120μl
1x20ml
WashBuffer
(25×concentrate)
TMBSubstrate1x10ml
StopSolution1x10ml
STORAGE
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1.Unopenedtestkitsshouldbestoredat2-8°Cuponreceipt
andthemicrotiterplateshouldbekeptinasealedbag.The
testkitmaybeusedthroughouttheexpirationdateofthekit,
provideditisstoredasprescribedabove.Refertothe
packagelabelfortheexpirationdate.
2.Openedtestplateshouldbestoredat2-8°Cinthealuminum
foilbagwithdesiccantstominimizeexposuretodampair.The
kitswillremainstableuntiltheexpiringdateshown,providedit
isstoredasprescribedabove.
3.Amicrotiterplatereaderwithabandwidthof10nmorless
andanopticaldensityrangeof0-3ODorgreaterat450nm
wavelengthisacceptableforuseinabsorbance
measurement.
REAGENTPREPARATION
Bringallreagentstoroomtemperaturebeforeuse.
1.WashBufferIfcrystalshaveformedintheconcentrate,
warmuptoroomtemperatureandmixgentlyuntilthe
crystalshavecompletelydissolved.Dilute20mlofWash
BufferConcentrateintodeionizedordistilledwatertoprepare
500mlofWashBuffer.
2.StandardCentrifugethestandardvialat6000-10000rpm
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for30s.ReconstitutetheStandardwith1.0mlofSample
Diluent.Thisreconstitutionproducesastocksolutionof200
U/ml.Allowthestandardtositforaminimumof15minutes
withgentleagitationpriortomakingserialdilutions.The
undilutedstandardservesasthehighstandard(200U/ml).
TheSampleDiluentservesasthezerostandard(0U/ml).
Preparefreshforeachassay.Usewithin4hoursanddiscard
afteruse.
3.Biotin-antibodyCentrifugethevialbeforeopening.Dilute
totheworkingconcentrationusingBiotin-antibody
Diluent(1:100),respectively.
4.HRP-avidinCentrifugethevialbeforeopening.Dilutetothe
workingconcentrationusingHRP-avidinDiluent(1:100),
respectively.
Precaution:TheStopSolutionprovidedwiththiskitisanacidsolution.Wear
eye,hand,face,andclothingprotectionwhenusingthismaterial.
OTHERSUPPLIESREQUIRED
Microplatereadercapableofmeasuringabsorbanceat450
nm,withthecorrectionwavelengthsetat540nmor570nm.
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Pipettesandpipettetips.
Deionizedordistilledwater.
Squirtbottle,manifolddispenser,orautomatedmicroplate
washer.
Anincubatorwhichcanprovidestableincubationconditions
upto37°C±0.5°C.
SAMPLECOLLECTIONANDSTORAGE
SerumUseaserumseparatortube(SST)andallow
samplestoclotfor30minutesbeforecentrifugationfor15
minutesat1000g.Removeserumandassayimmediatelyor
aliquotandstoresamplesat-20°C.Centrifugethesample
againafterthawingbeforetheassay.Avoidrepeated
freeze-thawcycles.
PlasmaCollectplasmausingcitrate,EDTA,orheparinas
ananticoagulant.Centrifugefor15minutesat1000gwithin
30minutesofcollection.Assayimmediatelyoraliquotand
storesamplesat-20°C.Centrifugethesampleagainafter
thawingbeforetheassay.Avoidrepeatedfreeze-thawcycles.
Note:Grosslyhemolyzedsamplesarenotsuitableforuseinthisassay.
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ASSAYPROCEDURE
Bringallreagentsandsamplestoroomtemperaturebeforeuse.Itis
recommendedthatallsamples,standards,andcontrolsbeassayedinduplicate.
Allthereagentsshouldbeaddeddirectlytotheliquidlevelinthewell.The
pipetteshouldavoidcontactingtheinnerwallofthewell.
1.Add100μlofStandard,Blank,orSampleperwell.Coverwith
theadhesivestrip.Incubatefor2hoursat37°C.
2.Removetheliquidofeachwell,don’twash.
3.Add100μlofBiotin-antibodyworkingsolutiontoeachwell.
Incubatefor1hourat37°C.Biotin-antibodyworking
solutionmayappearcloudy.Warmuptoroomtemperature
andmixgentlyuntilsolutionappearsuniform.
4.Aspirateeachwellandwash,repeatingtheprocessthree
timesforatotalofthreewashes.Wash:Filleachwellwith
WashBuffer(200μl)andletitstandfor2minutes,then
removetheliquidbyflickingtheplateoverasink.The
remainingdropsareremovedbypattingtheplateonapaper
towel.Completeremovalofliquidateachstepisessentialto
goodperformance.
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5.Add100μlofHRP-avidinworkingsolutiontoeachwell.
Coverthemicrotiterplatewithanewadhesivestrip.Incubate
for1hourat37°C.
6.Repeattheaspirationandwashfivetimesasstep4.
7.Add90μlofTMBSubstratetoeachwell.Incubatefor10-30
minutesat37°C.Keepingtheplateawayfromdraftsand
othertemperaturefluctuationsinthedark.
8.Add50μlofStopSolutiontoeachwellwhenthefirstfour
wellscontainingthehighestconcentrationofstandards
developobviousbluecolor.Ifcolorchangedoesnotappear
uniform,gentlytaptheplatetoensurethoroughmixing.
9.Determinetheopticaldensityofeachwellwithin30minutes,
usingamicroplatereadersetto450nm.
CALCULATIONOFRESULTS
Usingtheprofessionalsoft"CurveExert1.3"tomakeastandardcurveis
recommended,whichcanbedownloadedfromourweb.
Averagetheduplicatereadingsforeachstandard,control,and
sampleandsubtracttheaveragezerostandardopticaldensity.
Createastandardcurvebyreducingthedatausingcomputer
softwarecapableofgeneratingafourparameterlogistic(4-PL)
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curve-fit.Asanalternative,constructastandardcurvebyplotting
themeanabsorbanceforeachstandardonthey-axisagainst
theconcentrationonthex-axisanddrawabestfitcurvethrough
thepointsonthegraph.Thedatamaybelinearizedbyplotting
thelogoftheACEconcentrationsversusthelogoftheO.D.and
thebestfitlinecanbedeterminedbyregressionanalysis.This
procedurewillproduceanadequatebutlessprecisefitofthe
data.Ifsampleshavebeendiluted,theconcentrationreadfrom
thestandardcurvemustbemultipliedbythedilutionfactor.
LIMITATIONSOFTHEPROCEDURE
Thekitshouldnotbeusedbeyondtheexpirationdateonthe
kitlabel.
Donotmixorsubstitutereagentswiththosefromotherlotsor
sources.
ItisimportantthattheStandardDiluentselectedforthe
standardcurvebeconsistentwiththesamplesbeing
assayed.
Ifsamplesgeneratevalueshigherthanthehigheststandard,
dilutethesampleswiththeappropriateStandardDiluentand
repeattheassay.
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AnyvariationinStandardDiluent,operator,pipetting
technique,washingtechnique,incubationtimeor
temperature,andkitagecancausevariationinbinding.
Thisassayisdesignedtoeliminateinterferencebysoluble
receptors,bindingproteins,andotherfactorspresentin
biologicalsamples.Untilallfactorshavebeentestedinthe
QuantikineImmunoassay,thepossibilityofinterference
cannotbeexcluded.
TECHNICALHINTS
Centrifugevialsbeforeopeningtocollectcontents.
Whenmixingorreconstitutingproteinsolutions,alwaysavoid
foaming.
Toavoidcross-contamination,changepipettetipsbetween
additionsofeachstandardlevel,betweensampleadditions,
andbetweenreagentadditions.Also,useseparatereservoirs
foreachreagent.
Whenusinganautomatedplatewasher,addinga30second
soakperiodfollowingtheadditionofwashbuffer,and/or
rotatingtheplate180degreesbetweenwashstepsmay
improveassayprecision.
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Toensureaccurateresults,properadhesionofplatesealers
duringincubationstepsisnecessary.
SubstrateSolutionshouldremaincolorlessorlightblueuntil
addedtotheplate.KeepSubstrateSolutionprotectedfrom
light.SubstrateSolutionshouldchangefromcolorlessorlight
bluetogradationsofblue.
StopSolutionshouldbeaddedtotheplateinthesameorder
astheSubstrateSolution.Thecolordevelopedinthewells
willturnfrombluetoyellowuponadditionoftheStopSolution.
WellsthataregreenincolorindicatethattheStopSolution
hasnotmixedthoroughlywiththeSubstrateSolution.
人血管紧张素转化酶(ACE)ELISA试剂盒
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