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蚂蚁淘在线 / 品牌中心 / Everest Biotech / Everest Biotech/Human Adiponectin ELISA Kit 1 Plate/2900040015/1 Plate
商品描述
TypicalDataObtainedfromHumanADIponectin
(TMBreactionincubateat37°Cfor25min)
Concentration(ng/ml) | 0.0 | 1.56 | 3.12 | 6.25 | 12.5 | 25 | 50 | 100 |
O.D | 0.002 | 0.401 | 0.672 | 1.070 | 1.566 | 1.865 | 2.037 | 2.176 |
TypicalHumanAdiponectinELISAKitStandardCurveThisstandardcurvewasgeneratedatEtonfordemonstrationpurposeonly.Astandardcurvemustberunwitheachassay.
Range1.56ng/ml-100ng/ml
Sensitivity<60pg/ml
SpecificityNaturalandrecombinanthumantotalAdiponectin
Cross-reactivityNodetectablecross-reactivitywithotherrelevantproteins
Storage
Storeat4°Cfor6months,at-20°Cfor12months.Avoidmultiplefreeze-thawcycles(Shippedwithwetice.)
Principle
Eton’shumanAdiponectinELISAKitwasbasedonstandardsandwichenzyme-linkedimmune-sorbentassaytechnology.AmonoclonalantibodyfrommousespecificforAdiponectinhasbeenprecoatedonto96-wellplates.Standards(NSO,E19-N244)andtestsamplesareaddedtothewells,abiotinylateddetectionpolyclonalantibodyfromgoatspecificforAdiponectinisaddedsubsequentlyandthenfollowedbywashingwithPBSorTBSbuffer.Avidin-Biotin-PeroxidaseComplexwasaddedandunboundconjugateswerewashedawaywithPBSorTBSbuffer.HRPsubstrateTMBwasusedtovisualizeHRPenzymaticreaction.TMBwascatalyzedbyHRPtoproduceabluecolorproductthatchangedintoyellowafteraddingacidicstopsolution.ThedensityofyellowisproportionaltothehumanAdiponectinamountofsamplecapturedinplate.
KitComponents
Description | Quantity |
96-wellplateprecoatedwithanti-humanAdiponectinantibody | 1 |
LyophilizedrecombinanthumanAdiponectinstandard | 100ng/tube×2 |
Biotinylatedanti-humanAdiponectinantibody | 130μl(dilution1:100) |
Avidin-Biotin-PeroxidaseComplex(ABC) | 130μl(dilution1:100) |
Samplediluentbuffer | 30ml |
Antibodydiluentbuffer | 12ml |
ABCdiluentbuffer | 12ml |
TMBcolordevelopingagent | 10ml |
TMBstopsolution | 10ml |
MaterialRequiredButNotProvided
1.Microplatereaderinstandardsize.
2.Automatedplatewasher.
3.AdjustablePipettesandpipettetips.Multichannelpipettesarerecommendedintheconditionoflargeamountofsamplesinthedetection.
4.CleantubesandEppendorftubes.
5.Washingbuffer(neutralPBSorTBS).
Preparationof0.01MTBS:Add1.2gTris,8.5gNacl;450μlofpurifiedaceticacidor700μlofconcentratedhydrochloricacidto1000mlH2OandadjustpHto7.2-7.6.Finally,adjustthetotalvolumeto1L.
NoticeforApplicationofKitPreparationof0.01MPBS:Add8.5gsodiumchloride,1.4gNa2HPO4and0.2gNaH2PO4to1000mldistilledwaterandadjustpHto7.2-7.6.Finally,adjustthetotalvolumeto1L.
1.Toinspectthevalidityofexperimentoperationandtheappropriatenessofsampledilutionproportion,pilotexperimentusingstandardsandasmallnumberofsamplesisrecommended.
2.TheTMBColorDevelopingagentiscolorlessandtransparentbeforeusing,contactusfreelyifitisnotthecase.
3.BeforeusingtheKit,spintubesandbringdownallcomponentstothebottomoftubes.
4.Duplicatewellassayisrecommendedforbothstandardandsampletesting.
5.Don’tlet96-wellplatedry,fordryplatewillinactivateactivecomponentsonplate.
6.Don’treusetipsandtubestoavoidcrosscontamination.
7.Toavoidtousethereagentsfromdifferentbatchestogether.
8.Inordertoavoidmarginaleffectofplateincubationduetotemperaturedifference(reactionmaybestrongerinthemarginalwells),itissuggestedthatthedilutedABCandTMBsolutionwillbepre-warmedin37°Cfor30minbeforeusing.
Preparation
1.SamplePreparationandStorage
Storesamplestobeassayedwithin24hoursat2-8°C.Forlong-termstorage,aliquotandfreezesamplesat-20°C.Avoidrepeatedfreeze-thawcycles.
Cellculturesupernates:Removeparticulatesbycentrifugation,assayimmediatelyoraliquotandstoresamples
at-20°C.
Serum:Allowtheserumtoclotinaserumseparatortube(about4hours)atroomtemperature.Centrifugeatapproximately1000Xgfor15min.Analyzetheserumimmediatelyoraliquotandstoresamplesat-20°C.
Plasma:CollectplasmausingheparinorEDTAasananticoagulant.Centrifugefor15minat1500xgwithin30minofcollection.Assayimmediatelyoraliquotandstoresamplesat-20°C.
2.SampleDilutionGuideline
Theuserneedstoestimatetheconcentrationofthetargetproteininthesampleandselectaproperdilutionfactorsothatthedilutedtargetproteinconcentrationfallsnearthemiddleofthelinearregimeinthestandardcurve.Dilutethesampleusingtheprovideddiluentbuffer.Thefollowingisaguidelineforsampledilution.Severaltrialsmaybenecessaryinpractice.Thesamplemustbewellmixedwiththediluentsbuffer.
Hightargetproteinconcentration(1μg-10μg/ml).Theworkingdilutionis1:100.i.e.Add3μlsampleinto297μlsamplediluentbuffer.
Mediumtargetproteinconcentration(100-1000ng/ml).Theworkingdilutionis1:10.i.e.Add25μlsampleinto225μlsamplediluentbuffer.
Lowtargetproteinconcentration(1.56-100ng/ml).Theworkingdilutionis1:2.i.e.Add100μlsampleto100μlsamplediluentbuffer.
VeryLowtargetproteinconcentration(≤1.56ng/ml).Nodilutionnecessary,ortheworkingdilutionis1:2.
3.ReagentPreparationandStorage
A.ReconstitutionofthehumanAdiponectinstandard:Adiponectinstandardsolutionshouldbepreparednomorethan2hourspriortotheexperiment.TwotubesofAdiponectinstandard(100ngpertube)areincludedineachkit.Useonetubeforeachexperiment.
a.100ng/mlofhumanAdiponectinstandardsolution:Add1mlsamplediluentbufferintoonetube,keepthetubeatroomtemperaturefor10minandmixthoroughly.
b.50ng/ml→1.56ng/mlofhumanAdiponectinstandardsolutions:Label6Eppendorftubeswith50ng/ml,25ng/ml,12.5ng/ml,6.25ng/ml,3.12ng/ml,1.56ng/mlrespectively.Aliquot0.3mlofthesamplediluentbufferintoeachtube.Add0.3mloftheabove100ng/mlAdiponectinstandardsolutioninto1sttubeandmix.Transfer0.3mlfrom1sttubeto2ndtubeandmix.Transfer0.3mlfrom2ndtubeto3rdtubeandmix,andsoon.
Note:Thestandardsolutionsarebestusedwithin2hours.The100ng/mlstandardsolutionshouldbestoredat4°Cforupto12hours,orat-20°Cforupto48hours.Avoidrepeatedfreeze-thawcycles.
B.Preparationofbiotinylatedanti-humanAdiponectinantibodyworkingsolution:Thesolutionshouldbepreparednomorethan2hourspriortotheexperiment.
a.Thetotalvolumeshouldbe:0.1ml/wellx(thenumberofwells).(Allowing0.1-0.2mlmorethantotalvolume)
b.Biotinylatedanti-humanAdiponectinantibodyshouldbedilutedin1:100withtheantibodydiluentbufferandmixedthoroughly.(i.e.Add1μlBiotinylatedanti-humanAdiponectinantibodyto99μlantibodydiluentbuffer.)
C.PreparationofAvidin-Biotin-PeroxidaseComplex(ABC)workingsolution:Thesolutionshouldbepreparednomorethan1hourpriortotheexperiment.
a.Thetotalvolumeshouldbe:0.1ml/wellx(thenumberofwells).(Allowing0.1-0.2mlmorethantotalvolume)
b.Avidin-Biotin-PeroxidaseComplex(ABC)shouldbedilutedin1:100withtheABCdilutionbufferandmixedthoroughly.(i.e.Add1μlABCto99μlABCdiluentbuffer.)
AssayProcedure
TheABCworkingsolutionandTMBcolordevelopingagentmustbekeptwarmat37°Cfor30minbeforeuse.Whendilutingsamplesandreagents,theymustbemixedcompletelyandevenly.StandardAdiponectindetectioncurveshouldbepreparedforeachexperiment.TheuserwilldecidesampledilutionfoldbycrudeestimationofAdiponectinamountinsamples.
1.Aliquot0.1mlperwellofthe100ng/ml,50ng/ml,25ng/ml,12.5ng/ml,6.25ng/ml,3.12ng/ml,1.56ng/mlhumanAdiponectinstandardsolutionsintotheprecoated96-wellplate.Add0.1mlofthesamplediluentbufferintothecontrolwell(Zerowell).Add0.1mlofeachproperlydilutedsampleofhumancellculturesupernates,serumorplasma(heparin,EDTA)toeachemptywell.See“SampleDilutionGuideline”abovefordetails.ItisrecommendedthateachhumanAdiponectinstandardsolutionandeachsamplebemeasuredinduplicate.
2.Sealtheplatewiththecoverandincubateat37°Cfor90min.
3.Removethecover,discardplatecontent,andblottheplateontopapertowelsorotherabsorbentmaterial.DoNOTletthewellscompletelydryatanytime.
4.Add0.1mlofbiotinylatedanti-humanAdiponectinantibodyworkingsolutionintoeachwellandincubatetheplateat37°Cfor60min.
5.Washplate3timeswith0.01MTBSor0.01MPBS,andeachtimeletwashingbufferstayinthewellsfor1min.Discardthewashingbufferandblottheplateontopapertowelsorotherabsorbentmaterial.(PlateWashingMethod:Discardthesolutionintheplatewithouttouchingthesidewalls.Blottheplateontopapertowelsorotherabsorbentmaterial.Soakeachwellwithatleast0.3mlPBSorTBSbufferfor1~2minutes.RepeatthisprocesstwoadditionaltimesforatotalofTHREEwashes.Note:Forautomatedwashing,aspirateallwellsandwashTHREEtimeswithPBSorTBSbuffer,overfillingwellswithPBSorTBSbuffer.Blottheplateontopapertowelsorotherabsorbentmaterial.)
6.Add0.1mlofpreparedABCworkingsolutionintoeachwellandincubatetheplateat37°Cfor30min.
7.Washplate5timeswith0.01MTBSor0.01MPBS,andeachtimeletwashingbufferstayinthewellsfor1-2min.Discardthewashingbufferandblottheplateontopapertowelsorotherabsorbentmaterial.(SeeStep5forplatewashingmethod).
8.Add90μlofpreparedTMBcolordevelopingagentintoeachwellandincubateplateat37°Cindarkfor
25-30min(Note:Forreferenceonly,theoptimalincubationtimeshouldbedeterminedbyenduser.AndtheshadesofbluecanbeseeninthewellswiththefourmostconcentratedhumanAdiponectinstandardsolutions;theotherwellsshownoobviouscolor).
9.Add0.1mlofpreparedTMBstopsolutionintoeachwell.Thecolorchangesintoyellowimmediately.
10.ReadtheO.D.absorbanceat450nminamicroplatereaderwithin30minafteraddingthestopsolution.
Forcalculation,(therelativeO.D.450)=(theO.D.450ofeachwell)–(theO.D.450ofZerowell).ThestandardcurvecanbeplottedastherelativeO.D.450ofeachstandardsolution(Y)vs.therespectiveconcentrationofthestandardsolution(X).ThehumanAdiponectinconcentrationofthesamplescanbeinterpolatedfromthestandardcurve.
Note:ifthesamplesmeasuredwerediluted,multiplythedilutionfactortotheconcentrationsfrominterpolationtoobtaintheconcentrationbeforedilution.
Summary
1.Addsamplesandstandardsandincubatetheplateat37°Cfor90min.Donotwash.
2.Addbiotinylatedantibodiesandincubatetheplateat37°Cfor60min.Washplate3timeswith0.01MTBS.
3.AddABCworkingsolutionandincubatetheplateat37°Cfor30min.Washplate5timeswith0.01MTBS.
4.AddTMBcolordevelopingagentandincubatetheplateat37°Cindarkfor25-30min.
5.AddTMBstopsolutionandread.
1.Yamauchi,T.;Kamon,J.;Minokoshi,Y.;Ito,Y.;Waki,H.;Uchida,S.;Yamashita,S.;Noda,M.;Kita,S.;Ueki,K.;Eto,K.;Akanuma,Y.;Froguel,P.;Foufelle,F.;Ferre,P.;Carling,D.;Nagai,R.;Kimura,S.;Kahn,B.B.;Kadowaki,T.:Adiponectinstimulatesglucoseutilizationandfatty-acidoxidationbyactivatingAMP-activatedproteinkinase.NatureMed.8:1288-1295,2002.
2.Yokota,T.;Oritani,K.;Takahashi,I.;Ishikawa,J.;Matsuyama,A.;Ouchi,N.;Kihara,S.;Funahashi,T.;Tenner,A.J.;Tomiyama,Y.;Matsuzawa,Y.:Adiponectin,anewmemberofthefamilyofsolubledefensecollagens,negativelyregulatesthegrowthofmyelomonocyticProgenitorsandthefunctionsofmacrophages.Blood96:1723-1732,2000.
3.Takemura,Y.;Ouchi,N.;Shibata,R.;Aprahamian,T.;Kirber,M.T.;Summer,R.S.;Kihara,S.;Walsh,K.:Adiponectinmodulatesinflammatoryreactionsviacalreticulinreceptor-dependentclearanceofearlyapoptoticbodies.J.Clin.Invest.117:375-386,2007.
