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商品介绍
ForquantitativedetectionofhumanANGPTL3incellculturesupernates,serumandplasma(heparin,EDTA).
TypicalDataObtainedfromHumanANGPTL3
(TMBreactionincubateat37°Cfor15min)
Concentration(pg/ml) | 0 | 312 | 625 | 1250 | 2500 | 5000 | 10,000 | 20,000 |
O.D | 0.096 | 0.220 | 0.286 | 0.505 | 0.814 | 1.279 | 1.970 | 2.589 |
TypicalHumanANGPTL3ELISAKitStandardCurve
ThisstandardcurvewasgeneratedatEtonfordemonstrationpurposeonly.Astandardcurvemustberunwitheachassay.
Range312pg/ml-20,000pg/ml
Sensitivity<10pg/ml
SpecificityNaturalandrecombinanthumanANGPTL3
Cross-reactivityNodetectablecross-reactivitywithotherrelevantproteins
Storage
Storeat4°Cfor6months,at-20°Cfor12months.Avoidmultiplefreeze-thawcycles(Shippedwithwetice.)
Precision
Intra-AssayPrecision(Precisionwithinanassay)Threesamplesofknownconcentrationweretestedononeplatetoassessintra-assayprecision.
Inter-AssayPrecision(Precisionbetweenassays)Threesamplesofknownconcentrationweretestedinseparateassaystoassessinter-assayprecision.
Intra-AssayPrecision | Inter-AssayPrecision | |||||
Sample | 1 | 2 | 3 | 1 | 2 | 3 |
n | 16 | 16 | 16 | 24 | 24 | 24 |
Mean(ng/ml) | 3.2 | 5.8 | 10.3 | 3.5 | 6.2 | 12.1 |
Standarddeviation | 0.144 | 0.273 | 0.299 | 0.238 | 0.459 | 0.762 |
CV(%) | 4.5 | 4.7 | 2.9 | 6.8 | 7.4 | 6.3 |
Principle
Eton’shumanANGPTL3ELISAKitwasbasedonstandardsandwichenzyme-linkedimmune-sorbentassaytechnology.AmonoclonalantibodyfrommousespecificforANGPTL3hasbeenprecoatedonto96-wellplates.Standards(sf21,S17-E460)andtestsamplesareaddedtothewells,abiotinylateddetectionpolyclonalantibodyfromgoatspecificforANGPTL3isaddedsubsequentlyandthenfollowedbywashingwithPBSorTBSbuffer.Avidin-Biotin-PeroxidaseComplexwasaddedandunboundconjugateswerewashedawaywithPBSorTBSbuffer.HRPsubstrateTMBwasusedtovisualizeHRPenzymaticreaction.TMBwascatalyzedbyHRPtoproduceabluecolorproductthatchangedintoyellowafteraddingacidicstopsolution.ThedensityofyellowisproportionaltothehumanANGPTL3amountofsamplecapturedinplate.
KitComponents
Description | Quantity |
96-wellplateprecoatedwithanti-humanANGPTL3antibody | 1 |
LyophilizedrecombinanthumanANGPTL3standard | 20ng/tube×2 |
Biotinylatedanti-humanANGPTL3antibody | 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.
Preparationof0.01MPBS:Add8.5gsodiumchloride,1.4gNa2HPO4and0.2gNaH2PO4to1000mldistilledwaterandadjustpHto7.2-7.6.Finally,adjustthetotalvolumeto1L.
NoticeforApplicationofKit
1.Toinspectthevalidityofexperimentoperationandtheappropriatenessofsampledilutionproportion,pilotexperimentusingstandardsandasmallnumberofsamplesisrecommended.
2.TheTMBColorDevelopingagentiscolorlessandtransparentbeforeusing,contactusfreelyifitisnotthecase.
3.BeforeusingtheKit,spintubesandbringdownallcomponentstothebottomoftubes.
4.Duplicatewellassayisrecommendedforbothstandardandsampletesting.
5.Don’tlet96-wellplatedry,fordryplatewillinactivateactivecomponentsonplate.
6.Don’treusetipsandtubestoavoidcrosscontamination.
7.Avoidusingthereagentsfromdifferentbatchestogether.
8.Inordertoavoidmarginaleffectofplateincubationduetotemperaturedifference(reactionmaybestrongerinthemarginalwells),itissuggestedthatthedilutedABCandTMBsolutionwillbepre-warmedin37°Cfor30minbeforeusing.
Preparation
1.SamplePreparationandStorage
Storesamplestobeassayedwithin24hoursat2-8°C.Forlong-termstorage,aliquotandfreezesamplesat-20°C.Avoidrepeatedfreeze-thawcycles.
Cellculturesupernates:Removeparticulatesbycentrifugation,assayimmediatelyoraliquotandstoresamplesat
-20°C.
Serum:Allowtheserumtoclotinaserumseparatortube(about4hours)atroomtemperature.Centrifugeatapproximately1000Xgfor15min.Analyzetheserumimmediatelyoraliquotandstoresamplesat-20°C.
Plasma:CollectplasmausingheparinorEDTAasananticoagulant.Centrifugefor15minat1500xgwithin30minofcollection.Assayimmediatelyoraliquotandstoresamplesat-20°C.
2.SampleDilutionGuideline
Theuserneedstoestimatetheconcentrationofthetargetproteininthesampleandselectaproperdilutionfactorsothatthedilutedtargetproteinconcentrationfallsnearthemiddleofthelinearregimeinthestandardcurve.Dilutethesampleusingtheprovideddiluentbuffer.Thefollowingisaguidelineforsampledilution.Severaltrialsmaybenecessaryinpractice.Thesamplemustbewellmixedwiththediluentsbuffer.
Hightargetproteinconcentration(200-2000ng/ml).Theworkingdilutionis1:100.i.e.Add1μlsampleinto99μlsamplediluentbuffer.
Mediumtargetproteinconcentration(20-200ng/ml).Theworkingdilutionis1:10.i.e.Add10μlsampleinto90μlsamplediluentbuffer.
Lowtargetproteinconcentration(312-20,000pg/ml).Theworkingdilutionis1:2.i.e.Add50μlsampleto50μlsamplediluentbuffer.
VeryLowtargetproteinconcentration(≤312pg/ml).Nodilutionnecessary,ortheworkingdilutionis1:2.
3.ReagentPreparationandStorage
A.ReconstitutionofthehumanANGPTL3standard：ANGPTL3standardsolutionshouldbepreparednomorethan2hourspriortotheexperiment.TwotubesofANGPTL3standard(20ngpertube)areincludedineachkit.Useonetubeforeachexperiment.
a.20,000pg/mlofhumanANGPTL3standardsolution:Add1mlsamplediluentbufferintoonetube,keepthetubeatroomtemperaturefor10minandmixthoroughly.
b.10,000pg/ml→312pg/mlofhumanANGPTL3standardsolutions:Label6Eppendorftubeswith10,000pg/ml,5000pg/ml,2500pg/ml,1250pg/ml,625pg/ml,312pg/mlrespectively.Aliquot0.3mlofthesamplediluentbufferintoeachtube.Add0.3mloftheabove20,000pg/mlANGPTL3standardsolutioninto1sttubeandmix.Transfer0.3mlfrom1sttubeto2ndtubeandmix.Transfer0.3mlfrom2ndtubeto3rdtubeandmix,andsoon.
Note:Thestandardsolutionsarebestusedwithin2hours.The20ng/mlstandardsolutionshouldbestoredat4°Cforupto12hours,orat-20°Cforupto48hours.Avoidrepeatedfreeze-thawcycles.
B.Preparationofbiotinylatedanti-humanANGPTL3antibodyworkingsolution:Thesolutionshouldbepreparednomorethan2hourspriortotheexperiment.
a.Thetotalvolumeshouldbe:0.1ml/wellx(thenumberofwells).(Allowing0.1-0.2mlmorethantotalvolume)
b.Biotinylatedanti-humanANGPTL3antibodyshouldbedilutedin1:100withtheantibodydiluentbufferandmixedthoroughly.(i.e.Add1μlBiotinylatedanti-humanANGPTL3antibodyto99μlantibodydiluentbuffer.)
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.StandardANGPTL3detectioncurveshouldbepreparedforeachexperiment.TheuserwilldecidesampledilutionfoldbycrudeestimationofANGPTL3amountinsamples.
1.Aliquot0.1mlperwellofthe20,000pg/ml,10,000pg/ml,5000pg/ml,2500pg/ml,1250pg/ml,625pg/ml,312pg/mlhumanANGPTL3standardsolutionsintotheprecoated96-wellplate.Add0.1mlofthesamplediluentbufferintothecontrolwell(Zerowell).Add0.1mlofeachproperlydilutedsampleofhumancellculturesupernates,serumorplasma(heparin,EDTA)toeachemptywell.See“SampleDiluti
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