ForquantitativedetectionofhumanA2Mincellculturesupernates,serumandplasma(heparin,EDTA).
TypicalDataObtainedfromHumanA2M
(TMBreactionincubateat37°Cfor20min)
Concentration(pg/ml) | 0.0 | 625 | 1,250 | 2,500 | 5,000 | 10,000 | 20,000 | 40,000 |
| | | | | | | | |
O.D | 0.034 | 0.090 | 0.146 | 0.249 | 0.497 | 0.931 | 1.489 | 2.095 |
| | | | | | | | |
TypicalHumanA2MELISAKitStandardCurve ThisstandardcurvewasgeneratedatEtonfordemonstrationpurposeonly.Astandardcurvemustberunwitheachassay.
Range625pg/ml-40,000pg/ml
Sensitivity<20pg/ml
SpecificityNaturalhumanA2M
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) | 6.18 | 12.04 | 25.21 | 6.72 | 13.68 | 26.81 |
| | | | | | |
Standarddeviation | 0.377 | 0.771 | 0.174 | 0.457 | 0.985 | 2.12 |
| | | | | | |
CV(%) | 6.1 | 6.4 | 6.9 | 6.8 | 7.2 | 7.9 |
| | | | | | |
Principle
Eton’shumanA2MELISAKitwasbasedonstandardsandwichenzyme-linkedimmune-sorbentassaytechnology.AmonoclonalantibodyfrommousespecificforA2Mhasbeenprecoatedonto96-wellplates.Standards(fromhumanplasma)andtestsamplesareaddedtothewells,abiotinylateddetectionpolyclonalantibodyfromgoatspecificforA2MisaddedsubsequentlyandthenfollowedbywashingwithPBSorTBSbuffer.Avidin-Biotin-PeroxidaseComplexwasaddedandunboundconjugateswerewashedawaywithPBSorTBSbuffer.HRPsubstrateTMBwasusedtovisualizeHRPenzymaticreaction.TMBwascatalyzedbyHRPtoproduceabluecolorproductthatchangedintoyellowafteraddingacidicstopsolution.ThedensityofyellowisproportionaltothehumanA2Mamountofsamplecapturedinplate.
KitComponents
Description | Quantity |
| |
96-wellplateprecoatedwithanti-humanA2Mantibody | 1 |
| |
LyophilizedhumanA2Mstandard | 40ng/tube×2 |
| |
Biotinylatedanti-humanA2Mantibody | 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. 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℃for30minbeforeusing.
Preparation
1.SamplePreparationandStorage
Storesamplestobeassayedwithin24hoursat2-8°C.Forlong-termstorage,aliquotandfreezesamplesat-20°C.Avoidrepeatedfreeze-thawcycles.
Cellculturesupernates:Removeparticulatesbycentrifugation,assayimmediatelyoraliquotandstoresamplesat
-20°C.
Serum:Allowtheserumtoclotinaserumseparatortube(about4hours)atroomtemperature.Centrifugeatapproximately1000Xgfor15min.Analyzetheserumimmediatelyoraliquotandstorefrozenat-70°C.
Plasma:CollectplasmausingheparinorEDTAasananticoagulant.Centrifugefor15minat1000xgwithin30minofcollection.Analyzeimmediatelyoraliquotandstorefrozenat-70°C.Citrateisnotrecommendedastheanticoagulant.
2.SampleDilutionGuideline
Theuserneedstoestimatetheconcentrationofthetargetproteininthesampleandselectaproperdilutionfactorsothatthedilutedtargetproteinconcentrationfallsnearthemiddleofthelinearregimeinthestandardcurve.Dilutethesampleusingtheprovideddiluentbuffer.Thefollowingisaguidelineforsampledilution.Severaltrialsmaybenecessaryinpractice.Thesamplemustbewellmixedwiththediluentsbuffer.
Hightargetproteinconcentration(400-4000ng/ml).Theworkingdilutionis1:100.i.e.Add3μlsampleinto297μlsamplediluentbuffer.
Mediumtargetproteinconcentration(40-400ng/ml).Theworkingdilutionis1:10.i.e.Add25μlsampleinto225μlsamplediluentbuffer.
Lowtargetproteinconcentration(625-40,000pg/ml).Theworkingdilutionis1:2.i.e.Add100μlsampleto100μlsamplediluentbuffer.
VeryLowtargetproteinconcentration(≤625pg/ml).Nodilutionnecessary,ortheworkingdilutionis1:2.
3.ReagentPreparationandStorage
A.ReconstitutionofthehumanA2Mstandard:A2Mstandardsolutionshouldbepreparednomorethan2hourspriortotheexperiment.TwotubesofA2Mstandard(40ngpertube)areincludedineachkit.Useonetubeforeachexperiment.
a.40,000pg/mlofhumanA2Mstandardsolution:Add1mlsamplediluentbufferintoonetube,keepthetubeatroomtemperaturefor10minandmixthoroughly.
b.20,000pg/ml→625pg/mlofhumanA2Mstandardsolutions:Label6Eppendorftubeswith20,000pg/ml,10,000pg/ml,5,000pg/ml,2,500pg/ml,1,250pg/ml,625pg/mlrespectively.Aliquot0.3m
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