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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.

NoticeforApplicationofKit

Preparationof0.01MPBS: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.

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