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:Collectplasmausingheparinasananticoagulant.Centrifugefor15minat1500xgwithin30minofcollection.Assayimmediatelyoraliquotandstoresamplesat-20°C.
Saliva:CollectsalivausingacollectiondevicewithoutanyproteinbindingorfilteringcapABIlitiessuchasaSalivetteoraliquotandstoresamplesat-20°C.
2.SampleDilutionGuideline
Theuserneedstoestimatetheconcentrationofthetargetproteininthesampleandselectaproperdilutionfactorsothatthedilutedtargetproteinconcentrationfallsnearthemiddleofthelinearregimeinthestandardcurve.Dilutethesampleusingtheprovideddiluentbuffer.Thefollowingisaguidelineforsampledilution.Severaltrialsmaybenecessaryinpractice.Thesamplemustbewellmixedwiththediluentsbuffer.
Hightargetproteinconcentration(100-1000ng/ml).Theworkingdilutionis1:100.i.e.Add1μlsampleinto99μlsamplediluentbuffer.
Mediumtargetproteinconcentration(10-100ng/ml).Theworkingdilutionis1:10.i.e.Add10μlsampleinto90μlsamplediluentbuffer.
Lowtargetproteinconcentration(156-10,000pg/ml).Theworkingdilutionis1:2.i.e.Add50μlsampleto50μlsamplediluentbuffer.
VeryLowtargetproteinconcentration(≤156pg/ml).Nodilutionnecessary,ortheworkingdilutionis1:2.
3.ReagentPreparationandStorage
A.ReconstitutionofthehumanACEstandard:ACEstandardsolutionshouldbepreparednomorethan2hourspriortotheexperiment.TwotubesofACEstandard(10ngpertube)areincludedineachkit.Useonetubeforeachexperiment.
a.10,000pg/mlofhumanACEstandardsolution:Add1mlsamplediluentbufferintoonetube,keepthetubeatroomtemperaturefor10minandmixthoroughly.
b.5000pg/ml→156pg/mlofhumanACEstandardsolutions:Label6Eppendorftubeswith5000pg/ml,2500pg/ml,1250pg/ml,625pg/ml,312pg/ml,156pg/mlrespectively.Aliquot0.3mlofthesamplediluentbufferintoeachtube.Add0.3mloftheabove10,000pg/mlACEstandardsolutioninto1sttubeandmix.Transfer0.3mlfrom1sttubeto2ndtubeandmix.Transfer0.3mlfrom2ndtubeto3rdtubeandmix,andsoon.
Note:Thestandardsolutionsarebestusedwithin2hours.The10ng/mlstandardsolutionshouldbestoredat4°Cforupto12hours,orat-20°Cforupto48hours.Avoidrepeatedfreeze-thawcycles.
B.Preparationofbiotinylatedanti-humanACEantibodyworkingsolution:Thesolutionshouldbepreparednomorethan2hourspriortotheexperiment.
a.Thetotalvolumeshouldbe:0.1ml/wellx(thenumberofwells).(Allowing0.1-0.2mlmorethantotalvolume)
b.Biotinylatedanti-humanACEantibodyshouldbedilutedin1:100withtheantibodydiluentbufferandmixedthoroughly.(i.e.Add1μlBiotinylatedanti-humanACEantibodyto99μlantibodydiluentbuffer.)
C.PreparationofAvidin-Biotin-PeroxidaseComplex(ABC)workingsolution:Thesolutionshouldbepreparedno
morethan1hourpriortotheexperiment.
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.StandardACEdetectioncurveshouldbepreparedforeachexperiment.TheuserwilldecidesampledilutionfoldbycrudeestimationofACEamountinsamples.
1.Aliquot0.1mlperwellofthe10,000pg/ml,5000pg/ml,2500pg/ml,1250pg/ml,625pg/ml,313pg/ml,156pg/mlhumanACEstandardsolutionsintotheprecoated96-wellplate.Add0.1mlofthesamplediluentbufferintothecontrolwell(Zerowell).Add0.1mlofeachproperlydilutedsampleofhumancellculturesupernatants,serum,plasma(heparin)orsalivatoeachemptywell.See“SampleDilutionGuideline”abovefordetails.WerecommendthateachhumanACEstandardsolutionandeachsampleismeasuredinduplicate.
2.Sealtheplatewiththecoverandincubateat37°Cfor90min.
3.Removethecover,discardplatecontent,andblottheplateontopapertowelsorotherabsorbentmaterial.DoNOTletthewellscompletelydryatanytime.
4.Add0.1mlofbiotinylatedanti-humanACEantibodyworkingsolutionintoeachwellandincubatetheplateat37°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
20-25min(Note:Forreferenceonly,theoptimalincubationtimeshouldbedeterminedbyenduser.AndtheshadesofbluecanbeseeninthewellswiththefourmostconcentratedhumanACEstandardsolutions;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).ThehumanACEconcentrationofthesamplescanbeinterpolatedfromthestandardcurve.
Note:ifthesamplesmeasuredwerediluted,multiplythedilutionfactortotheconcentrationsfrominterpolationtoobtaintheconcentrationbeforedilution.
Summary
1.Addsamplesandstandardsandincubatetheplateat37°Cfor90min.Donotwash.
2.Addbiotinylatedantibodiesandincubatetheplateat37°Cfor60min.Washplate3timeswith0.01MTBS.
3.AddABCworkingsolutionandincubatetheplateat37°Cfor30min.Washplate5timeswith0.01MTBS.
4.AddTMBcolordevelopingagentandincubatetheplateat37°Cindarkfor20-25min.
5.AddTMBstopsolutionandread.
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2.Ramaraj,P.;Kessler,S.P.;Colmenares,C.;Sen,G.C.Selectiverestorationofmalefertilityinmicelackingangiotensin-convertingenzymesbysperm-specificexpressionofthetesticularisozyme.J.Clin.Invest.102:371-378,1998.
