ELISA Inhibition Assay
来自 : 蚂蚁淘
Sensitizea96-wellmicrotiterplatewithpurifiedantigen.- Prepareasolutionofthepurifiedantigenofinterestinphosphatebuffer(seerecipebelow)suchthataconcentrationofapproximately10µg/mlisachieved.Pipette100µlofthissolutiontoallwellsoftheplatewiththeexceptionofthoseintheoutermostrowsandcolumns(theseareasoftheplatearepronetoevaporation).
- CovertheplatewithParafilmandincubatefor2hoursat37°C,followedbyanovernightincubationat4°C.Covertheplatetightlytoavoidevaporation.Storeat4°C.Theplate(s)willremainusableforaboutonemonth.
- Blockthesensitizedplate.
- Whenreadytouse,discardtheantigensolutioncontainedinthewellsofthesensitizedplate.Pattheinvertedplatesharplyseveraltimesagainstapadofpapertowelstoinsurecompleteremovalofthesolution.
- Washthewellsthreetimeswith1xPBS/0.05%-Tween20.Pattodrycompletelyafterthefinalwash,asabove.
- Pipette100µlof5%skimmilk/PBS/0.005%thimerasol(addedtoretardbacterialgrowth)toeachwell.Incubatetheplate,coveredwithParafilm,at37°Cfor2hourstoovernight.Itisconvenientduringthisincubationsteptobeginpreparationofthesamplestobetested,outlinedbelow.
- WashthewellsthreetimeswithPBS/Tween,dryasbeforeafterthelastwash.
- Preparethesamplestobetested.
- UsingaSharpieorothersuitablepermenantMarker,markasecond,unsensitizedmicrotiterplatetoidentifytheintendedlocationofallsamplestobetested,andthelocationofthepositiveandnegativecontrols(thepositivecontrolinthisassayisapurifiedsampleoftheantigenofinterest;thesameantigenwithwhichthepreviousplatehasbeensensitized.ThenegativecontrolisPBS).Donotusetheoutermostwellsoftheplate,asthesehavenotbeensensitizedinthepreviousplate.Seediagram.
- Pipette60µlof5%skimmilk/PBS/Tweentoallexperimentalwellsexceptthefirstcolumnofthesewells,whichwillcontainundilutedaliquotsofthesamplestobetested.
- Add60µlofthesamplestobetestedtoboththefirst(empty)andsecondwellsoftheexperimentalareaoftheplate.Mixthe1:2dilution(secondwell)bypipettingupanddownseveraltimes.
- Usingafreshpipettetip,transfer60ulfromwell2(1:2dilution)towell3,creatinga1:4dilutioninthiswell.Mixbypipettingseveraltimes.Seriallydilutethesamplesinthismanneruntilalldesireddilutionsareachieved.Itiscriticallyimportanttouseafreshpipettetipforeachtransfer.
- Place60µlofPBSinthenegativecontrolwellsand60µlofthepurifiedantigensampleinthepositivecontrolwells.
- Diluteantiseratotheantigenofinterestappropriatelyin5%skimmilk/PBS/Tween(i.e.1:1000,optimaldilutionmayvarydependingonantigen-antisera.Finaldilutionunderexperimentalconditionswillbedoubled)andpipette60µlofthedilutedantiseratoallwells(experimentalandcontrol).
- Incubatetheplate,coveredwithParafilmtominimizeevaporation,at37°Cfor2hourstoovernight.
- Testingthesamples.
- RetrievethesensitizedandblockedmicrotiterplatepreparedinsectionsA&B,above.Duplicateexactlythepatterndrawnonthenon-sensitizedplateonthisplate.
- Transfer100µloftheantisera-antigensolutionsfromthenon-sensitizedplatetotheircorrespondingwellsonthesensitized,blockedplate.Incubatethisplate,covered,at37°Cfor1.5hours.
- Discardthesolutionsfromthewellsoftheplateafterincubation.WashallwellsthreetimeswithPBS/Tweenasbefore.Dryafterlastwash.
- Addtoeachwell100µlofconjugateantibody(i.e.goatantirabbitalkalinephosphatase,ifantiserawasraisedinrabbits)dilutedappropriatelyinskimmilk/PBS/Tween(i.e.1:2000).Incubatethecoveredplateat37°Cfor1.5hours.
- Discardtheconjugateantibodysolutionfromthewells.WashthreetimeswithPBS/Tweenasbefore.
- Addtoeachwell100µlofsubstratesolutionappropriatefortheconjugateantibodyused.Foralkalinephosphataseconjugate,usep-nitrophenylphosphatedisodium(Sigma104)dissolvedinsubstratebuffer(795mgNa2CO3,1.456gNaHCO3,100mgNaN3,50mgMgCl2inafinalvolumeof500mlsdH2O).
- Readabsorbencevaluesatappropriatetimeintervals(usuallyevery15minutes,ashorterintervalisrequiredifthecolorreactionisquicker;watchcolordevelopment).
PhosphateBufferSkimMilkSolutionsSolution1Solution250.0gskimmilkpowderNa2HPO42.84gNaH2PO42.76g100.0ml10xPBSdH2Oto100mldH2Oto100ml1.0ml5%thimerosalsteriledH2Otofinalvolumeof1LCombine6.1mlofsolution1and3.9mlofsolution2.Add40mldH2O.Dividesolutioninto500mlaliquots.Toonealiquot,add250µlTween-20.Pasturizeat65°Cfor30minutes.
10xPhosphateBufferedSaline(PBS)NaCl80.0gKCl2.0gNa2HPO414.4gKH2PO42.4gDissolvereagentsin800mldH20.AdjustpHto7.4withHCl.Autoclavetosterilize.Diluteto1xbeforeuse.Add250µlTweento500ml1xPBSforthePBS/Tweenwashsolution.
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