ELISA Illustrated Assay.docx
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ELISA Illustrated Assay.docx
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ELISAIllustratedAssay
ELISAIllustratedAssay
ELISATheoryOverview:
∙ELISAisactuallyanacronym:
EnzymeLinkedImmunoSorbentAssay.
∙SimilartoWesternBlots,antibodiesareusedtodetectthepresenceofproteinsorotherantibodies,knownas'antigens'.
∙UnlikeWesternBlots,theproteinorantibodyis“bound”toawell,andhundredsofsamplescanbeanalyzedquickly.
ELISATypeOverview:
TherearethreemajortypesofELISAs:
Indirect,Sandwich and Competitive
∙Indirect:
theproteinsampleisboundthroughadsorption,directly(andnon-specifically)tothewell.Next,anantibodyisusedtodetectthepresenceofoneoftheproteinscontainedinthesample,knownastheantigen.
∙Sandwich:
a'capture'antibodyisboundtothewellfirst,andwhenthesampleisadded,onlyproteinstheantibodyrecognizesare'captured'.Next,asecond'detection'antibodyisusedtodetecttheboundprotein.The'capture'and'detection'antibodiesarecommonlycalled'matched-pairs'.Finally,athird,enzyme-labeledantibodyisaddedtodetectthe'detection'antibody.
∙Competitive:
aprimaryantibodyisincubatedwiththesample,whichformsacomplex.Thecomplexisthenadsorbedtothewells.Next,asecondaryantibodyisaddedtothewells,whichrecognizestheprimaryantibodyonlyifitisnotboundtotheantigen.Therefore,thesecondaryantibody'competes'withtheantigen.
IndirectELISA
First,samplesareprepared,usuallybyserialdilutionwithPBS.Thesamplescanbecomplexproteinmixturessuchascelllysates,orcontainantibodies/proteinsofinterestinotherformats,suchasbloodaliquotsfromsubjects.
Samplesarethenaddedtothewellsofaplatesuitableforantigenbindingandincubatedsothattheantigenwillbethoroughlyadsorbedtothewellsurface.
Afterincubation,thewellsmustbewashedtoremoveanyunbound,orpoorlyboundantigen.Thisstepisveryimportantandwilloccuraftereveryotherstep.
Next,thewellsmustbefilledwith'blocking'solution,whichisnon-specificproteinthatbindstotheexposedsurfacesinthewellandkeepstheprimaryantibodyfrombindingnon-specificallytothewell.
Followingblocking,theplateiswashedagainandtheprimaryantibody,indilution,isaddedtothewell.Theantibodywillonlyrecognizeoneantigen,ideally.Theprimaryantibodyisusuallyaddedinarangeofdilutions,andeachdilutionisusuallytestedinduplicateortriplicate.
Afterprimaryantibodyincubation,anotherseriesofwashesareperformedandthenconjugatedsecondaryantibodyisaddedtoeachwell,ataconstantdilution.
Finally,thewellsarewashedagain,andtheenzyme-conjugatedsecondaryantibodyiscausedtoreact,givingoffasignal,whichisreadona'platereader'.The'substrate'usedvariesaccordingtotheenzymethesecondaryantibodyisconjugatedto.
IndirectELISAResults
∙Thepresenceofasignalfromthesecondaryantibodymeanstheantigenofinterestispresent.
∙Inordertodetermineantigenconcentration,astandardcurveofknownantigenconcentrationmustberunonthesameplate.
∙Negativecontrolsshouldalsoberuntomakesureyourantibodiesarebindingspecificallytotheantigenonly.
IndirectELISAPros/Cons
∙ThistypeofELISAiscommonlyusedtodeterminetheidealconcentration/dilutionofprimaryantibodytouseinotherexperiments.
∙ThisisdonebyrunningtheELISAagainstaknownconcentrationofantigen,andperformingserialdilutionsoftheprimaryantibody.
∙Thedilutionsthatresultinasignal,showtherangetheprimaryantibodyiseffectivein,accordingtotheconcentrationoftheantigen.
∙Becausethedesiredantigenmaybepresentinextremelysmallquantitiesrelativetothepresenceofotherproteins,andbecauseallproteinsinasamplewillbindtothewell,IndirectELISAmaybeunabletodetectthepresenceofantigeninasample.
∙Inthiscase,theantigenisusuallypurifiedoutofthesample,sothatitcanbemorereadilydetected.
∙TheprimaryandsecondaryantibodiesusedinthistypeofELISAcanalsobeusedinthesamedilutionsagainstthesamesamplesinWesternBlot,thereforeeliminatingtheneedtopurchaseandtestotherantibodies.
SandwichELISA
First,thesamplesareprepared,usuallybyserialdilutionwithPBS.Thesamplescanbecomplexproteinmixturessuchascelllysates,orcontainantigensofinterestinotherformats,suchasbloodaliquotsfromsubjects.
The'capture'antibody,alsoknownasthefirstprimaryantibody,isaddedtoeachwell,andincubated,toallowtheantibodytoadsorbtothesurfaceofthewell.
Afterincubationofthecaptureantibody,thewellsarewashedtoremoveanyunboundantibody.
Next,'blocking'solutionisadded.Thisisnon-specificproteinthatbindstotheopensitesinthewellandkeepsthenon-specificproteinsinthesamplefrombindingtothewell.
Next,samplesofunknownsareaddedtoeachantibody-coatedwellandagainallowedtoincubateandbindtothecaptureantibody.
Afteranotherwashcycle,the'detection'antibodyisaddedtothewellsandallowedtoincubateanddetecttheantigen.Thedetectionantibodymustbea'matched-pair'withthecaptureantibody,tomakesurethattheantibodiesdon'trecognizeeachother,orthesamesiteontheantigenofinterest.
Afteranotherwash,theenzyme-linkedantibody(sometimesreferredtoasasecondary,eventhoughitistertiaryinthiscase)isaddedtoeachwellanditdetectsthe'detection'antibody.
Finally,anotherwashcycleisperformed,andtheenzymeonthetertiaryantibodyisreacted,togiveoffasignal,whichcanbereadonaplatereader.
SandwichELISAResults
∙Thepresenceofsignalmeanstheantigenofinterestispresent.
∙AstandardcurvemustbeincludedonthesameplatetomakethisELISAquantitative.
∙Negativecontrolsshouldalsoberuntomakesureonlyspecificbindingisoccurringamongthemanyantibodiesused.
SandwichELISAPros/Cons
∙UnlikeIndirectELISAs,antigensofveryloworunknownconcentrationinthesamplecanbedetectedbecausethe'capture'antibodyonlygrabstheantigenofinterestandalltheotherproteinsinthesamplearewashedaway.
∙Itisnotnecessarytouseatertiaryenzyme-linkedantibody,ifthe'detection'antibodyisalreadyenzyme-linked.However,itcanbeverydifficult,ifnotimpossibletofindamatched-pairwherethe'detection'antibodyisalreadyconjugated.
∙Theend-usercanconjugatethe'detection'antibodythemselves,ifsoinclined.
∙Onlymonoclonalantibodiescanbeusedasmatchedpairs,becauseonlymonoclonalsrecognizeonespecificsiteonanantigen(knownasthe'epitope').
∙Monoclonalantibodiescanbemoreexpensivethanpolyclonalantibodiesandmatched-pairantibodiescanbeverydifficulttofind.
NovusBiologicalscanhelpyoufindmatched-pairantibodies,ifyoucan'tfindwhatyouarelookingfor!
Email:
novus@
CompetitiveELISA
Tobeginwith,thesamplesarepreparedbyincubatingtheprimaryantibodywiththesamplesintubes,wheretheantigenofinterestformsa'complex'withtheantibody.
Thesampleisaddedtothewell,andallowedtoincubatesothatitadsorbstothesurfaceofthewell.The'complex',anyunboundantibody,andotherproteinscanalladsorb.
Aftersampleincubation,thewellsarewashedtoremoveanyunboundproteinorantibody/antigen'complex'.
Thewellsarethencoatedwithblockingsolution,whichwillkeepthesecondaryantibodyfromnon-specificallybindingtothewells.
Afteranotherwashcycle,theconjugatedsecondaryantibodyisallowedtoincubateand'compete'withtheantigenofinterestforcontinuedbindingtotheprimaryantibody.
Afterafinalwashcycle,theconjugatedsecondaryenzymeisreactedtoproduceasignal,whichcanbereadonaplatereader.
CompetitiveELISAResults
∙Thestrengthofthesignalisinverselyrelatedtothequantityofantigenpresent.Themoreantigenpresent,themoredifficultitisforthesecondaryantibodytobindtotheprimaryantibodyandviceversa.
∙JustasinotherELISAs,knownstandardscanberuntodetermineconcentration,butremembertheinverserule.
∙LikeSandwichELISA,thisformofELISAcandetectsmallerquantitiesofantigenpresentthanIndirectELISAscan.
∙ThistypeofELISAdoesnotrequiretheuseofmatched-pairantibodies,asSandwichELISAsdo.
∙Insteadofaconjugatedsecondaryantibodybeingusedasthecompetitor,anotherconjugatedantigencanbeusedthattheprimaryantibodyalsorecognizes.
∙Inotherwords,useaproteinthattheprimaryantibodyalsorecognizes,thatisnotthesameastheantigenofinterest.
∙Thebenefitofusingthismethodisthatyoudonothavetouseasecondaryantibody.
∙Thedisadvantageisthatyoumayhavedifficultyfindinganotherproteinyourprimaryantibodyrecognizes,andyouwillalsolikelyhavetoconjugatetheproteinyourself.
∙Theprimaryantibodyusedcanbeunpurified,andpolyclonal.
∙TheantigenofinterestthatisidealforthistypeofELISAcontainsonlyonerecognizableepitopebytheprimaryantibody.
CommonProblems
∙Thenegativecontrolscangivepositiveresultswhentheblockingsolutionisn'teffective,thereforethesecondaryantibodyorantigenofinterestcanbindtotheopensitesinthewell.
∙Ifthepositivecontrolsorstandardsgivenosignal,checkyourchemicalsandbeawarethattheenzymereactionisshort-term,sotheplateshouldbereadasquicklyaspossible.
∙Runningyoursamplesinduplicateandtriplicatewillallowforamoreaccuratedeterminationofconcentration.
∙Applyingyourprimaryantibodyinadilutionrangeincreasesthelikelihoodthatyouwillgetasignalthatisneithertooweaknortoostrong.
∙Pastacertainlimit,thes
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- ELISA Illustrated Assay