TheassayisbasedonthesandwichELISAtechniquewhereamonoclonalantibody(mab)specificforaconformationalepitopeonassembledAAVcapsidsiscoatedontotheplateandisusedtocaptureAAVparticlesfromthespecimen.

ThedetectionofcapturedAAVparticlesisatwo-stepprocess.

1. Abiotin-conjugatedmabisboundtothecapturedAAVparticles.
2. Astreptavidinperoxidaseconjugatereactswiththebiotinmolecules.Theadditionofthesubstrateresultsinacolorreactionwhichisproportionaltotheamountofspecificallyboundviralparticles.

ComparisonofAAVQuantificationMethods: 

Eachofthecommonlyusedquantificationmethodshasitsprosandcons:

• qPCR iswidelyused,butsuffersfromseveralissuessuchassamplepreparation,primerdesign,orPCRefficiencythatcanleadtohighinter-laboratoryvariationofresults.
• DigitaldropletPCR methodsovercomesomeofthelimitationsofqPCR.However,variationsbetweenlabscanstilloccurduetodifferentsampleprocessingprotocols.
• Dotblot isasimpleandquantitativemethod,ifreliablereferencematerialisused.However,itsuffersfromthelimitedlinearityanddynamicrangeofwesternblottingingeneral.

Giventhepracticaldrawbacksoftheaforementionedtechniques,aconventionalsandwichELISAcurrentlyappearstobesuperiorintermsofinter-andintralaboratoryvariationaswellaseaseofuse.Therefore,itrepresentsthebestformatforreliableandreproduciblequantificationoftotalrAVVcapsidtiters.

Useofshuffled/mutatedAAV:

Therecognitionofshuffled/mutatedAAVvectorsdependonthespecificcapsidregionwhichisaffectedbytheshuffling/mutation.ThecaptureantibodiesusedforProgen’sAAVELISAsbindspecificand,insomecases,welldefinedconformationalepitopes.TheseepitopesaregeneratedbythecapsidassemblyofthecorrespondingAAVSEROtypes.AfirstindicationthattheELISAmightrecognizeyourshuffled/mutatedAAVvectoristhepresenceoftheantibody-bindingepitope.However,changesintheproteinsequencesofthecapsidproteinsmightalsoinfluenceconformationoftheproteins,hencetheconformationoftheepitopespresentedontheAAVcapsid.Thismightinfluencebindingaffinityoftheantibodyandaffectdeterminationofthetiterbasedonthe(non-shuffled)KitControlprovidedwiththeAAVELISAkit.Sincethesepropertiesstronglydependonthespecificshuffling/mutationperformed,PROGENcannotguaranteesuccessfulandprecisequantificationofyourshuffled/mutatedAAVvector.Eveniftheantibodybindingepitopesarestillpresentonyourshuffled/mutatedAAVcapsid,yourassayneedstobetestedandoptimizedforyourspecificAAVvector.

PROGENhighlyrecommendstheproductionandcalibrationofasuitable(shuffled/mutated)KitControl,toensurereliabletiterdeterminationofyourindividuallyshuffled/mutatedAAVvectorwithPROGENELISAkits.

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