ng protein binding in nitrocellulose membranes.docx
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ng protein binding in nitrocellulose membranes.docx
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ngproteinbindinginnitrocellulosemembranes
Troubleshootingproteinbindinginnitrocellulosemembranes
Part1:
Principles
KevinD.Jones
Developersofmembrane-basedassaysshouldhaveafirmgrasponthevariousfactorsthatcaninfluenceproteinbinding—includingthoseinherentinthematerialsandprocessingusedfortheirtests.
Thenumberofmembrane-basedrapidimmunochromatographicdevicesonthemarketiscontinuingtoincreaseataveryquickpace.Majorfactorsthatarecontributingtothisgrowthincludeimprovementsinconjugatetechnologyandagrowingunderstandingamongproductdevelopersofthegeneraldesignprinciplesinvolved.
Althoughtoday'simmunochromatographicdevicescomeinawidevarietyofdesignswithadiverseassortmentofhousings,mostcommerciallyavailabletestsarebasedononeoftwosimpleformats.Themostcommonformatisthelateral-flowordipstickdesign,whichhasbecomefamiliarthroughitsuseinphysician-officeassaysaswellasinover-the-countertests(e.g.,Unipath'sClearBluepregnancytest).Alesswidespreadformatistheflow-throughortransverse-flowdesign,whichrequiresgreateroperatorskillandisthereforeusuallyrestrictedtoprofessionaluse(e.g.,Medmira'srapidHIVscreen).
Figure1.Achievingacrisp,cleartestresult,suchasinthesamplesshownhere,dependsoncorrectbindingofthecapturereagenttothemembrane.
Regardlessoftheformatbeingused,achievingasensitiveandreproducibletestrequiresthemanufacturertohaveanefficientprocedureforapplyingthecapture-linereagent.Companiesinvolvedintherapiddiagnosticindustryhavebeenactiveinpublishinginformationabouthowtooptimizecapture-lineapplication.1–5Thisarticleoffersfurtheraidtoproductdevelopers,discussingthebasicprinciplesinvolvedinapplyingproteincapturelinestonitrocellulosemembranes,andhighlightingsomeofthecommonproblemsthatcanbeencounteredduringthedevelopmentofanimmunochromatographicassay.Becausetheproblemsassociatedwithproteinbindingaremoreprevalentinlateral-flowassays,thisarticlewillfocusespeciallyonissuesrelatingtosuchsystems.
TheImportanceofProteinBinding
Inimmunochromatographicassays,theprimaryfunctionofaproteinappliedtoamembraneistoactasacapturereagentforthetargetanalyteinasample.Becausethetestresultistotallydependentuponachievingagoodbindingofthecapturereagenttothemembrane,theimportanceofachievingahighandconsistentlevelofproteinbindingcannotbeoverstressed(seeFigure1).
Despitetheconsiderableamountofresearchthathasbeenconductedsincenitrocellulosewasfirstusedasaprotein-bindingmembrane,theexactmechanismofthatbindingremainsunknown.6Itisknownthatanumberofforcesareatwork—specifically,hydrophobicinteractions,hydrogenbonding,andelectrostaticinteractions—butaclearunderstandingoftheexacteffectandsignificanceofeachforcehasremainedelusive.Tworeasonablemodelshavebeenproposed.Thefirstmodelsuggeststhatproteinsareinitiallyattractedtoamembranesurfacebyelectrostaticinteraction,whilelong-termattachmentisaccomplishedbyacombinationofhydrogenbondingandhydrophobicinteractions.Althoughextremelydifficulttoprove,thismodeloftheinteractionfitsthepublishedexperimentaldataandisoftentheacceptedmodeofinteraction.1,7–11
Figure2.Problemswithproteinbindingaretypicallyvisibleinthecapturelineofanassay'stestresult,asintheseexamples.
Asecondmodelsuggeststhattheinitialattachmentoftheproteiniscausedbyhydrophobicinteractions,withlong-termbindingaccomplishedbyelectrostaticforces.Thismodelalsoagreeswithmuchofthepublisheddata.However,theelectrostaticpartitionmechanismmaynotprovideafullexplanationforthelong-termstabilityconferredonproteinattachmentbydryingortheuseofanalcoholfixationstep.3,6
Whateverthebalanceofforcesresponsibleforproteinbinding,itiswidelyagreedthatproductdevelopersshouldconsiderallsuchforceswhentheyareseekingtooptimizethebindingofproteinstoaparticularmembrane.Suchconsiderationswillinevitablyhaveimplicationsforboth
theselectionofmaterialstobeused,andthewaysthattheywillbeprocessed.Forinstance,iftheproductdeveloperselectsabufferthattoogreatlyreduceseitherhydrophobicorelectrostaticinteractions,thelevelofproteinbindingcouldbedramaticallyreduced.Similarly,itiswidelyrecognizedthatadequatedryingofthemembraneafterproteinapplicationisanimportantpracticeforensuringthelong-termstabilityoftheprotein–membranebond.1–4,6
Figure3.Aweakcapturelineindicatesthattheamountofproteinboundtothemembraneistoolow.
Themanufacturer'sselectionofmaterialscanhaveaneffectonthebindingofproteinstonitrocellulosemembranes.Materialsthatinterferewithproteinbindingcanbedividedintothreegeneraltypes:
nonspecificproteins,materialsthatinterferewithelectrostaticinteractions,andmaterialsthatinterferewithhydrophobicinteractions.Commonlyusedmaterialsthatreduceproteinattachmentincludethosethatcompeteforbindingsites,suchastheclassicbulkingproteins(e.g.,BSA,animalsera),aswellasthosethatinterferewithhydrogenbonding(e.g.,formamide,urea)andthosethatinterferewithhydrophobicbonding(e.g.,Tween,Triton,orBrij).Man-madepolymerssuchaspolyvinylalcohol(PVA),polyethyleneglycol(PEG),andpolyvinylpyrrolidone(PVP)canalsointerferewithproteinbinding.Theirmodeofactionmaybeacombinationofeffectsthatinhibitoneormoreoftheforcesessentialtoprotein–membranebinding.
Ifaninsufficientamountofproteinbindstothemembrane,oriftheproteindoesnotbondtothemembranewiththenecessarystrength,somesignificantproblemscanarise.Theseproblemsaretypicallyvisibleinthecapturelineofanassay'stestresult(seeFigure2).Iftheamountofproteinboundtothemembraneistoolow,theresultingcapturelinewillbeweakandtestsensitivitywill
bereduced(seeFigure3).Ifbindingisinefficient,theproteincandiffusebeforefinallybecomingimmobilizedonthemembrane.Theresultingcapturelinewillbebroadandweakinsteadofcrispandclear,makingtestresultsdifficulttointerpret.Inextremecaseswherethephysicalattachmentoftheproteintothemembraneistooweak,thepassageofanalyteproteinsandsurfactantsolutionscanactuallywashcapturereagentsoffthemembrane.Insuchcasestheassaywilldisplayabroadline—ornoclearlineatall—againmakingitdifficulttointerpretthetestresults(seeFigure4).
Figure4.Adiffusecapturelinecanresultwhenthecapturereagentiswashedawaybythepassageofanalyteproteinsandsurfactantsolutions.
ProblemssuchastheseareregularlyseenbyproductdevelopersintheIVDindustry,andcansignificantlyslowthedevelopmentofasuccessfulimmunochromatographicassay.Tounderstandhowtogoaboutresolvingsuchproblems,developersshouldfirsthaveafirmgrasponthevariousfactorsthatcaninfluenceprotein–membranebinding,includingthoseinherentinthematerialsandprocessingusedfortheirtests.Theseelementswillbediscussedinthefirstinstallmentofthisarticle.Typicaltechniquesforsolvingsuchproblemswillbegiveninthesecondinstallment,whichwillappearinafutureissueofIVDTechnology.
FactorsThatInfluenceProteinBinding
Wheninvestigatingthebindingofproteincapturereagentstonitrocellulosemembranes,productdevelopersshouldconsidereachofthefollowingfivecriticalareasthatcanhaveaneffectonthebindingmechanism.
∙Theapplicationbufferinwhichthecapturereagentisdissolved.
∙Themembranetowhichthecapturereagentisapplied.
∙Thecapturereagentitself.
∙Thesystemusedforapplyingtheproteintothemembrane.
∙Theambienthumidityatthetimeofproteinapplication.
Althoughmanydevelopmentlabsdoagoodjobofstudyingandcharacterizingtheapplicationbuffersandmembranesusedintheirtests,theyarelesslikelytofullyinvestigateoroptimizethecapturereagentsandapplicationsystemstheyemploy.Suchanomissionisoftenduetothefactthatthelatterelementsarefrequentlyconsideredsetevenbeforethebeginningofthedevelopmentprocess,leavinglittleopportunityforchangestobemade.Withthosefactorsoutofconsideration,productdevelopersoftenhavenochoicebuttofocusonoptimizingtheotherelementsthatarestillwithintheirdiscretion.
CaptureReagents.Theproteinsusedascapturereagentsvaryfromtesttotest.Howeversubtletheirdifferences,nosinglecapturereagentisabsolutelyidenticaltoanother.Perhapsmoreimportant,differentproteinsexhibitvaryinglevelsofattachmenttodifferentmembranes(seeFigure5).5Theprocessofoptimizingbindingismoststraightforwardwithamonoclonalantibody,wheretheproteinisahomogeneousmaterial.Optimizationismoredifficultinthecaseofpolyclonalantibodiesbecausethereareavarietyofepitopespresent,andideallyeachrequiresslightlydifferentbindingconditions.SpeciessuchasIgAorIgMcanpresentanevengreaterchallengebecauseofthepotentialforstructuralorstericproblems.OtherproteinssuchasBSA,proteinA,orproteinGcancausesignificantdifficultiesdueeithertotheirchemistryortheirsize(largemoleculesaremorelikelytoremainattachedtoasolidphasethansmallerones).
ApplicationEquipment.Althoughsystemsusedtoapplycapturereagentscanalsopresentproblems,mostcommerciallyavailableequipmenthasbothadvantagesanddisadvantages.Variablescanincludetheabilityorinabilitytodispensemea
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