ng protein binding in nitrocellulose membranes.docx

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