Antibodiestosmallpeptideshavebecomeanessentialtoolinlifescienceresearch,withapplicationsincludinggeneproductdetectionandidentification,proteinprocessingstudies,diagnostictests,proteinlocalization,activesitedetermination,proteinhomologystudiesandproteinpurification.Whileitisquiteeasytogenerateanti-peptideantibodies,itisimportanttocarefullyconsidertheultimateusefortheantibodyandthesequenceusedtoensuresuccess.Thistechsheetwillbrieflyexplorepeptideselectionanddesign,couplingstrategy,andcarrierproteinswhichareimportantfactorsinanti-peptideantiserageneration.Serumpurificationwillalsobediscussed.Formorecompletecoverageofantigendesign,pleaserefertotheReferences1,2.

PeptideSelectionandDesign

Thefirststepintheprocessistheselectionoftheappropriatepeptidesequence.Atthissteptheultimateusefortheantibodymustbeconsidered.Iftheantibodyisneededtoprobeaspecificproteindomainthenthechoiceissimple.Forexample,ifoneisstudyingproteolyticprocessingofanN-terminalprecursor,antibodiesagainsttheN-terminalregionofinterestwouldberaised.Likewiseifthegoalistomonitorthephosphorylationstateofaspecificsequence,antibodiestothephosphorylatedsequencecanbeused.

Ifthegoalistoraiseantibodiesthatwillrecognizetheproteininitsnativestate,theproblembecomesmorecomplex.Anti-peptideantibodieswillalwaysrecognizethepeptide.However,thesameantibodymaynotrecognizethesequencewithinthefoldedintactprotein.Sequenceepitopesinproteinsgenerallyconsistof6-12aminoacidsandcanbeclassifiedascontinuousanddiscontinuous.Continuousepitopesarecomposedofacontiguoussequenceofaminoacidsinaprotein.Anti-peptideantibodieswillbindtothesetypesofepitopesinthenativeproteinprovidedthesequenceisnotburiedintheinterioroftheprotein.Discontinuousepitopesconsistofagroupofaminoacidsthatarenotcontiguousbutarebroughttogetherbyfoldingofthepeptidechainorbythejuxtapositionoftwoseparatepolypeptidechains.Anti-peptideantibodiesmayormaynotrecognizethisclassofepitopedependingonwhetherthepeptideusedforantiseragenerationhassecondarystructuresimilartotheepitopeand/oriftheproteinepitopehasenoughcontinuoussequencefortheantibodytobindwithaloweraffinity.

Whenexaminingaproteinsequenceforpotentialantigenicepitopes,itisimportanttochoosesequenceswhicharehydrophilic,surface-oriented,andflexIBLe3.Mostnaturallyoccurringproteinsinaqueoussolutionshavetheirhydrophilicresiduesonthesurfaceandtheirhydrophobicresiduesburiedintheinterior.Antibodiesbindtoepitopesonthesurfaceofproteins.Additionally,ithasbeenshownthatepitopeshaveahighdegreeofmobility4.

BecausetheC-terminiofproteinsareoftenexposedandhaveahighdegreeofflexibilitytheyareusuallyagoodchoiceforgeneratinganti-peptideantibodiesdirectedagainsttheintactprotein.IftheproteinisanintegralmembraneproteinandtheC-terminusispartofthetransmembranesegment,thissequencewillbetoohydrophobicandnotagoodchoice.

LiketheC-terminus,theN-terminusisalsofrequentlyexposedandonthesurfaceoftheproteinmakingitanidealcandidateforantibodygeneration.IfaproteinsequenceisderivedfromtheCDNAsequence,theleadersequenceshouldnotbeincludedinthesequenceselectedforantibodygeneration.

Algorithmsforpredictingproteincharacteristicssuchashydrophilicity/hydrophobicityandsecondarystructureregionssuchasalpha-helix,beta-sheetandbeta-turnaidselectionofapotentiallyexposed,immunogenicinternalsequenceforantibodygeneration.

HydrophilicityplotsasdescribedbyHoppandWoods5assignanaveragehydrophilicityvalueforeachresidueinthesequence.Thehighestpointofaveragehydrophilicityforaseriesofcontiguousresiduesisusuallyatornearanantigenicdeterminant.AslightlydifferentalgorithmdescribedbyKyteandDoolittle6evaluatesthehydrophilicandhydrophobictendenciesofthesequence.Thisprofileisusefulforpredictingexteriorvs.interiorregionsofthenativeprotein.SecondarystructurecanbeidentifiedbytheuseofalgorithmsdevelopedbyChouandFasman7orLim8.Surfaceregionsorregionsofhighaccessibilityoftenborderhelicalorextendedsecondarystructureregions.Inaddition,sequenceregionswithbeta-turnoramphipthichelixcharacterhavebeenfoundtobeantigenic9.

ManycommercialsoftwarepackagessuchasMacVectorTM,DNAStarTM,andPC-GeneTMincorporatethesealgorithms.Tobesucessful,noneofthealgorithmsshouldbeusedalone.Combineduseofthepredictivemethodsmayresultinasuccessrateashighas86inpredictingantigenicdeterminants9,10.

Oncetheproteinregionofinteresthasbeenidentified,thelengthofthepeptidemustbeselected.Therearetwodifferingthoughtsonthetopicofpeptidelength.Onesuggeststhatlongpeptides(20-40aminoacidsinlength)areoptimalbecauseitincreasesthenumberofpossibleepitopes.Theothersuggeststhatsmallerpeptidesaresufficient,andtheiruseensuresthatthesite-specificcharacterofanti-peptideantibodiesisretained.Clearly,anypeptideselectedmustbechemicallysynthesizableandshouldbesolubleinaqueousbufferforconjugationtothecarrierprotein.Peptideslongerthan20residuesinlengthareoftenmoredifficulttosynthesizewithhighpuritybecausethereisgreaterpotentialforsidereactions,andtheyarelikelytocontaindeletionsequences.Ontheotherhand,shortpeptides(10aminoacids)maygenerateantibodiesthataresospecificintheirrecognitionthattheycannotrecognizethenativeproteinordosowithlowaffinity.Thetypicallengthforgeneratinganti-peptideantibodiesisintherangeof10-20residues.Peptidesequencesofthislengthminimizesynthesisproblems,arereasonablysolubleinaqueoussolutionandmayhavesomedegreeofsecondarystructure.

CouplingStrategy

Afactorthatisoftenover-lookedwhendesigningasyntheticpeptideisthemethodofcouplingthepeptidetothecarrierprotein.Forexample,N-terminalsequencesshouldbecoupledthroughtheC-terminalaminoacidandviceversaforC-terminalsequences.Internalsequencescanbecoupledateitherend.Anotherconsiderationforinternalsequencesistoacetlyateoramidatetheunconjugatedendasthesequenceinthenativeproteinmoleculewouldnotcontainachargedterminus.

Themostcommoncouplingmethodsrelyonthepresenceoffreeamino(alph-aminoorLys),sufhydryl(Cys),orcarboxylicacidgroups(Asp,Glu,oralpha-carboxyl).Couplingmethodsshouldbeusedthatlinkthepeptidetothecarrierproteinviathecarboxy-oramino-terminalresidue.Thesequencechosenshouldnothavemultipleresiduesthatmayreactwiththechosenchemistry.Ifmultiplereactivesitesarepresent,trytoshortenthepeptideorchoosethesequencesotheyarealllocalizedateithertheaminoorthecarboxylterminusofthepeptide.Forinternalsequencestheendfurthestfromthepredictedepitopeisnormallyfavoredasthisavoidspotentialmaskingproblems.

TheEDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride)orcarbodiimidemethodisroutinelyusedintheSigma-Genosyslaboratoryunlessotherwisestatedbytheresearcher.Carbodiimidescanactivatethesidechaincarboxylicgroupsofasparticandglutamicacidaswellasthecarbooxylterminalgrouptomakethemreactivesitesforcouplingwithprimaryamines.Theactivatedpeptidesaremixedwiththecarrierproteintoproducethefinalconjugate.Ifthecarrierproteinisactivatedfirst,theEDCmethodwillcouplethecarrierproteinthroughtheN-terminalalphaamineandpossiblythroughtheamineintheside-chainofLysine,ifpresentinthesequence.

Them-Maleimidobenzoyl-N-hydroxysuccinimideester(MBS)isaheterobifunctionalreagentthatcanbeusedtolinkpeptidestocarrierproteinsviacysteines.Thecouplingtakesplacewiththethiolgroupofcysteineresidues.IfthechosensequencedoesnotcontainCysitiscommontoplaceaCysresidueattheN-orC-terminustoobtainhighlycontrolledlinkingofthepeptidetothecarrierprotein.ForsynthesispurposeswerecommendthattheplacementofcysteinebeattheN-terminusofthepeptideifpossible.

Glutaraldehydeisabifunctionalcouplingreagentthatlinkstwocompoundsthroughtheiraminogroups.Glutaraldehydeprovidesahighlyflexiblespacerbetweenthepeptideandcarrierproteinforfavorablepresentationtotheimmunesystem.Unfortunately,glutaraldehydeisaveryreactivecompoundandwillreactwithCys,TyrandHistoalimitedextent.Theresultisapoorlydefinedconjugate.Theglutaraldehydemethodisparticularlyusefulwhenapeptidecontainsonlyasinglefreeaminogroupatitsaminoterminus.Ifthepeptidecontainsmorethanonefreeaminogorup,largemultimericcomplexescanbeformed,whicharenotwelldefined,butarehighlyimmunogenic.

SelectingtheProteinCarrier

Conjugationtoacarrierproteinisimportantbecausepeptidesaresmallmolecules,thatalonedonottendtobeimmunogenic,thuspossiblyelicitingaweakimmuneresponse.ThecarrierproteincontainsmanyepitopesthatstimulateT-helpercells,whichhelpinducetheB-cellresponse.Manydifferentcarrierproteinscanbeusedforcouplingtosyntheticpeptides.Themostcommonlyselectedcarriersarekeyholelimpethemacyanin(KLH)andbovineserumalbumin(BSA).ThehigherimmunogenicityofKLHoftenmakesitthepreferredchoice.AnotheradvantageofchoosingKLHoverBSAisthatBSAisusedasablockingagentinmanyexperimentalassays.BecauseantiseraraisedagainstpeptidesconjugatedtoBSAwillalsocontainantibodiestoBSA,falsepositivesmayresult.AlthoughKLHislargeandimmunogenic,itmayprecipitateduringcross-linking,makingitdifficulttohandleinsomecases.

Ovalbumin(OVA)isanotherusefulcarrierprotein.Itisagoodchoiceasasecondcarrierproteinwhenverifyingwhetherantibodiesarespecificforthepeptidealoneandnotthecarrier.RabbitSerumAlbumin(RSA)maybeusedwhentheantibodyresponsetothecarrierproteinmustbekepttoaminimum.RabbitsimmunizedwithRSAconjugatearelesslikelytoraiseantibodiestothecarrier,astheRSAisrecognizedas"self."IftheRSAconjugatewereinjectedintoanotherhost,theproteinwouldnotberecognizedasself.

Itisimportanttorecognizethattheimmunesystemreactstothepeptide-proteincarrierasawholeandthattherewillbeaportionofresponsedirectedagainsttheconjugatedpeptideaswellasthelinkerandthecarrierprotein1.WhenscreeningbyELISAitisadvisabletouseapeptideconjugatepreparedusingadifferentcarrierprotein.ThisisnotnecessaryifperformingELISAassayswheretheplatesarecoateddirectlywithunconjugatedpeptide.

MultipleAntigenicPeptides(MAPs)

TheMAPsystemrepresentsauniqueapproachtoanti-peptideantibodygeneration11.Thesystemisbasedonasmallimmunogenicallyinertbranchedlysinecoreontowhichmultipepeptidesaresynthesizedinparallel.Theresultaftersynthesisisathree-dimensionalmolecule,whichhasahighmolarratioofpeptideantigentocoremoleculeandthereforedoesnotrequiretheuseofacarrierproteintoinduceanantibodyresponse.Eachcoremoleculemaycontainfouridenticalpeptides.Intheory,MAPhasanadvantagewhencomparedtoitsmonomericcounterpartattachedtoacarrierproteininthatthelysinecoreofaMAPissmallcomparedwiththepeptideantigen.Therefore,theconcentrationofantigenisatamaximum.TheresultisahighlyimmunogenicMAP,whichexhibitssignificantlyhighertiterswhencomparedtoitsmonomericcounterpartattachedtoacarrierprotein.

ItshouldbenotedthattherearesomesynthesisconcernswhenmakingaMAPcomplex.Thebranchednatureofthelysinecoreallowsformultiplecopiesofthepeptidetobesynthesized;however,sterichindrancebecomesaproblemduringthesynthesisoflongpeptides,resultinginsomearmsofthedendrimerbeingdeletionproducts.Thehighmolecularweightofthecomplexdoesnotlenditselftogoodqualitycontrolmeasures(massspecand/oranalyticalHPLC).AnindirectsynthesisoftheMAPcaneliminateanalysisproblems.Intheindirectmethod,thepeptideisfirstsynthesized,purifiedthenanalyzedusingmassspecandanalyticalHPLC.ThepeptideantigenisthencoupledthroughaCystoafunctionalizedlysinecore.

ChoiceofHost

Whenattemptingtoraiseanantibody,chooseananimalthatisgeneticallyverydifferentfromthesourceofimmunogen.Inordertoachievemaximumimmuneresponse,itisimportanttoavoidself-recognitionoftheimmunogenbythehostanimal.Asanexample,whenraisingantibodiesagainstahumanprotein,itismoresuitabletousearabbitormousehostthanamonkey.Forhighlyconservedmammalianproteins,raisingantibodiesintheavian(chicken)systemisoftenapreferredalternative.

Adjuvant,Immunization,&SeraCollection

Sigma-GenosysroutinelyusesFreund'sadjuvantforimmunizationpurposes.ThefirstinjectionisgiveninCompleteFreund'sadjuvant.Adjuvantiscombinedwiththeantigentoimprovetheimmuneresponsesothatlessvaccineisneededtoproducemoreantibodies.Theadjuvantallowsaslowreleaseoftheantigenwhichallowsforcontinualstimulation.Injectionsareroutinelyperformedsubcutaneouslyatmultiplesites.Apre-immunebleedshouldbedrawnfromeachhostanimaltoproduceabaselinetowhichtheproductionbleedscanbecompared.Thedrawnserawillcontainanumberofdifferenttypes(IgG,IgM,IgA)andsubclasses(Ig1,Ig2a,Ig2b,Ig3).Sodiumazide(0.1)canbeaddedtothesera.Sodiumazideisabroad-spectrumenzymeinhibitorandactsasanantimicrobialagent.Sodiumazideshouldnotbeaddedtoserawhenusingincellcultureorinvivostudies.

AntiseraPurification

Ifahighbackgroundisobservedinassaysusingtheantisera,variouspurificationtechniquesareavailable.Itisimportanttofirstcheckthatthebackgroundisnon-specificandnotduetotheresponseagainstthepeptide.Thiscanbedeterminedbyperformingacompetitivepeptideblockingstudy.Peptideblockingstudiescheckthattheresponseagainstthetargetproteinisnotabackgroundartifact.

AmmoniumSulfatePrecipitation

Ammoniumsulfateprecipitationisacommonlyusedmethodforremovingproteinfromsolution.Themethodisafairlycrude,non-specificpurificationthatremovesthemajorityofplasmaproteinsandleavestheimmunoglobulinfraction.Wheninsolution,proteinsformhydrogenbondswithwaterthroughtheirexposedpolarandionicgroups.Addingsmallionssuchasammoniumorsulfateremoveswatermoleculesfromtheprotein,resultinginprecipitationoftheproteinoutofsolution.Itshouldbestatedthatammoniumsulfateprecipitationwillnotresultinhighlypurifiedantibodies.Thecontaminantswillconsistofotherhigh-molecular-weightproteinsandproteinsthataretrappedinthelargeflocculentprecipitates.Itisrecommendedthatammoniumsulfateprecipitationbeusedaspartofapurificationschemeinvolvingfurtherpurificationsteps.

ProteinA/G

ProteinAorProteinGpurificationremovestheIgGfractionbasedonthespecificityoftheseproteinsfortheFcportionoftheIgG.ProteinAisproducedfromStaphylococcusaureus.IthasthecapacitytobindatleasttwomoleculesofIgG.ThebindingisspecifictotheFcportionanddoesnotaffecttheantigenbindingsites.ProteinGisisolatedfromGroupGstreptococciandbindstheFcregionoftheIgGinasimilarmannertoProteinA.ProteinAandGhavedifferingbindingefficienciesforIgGfromdifferentspecies.Itisimportanttocheckthisbeforedecidingwhichmethodtouse.Forexample,ProteinGworkswellwithsheepIg,butProteinAdoesnot.NeitherProteinAnorProteinGwillbindtochickenIg.Careshouldbetakenwhenelutingtheantibodyfromthecolumntoavoiddenaturationoftheantibody.

ImmunoaffinityPurification

Themostcommonlyusedmethodtopurifyantigen-specificantibodiesfromcrudeseraisimmunoaffinitypurification.UnlikeProteinAorG,thenon-specificIgfractionisnotretained.Inthisprocedure,peptideantigenisboundcovalentlytoasolidsupport.Theantibodieswithinthepolyclonalsamplethatarespecificforthepeptideantigenbindtothesupportcolumn.Theunboundantibodiesareremovedfromthecolumnbywashingandthespecificantibodiesareelutedfromthecolumn.Theproductofimmunoaffinitypurificationishighlyspecificantibodies.Immunoaffinitypurificationcanoccasionallycausedenaturationoftheantibodyduetotheconditionsusedtoelutetheboundantibodyfromthecolumn.Itisimportanttocomparetheresponsegeneratedbythepurifiedsampleagainsttheresponsegeneratedbythecrudesera.

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