AntigenDesign&SeraPurificationTechSheet 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.Combineduseofthepredictivemethodsmayresultinasuccessrateashighas86%inpredictingantigenicdeterminants9,10. 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. References 1. VanRegenmortel,M.H.V.,1988,SyntheticPolypeptidesasAntigens,Elsevier,Amsterdam. 2. Halow,E.,1988,Antibodies;ALaboratoryManual,ColdSpringHarbor,NewYork. 3. VanRegenmortel,M.H.V.,1986,TrendsinBiochemistry,11:36-39. 4. Westof,E.,1984,Nature,411:123-126. 5. Hopp,T.P.andWoods,K.R.,1981,Proc.Natl.Acad.Sci.U.S.A.,78:3824-3828. 6. Kyte,J.andDoolittle,R.F.,1982,J.Mol.Biol.,157:105-132. 7. Chou,P.Y.andFasman,G.D.,1974,Biochemistry,13:222-245. 8. Lim,V.I.,d1974,J.Mol.Biol.,88:873-894. 9. Parker,J.M.R.andHodges,R.S.,1991,PeptideRes.,4:347-354. 10. Parker,J.M.R.andHodges,R.S.,1991,PeptideRes.,4:355-363. 11. Tam,J.P.,1988,Proc.Natl.Acad.Sci.U.S.A.,85:5409-5413.