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AntigenDesignAntibodiestosmallpeptideshavebecomeanessentialtoolinlifescienceresearch,withapplicationsincludinggeneproductdetectionandidentification,proteinprocessingstudies,diagnostictests,proteinlocalization,activesitedetermination,proteinhomologystudiesandproteinpurification.Whileitisquiteeasytogenerateanti-peptideantibodies,itisimportanttocarefullyconsidertheultimateusefortheantibodyandthesequenceusedtoensuresuccess.Thistechsheetwillbrieflyexplorepeptideselectionanddesign,couplingstrategy,andcarrierproteinswhichareimportantfactorsinanti-peptideantiserageneration.Serumpurificationwillalsobediscussed.PeptideSelectionandDesignThefirststepintheprocessistheselectionoftheappropriatepeptidesequence.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,ithasbeenshownthatepitopeshaveahighdegreeofmobility.BecausetheC-terminiofproteinsareoftenexposedandhaveahighdegreeofflexibilitytheyareusuallyagoodchoiceforgeneratinganti-peptideantibodiesdirectedagainsttheintactprotein.IftheproteinisanintegralmembraneproteinandtheC-terminusispartofthetransmembranesegment,thissequencewillbetoohydrophobicandnotagoodchoice.LiketheC-terminus,theN-terminusisalsofrequentlyexposedandonthesurfaceoftheproteinmakingitanidealcandidateforantibodygeneration.IfaproteinsequenceisderivedfromthecDNAsequence,theleadersequenceshouldnotbeincludedinthesequenceselectedforantibodygeneration.Algorithmsforpredictingproteincharacteristicssuchashydrophilicity/hydrophobicityandsecondarystructureregionssuchasalpha-helix,beta-sheetandbeta-turnaidselectionofapotentiallyexposed,immunogenicinternalsequenceforantibodygeneration.HydrophilicityplotsasdescribedbyHoppandWoodsassignanaveragehydrophilicityvalueforeachresidueinthesequence.Thehighestpointofaveragehydrophilicityforaseriesofcontiguousresiduesisusuallyatornearanantigenicdeterminant.AslightlydifferentalgorithmdescribedbyKyteandDoolittle6evaluatesthehydrophilicandhydrophobictendenciesofthesequence.Thisprofileisusefulforpredictingexteriorvs.interiorregionsofthenativeprotein.SecondarystructurecanbeidentifiedbytheuseofalgorithmsdevelopedbyChouandFasmanorLim.Surfaceregionsorregionsofhighaccessibilityoftenborderhelicalorextendedsecondarystructureregions.Inaddition,sequenceregionswithbeta-turnoramphipthichelixcharacterhavebeenfoundtobeantigenic.ManycommercialsoftwarepackagessuchasMacVectorTM,DNAStarTM,andPC-GeneTMincorporatethesealgorithms.Tobesucessful,noneofthealgorithmsshouldbeusedalone.Combineduseofthepredictivemethodsmayresultinasuccessrateashighas86%inpredictingantigenicdeterminants.Oncetheproteinregionofinteresthasbeenidentified,thelengthofthepeptidemustbeselected.Therearetwodifferingthoughtsonthetopicofpeptidelength.Onesuggeststhatlongpeptides(20-40aminoacidsinlength)areoptimalbecauseitincreasesthenumberofpossibleepitopes.Theothersuggeststhatsmallerpeptidesaresufficient,andtheiruseensuresthatthesite-specificcharacterofanti-peptideantibodiesisretained.Clearly,anypeptideselectedmustbechemicallysynthesizableandshouldbesolubleinaqueousbufferforconjugationtothecarrierprotein.Peptideslongerthan20residuesinlengthareoftenmoredifficulttosynthesizewithhighpuritybecausethereisgreaterpotentialforsidereactions,andtheyarelikelytocontaindeletionsequences.Ontheotherhand,shortpeptides(10aminoacids)maygenerateantibodiesthataresospecificintheirrecognitionthattheycannotrecognizethenativeproteinordosowithlowaffinity.Thetypicallengthforgeneratinganti-peptidea