Production of polyclonal antibodies against peptide antigens using polystyrene beads as a carrier

ORIGINALRESEARCHPAPER

Productionofpolyclonalantibodiesagainstpeptideantigensusingpolystyrenebeadsasacarrier

MiheeKimÆChul-HoYunÆSeong-KiParkÆJung-HoonSeoÆTaehoAhn

Received:14March2007/Revised:14June2007/Accepted:18June2007/Publishedonline:13July2007ÓSpringerScience+BusinessMediaB.V.2007

AbstractWedescribeamethodforproducingpolyclonalantibody(PAb)againstpeptideantigen(theepitoperegionofcytochromesP4501A2and3A4,HAtag,FLAGtag,andc-myctag)usingapolymerbeadasacarrier.Carboxylatedpolystyrenebeads(0.05,0.5,1,and2mmdiam)wereconjugatedwiththepeptide,whichisknownasanepitoperegion,usingachemicalcross-linkerandinjectedintorabbitswithadjuvant.Byimmunizingtheanimalsintradermallytwiceattwoweekintervals,animmunoblotassaywithanti-serashowedtheefficientgenerationofthePAb.

KeywordsCarrierÁConjugationÁPeptideantigenÁPolyclonalantibodyÁPolystyrenebead

Introduction

Bothnaturalandrecombinantproteinshavebeenusedtoproducepolyclonalantibodies(PAb).How-ever,proteinsarerarelyavailableinapureformandantibodiesareoftensimultaneouslyraisedagainstcontaminatingprotein(s).Recombinantpolypeptidesalsohavelimitationstobewidelyusedasidealantigensdueto:(1)thedifficultyoftheirexpressioninhostcells,particularlyinthecaseofmembraneproteins,(2)correctfoldingproblems,suchastheformationofinclusionbodies,and(3)purificationprocesses.

Syntheticpeptidesareavailableinahighlypureform,whichmakesitpossibletoproduceantibodiesagainstproteinsthathavenotbeenpurifiedandcanbeidentifiedonlybycDNAorESTclones(Angeletti1999).Peptideantigenscanalsotargetthespecificregion(s)ofthewholeproteinandmayreducethecross-reactivityoftheantibodiesproducedwithotherantigensduetominimalepitopicnumbers.However,peptidesalonecannotgenerateantibodieswheninjectedintoananimalduetotheirlowimmunoge-nicity.Therefore,thepeptideepitopesareusuallyconjugatedtoalargeproteinmoleculeknownasa‘‘carrier’’suchaskeyholelimpethemocyanin(KLH),bovineserumalbumin(BSA),ovalbumin,andTet-anustoxoid(HarlowandLane1988).Theincreasedsizeofthepeptide-carriercomplexisrecognizedandengulfedbytheantigenpresentingcells,whichstimulatestheimmunesystem.

M.Kim

DepartmentofMolecularEndocrinology,ChonnamNationalUniversity,Gwangju500-757,Korea

C.-H.Yun

SchoolofBiologicalSciencesandTechnology,

ChonnamNationalUniversity,Gwangju500-757,KoreaS.-K.ParkÁJ.-H.SeoÁT.Ahn(&)

DepartmentofBiochemistry,CollegeofVeterinaryMedicine,ChonnamNationalUniversity,

300Yongbong-dong,Buk-gu,Gwangju500-757,Koreae-mail:thahn@chonnam.ac.kr

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1736Althoughthecarrierproteinsarewidelyusedtofacilitatetheproductionofantibodiesagainstpeptideantigens,theirapplicationislimitedasaresultofthegenerationofantibodiestothecarrierproteinitself,theirlowsolubilityinwaterandthedifficultyindeterminingthecouplingefficiency(thenumberofpeptidesconjugatedtoacarrierprotein).Therefore,somelaboratoriesprefertousesyntheticpolymersasapeptide-conjugatesincethesemaybemoreimmu-nologicallyneutralthanconventionalcarrierproteins(HarlowandLane1988).

ThisstudysuggeststhatpolystyrenebeadscanbeusedascarriersforthePAbproductionagainstpeptideantigens.Usingthismethod,theantibodiesweregeneratedefficientlyinthelaboratoryanimalsuchasrabbit.

MaterialsandmethodsMaterials

ThecarboxylatedpolystyrenebeadswerepurchasedfromPolysciencesInc.(Warrington,PA).1-Ethyl-(3-dimethylaminopropyl)carbodiimide(EDC),incompleteFreund’sadjuvants,andfluorescaminewereobtainedfromSigma.ThePosi-TagEpitopeTagcontrolproteinwasacquiredfromCRPInc.(Berkeley,CA).ThePVDFandECLkitweresuppliedbyAmershamPharmaciaBiotech(Piscat-away,NJ).TheNewZealandwhiterabbits,between1.4kgand1.6kg,werepurchasedfromBiogenom-ics(Gwangju,Korea).Thepeptideantigens(Table1)werechemicallysynthesizedbyPepTronInc.(Daejon,Korea).

Table1AminoacidsequencesofthepeptideantigensusedinthisstudyPeptideSequenceReference

CYP1A2SENWKDNEdwardsetal.(1995)CYP3A4LEDTQKHWangetal.(1999)HAtagYPYDVPDYAFieldetal.(1988)

FLAGtagDYKDDDDDKKnappikandPluckthun(1994)c-myctag

EQKLISEEDL

Evanetal.(1985)

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Conjugationbetweenpeptidesandpolystyrenebeads

Peptide-carrierbeadconjugationwaspreparedbyusingthemethodasdescribedelsewhere(Fifisetal.2004).Briefly,carboxylatedpolystyrenebeads(2.6%solids-latex,200mlbedvolume)withdifferentdiameters(0.05,0.5,1,2mm)weremixedat1:1ratio(v/v)witheachpeptide(2mg/ml)dissolvedin100mMMESbuffer,pH6.0,andincubatedfor30minat25°C.EDCdissolvedin50mMMESatpH6.0wasaddedtogive4.5mg/mlandwasfurtherincubatedfor4hat25°C.Thesamplesolutionwasthencentrifugedtoprecipitatethepeptide-polysty-renecomplex.Thesupernatantwastakenandtheconcentrationoffreepeptidewasdeterminedusingafluorescamineassay(Castelletal.1979).Thecouplingefficiencywasmorethan90%regardlessofthetypeofpeptideantigensused.Theconjugationreactionwasquenchedbyaddingglycinetogive100mMandthesamplesweredialyzedagainstPBSfor12hat4°C.

Immunization

Theinjectionsampleswerepreparedbymixingthepeptide-beadcomplexwithincompleteFreund’sadjuvantasa1:1(v/v)emulsion.Theinjectionvolumeforanimmunizationwasapproximately0.5mlperarabbit.Theantigenwasadministeredintradermally(ID)totheshavedbackskinofarabbit(fiverabbitspergroup)infiveinjections(100ml/injection)usingastandarddisposable28-gaugesyringe.Tocomparetheeffectofadministrationroutesontheantibodyproduction,thesubcutaneousandintramuscularinjectionoftheantigenwerealsoused.Theanimalswereboosted2weekslaterusingthesamemethodsdescribedforthefirstimmuniza-tion.Bloodsamplesweretakenfromtheearveinoftherabbit(totalbleedingvolumeofapproximately300ml)at2weeksafterthefinalinjection.Westernblotassay

ThegenerationofantibodieswasexaminedbyWesternblottingwitheithertheTagcontrolproteinorhumanlivermicrosomesforthedetectionofcytochromesP4501A2(CYP1A2)and3A4proteins

BiotechnolLett(2007)29:1735–1740(CYP3A4).ThemicrosomeswerekindlyprovidedbyProf.F.PeterGuengerich(Nashville,TN).ELISA

InadditiontotheWesternblotanalysis,thelevelofanti-peptideantibodieswasdeterminedusingcon-ventionalELISAmethod.Forthisassay,eachpeptidewasfirstconjugatedtotheactivatedbovineserumalbumin(ImjectMaleimideActivatedBSA,PierceBiotechnology,Inc.,Rockford,IL)accordingtothemanufacturer’sinstructions,andthepeptide-BSAcomplexwasthenusedasanELISAantigen.

Resultsanddiscussion

Figure1showstheresultsofimmunoblotanalysisofhumanlivermicrosomesusingtheanti-serapreparedfromarabbitinjectedwiththepolystyrene-CYP3A4peptideconjugate.Theanti-seraweredrawnfromtheearveinoftheanimal2weeksafterthesecondimmunization.TheantibodyproducedappearedtospecificallyrecognizetheCYP3A4proteininthemicrosomesatadilutionfactorof1,000(Fig.1A).Theresultobtainedfromtwothousanddilutionsofanti-serawasalsocomparablewiththatshowninFig.1A(Fig.1B).Basedonthisresult,itisanticipatedthatfurtherdilutionsoftheanti-seracanbeusedintheimmunoblotanalysisfordetectingtheCYP3A4protein.

Fig.1Westernblotanalysisofhumanlivermicrosomesusingtheanti-serapreparedfromaCYP3A4peptide-polystyrenebeadconjugate.MicrosomeswithincreasingamountsofthetotalproteinswereseparatedbySDS-PAGE,transferredtoaPVDFmembrane,andincubatedwitheithera1:1,000(A)or1:2,000(B)dilutionofrabbitanti-serum.TheblotswereincubatedwiththeTMappropriatesecondaryantibodyanddevelopedbyECLaccordingtotheconventionalmethod.Lanes1,2,3,and4representthetotalamountofproteinswith30,60,90,and120mg,respectively

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Asacontrolexperiment,whenthesameprocedurewascarriedoutwiththepurifiedCYP3A4protein(Kimetal.2003)insteadofthemicrosomes,theresultwasconsistentwiththecaseofthemicrosomes,wherethegeneratedantibodyspecificallyrecognizedtheproteinantigen(resultnotshown).ThisresultshowsthatthespecificantibodyagainstthepeptidecorrespondingtotheCYP3A4proteinwasefficientlyproducedwithin4weeks,andapolystyrenebeadcanplaytheroleofaconventionalproteincarriertopreparethePAbs.

Inordertoapplythismethodtotheproductionofotherpeptideantigens,thepeptideepitopescorre-spondingtotheCYP1A2proteinandthreetagpeptides(Table1)wereconjugatedwiththepolysty-renebeadsseparately.Theanti-seraagainstthesepeptideantigenswerepreparedusingthesamemethodandthesametimeschedule.Whenassayedbyimmunoblotting,theCYP1A2proteininthemicrosomesandthetagcontrolprotein(totalextractofEscherichiacolicontainingrecombinantlyex-pressedTagprotein)includingeachtagpeptideregionwerespecificallyrecognizedbythecorre-spondinganti-seraatadilutionof2,000fold(Fig.2AandB).However,thenonspecificband(atthelowerregionthanthatofCYP1A2)wasalsodetectedbytheanti-seraagainstCYP1A2peptide.Inthesamemanner,theantibodygeneratedwasconfirmedwiththepurifiedCYP1A2protein(Ahnetal.2005)asanimmunoblotantigen(resultnotshown).Overall,theseresultssuggestthatthepresentmethodcanbeappliedtoanypeptide,facilitatingdirectuseofpeptidesasimmunogens.Asanegativecontrol,whenallimmunoblotexperimentswererepeatedwithanti-seraobtainedfromunimmunizedrabbitsunderthesameconditions,thecorrespondingbandstothespecificantigenswerenotdetected(resultsnotshown).

Similarmethodusingnano-beadshasbeenalreadyutilizedtoinduceimmuneresponses(Fifisetal.2004;Scheerlincketal.2006).Inthosepapers,polystyrenenano-beads(ormicrospheres)werecon-jugatedwiththepeptide/proteinandusedasantigencarriersinmucosalimmunizationorasanadjuvantforthecell-mediatedimmuneresponsesalthoughpolystyreneparticlescouldresultintoxicityinanimals(Olivieretal.2003).Inadditiontothepolystyrenebeads,otherpolymershavebeenusedwidelyinthedeliveryofantigens(Slobbeetal.

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Fig.2WesternblotanalysisofhumanlivermicrosomesandtheTagcontrolprotein.Theproductionofantibodiesagainstthepeptideantigens(CYP1A2,HA,FLAG,andc-myctag)wasanalyzedbyWesternblottingwiththesamemethoddescribedintheFig.1legend.A,Lanes1,2,and3representthetotalamountofproteinswith30,60,and90mg,respectively.InPanelB,theappropriateamountofTagcontrolproteinwasusedasrecommendedbythesupplier

¨holm2002),2003),adjuvants(WilingssonandSjo

andasasubstituteforcarrierproteins(Menetal.1996).Therefore,ourresultsparallelthosereportedpreviouslyandhighlightthepotentialapplicationsofthenano(ormicro)beadsfortheproductionofPAbsagainstvariouspeptides,eventhoughtheotherpolymerswerenottestedinthisstudy.

Usingthepresentmethod,itshouldbealsoconsideredthatEDCasachemicalcross-linkerreactswiththecarboxylicacidgroup,allowingittobecoupledtotheaminegroupsthatarepresentonbothLysresiduesandtheN-terminusofpeptides(correspondingtoCYP1A2,3A4,FLAG,andc-myctagpeptides).Therefore,thecross-linkingmayaffectthepropertiesoftheepitopesused.Atpresent,however,itisnotclearastohowthisreactionchemistryhasaneffectontheproductionofpolyclonalantibodiesagainstpeptideantigens.

Figure3showstheamountsofserumimmuno-globulinaftertheimmunizationwithdifferentsizesofpolystyrene-peptidecomplex.Theadministrationofa1mmparticleelicitedthestrongestantibodyresponse.

Moreover,inadditiontotheeffectofpolymersize,theadministrationroutewasalsofoundtobeimportantintheproductionofantibodies,andanIDinjectionoftheantigens(thepeptidesconjugatedto1mmpolystyrenebead)inducedthehighestamount

4.03.5CYP1A2CYP3A4HA-tagSpecific Absorbance (405 nm)3.02.52.01.51.00.50.00.05 µm 0.5 µm1.0 µm2.0 µmFig.3Sizeeffectofthepolystyrenebeadsontheamountofantibodyproduced.Thesameamountofantigenswithdifferentpolymersizeswasusedfortheproductionofantibodiesunderthesameexperimentalconditionsinallsamples.Aftersamplingthebloodfromimmunizedrabbits,serumsamplesdilutedby1,000foldwerethenanalyzedbyconventionalELISAmethodusingthepeptide-BSAconjugateasanELISAantigenasdescribedin‘Materialsandmethods’.Theanti-rabbitIgGperoxidaseconjugateand2,20-azino-di-(3ethyl-benzthiazolinesulfonicacid)wereusedasasecondaryantibodyandtheperoxidasesubstrate,respectively.Non-specificbindingofantigentothewellswithoutantigenimmobilizationwassubtracted.Theresultisexpressedasamean±standarddeviation(n=5)foreachgroup123

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4.5SC4.0IMID)m3.5n 5043.0( ecna2.5brosb2.0A cif1.5icepS1.00.50.0CYP1A2 CYP3A4 HA-tagFig.4Effectofadministrationrouteofantigensonantibodyproduction.Thepeptidesconjugatedto1.0mmpolystyrenewereinjectedintorabbitsbytheSC,IM,andIDroutesunderthesameexperimentalconditionsandtheefficiencyofPAbproductionwasassayedbyELISAasdescribedinthelegendofFig.3ofantibodies(Fig.4).Althoughsubcutaneous(SC)andintramuscular(IM)administrationhavebeenfrequentlyusedforPAbproductionthisresultsuggeststhattheIDrouteismoreefficientthanSCorIMmethodwhenusingthepolystyrene-peptidecomplexasanantigenunderthesameexperimentalconditions.

Thereareseveraladvantagesofusingapolymerbeadfortheproductionofantibodiesasacarrier.Inadditiontotheeasyprocessforpreparingantigensamples,thedeterminationoftheefficiencyforthepeptide-carrierconjugationandantibodypurificationwassimplifiedusingthepeptide-polymercomplexitselfasanaffinitycolumn.Moreover,asdescribed,thepolymermightbemoreimmunologicallyneutralthanconventionalcarrierproteins.Althoughasys-temicinvestigationwasnotperformedandtheresultsusingtheconventionalcarrierproteinsuchasKLHandBSAforcomparingtheefficiencyofPAbproductionwerenotincludedinthisstudy,itisanticipatedthatthewholeprocessesforpreparinganti-serumagainstvariousantigensmightbesimplerandmoreefficientthanthoseusedinpeptide-carrierproteinconjugation.

Inconclusion,theconjugationofapeptidetoapolystyrenemicrospherebead(approx.1mmsize)

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andtheintradermaladministrationoftheantigenintoananimalwiththeadjuvantistheoptimummethodforobtainingpolyclonalantibodiesagainstpeptideantigenswithin4weeks.

AcknowledgementThisworkwassupportedbyaKoreaResearchFoundationGrantKRF-2005-070-C00081.

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