You"llneedtocouplethesyntheticpeptidehaptentoaproteincarrier,withabifunctionalreagent.

Forantibodydevelopment,wedon"tbothertousehighlypurifiedpeptide.Crude(unpurified)peptideorpartiallypurifiedpeptidedoesjustfine;afterall,theimpuritiesinunpurifiedpeptidesareprincipallyincomplete,partialversionsofthedesiredpeptide.Sinceyouneedtousequiteabitofpeptidetogetgoodcouplingandantibodyproduction(10sofmg),it"sprobablymosteconomicaltouseunpurifiedsyntheticpeptides.

Themostusefulcouplingprotocolsarein:RFDoolittle.OfUrfsandOrfs.APrimeronHowtoAnalyzeDerviedAminoAcidSequences.UniversityScienceBooks,1987.

Goodcarriersincludebovineserumalbumin,orkeyholelimpethemocyanin(ourfavorite,fromCalbiochem#374817).

Goodcouplingmethodsareglutaraldehyde(aminotoamino),MBS(=m-Maleimidobenzoicacid-N-hydroxysuccinimide;aminotosulfhydryl),BDB(bisdiazobenzidine;couplestyrtotyr),EDAC(carbodiimide;couplesaminotocarboxyl).

Weoftenaddanextra("adventitious")tyrtotheN-orC-terminusofthesyntheticpeptide,forBDBcoupling.We"vedonethismoreoftenthannot.Trytoputtheextratyrattheleast"interesting"endofthepeptide,sinceitwillbeattached(buried)tothecarier,andhenceyou"lltendtogetantibodytothemostinterestingendofthepeptide(awayfromthecarrier).

It"susefultohaveaverylowspecificactivity(say,10,000cpm/mgpeptide)14Clabelinaglyoralaresidueinthepeptide.Youcanthenfollowcouplingefficiencyofthepeptideintothecarrier.

WetypicallyimmunizerabbitsbytheVaitukaitisprotocol,withmultipleintradermalimmunizationsatonce.Weshavetheupperbackoftherabbitpriortoimmunization.Wetendtouse1mgofpeptideperimmunization(3-5intradermalsitesateachimmunization),andre-immunizeat2-6weekintervals.Intradermal(intracutaneous)means:raiseawheal(injectintotheskin,justammorsobeneeaththesurface).Aftertheinitialimmunizationand2-3boosts,webleed3-4weekslater,andtesttheantibodies,bywesternblot(immunoblot)orimmunoprecipitationof[125-I]-labeledantigen(eitherthepeptide,orthewholemoleculefromwhichthepeptidederives).

Fortheinitialimmunization,wemixtheimmunogen(hapten-carrierconjugate)withanequalvolumeofcompleteFreund"sadjuvant.Forboosts,weuseincompleteFreund"sadjuvant.Mixingtheoilyadjuvantwiththeaqueousconjugateisbestdonewithamixingneedleandtwoattachedsyringes.MixingresultsinasUSPension.

Bythiskindofprotocol,weALWAYSobtainsyntheticpeptideantibodieswhichrecognizetheantigen(intactfulllengthmolecule,fromwhichthepeptidewasderived),especiallyonimmunoblot.Sometimestheepitopeisweak,andtheantobodiesareoflowtiter(e.g.,1:100forimmunoblot).Butwealwaysgetausableantibody.

Citationswherewe"veusedtheseprocedures:

BarbosaJA,GillBM,TakiyyuddinMA,O"ConnorDT:ChromograninA:posttranslationalmodificationsinsecretorygranules.Endocrinology128:174-190,1991.Here,weusedantibodiestochromograninAsyntheticpeptidestoexaminerouteofproteolysis(post-translationalprocessing)ofthemolecule.

GillBM,BarbosaJA,DinhTQ,GarrodS,O"ConnorDT:ChromograninB:isolationfrompheochromocytoma,N-terminalsequence,tissuedistributionandsecretoryvesicleprocessing.RegulPeptides33:223-35,1991.Here,weusedantibodiestochromograninBsyntheticpeptidestoexaminerouteofproteolysisofthemolecule.

GillBM,BarbosaJA,Hogue-AngelettiR,VarkiN,O"ConnorDT:ChromograninAepitopes:cluesfromsyntheticpeptidesandpeptidemapping.Neuropeptides21:105-18,1992.Here,weusedantibodiestochromograninAsyntheticpeptidestoimmunoprecipitatethemolecule.

TakiyyuddinMA,DeNicolaL,GabbaiFB,DinhTQ,KennedyB,ZieglerMG,SabbanEL,ParmerRJ,O"ConnorDT:Catecholaminesecretoryvesicles.Augmentedchromograninsandaminesinsecondaryhypertension.Hypertension21:674-9,1993.HerewedevelopedanRIAforchromograninA,basedonanN-terminalsyntheticpeptide.

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