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ANANTIGENMICROARRAYIMMUNOASSAYFORMULTIPLEXSCREENINGOFMOUSEMONOCLONALANTIBODIESBYMANLIODICRISTINA1,LUISANUNZIANGELI1,MARIAANGELAGIUBILEI1,BARBARACAPUCCINI1,LORENZODEPISCOPO2,ROBERTASPACCAPELO1ANDANDREACRISANTI1,21UNIVERSITYOFPERUGIA,DEPARTMENTOFEXPERIMENTALMEDICINEMICROBIOLOGYSECTIONVIADELGIOCHETTO,PERUGIA,ITALY2DIVISIONOFMOLECULARANDCELLBIOLOGY,IMPERIALCOLLEGE,IMPERIALCOLLEGEROAD,SW72AZLONDON,UNITEDKINGDOMKEYWORDSMULTIPLEXIMMUNIZATIONHYBRIDOMAMONOCLONALANTIBODYMICROARRAYIMMUNOASSAYCORRESPONDINGAUTHOREMAILACRSIMPERIALACUKPHONE442075945426FAX442075945439ABSTRACTTHEMOUSEMONOCLONALANTIBODYTECHNOLOGYSTILLREPRESENTSAKEYSOURCEOFREAGENTSFORRESEARCHANDCLINICALDIAGNOSISTHOUGHITISRELATIVELYINEFFICIENTANDEXPENSIVEANDTHEREFOREUNSUITABLEFORHIGHTHROUGHPUTPRODUCTIONAGAINSTAVASTREPERTOIREOFANTIGENSHEREWEDESCRIBEAPROTOCOLTHATCOMBINESTHEIMMUNIZATIONOFINDIVIDUALMICEWITHCOMPLEXMIXTURESOFINFLUENZAVIRUSSTRAINSANDAMICROARRAYBASEDIMMUNOASSAYPROCEDURETOPERFORMAPARALLELSCREENINGAGAINSTTHEVIRALANTIGENSTHEPROTOCOLINVOLVESTESTINGTHESUPERNATANTSOFSOMATICCELLHYBRIDSAGAINSTACAPTURESUBSTRATUMCONTAININGANARRAYOFDIFFERENTANTIGENSFOREACHFUSIONEXPERIMENTWECARRIEDOUTMORETHAN25,000ANTIGENANTIBODYREACTIVITYTESTSINLESSTHANAWEEK,ATHROUGHPUTTHATISTWOORDERSHIGHERTHANTRADITIONALANTIBODYDETECTIONASSAYSSUCHASELISAANDIMMUNOFLUORESCENCEUSINGALIMITEDNUMBEROFMICEWECOULDDEVELOPAVASTREPERTOIREOFMONOCLONALANTIBODIESDIRECTEDAGAINSTNUCLEARANDSURFACEPROTEINSOFSEVERALHUMANANDAVIANINFLUENZAVIRUSSTRAINSINTRODUCTIONASSEQUENCINGPROJECTSOFMICROBIALORGANISMSAREBEINGCOMPLETEDALARGENUMBEROFGENESOFPOTENTIALBIOLOGICALANDMEDICALRELEVANCEARECLASSIFIEDAS“UNKNOWN”BECAUSETHEIRUNIQUESEQUENCEANDSTRUCTURALFEATURESELUDEBIOINFORMATICPREDICTIONSTHEBIOCHEMICALANDFUNCTIONALCHARACTERIZATIONOFTHESEGENESISACHALLENGINGTASKTHATREQUIRESTHEADHOCDEVELOPMENTOFAGREATNUMBEROFHIGHLYSPECIFICANTIBODIESDIRECTEDAGAINSTINDIVIDUALCOMPONENTSOFTHEMICROBIALPROTEOMESTHESOMATICCELLHYBRIDTECHNOLOGYTHATISUTILIZEDFORGENERATINGMOUSEMONOCLONALANTIBODIESMABS1HASBEENSUBSTANTIALLYIMPROVEDOVERTHELASTTWENTYYEARSBUTSTILLREMAINSACOMPLEXANDRELATIVELYINEFFICIENTPROCEDURETHEMOSTIMPORTANTLIMITINGFACTORINTHETECHNOLOGYISTHETHROUGHPUTOFTHEANTIBODYSCREENINGASSAYTHATINTURNDETERMINESTHENUMBEROFDISTINCTANTIGENSPECIFICMABSTHATCANBEIDENTIFIEDATANYGIVENTIMEINATYPICALEXPERIMENTANTIBODYPRODUCINGLYMPHOCYTESCOLLECTEDFROMTHESPLEENOFANTIGENIMMUNIZEDMICEAREFUSEDWITHMYELOMACELLSTOGENERATEHYBRIDSTHATPRODUCEANTIGENSPECIFICMABANDMULTIPLYINDEFINITELY2THECONCOMITANTGROWTHOFTHOUSANDSOFCELLHYBRIDSINSMALLWELLSDICTATESTHENEEDTORAPIDLYIDENTIFYTHOSECULTURESPRODUCINGTHERELEVANTANTIBODIESFORFURTHEREXPANSIONANDCLONING3NORMALLYTHESCREENINGPROCEDUREISCARRIEDOUTUSINGIMMUNOASSAYSSUCHASTHEENZYMELINKEDIMMUNOSORBENTASSAYELISA,IMMUNOFLUORESCENCEANDCYTOFLUORIMETRY410THESEASSAYSAREONLYSUITABLEFORINVESTIGATINGANTIBODYSPECIFICITYAGAINSTALIMITEDNUMBEROFANTIGENSATONCETIMEBECAUSETHEYARELABORINTENSIVE,REQUIRELARGEVOLUMESOFSAMPLESANDHAVEALOWTHROUGHPUTTHEPRODUCTIONOFMABLIBRARIESDIRECTEDAGAINSTAVASTANTIGENREPERTOIREINATIMELYMANNERREQUIRESASSAYSYSTEMSCAPABLEOFPERFORMINGTHOUSANDSOFIMMUNOASSAYSINPARALLELTHISREQUIREMENTISFARBEYONDTHETHROUGHPUTOFCONVENTIONALIMMUNOASSAYSANDTHECAPABILITYOFMOSTRESEARCHLABORATORIESINTERMOFLOGISTICS,RESOURCESANDPERSONNEL1115THEDEVELOPMENTANDVALIDATIONOFMULTIPLEXMICROARRAYIMMUNOASSAYS1618HASOVERTHELASTYEARSDRAMATICALLYENHANCEDTHECAPABILITYOFSOMATICCELLHYBRIDTECHNOLOGYTODEVELOPMABLIBRARIESWITHAVASTANTIGENSPECIFICITYREPERTOIREAPPROACHESUSINGMICROARRAYTECHNOLOGYHAVEALSOBEENEXPLOITEDTOEXPEDITETHESCREENINGOFMABOFDESIREDSPECIFICITYONEAPPROACHEMPLOYSASOFTLITHOGRAPHICPROCESSCALLEDMICROENGRAVINGTOSCREENANDRETRIEVEINDIVIDUALANTIBODYSECRETINGCELLS19SINGLEANTIBODYSECRETINGCELLSAREDISTRIBUTEDONTOMICROSCALEWELLSTOGENERATEANANTIBODYARRAYSUBSTRATETHATISINCUBATEDWITHDIFFERENTANTIGENSTHISSTRATEGYALLOWSTHEISOLATIONOFACLONALLINEOFHYBRIDOMASTHATPRODUCESTHEANTIBODYOFINTERESTDIRECTLYWITHOUTTHENEEDFORADDITIONALCLONINGALTHOUGHTHISMETHODTYPICALLYYIELDSLARGENUMBERSOFCLONESTHATPRODUCEANTIBODIESSPECIFICFORTHETARGETOFINTERESTITISNOTAPPROPRIATEFORIMMUNIZATIONWITHCOMPLEXMIXTURESOFANTIGENS,SUCHASWHOLEMICROORGANISMSAMETHODCOMBININGIMMUNIZATIONWITHMULTIPLERECOMBINANTANTIGENSANDANANTIGENCOATEDMICROARRAYSCREENINGASSAYAMAHASBEENEMPLOYEDTOGENERATEMONOCLONALANTIBODIESDIRECTEDTODIFFERENTTARGETS20THISMETHODUTILISESCHIPSHOMOGENEOUSLYCOATEDWITHASINGLEANTIGENTHATARESUBSEQUENTLYARRAYEDWITHHYBRIDOMASUPERNATANTSTHISAPPROACHREPRESENTSAUSEFULTOOLTOSCREENMONOCLONALANTIBODIESALTHOUGHISDIFFICULTTOSTANDARDISEANDQUITEIMPRACTICALWHENHYBRIDOMASMUSTBESCREENEDAGAINSTMULTIPLEANTIGENSMOREOVER,AMAREQUIRESAHIGHAMOUNTOFMATERIALTOCOATTHEWHOLESURFACEOFASLIDE5GPERSLIDEINPREVIOUSWORKSWEHAVESHOWNHOWIMMUNOASSAYSTHATUTILIZEANANTIGENMICROARRAYASSUBSTRATUMCOULDBEUSEDTODETECTSERUMANTIBODIESDIRECTEDTOMICROBIALORGANISMSSUCHASTOXOPLASMA,RUBELLAANDHSV2123,TOASSAYALLERGENS24,25ANDTOINVESTIGATEHUMANIMMUNERESPONSEAGAINSTAVARIETYOFPLASMODIUMFALCIPARUMANTIGENS26THEPOSSIBILITYTODISTRIBUTEAGREATNUMBEROFCAPTUREMOLECULESPACKEDINASMALLAREAONTHESAMEASSAYSUBSTRATUMALLOWSTHOUSANDSOFDISTINCTANTIGENANTIBODYREACTIONSTOBEPERFORMEDSIMULTANEOUSLYUSINGSMALLQUANTITIESOFCULTURESUPERNATANTSANDREAGENTSTHISASSAYFORMATTHUSINCORPORATESKEYFEATURESSUCHASTRUEPARALLELISM,MINIATURIZATIONANDHIGHTHROUGHPUTTHEREBYOVERCOMINGMOSTOFTHELIMITATIONSOFTRADITIONALIMMUNOASSAYS2325THEPROTOCOLPRESENTEDHEREALLOWSTHEOPERATORTOOBTAIN,FROMASINGLEASSAY,INFORMATIONNOTONLYONTHEREACTIVITYOFAMONOCLONALANTIBODYTOANANTIGENORMICROORGANISM,BUTWHETHERITSSPECIFICITYISRESTRICTEDTOASINGLEMOLECULE/ORGANISMORCROSSREACTSTOOTHERSTHEARRAYCANBEDESIGNEDTOINCLUDEAVARIETYOFDIFFERENTANTIGENSORWHOLEMICROORGANISMSRESULTINGINAHIGHLYINFORMATIVEASSAYTHATDRIVESANEARLYDECISIONONWHETHERORNOTTOSELECTADETERMINEDHYBRIDOMA,DRASTICALLYINCREASINGTHEEFFICIENCYOFTHEPROCESSANDREDUCINGTHEWORKLOADBYELIMINATINGUNWANTEDCELLCULTURESATAVERYEARLYSTAGETHISPROTOCOLMAYBEPARTICULARLYSUITABLEFORSEEKINGMONOCLONALANTIBODIESRECOGNIZINGCONSERVEDEPITOPESOFHIGHLYVARIABLEANTIGENSORORTHOLOGOUSPROTEINSFROMDIFFERENTMICROORGANISMSATTHESAMETIME,THETECHNOLOGYDESCRIBEDHEREISEQUALLYAPPROPRIATEFORSELECTINGMONOCLONALANTIBODIESREACTINGEXCLUSIVELYWITHONESPECIFICSTRAIN,TYPE,SUBTYPEORISOLATEOFONEMICROORGANISMMAYBEIDENTIFIEDMOREOVER,INOURHANDS,IMMUNIZATIONWITHWHOLEMICROORGANISMS,INSTEADOFRECOMBINANTANTIGENS,HASSHOWNTOBEAVALIDSTRATEGYTOGENERATEHYBRIDOMASTHATPRODUCEANTIBODIESDIRECTEDTOGLYCOSYLMOIETIESTHEPRINCIPALLIMITATIONOFTHISAPPROACHISTHENUMBEROFSLIDESTOBEPROCESSEDDURINGTHEHYBRIDOMASCREENINGMORETHANONEHUNDREDSLIDESARENECESSARYTOCOMPLETETHESCREENINGOFINDIVIDUALHYBRIDOMALIBRARIESRESULTINGFROMONEFUSIONPROCEDUREOFCOURSE,ANINSTRUMENTABLETOAUTOMATICALLYPROCESSSLIDESWOULDOVERCOMETHEINTENSEWORKOFTHISPHASEOFTHEPROTOCOLTHEHETEROGENEOUSNATUREOFTHEANTIGENSPROTEIN,SUGARRESIDUESANDLIPIDSPRESENTSMANYCHALLENGESINALLASPECTSOFDEVELOPINGSUCHARRAYS,FROMIMMOBILIZATIONOFTHECAPTUREMOLECULETODETECTIONOFTHEBOUNDLIGANDINADDITION,THEREISNOSIMPLEMETHODOFANTIGENAMPLIFICATIONSUCHASPCRFORNUCLEICACIDS,ANDSTABILIZATIONISYETAFURTHERMAJORCONSIDERATIONDIFFERENTIMMOBILIZATIONPROTOCOLSHAVEBEENEXPERIMENTALLYVALIDATEDUSINGCOMBINATIONOFBUFFERSANDIMMOBILIZATIONSUBSTRATESEGPOLYLYSINE,ALDEHYDE,SYLILATEDALDEHYDEORSILANATEDAMINEARRAYSOFPROTEINSCOVALENTLYBOUNDTOALDEHYDECOATEDGLASSSLIDESWERESHOWNTORETAINTHEIRABILITYTOINTERACTSPECIFICALLYWITHOTHERPROTEINSORWITHSMALLMOLECULESINSOLUTIONS27INTHEPROTOCOLPRESENTEDHEREWEHAVESHOWNTHATWHOLEMICROORGANISMS,SUCHASINFLUENZAVIRUSES,CANBEPRINTEDINARRAYSANDUSEDASCAPTURESUBSTRATESOVERCOMINGDIFFICULTIESASSOCIATEDWITHPRODUCINGRECOMBINANTANTIGENSORWITHPURIFYINGMOLECULESFROMTHEIRSOURCESWITHREGARDSTOARRAYSTABILIZATION,WEOBSERVEDTHATBINDINGOFSUBSTRATESONTOALDEHYDEGLASSSLIDESREQUIRED24HOURSTOBECOMESTABLEANDNOSIGNIFICANTDIFFERENCESWEREOBSERVEDINTHEASSAYPERFORMANCEFORATLEASTSIXMONTHS,THEREAFTERWEEXPERIENCEDAPROGRESSIVEDETERIORATIONOFTHEARRAYSNOTWITHSTANDINGTHESECHALLENGES,ANTIGENIMMUNOASSAYSREPRESENTASUITABLETOOLTOTRANSLATEGENOMICINFORMATIONORIGINATINGFROMSEQUENCINGPROJECTSOFMICROBIALORGANISMSINTOFUNCTIONALIMMUNOLOGICALKNOWLEDGEHERE,WEDESCRIBETHESTEPBYSTEPPROTOCOLTOPRODUCEAMONOCLONALANTIBODYLIBRARYDIRECTEDAGAINSTDISTINCTINFLUENZAVIRUSTYPESANDSUBTYPESEMPLOYINGMULTIPLEXIMMUNIZATION28WITHWHOLEINACTIVATEDMICROORGANISMSINTHISEXAMPLEWHOLEINACTIVATEDVIRUSESASSOURCEOFANTIGENSANDANIMMUNOASSAYTHATEMPLOYSASCAPTURESUBSTRATUMAMICROARRAYOFINFLUENZAANTIGENSTHISPROTOCOLISPARTICULARLYUSEFULWHENTHEOBJECTIVEISTOPRODUCESEVERALMABSEACHDIRECTEDTOADISTINCTCOMPONENTOFCELLULARFRACTIONS,MICROBIALORGANISMSANDPARASITESWEILLUSTRATEHOWCOMBININGMICROARRAYIMMUNOASSAYANDSOMATICCELLHYBRIDMABTECHNOLOGYISPOSSIBLETORAPIDLYIDENTIFYALARGENUMBEROFANTIBODIESDISPLAYINGARANGEOFDIFFERENTSPECIFICITIESAGAINSTTHEMICROORGANISMSUSEDINTHEMULTIPLEXIMMUNIZATIONREGIMENEXPERIMENTALDESIGNIMMUNIZATIONREGIMENTHEPROTOCOLALLOWSFORTHERAPIDDEVELOPMENTOFMABSOFDIFFERENTSPECIFICITIESSTARTINGFROMANIMALSIMMUNIZEDWITHWHOLEMICROORGANISMS,CELLEXTRACTSANDCELLORGANELLESASANEXAMPLE,WEIMMUNISEDSEVENGROUPSOFMICEWITHSIXSUBTYPESOFWHOLEINFLUENZAVIRUSESINACTIVATEDBYEITHERFORMALDEHYDEORPROPIOLACTONETREATMENTTHEFIRSTGROUPOFANIMALSWASINJECTEDWITHAMIXTUREOFVIRUSESCONTAININGTHEASUBTYPESH1N1ANDH5N3ANDONEBSTRAINOFINFLUENZAVIRUSVICTORIALINEAGETHESECONDGROUPWASIMMUNISEDWITHAMIXTUREOFH3N2ANDH7N3SUBTYPESOFINFLUENZAAVIRUS,WHEREASOTHERFIVEGROUPSOFANIMALSWEREEACHINJECTEDWITHINDIVIDUALVIRUSSUBTYPESH5N1,H1N1,H3N2,H7N3ANDH5N3TABLE1MOUSEIMMUNIZATIONISNORMALLYPERFORMEDBYINJECTING50100GOFANTIGENFOR45TIMESAT15DAYINTERVALSASWEIMMUNIZEDMICEWITHWHOLEINFLUENZAVIRUSES,WEINJECTEDTHEMAXIMUMAMOUNTOFVIRUSTOLERATEDBYTHEANIMALTODELIVERASUFFICIENTAMOUNTOFEACHVIRALPROTEINTHEOVERALLAMOUNTOFVIRUSTOLERATEDBYTHEMICEWASTHEEQUIVALENTOF21GOFTOTALHEMAGGLUTININWEMIXED250LOFVIRUSSOLUTIONWITH250LOFADJUVANTSOR1XPHOSPHATESALINEBUFFERPBSTOBEINJECTEDINTOEACHMOUSEANTIGENMICROARRAYPRODUCTIONANTIGENSELECTIONTHENATUREANDTHEPURITYOFTHEARRAYEDANTIGENSWILLLARGELYDETERMINETHECAPABILITYOFIDENTIFYINGTHEANTIBODYWITHTHEDESIREDREACTIVEPROFILEASCAPTURESUBSTRATUMWEEMPLOYEDANANTIGENMICROARRAYCONTAININGAVASTCOMBINAT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