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NRC Publications Archive Archives des publications du CNRC Single-domain antibodies and their utility Baral, Toya Nath; MacKenzie, Roger; Ghahroudi, Mehdi Arbabi For the publisher’s version, please access the DOI link below./ Pour consulter la version de l’éditeur, utilisez le lien DOI ci-dessous. Publisher’s version / Version de l'éditeur: http://doi.org/10.1002/0471142735.im0217s103 Current Protocols in Immunology, SUPPL 103, pp. 1-57, 2013-11-18 NRC Publications Record / Notice d'Archives des publications de CNRC: http://nparc.cisti-icist.nrc-cnrc.gc.ca/eng/view/object/?id=e552ed0a-502f-47b3-a6f6-9daf5bd955d3 http://nparc.cisti-icist.nrc-cnrc.gc.ca/fra/voir/objet/?id=e552ed0a-502f-47b3-a6f6-9daf5bd955d3 Access and use of this website and the material on it are subject to the Terms and Conditions set forth at http://nparc.cisti-icist.nrc-cnrc.gc.ca/eng/copyright READ THESE TERMS AND CONDITIONS CAREFULLY BEFORE USING THIS WEBSITE. L’accès à ce site Web et l’utilisation de son contenu sont assujettis aux conditions présentées dans le site http://nparc.cisti-icist.nrc-cnrc.gc.ca/fra/droits LISEZ CES CONDITIONS ATTENTIVEMENT AVANT D’UTILISER CE SITE WEB. Questions? Contact the NRC Publications Archive team at [email protected]. If you wish to email the authors directly, please see the first page of the publication for their contact information. Vous avez des questions? Nous pouvons vous aider. Pour communiquer directement avec un auteur, consultez la première page de la revue dans laquelle son article a été publié afin de trouver ses coordonnées. Si vous n’arrivez pas à les repérer, communiquez avec nous à [email protected]. Single-Domain Antibodies and Their UNIT2.17 Utility ToyaNathBaral,1 RogerMacKenzie,1,2 andMehdiArbabiGhahroudi1,2,3 1Human Health Therapeutics, Life Sciences Division, National Research Council Canada, Ottawa,Ontario,Canada 2UniversityofGuelph,Guelph,Ontario,Canada 3DepartmentofBiology,CarletonUniversity,Ottawa,Ontario,Canada ABSTRACT Engineeredmonoclonalantibodyfragmentshavegainedmarketattentionduetotheirver- satilityandtailor-madepotentialandarenowconsideredtobeanimportantpartoffuture immunobiotherapeutics. Single-domain antibodies (sdAbs), also known as nanobodies, arederivedfromVHHs[variabledomains(V)ofheavy-chain-onlyantibodies(HCAb)] ofcamelidheavy-chainantibodies.Thesenature-madesdAbsarewellsuitedforvarious applications due to their favorable characteristics such as small size, ease of genetic manipulation,highaffinityandsolubility,overallstability,resistancetoharshconditions (e.g.,lowpH,hightemperature),andlowimmunogenicity.Mostimportantly,sdAbshave the feature of penetrating into cavities and recognizing hidden epitopes normally inac- cessible to conventional antibodies, mainly due to their protruding CDR3/H3 loops. In thisunit,wewillpresentanddiscusscomprehensiveandstep-by-stepprotocolsroutinely practicedinourlaboratoryforisolatingsdAbsfromimmunizedllamas(orothermembers of the Camelidae family) against target antigens using phage-display technology. Ex- pression,purification,andcharacterizationoftheisolatedsdAbswillthenbedescribed, followed by presentation of several examples of applications of sdAbs previouslychar- acterized in our laboratory and elsewhere. Curr. Protoc. Immunol. 103:2.17.1-2.17.57. (cid:2)C2013byJohnWiley&Sons,Inc. (cid:2) (cid:2) (cid:2) Keywords:single-domainantibody heavy-chainantibodies VHH antibody (cid:2) (cid:2) fragmentlibrariesdimericVHH pentabodies multivalency INTRODUCTION Antibodies are becoming very useful both in research and in biotechnological applica- tions, and these molecules have proven to be an excellent paradigm for the design of high-affinity, protein-based binding reagents. The discovery of mouse hybridoma tech- nologytogeneratemonoclonalantibodies(mAbs)openedanewerainantibodyresearch. Subsequently, innovative recombinant DNA technologies, including chimerization and humanization, have enhanced the clinical efficiency of murine mAbs, and have led to regulatory approval as treatment for solid tumors and hematological malignancies, in- fectious disease, and inflammatory disease (Hudson and Souriau, 2003; Holliger and Hudson,2005;Scottetal.,2012). Antibodies have two distinct functions: binding to antigen, carried out by the antigen- binding fragments (Fabs), and interaction with the receptors on different cells, carried out by the fragment crystallizable (Fc) region. Minimizing the size of antigen-binding proteins to a single immunoglobulin domain with high affinity to a target antigen has beenoneofthemajorgoalsofantibodyengineering(Revetsetal.,2005).Smallerrecom- binantantibodyfragmentssuchasFabs,single-chainFvs(scFvs),andotherengineered variants that are sometimes termed third-generation (3G) molecules (Nelson, 2010), suchasdiabodies,minibodies,andsdAbs,areemergingascrediblealternatives.Genetic Inductionof Immune engineering methods have been successfully applied to improve several issues that are Responses CurrentProtocolsinImmunology2.17.1-2.17.57,November2013 2.17.1 PublishedonlineNovember2013inWileyOnlineLibrary(wileyonlinelibrary.com). DOI:10.1002/0471142735.im0217s103 Supplement103 Copyright(cid:2)C 2013JohnWiley&Sons,Inc. associatedwithunmodifiedIgG,suchassizeandimmunogenicity.Ithasbeenclearthat engineeredantibodieshavecomeofageasbiopharmaceuticals. STRATEGICPLANNING In this unit, we provide detailed protocols for the isolation and characterization of antigen-specific VHHs. Specifically, we describe the protocols for llama immunization andserumresponsemonitoring(BasicProtocol1andAlternateProtocol1),VHHphage- displaylibraryconstructionfromheavy-chainrepertoires(BasicProtocol2),construction of non-immune na¨ıve libraries (Alternate Protocol 2), rescue of phagemid library and panning (Basic Protocol 3), monitoring the progress of biopanning by phage ELISA and colony PCR (Basic Protocol 4), subcloning of VHH fragments in bacterial vectors (BasicProtocol5),expressionofsingle-domainantibodiesinbacterialsystemsandpu- rification of single-domain antibodies (Basic Protocols 6 and 7, respectively), and gel filtration/size-exclusion chromatography (Basic Protocol 8). Methods used to increase functional affinity and potency of VHHs (Basic Protocol 9), preparing VHH Fc fusion proteins (Basic Protocol 10), and characterizing antigen-specific VHHs (Basic Proto- col 11) are also included. Finally, a set of Support Protocols are included for necessary ELISAs (Support Protocol 1), serum fractionation (Support Protocol 2), purification of DNAfragmentsfromagarosegel(SupportProtocol3),preparationofelectrocompetent bacterialcells(SupportProtocol4),SDS-PAGEandwesternblottingofsamples(Support Protocol 5), pentamerization of VHHs (Support Protocol 6), and surface plasmon reso- nanceandepitopemapping(SupportProtocol7).Eachoftheseprotocolsinvolvessome critical considerations, which are described in Critical Parameters in the Commentary. For immunization, it is important to pay attention to the route of immunization, nature and quality of antigen, and quantity of the antigen available. For library construction, it is very important to process the lymphocytes as soon as possible to purify RNA and makecDNA.Ifthelymphocytesarekeptfortoolong,thequalityofRNAandallfurther stepswillbecompromised.Anotherimportantparameteristhesizeanddiversityofthe library, as this will dictate the types of binders that can be isolated. During panning, consideration must be given as to whether the panning will be done in solution or on a coatedsurface.Theamountofantigentobeusedineachroundofpanning,thewashing stringency, and subsequent enrichment in each round of panning are critical aspect to considerandobserve.Forbetter expressionandpurification,itisimportanttoselectan easyandconvenientexpressionsystemintermsofvectorandhost.Forcertaintypesof antibodyformatsorapplications,eukaryoticexpressionsystemsarepreferredoradvan- tageous. Antibody purity is a critical factor for accurate binding and functional studies, andthereforepropersignalsequencesfordirectingantibodiestothebacterialperiplasm orothercellcompartments,aswellasappropriatetagsequencesattheN-orC-terminals, arekeyparameterstobeconsideredandplannedinadvance. BASIC IMMUNIZATIONOFLLAMAS PROTOCOL1 Immunizationisacriticalstepintheisolationofhigh-affinityantibodies.Itiscommon practice in many research laboratories to use emulsions of purified immunogen and Freund’scomplete/incompleteadjuvants.Theadjuvantformsastableemulsionwiththe immunogenandresultsinitssustainedpresentationtotheanimalimmunesystem,thereby stimulating and enhancing the immune response to the immunogen. Other complex materialssuchasfractionatedmembranesorevencelllinesexpressingantigenofinterest can be also used for immunization (Baral et al., 2011). As immunization plans, short and long llama immunization schedules (see Table 2.17.1) are provided here and both have worked well in our laboratory. The short immunization protocol works well for solubleandaccessibleproteinantigens,andsavesconsiderabletimeontheimmunization. SingleDomain However,forlessimmunogenicantigensincludinghaptens,itisrecommendedtofollow Antibodies thelongimmunizationprotocol. 2.17.2 Supplement103 CurrentProtocolsinImmunology Table2.17.1 LongandShortImmunizationSchedules Time Activity Longimmunizationschedule Day1 Pre-immunebleed Day1 Immunization(CFAasanadjuvant) Day22 Boostno.1(IFAasanadjuvant) Day29 Testbleed1 Day36 Boostno.2(IFAasanadjuvant) Day43 Testbleed2 Day50 Boostno.3(IFAasanadjuvant)andtestbleed2 Day57 Testbleed3 Day64 Boostno.4(noadjuvant) Day71 Productionbleed Shortimmunizationschedule Day0 Pre-immunebleed Day0 Immunization(CFAasanadjuvant) Day21 Boostno.1(IFAasanadjuvant) Day28 Boostno.2(IFAasanadjuvant) Day35 Testbleed Day35 Boostno.3(noadjuvant) Day42 Productionbleed Subcutaneousinjectioninthelowerbackorintramuscularinjectionatthelowerrumpare regularroutesofimmunization.Weroutinelyusellamas(Lamaglama)forimmunization due to their availability; however, other camelid members such as alpacas, camels, or dromedariescanbeusedforthispurposeshouldtheybeavailable. Materials Llama(Lamaglama)(CedarlaneLaboratories) Antigen(1mgrequired) Phosphate-bufferedsaline(PBS;seerecipe) Freund’scompleteandincompleteadjuvant(Sigma;alsoseeUNIT2.4) 1-to2-mlsyringeswith21-G,1-to1.5-in.longneedlesforllamaimmunization and10-to15-mlVacutainerforbloodcollection Heparin-coatedtubes(BectonDickinson) AdditionalreagentsandequipmentforemulsifyingantigenusingFreund’s adjuvant(UNIT2.4),isolationoflymphocytes(UNIT7.1),ELISA(Support Protocol1),andfractionationofsera(SupportProtocol2) 1. OnDay1,conductapre-immunebleed(10to15ml)andthenimmunizetheanimal with100to200µgofeachantigen(ifmultipleantigensareused).Dilutetheantigen in PBS to 1 ml total and filter sterilize. Mix the antigen solution well with 1 ml of completeFreund’sadjuvant(CFA)tomakeatotalimmunizationvolumeof2ml(see UNIT2.4). Intramuscular injections (i.m.) at multiple sites in the lower rump on either side of the Inductionof backlegareusedfortheinitialandsubsequentimmunization. Immune Responses UNIT2.4providesguidanceonhowtoemulsifyantigenusingFreund’sadjuvant. 2.17.3 CurrentProtocolsinImmunology Supplement103 All immunizations are performed at Cedarlane Facility in Burlington, Ontario, Canada (http://www.cedarlane.ca)andarebasedontheprotocolprovidedhereandfollowingthe guidelinessetbytheCanadianCouncilonAnimalCare(CCAC).Iffeasible,usetwollamas forimmunization,asthequalityoftheimmuneresponseisdependentontheindividuals. AccesstoalpacasisalsopossibleatCedarlane. 2. On Days 22, 36, and 50, immunize with 100 µg of the same antigens (diluted in PBSto1mltotalandfilter-sterilized),mixedwellwith1mlofincompleteFreund’s adjuvant(IFA;alsoseeUNIT2.4). 3. OnDay64,immunizewith100µgofeachantigen(dilutedinPBSto1mltotal)with noadjuvant. 4. Collect blood (10 to 15 ml) into heparin-coated tubes on Days 29, 43, 57 as test bleed, and collect 50 ml of blood on Day 71 as production bleed. Place the blood immediatelyonice. Itisimportanttohaveapre-immunebleedonDay1(seestep1),whichwillbeusedasa non-immunizedcontrolforasubsequentELISA(SupportProtocol1). 5. Store the collected blood from each bleed overnight at 4°C. Prepare the serum the nextdaybycentrifuging10minat2700×g,4°C,andstoreandtheseraat4°C. 6. Isolatethelymphocytes(UNIT7.1)frombloodcollectedonDay71anduseforphage- displaylibraryconstruction(BasicProtocol2). 7. Following immunizations, analyze llama to monitor antigen-specific heavy-chain antibodyresponsesbyELISA(SupportProtocols1and2). 8. Fractionateserumfromproductionbleed(SupportProtocol2). ALTERNATE IMMUNIZATIONOFLLAMASUSINGSHORTIMMUNIZATION PROTOCOL1 PROTOCOL The short immunization protocol was adapted in our laboratory for easy-to-access and soluble protein antigens. It has the advantage of saving immunization time, requiring loweramountsofantigen,andrequiringfewerboostingstepsfortheanimals. Formaterials,seeBasicProtocol1. 1. OnDay1,conductapre-immunebleed(10to15ml),andthenimmunizetheanimal with200µgofeachantigen(ifmultipleantigensareused).DilutetheantigeninPBS to1mltotalandfiltersterilize.Mixtheantigensolutionwellwith1mlofcomplete Freund’sadjuvant(CFA)tomakeatotalimmunizationvolumeof2ml(seeUNIT2.4). Intramuscular injections (i.m.) at multiple sites in the lower rump on either side of the backlegareusedfortheinitialandsubsequentimmunization. UNIT2.4providesguidanceonhowtoemulsifyantigenusingFreund’sadjuvant. All immunizations are performed at Cedarlane Facility in Burlington, Ontario, Canada (http://www.cedarlane.ca)andarebasedontheprotocolprovidedhereandfollowingthe guidelinessetbytheCanadianCouncilonAnimalCare(CCAC).Iffeasible,usetwollamas forimmunization,asthequalityoftheimmuneresponseisdependentontheindividuals. AccesstoalpacasisalsopossibleatCedarlane. 2. On Days 21 and 28, immunize with 100 µg of the same antigens (diluted in PBS to 1mltotalandfiltersterilized),mixedwellwith1mlofincompleteFreund’sadjuvant (IFA;alsoseeUNIT2.4). SingleDomain 3. OnDay35,immunizewith100µgofeachantigen(dilutedinPBSto1mltotal)with Antibodies noadjuvant. 2.17.4 Supplement103 CurrentProtocolsinImmunology 4. Collectblood(10to15ml)intoheparin-coatedtubesonDay35and50mlofblood onDay42(productionbleed).Immediatelyplaceonice. Itisimportanttohaveapre-immunebleedonDay1,whichwillbeusedasanon-immunized controlforasubsequentELISA. 5. Store the collected blood from each bleed overnight at 4°C. Prepare the serum the nextdaybycentrifuging10minat2700×g,4°C,andstoreandtheserumat4°C. 6. Isolatethelymphocytes(UNIT7.1)frombloodcollectedonDay42anduseforphage- displaylibraryconstruction. 7. Followingimmunizations,analyzellamaseratomonitorantigen-specificheavy-chain antibodyresponsesbyELISA(SupportProtocols1and2). 8. Fractionateserumfromproductionbleed(SupportProtocol2). ENZYMELINKEDIMMUNOSORBENTASSAY(ELISA) SUPPORT PROTOCOL1 The enzyme-linked immunosorbent assay is a routine and widely practiced assay for analyzing the specific interaction between antigens and antibodies in a relatively short period of time. In order to correctly interpret the ELISA results, it is strongly recom- mendedtouseappropriatenegative/blankandpositivecontrols.Itisalsorecommended to examine the stability of the protein antigen before adding it to the ELISA well, in ordertodeterminetheappropriatecoatingconditions.Formostproteins,coatingat4°C isrecommended. Materials Antigenat5to10µg/ml 5µg/mlBSAinPBS(filtersterilizeandstoreat4°C) PBS(seerecipe) PBST:PBS(seerecipe)containing0.05%(v/v)Tween20 BlockingbufferA:3%(w/v)BSAinPBS-T Goatanti-llamaIgGandswineanti-goatIgGlabeledwithhorseradishperoxidase (HRP)(Cedarlane) HRPsubstratesolutionsforELISA(GEHealthcare) 1MH PO 3 4 96-wellMaxiSorpmicrotiterplates(VWR) Microtiterplatereader(Biochrom) AdditionalreagentsandequipmentforELISA(UNIT2.2) 1. ForELISA,coat96-wellmicrotiterplateswith100µloftheantigenat5to10µg/ml andBSAat5µg/mlasacontrolforovernightat4°C(UNIT2.2). UNIT2.2includesdetailedprotocolsforELISA. Theamountofantigenusedis1to5µg/wellinavolumeof100µlinPBS. 2. Thenextday,washthreetimeswithPBST,thenblockthewellswith200µl/wellof blockingbufferAfor2hrat37°C. 3. Washtheplateandaddserialdilutionsofpre-immunetotalserum(collectedonDay 1),post-immunetotalserumfromvariousbleeds(collectedonDays22,43,57,and 71), and fractionated serum (IgG3, IgG1 and IgG2a/b/c fractions) from Day 57 and Day71bleeds.MakethedilutionsinPBSanduse100µlperwell.Incubatefor1.5hr atroomtemperature. Inductionof Immune ThelastwellshouldcontainPBSonlyasacontrol. Responses 2.17.5 CurrentProtocolsinImmunology Supplement103 4. Washthewellsfivetimes,eachtimewith300µlPBST,andadd100µl/wellofgoat anti–llamaIgG-HRP(diluted1:10,000inPBS).Incubate1hrat37°C. Alternatively, goat anti–llama IgG (diluted 1:1,000 in PBS) can be used. Incubate 1 hr at37°Candthen,afterwashingtheplates,add100µl/wellofswineanti-goatIgG-HRP (diluted1:3,000inPBS)for1hrat37°C. 5. Wash the wells again as described in step 4, add HRP substrate (100 µl/well), and incubate at room temperature for 5 to 10 min. Stop the reaction with 1 M H PO 3 4 (100µl/well)andreadat450nmwithamicrotiterplatereader. IfapositiveimmuneresponseisdetectedfromtheIgG2a/b/cfraction,butnotfromtheIgG3 fraction,makesurethattheresponseintheIgG2a/b/cfractionisnotfromcontaminating IgM. SUPPORT LLAMASERUMFRACTIONATIONTOPREPAREPOOLEDIgG2a,IgG2b, PROTOCOL2 ANDIgG2cISOTYPES This protocol fractionates llama sera using protein G and an A¨KTA FPLC purification system followed by the use of a protein A column to prepare the heavy-chain IgG2 fraction,whichconsistsofIgG2a,IgG2b,andIgG2cisotypes. BelowisaproceduretofractionatetheserapreparedfrombloodcollectedonDay57and Day71ofBasicProtocol1(Day42ofAlternateProtocol1)toseparateconventionalIgG and HCAbs (Hamers-Casterman et al., 1993; Doyle et al., 2008). A schematic drawing oftheserumfractionworkflowispresentedinFigure2.17.1. Materials SerapreparedfrombloodcollectedonDay57andDay71ofBasicProtocol1or day42ofAlternateProtocol1 20mMNaPibuffer:8.46mlof1MNaH PO ,11.54mlof1MNa HPO ,diluted 2 4 2 4 in1literofdistilled,deionizedH O(pH7.0) 2 100mMcitratebuffer:3.11gcitricacid,1.53gsodiumcitrate,pH3.5,per200ml ofdistilled,deionizedH O—adjusttopH3.5with1MNaOH,filtersterilize, 2 andstoreat4°C. 1MTris·Cl,pH8.8(APPENDIX2A) fraction serum elute (pH 3.5) IgG3 protein G elute (pH 2.7) IgG1 flowthrough protein A elute (pH 4.5) IgG2 Figure2.17.1 SchematicdrawingoffractionationofllamaserumtoseparateHCAb(IgG2and SingleDomain IgG3)andconventionalAb(IgG1). Antibodies 2.17.6 Supplement103 CurrentProtocolsinImmunology 100mMglycinebuffer:1.5gglycine,pH2.7,per200mlofdistilledH O—adjust 2 topH2.7with3MHCl,filtersterilize,andstoreat4°C. 100mMsodiumacetatebuffer:2.7gsodiumacetate,pH4.5,per200mlof distilledH O—adjusttopH4.5withaceticacid,filtersterilize,andstoreat4°C. 2 Phosphate-bufferedsaline(PBS;seerecipe) DialysistubingwithMWCO8000(BiodesignInc.,http://biodesignofny.com 1-mlHiTrapProteinGHPandHiTrapProteinAHPcolumns(GEHealthcare) A¨KTAFPLCpurificationsystem(GEHealthcare) pHpaper Additionalreagentsandequipmentfordialysis(APPENDIX3H),SDS-PAGE(Support Protocol5;alsoseeUNIT8.4),andELISA(SupportProtocol1) 1. Dialyze 5 ml of llama serum overnight against 500 ml NaPi buffer using an 8000 MWCOmembrane(APPENDIX3H). 2. Centrifuge 5 min at 3600 × g, 4°C, using a bench-top centrifuge with a swinging- bucketrotorandpasssupernatantthrougha0.22-µmfilter. 3. Load1to2mlofthedialyzedllamaserumontoa1-mlHiTrapProteinGHPcolumn that has been equilibrated with 10 ml NaPi buffer, using A¨KTA FPLC purification system. ThecolumnbuffersandMilli-Qwatershouldbedegassedpriortocolumnchromatography. 4. Collecttheflowthrough,washtheproteinGcolumnwith10mlNaPibuffer,andelute theheavy-chainIgG3fractionwith1to2mlofcitratebuffer(pH3.5).Immediately neutralizetheelutedfractionbyadding0.5to1ml1MTris·Cl(pH8.8). It is recommended to check the pH of the eluate by pH paper in order to add the exact amountofTris·Clbuffer. 5. Perform a second elution with 2 to 4 ml of glycine buffer (pH 2.7) to elute the conventional IgG1 fraction. Neutralize the fraction by adding 1 to 2 ml 1 M Tris·Cl (pH8.8). 6. Next, load the flow-through from the protein G column onto a 1 ml HiTrap Protein A HP column equilibrated with 10 ml NaPi buffer. After loading, wash the protein A column with 10 ml of NaPi buffer and apply 1 to 2 ml of sodium acetate buffer (pH4.5)toelutetheheavy-chainIgG2fraction,whichconsistsofIgG2a,IgG2b,and IgG2cisotypes.Neutralizethefractionsbyadding0.1to0.2ml1MTris·Cl(pH8.8). 7. AnalyzetheelutedfractionsonanSDS-PAGEgelundernon-reducingandreducing conditions(SupportProtocol5;alsoseeUNIT8.4). ByrunninganSDS-PAGEgel,successfulseparationofheavy-chainIgGandconventional IgGantibodiesfromthellamaseracanbeconfirmed.Theexpectedmolecularweightsof conventional IgGs, IgG heavy chains, and IgG light chains are approximately 150 kDa, 55 kDa, and 25 kDa, respectively. The expected molecular weight of heavy-chain IgGs andtheheavychainsofHCAbareapproximately85kDaand42kDa,respectively. 8. TesttheseparatedfractionsbyELISA(SupportProtocol1)againsttheimmunogen. If a positive response is found within the heavy-chain IgG fractions, proceed with VHHphage-displaylibraryconstruction(BasicProtocol2). 9. Dialyze the correct fractions against PBS (using 2 to 10 ml of fraction and 2 liters of PBS with an 8000 MWCO dialysis membrane) and store them at 4°C for further analysisbyELISA. Inductionof ELISA is performed on total and fractionated sera to determine if there was an antigen Immune Responses specificheavy-chainantibodyimmuneresponse. 2.17.7 CurrentProtocolsinImmunology Supplement103 BASIC CONSTRUCTIONOFVHHLIBRARY PROTOCOL2 In the first steps of this protocol, peripheral blood lymphocytes (PBLs) are isolated fromtheblood.Thestorageperiodandstorageconditionsforthebloodareverycritical for isolation of good-quality RNA and cDNA synthesis. It is recommended to perform the PBL purification, RNA isolation, and cDNA synthesis as quickly as possible. In this step, PBLs are isolated from the blood obtained on Day 71 (or 42 in the short immunization protocol) post-immunization and used as a source of mRNA for library construction.Byreversetranscription,cDNAissynthesizedusingthemRNAastemplate and oligo(dT) or random hexamers as primers. The cDNA is used as template in PCR with a combination of IgG-specific 5′-end primers (VH/VHH framework 1 region) and 3′-endprimers(constantregion2;CH2).ThisPCRwillamplifybothVHandVHHfrom IgG1, IgG2, and IgG3 genes. The first PCR normally yields a DNA band of about 800 to 900 bp corresponding to VH-CH1-Hinge and almost the first half of the CH2 region (called VH-CH2) of conventional antibodies, and another DNA band of about 550 to 650bpcorrespondingtotheVHH-HingeandalmostthefirsthalfoftheCH2region(called VHH-CH2) of heavy-chain antibodies (Fig. 2.17.2). The VHH region of heavy-chain antibodies,whichisapoolfromIgG2andIgG3genes,isamplifiedinasecondPCRusing VHH-specificprimersanchoredwithSfiIrestrictionsites.TheVHHfragmentsarecloned intothepMED1phagemidvector(ArbabiGhahroudietal.,2009a;Fig.2.17.3A)between thetwoSfiIrestrictionsites,locatedatthe3′-endofthepelBleadersequenceand5′-end of the gene III coding sequence. This allows VHHs to be expressed as fusions to gene III, transported to the E. coli periplasmic space, and displayed on the surface of phage whenitissuper-infectedbyasuitablehelperphagesuchasM13KO7.Inprinciple,any other phagemid vector with similar features such as pHEN1(Clackson et al., 1991) or pCANTAB 5E (GE Healthcare) can be used. In constructing immune VHH libraries, a librarysizeof5×106 to5×107 individualcloneswithuniquesequencesshouldfairly representthelymphocytepopulationinllamablood(approximately1–5×106/mlblood; DeGenstetal.,2006a)usedfortheextractionofRNAandcDNAsynthesis.Generally, a sampling size of 50 to 100 colonies from the library provides a good estimate of its 1000 bp VH VHH 500 bp Figure2.17.2 ADNAagarosegelafterfirstPCRoflibraryconstructionshowingVHandVHH SingleDomain band(leftlane)andmolecularladder(rightlane). Antibodies 2.17.8 Supplement103 CurrentProtocolsinImmunology Figure2.17.3 SchematicdrawingofphagemidvectorpMED1andexpressionvector(pSJF2)withtheirrespective multiplecloningsites. complexity, which is expressed as the number clones with full inserts, each having a unique sequence. Finally, the library is grown in the presence of 2% to 4% glucose for anumberofhoursbeforemakingaglycerolstockofthebacterialcellsandfreezingthe aliquotsat–80°C. Materials LlamablooddrawnonDay71ofBasicProtocol1(orDay42ofAlternate Protocol1) RPMI1640medium(e.g.,LifeTechnologies) LymphoprepTube(Cedarlane) Trizol(LifeTechnologies) First-StrandcDNASynthesiskit(GEHealthcare) 10pmol/µlprimers:CH2FORTA4,CH2B3-F,MJ1,MJ2,MJ3,MJ7,MJ8,PN2, M13RP(seeTable2.17.2)—primerswerepurchasedfromIntegratedDNA Technology(IDT) 5U/µlTaqDNApolymeraseand10×PCRbuffer dNTPs:10mMeachofdTTP,dATP,dCTPanddGTP(NewEnglandBiolabs) QIAquickGelExtractionandQIAquickPCRpurificationkits(Qiagen) pMED1phagemidvector(ArbabiGhahroudietal.,2009a;thevectorisavailable fromtheauthorsuponrequest) Inductionof SfiI,XhoI,andPstIrestrictionendonucleasesandtheirrespective10×buffers Immune Responses LigaFastRapidDNALigationSystem(Promega) 2.17.9 CurrentProtocolsinImmunology Supplement103

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Single-domain antibodies (sdAbs), also known as nanobodies, are derived from Current Protocols in Immunology 2.17.1-2.17.57, November 2013.
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