Single-domain antibodies and their utility

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Single-domain antibodies and their utility
Baral, Toya Nath; MacKenzie, Roger; Ghahroudi, Mehdi Arbabi
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Current Protocols in Immunology, SUPPL 103, pp. 1-57, 2013-11-18
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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