Antibody Phage Display Derived Antibodies against BmR1 Antigen

21 Pages·2017·5.68 MB·English

International Journal o f
Molecular Sciences
Article
Antibody-Based Protective Immunity against
Helminth Infections: Antibody Phage Display
Derived Antibodies against BmR1 Antigen
AnizahRahumatullah1,IzzatiZahidahAbdulKarim1,RahmahNoordin1 ID and
TheamSoonLim1,2,*
1 InstituteforResearchinMolecularMedicine(INFORMM),UniversitiSainsMalaysia,
Minden11800,Penang,Malaysia;[email protected](A.R.);[email protected](I.Z.A.K.);
[email protected](R.N.)
2 AnalyticalBiochemistryResearchCentre,UniversitiSainsMalaysia,Minden11800,Penang,Malaysia
* Correspondence:[email protected];Tel.:[phone];Fax:[phone]
Received:5September2017;Accepted:2November2017;Published:22November2017
Abstract: Helminthparasiteinfectionsaresignificantlyimpactingglobalhealth,withmorethantwo
billioninfectionsworldwidewithahighmorbidityrate. Thecomplexlifecycleofthenematodes
hasmadehostimmuneresponsestudiesagainsttheseparasitesextremelydifficult. Inthisstudy,
weutilizedtwophageantibodylibraries;theimmuneandnaïvelibrarywereusedtoidentifysingle
chainfragmentvariable(scFv)clonesagainstaspecificfilarialantigen(BmR1). TheV-geneanalysisof
isolatedscFvcloneswillhelpshedlightonpreferentialVDJgenesegmentusageagainstthefilarial
BmR1antigeninhealthyandinfectedstates. Theimmunelibraryshowedtheusageofbothlambda
andkappalightchains. However,thenaïvelibraryshowedpreferentialuseofthelambdafamily
with different amino acid distributions. The binding characteristics of the scFv clones identified
fromthisworkwereanalyzedbyimmunoassayandimmunoaffinitypulldownofBmR1. Thework
highlightstheantibodygeneusagepatternofanaïveandimmuneantibodylibraryagainstthesame
antigenaswellastherobustnatureoftheenrichedantibodiesfordownstreamapplications.
Keywords: single chain fragment variable (scFv); monoclonal antibodies; BmR1; phage display;
immunelibrary;naïvelibrary
1. Introduction
Humanlymphaticfilariasisinfectionremainsamajorpublichealthproblem,mainlyintropical
countries. TheWorldHealthOrganization(WHO)hasidentifiedlymphaticfilariasisasthesecond
leading cause of permanent and long-term disability in the world, after leprosy. It is caused by
microscopic,thread-likeworms,namelyWuchereriabancrofti(W.bancrofti),Brugiamalayi(B.malayi),and
Brugiatimori(B.timori)[1]Thecomplexlifecycleofhumanfilarialnematodes,especiallyW.bancrofti
andB.malayi,involvesmosquitosastransmissionvectors[2]. Thecomplexlifecycleofthenematodes
ismainlyattributedtothecomplicatedhostimmuneresponse,adirectreflectionofthehost–parasite
interaction. Antibodyresponsesagainstparasiticinfectionsareaformofprotectionaffordedbythe
humanbody. Immunoglobulin(Ig)Eantibodiesaremainlyelevatedinfilarialinfectionsbutmostare
non-antigeninducedresponses. InadditiontoIgE,otherantibodyisotypesthataremainlyregulated
inresponsetofilarialinfectionsareIgG1andIgG4antibodies[3]. Studieswithmicemodelshave
showntheroleofIgEaswellasIgMinconferringprotectionagainstB.malayiinfections. Thereisalso
evidenceofIgGactingasapotentmediatorforantibody-mediatedpassiveimmunityinnaïveanimal
modelsusingpurifiedIgG[4]. Therefore,itislikelythatantibodyrepertoiresfromIgMandIgGcould
bepotentiallyusefulrepertoiresfortheisolationofantigen-specificantibodies.
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The flexibility of antibodies to counteract different parasites is evidenced by the exhaustive
variabilityofantibodygenesfoundinthebody. Thisistheresultofvariousgene-editingprocesses
likeVDJgenerearrangementsandsomatichypermutationthatincreasesthediversityofthemature
antibodysequences[5]. Inordertostudythedifferentantibodygeneusagepatternsofahealthyand
parasite-infectedpopulation,theIgMandIgGrepertoireswouldbeidealforuse. However,theIgG
andIgMrepertoirehastobereplicatedinvitroinordertoinvestigatethedifferencesexperimentally.
Thiscanbeachievedbyusingphagedisplaytechnologywheretherepertoireiscapturedinacollection
ofclonedantibodysequencesasafusiontophagecoatproteinstoproduceadiverseantibodylibrary
forpanning[6].Anaïveantibodylibrarywillberepresentativeofahealthyrepertoirewherenoclinical
symptomsofthediseaseareevident[7].However,thisdoesnotdiscountthefactthatsamplecollection
inareaswithhighincidencescouldhavebeenexposedtotheparasiteandrecoveredpriortocollection.
Therefore,memoryresponsesagainsttheexposedantigenswouldlikelystillpersistintherepertoire,
makingatrulynaïverepertoireimpossibletoachieve. Immunelibraries,however,wouldfocuson
therepertoirefromindividualsthatareinfectedandshowclinicalsymptoms[7],providingprimed
antibodyresponsesatthetimeofsamplecollection. Therefore,thenaïverepertoirewouldbewell
definedbytheIgMisotype,whereastheimmunerepertoirewouldbereflectedbytheIgGrepertoire.
As the diversity of the antibody repertoire is dependent on VDJ recombination, a number of
studieshavebeenconductedtostudythediversityofVDJsegmentsinresponsetovariousinfections,
malignancies,andautoimmunediseases[8–12]. Evenwiththeincreasedinterestinantibodygene
usage,thefocusonparasiticinfectionsisscarce.Inthisstudy,animmunelibraryforlymphaticfilariasis
(IgGisotype)[13]andanaïvelibrary(IgMisotype)[14]wasusedtocomparetheabilityofeachlibrary
repertoiretogeneratemonoclonalscFvclonesagainstaspecificfilarialtarget. Thetargetantigenused
inthisstudyisBmR1,aspecificantigenusedforthediagnosisoflymphaticfilariasis. Itisusedas
oneofthetargetantigensforthePanLFRapidtestkitthatisbeingusedbytheMalaysianMinistry
ofHealthinthelymphaticfilariasiseliminationprogram. TheBmR1antigenisagoodtargetasthe
modelproteinduetoitsspecificityandsensitivityinthediagnosisoflymphaticfilariasis. Theantigen
isexpressedbythegenepPROEXHT/Bm17DIII (GenBankaccessionNo. AF225296)andishighly
specificandsensitiveforIgG4assaystodetecttheinfectionofbothB.malayiandB.timori. Theantigen
hasbeenusedinvariousdiagnosticsetupswithpromisingsensitivitylevelsof93–100%. TheBmR1
antigenisalsohighlyspecific(99–100%)intermsofreactivitywithserafromnon-filarialinfections,
makingitanidealcandidateforthiswork. Thecomparativefocusonasingleantigenwouldallowfor
amoresystematiccomparisontodistinguishthegenesegmentuseandcomplementaritydetermining
region(CDR)mutationsofbothrepertoires.
The work allowed us to shed some light on antibody responses, in particular the VDJ gene
rearrangementandusageagainstaparticularparasiticantigen. Theisolatedcloneswerecharacterized
basedontheirgenesequencediversityandbindingfunctionality. ThescFvcloneswereevaluated
fortheirfunctionalitytoidentifytheBmR1antigen. Asequencecomparisonbetweentheantibodies
frombothlibrarieswasalsocarriedouttoidentifypreferentialgeneusagepatternsbyeachrepertoire.
Inconclusion,thestudyprovideduscriticalinsighttothepreferentialVDJgenearrangementaswell
ascomplementaritydeterminingregion(CDR)mutationandlengthdistributionofthescFvclones
againsttheBmR1antigen. Inaddition,theantibodiesdevelopedhavepotentialdiagnosticapplications,
evidentfromtheresultsofthebindingstudies.
2. Results
2.1. BiopanningandPolyclonalELISA
PhageparticlesdisplayingindividualhumanscFvantibodyfragmentswereselectedbythree
roundsofbiopanningonMaxisorpplatescoatedwithBmR1antigenusingbothnaïveandimmune
libraries. The naïve library was previously developed using the repertoire from a pool of healthy
donors. However,theimmunelibrarywasdevelopedusingtherepertoireobtainedfromindividuals
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donors. However, the immune library was developed using the repertoire obtained from
tinhdativwideurealcso nthfiartm ewderpeo scitoinvfeirwmiethd fiplaorsiiatisvise inwfeitchti ofnil.arTihaseiso bisnefrevcetidonin. cTrehaes eoibnsaerbvseodrb ainnccreevasaelu eins
ianbdsiocrabtaenscseu vccaelussefsu ilnednirciacthems seunctcoefsstfaurlg eentrsipcehcmifiecnst coFfv taprrgeeste snptiencgifipch sacgFev pparretsiecnletsinfgo rpehaacghe rpoaurntidcloesf
pfoarn neaincgh (rFoiugnudre o1fa ,pba).nAninsigg n(iFfiigcaunret i1nac,rbe)a. sAei nsiagbnsifoircbanant ciencvraelausee foinr raobusnordb3anpcaen nvianlugew faosr orbotuaninde d3
fpoarnbnointhg liwbraasr oiebst.aTinheedp foosri tbivoetha nlidbrnaergieast.i vTehceo nptorsoiltsivaer eanindd inceagtiavteivteh actotnhteroplsa nanrein igndwiacastsiuvecc tehsasft utlhlye
cpaarnrnieidngo uwtaws istuhcnceosbsfauclklyg rcoaurnridedis osuuet sw. ith no background issues.
TThhee ssttaarrttiinngg aammoouunntt ooff pphhaaggeep paarrtitciclelessf oforrt htheen naiaviveel iblirbarrayryw wasas1 .13.232× ×1 1001212p pfufua anndd3 3.8.855× × 11001100
ffoorr tthhee iimmmmuunnee lliibbrraarryy.. TThhee aammoouunntt ooff pphhaaggee rreeccoovveerreedd ffrroomm rroouunnddss 11 ttoo 33 ffoorr tthhee iimmmmuunnee lliibbrraarryy
wwaass 44..1155 ×× 110077,, 22..6655 ×× 11005,5 a,nandd 55.1.155 ×× 10180, 8r,ersepsepcetcivtievleyl.y W. Whhereeraesa,s ,ththee aammoouunnt tooff pphhaaggee rreeccoovveerreedd ffrroomm
rroouunnddss 11 ttoo 33 ffoorr tthhee nnaaïïvvee lliibbrraarryy wwaass 33..6611 ×× 110066, ,11.0.055 ×× 10140, 4a,nadn d6.261.2 ×1 1×071, 0r7e,srpeescpteivcetilvye. ly .
TThhee eennrriicchhmmeenntt rraattiioo foforrt htheei mimmmunuenel iblirbarrayryw awsa1s. 017.0×7 ×1 01−03−,3,0 .06.969× ×1 01−0−55,, aanndd 11.3.333× × 1100−−22,,
rreessppeeccttiivveellyy.. TThheee ennrricichhmmeennttr raatitoiof oforrt htheen naaiviveel ilbibrarrayryw wasas2 .27.373× ×1 100−−66,, 77..99 ×× 1100−9−, 9a,nadn 4d.74 .×7 1×0−15 0fo−r5 tfhoer
tthhereteh rreoeurnodusn, dres,spreescptievcetilvye. lTy.hTe heenreincrhimchemnet nfotrf otrhteh iemimmmunuen eliblirbarrayr yfrforomm rorouunndd 22 ttoo 33 ccoommppaarreedd ttoo
rroouunndd 11 iiss 00..00006644-- aanndd 1122..4422--ffoolldd.. TThhee eennrriicchhmmeenntt ffoorr tthhee nnaaïïvvee lliibbrraarryy iiss 00..00002299-- aanndd 1177..22--ffoolldd..

FFiigguurree 11.. PPoollyycclloonnaall pphhaaggee EELLIISSAA ooff BBmmRR11 aannttiiggeenn dduurriinngg tthhee ppaannnniinngg ccyycclleess.. ((aa)) IImmmmuunnee lliibbrraarryy
ppaannnniinngg;; ((bb)) nnaaiivvee lliibbrraarryy ppaannnniinngg.. TThhee ppoossiittiivvee ccoonnttrrooll iinncclluuddeedd wwaass ggrreeeenn flfluuoorreesscceennccee pprrootteeiinn
((eeggffpp)) totov avlaidliadtaeteth ethaes saasys.aByo. uBnodupnhda gpehsawgeerse wdeetreec tdedetbecyteindc ubbya tiinocnuwbaitthioHn RwPictho nHjuRgaPt ecdoannjutig-Mate1d3
aannttiib-Mod1y3 (adnitliubtoeddy1 :(5d0il0u0t)e.d 1:5000).
22..22.. MMoonnoocclloonnaall ssccFFvv EELLIISSAA
AA ttoottaallo off3 93292s csFcvFcvl ocnloesnefrso mfrobmo thbolitbhr alirbiersarwieesr ewaenrael yazneadlyuzseindg umsionngo cmloonnaolcslcoFnvalp hscaFgve EpLhIaSgAe.
TEhLeISmAo. nTohcel omnoanlosccFlovnpalh sacgFevE pLhISagAe rEeLsuISltAed reinsuslitxedp oinsi stiivxe pboisnitdiveres biidnednetrifis eiddefnotriftihede ifmorm thuen iemlimbruanrye
alinbdra1ry1 panosdi t1iv1e pboisnidtievres bfoinrdtheres nfaoïrv tehleib nraaïrvye( Fliibgruarreys (2Faig–udraens d2a3–ad– da)n.dP o3sai–tidv)e. Pcloosniteisvew celroendees cwideerde
wdeitchidaedcu wt-oitfhf Oa Dcu40t5-ovffa lOueDa40b5 ovvaelu0e.5 aabfotevret a0k.5i nagftienrt otackoinngsi dinetroa tcioonnstihdeerbaatciokngr tohuen bdavcaklgureo.uTnhde vraanlugee.
oTfhteh reanOgDe 4o0f5 tvhael OueDs4o05f vpaoluseitsi voef pcloosniteisvew calosn0e.6s 9wtaos 30..5689. toT h3.e58im. Tmheu nimemanudnen aanïvde nliabïvraer lyibwraerrye waberlee
taoblper otov idperosvaitdisef ascatotirsyfaecntorircyh menernicthomfBenmtR o1f- spBemcRifi1c-sspceFcvificcl osnceFsv. Tchloenoevs.e rTahlleb aocvkegrarollu nbdacrkegardoiunngds
orebatdaiinnegds ofrbotmainaelldt hfreocmlo anlel sthaere crloelnaetsiv aerley rleolwati(vFeiglyu rloews2 (aF–idguarneds 23aa––dd) a.nd 3a–d).
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FFiigguurree 22.. MMoonnoocclloonnaall pphhaaggee EELLIISSAA aannaallyyssiiss ooff ssccFFvv cclloonneess ffrroomm tthhee imimmmuunnee lilbibrraarryy aaggaaiinnsstt BBmmRR11
aannttiiggeenn.. BBoouunndd pphhaaggeess wweerree ddeetteecctteedd bbyy ininccuubbaattiioonn wwiitthh HHRRPPc coonnjujuggaatteedd aanntti-i-MM1133 aannttibibooddyy ((ddililuutteedd
11::55000000)).. ((aa)) PPllaattee 11 ooff mmoonnoocclloonnaall pphhaaggee EELLIISSAA,, ((bb)) PPllaattee 22 mmoonnoocclloonnaall pphhaaggee EELLIISSAA,, ((cc)) PPllaattee 33
mmoonnoocclloonnaall pphhaaggee EELLIISSAA, ,((dd) )PPlalatet e4 4mmonooncolcolnoanla plhpahgaeg EeLEILSAIS.A .
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Figure3.MonoclonalphageELISAanalysisofscFvclonesfromthenaïvelibraryagainstBmR1antigen.
Figure 3. Monoclonal phage ELISA analysis of scFv clones from the naïve library against BmR1
BoundphagesweredetectedbyincubationwithHRPconjugatedanti-M13antibody(diluted1:5000).
antigen. Bound phages were detected by incubation with HRP conjugated anti-M13 antibody
(a)Plate1ofmonoclonalphageELISA,(b)Plate2monoclonalphageELISA,(c)Plate3monoclonal
(diluted 1:5000). (a) Plate 1 of monoclonal phage ELISA, (b) Plate 2 monoclonal phage ELISA, (c) Plate 3
phageELISA,(d)Plate4monoclonalphageELISA.
monoclonal phage ELISA, (d) Plate 4 monoclonal phage ELISA.
2.3. Antibody Sequence and Gene Analysis
The positive scFv clones were then sequenced and analyzed using IMGT/VQUEST and VBASE2
to determine the human germline sequences of each clone. The sequence analysis showed that only
four out of six scFv clones for the immune library and eight out of 11 naïve scFv clones exhibited
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2.3. AntibodySequenceandGeneAnalysis
ThepositivescFvcloneswerethensequencedandanalyzedusingIMGT/VQUESTandVBASE2
tInot.d J.e Mteorl.m Sciin. 2e0t1h7,e 1h8,u 2m376a n germlinesequencesofeachclone. Thesequenceanalysisshowedthat6o onf l2y1
four out of six scFv clones for the immune library and eight out of 11 naïve scFv clones exhibited
ccoommpplleettee ggeennee sseeqquueenncceess wwiitthhoouutt aannyy mmuuttaattiioonnss.. TThhee rreemmaaiinniinngg sseeqquueenncceess ffoorr bbootthh lliibbrraarriieess hhaadd
mmuuttaattiioonnss rreessuullttiinngge eitihthereri nina far afmraemseh isfhtiofrt torru ntrcuatnicoant.ioTnh.e Tchloen celsowneitsh wpirtohb lpermoabtliecmseaqtiuc esnecqeusewneceres
twheerneo tmheintt eodmfirtotemd ffurortmhe fruarnthaelyr sains.alysis.
TThhee ssccFFvv cclloonneess ffrroomm tthhee iimmmmuunnee lliibbrraarryy hhaadd aann eeqquuaall rreepprreesseennttaattiioonn ooff IIggHHVV22––VVλλ33 ((5500%%)) aanndd
IIggHHVV22––VVκκ33 ((5500%%)) ((FFiigguurree 44aa)).. TThhee ssccFFvv cclloonneess ffrroomm tthhee nnaaïïvvee lliibbrraarryy sshhoowweedd vvaarriiaattiioonnss iinn ggeennee
ddiissttrriibbuuttiioonn wwiitthh ttwwoo ssccFFvv sseeqquueennccee ppaaiirriinnggss bbeeiinngg iiddeennttiififieedd.. SSiixx oouutt ooff eeiigghhtt cclloonneess wweerree ddeerriivveedd
ffrroomm tthhee IIggHHVV44––VVλλ33 ((7755%%)) ppaaiirriinngg aanndd tthhee ootthheerr ttwwoo sseeqquueenncceess sshhoowweedd IIggHHVV11––VVλλ11 ((2255%%)) ppaaiirriinngg
((FFiigguurree 44bb)).. TThhee cclloonneess ffrroomm tthhee iimmmmuunnee lliibbrraarryy sshhoowweedd aa pprreeffeerreennccee ffoorr tthhee VVHH22 ffaammiillyy oonnllyy..
HHoowweevveerr,, cclloonneess ffrroommt htheen naaïvïveel ilbibrararyrys hshoowwededn noor erperperseesnetnattaiotinono fotfh tehVe κVfκa mfaimlyi,lyw, iwthitahm a amjoarjiotyriotyf
colfo cnleosnuesti ulitziilnizginthge thIgeH IgVH4V–V4λ–V3λco3m cobminbaitnioanti.on.

FFiigguurree 44.. AAnnalaylysissiso fogfe ngeenpea iprianigrinfrge qfureenqucyenocfyh uomf ahnumscFanv asnctFivbo adnietsibsoedleiectse dsealegcatiendst aBgmaRin1sat nBtimgeRn1.
(aan)tIigmemn.u (nae) lIimbrmaruyn;e( bli)bnraarïvye; (lbib)r anrayï.veF ilgiburraersya. rFeigpurerseesn atered parseasepnetrecde natsa gae poefrcthenettaogtea lonfu tmheb etortoafl
sncuFmvbseeqr uoef nscceFdv. sequenced.
2.4. CDR Length Analysis
2.4. CDRLengthAnalysis
The antigen-binding site of antibodies is usually formed by the combination of six loops of
The antigen-binding site of antibodies is usually formed by the combination of six loops of
hypervariable sequences, three CDRs (CDR1, CDR2, and CDR3) from the HC and LC, respectively.
hypervariablesequences,threeCDRs(CDR1,CDR2,andCDR3)fromtheHCandLC,respectively.
In general, the CDR length for the VH is between seven and 22 amino acids. The HC CDR1 was
Ingeneral,theCDRlengthfortheVHisbetweensevenand22aminoacids. TheHCCDR1wasfound
found to be eight and 10 amino acids in length. CDR2 had only six and seven amino acids, whereas
tobeeightand10aminoacidsinlength. CDR2hadonlysixandsevenaminoacids,whereasforCDR3
for CDR3 the distribution was 12 and 22 amino acids. The LC analysis showed six and seven amino
thedistributionwas12and22aminoacids. TheLCanalysisshowedsixandsevenaminoacidsfor
acids for CDR1, only three amino acids for CDR2, and 9 and 11 amino acids for CDR3 (Figure 5a,b).
CDR1,onlythreeaminoacidsforCDR2,and9and11aminoacidsforCDR3(Figure5a,b).
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FiFgiguurere5 .5.A Ammininooa acciidd lleennggtthh vvaarriiaattiioonnss ooff IIggHHVV aanndd IIggLLVV ooff BBmmRR11 ssccFFvv cclolonnees.s .(a()a )ImImmmunuen eliblirbarrayr; y;
(b(b)n) anïvaeïvleib rliabrrya.rLye. nLgetnhgdtihs trdiibsutrtiibountoiofnV HofC VDHR 1C,CDDRR1,2 ,CaDndR2C, DaRn3d reCgDioRn3s srheogwioinnsg shhigohwliyndgi vheirgshifilye d
CdDivRe3rsriefpieedr tCoiDreRs3w reitpherdtiofifreerse nwtiltehn dgitfhfesr.ent lengths.
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2.5. Amino Acid Distribution and Polarity Analysis
The amino acid propensity of the enriched scFv clones showed a random distribution pattern
for antibody clones obtained from both libraries (Figure 6a,b). The abundance of each individual
Ianmt.Ji.nMoo al.cSicdi. f2o01r7 t,h18e, H23C76 and LC was assessed. Increase in the presence of different amino acids c8anof b21e
seen from CDR1 to CDR3. CDR3 showed the highest variation in amino acids compared to CDR1
and CDR2. The amino acid distribution pattern showed certain amino acids being significantly over
2.5. AminoAcidDistributionandPolarityAnalysis
represented, under represented or even absent in some cases. This variation highlights the diversity
of thTe haentaibmoidnyo raecpiderptoriorpe ewnistihtyino af sthmeaelln sruicbhseedt osfc Fclvoncleosn aegsasinhsotw ae pdaartircaunldaro mantdigisetnri.b utionpattern
for anTthibeo CdDyRc1lo onfe tshoeb HtaCin ferdomfr othme bimotmhulinber alirbierasr(yF icglounrees6 sah,bo)w. eTdh ea ahbiguhnedr arnecpereosfeneatacthioinn doifv siedruinael
a(Sm),i nfoolalocwidefdo rbtyh eglHycCinaen (dGL).C Hwoawseavsesre, sCseDdR. 2In ocrf etahsee HinCth weapsr epsreendcoemofindainfftelyre anstpaamrtiantoe a(Dci)d, swcaitnh
baergsieneinnef r(oRm) aCnDdR a1latnoiCneD (RA3). aCbDuRnd3asnhto wine CdDthRe3h oigf hthees tHvCar.i aOtino nthien oatmheinr ohaancidd,s tchoem CpDaRre1d otfo tCheD LRC1
ahnadd Ca DhRig2h.eTrh peearcmeinntoagaec iodf dseisrtirnieb u(Sti)o annpda ttyterronsisnheo (wYe).d Gcleyrctainine a(Gm)i nwoaas chidigshbeesitn ign sCigDnRifi2c aanndtl yseorvinere
r(eSp) rwesaesn pterde,duonmdienrarnetp froers eCnDteRd3o. rGelvuetanmabastee n(Eti)n wsaosm feoucansde st.oT bhei savbasreinatt ifornomhi gthheli gahnttsibtohdeyd irveepresrittyoioref
tohfe tahnet ibimodmyurnepe erlitboriraeryw ictlhoinneas.s mThaell sHuCbs eCtDofRc loanneaslyasgisa inosf tnaapïvaret icliublraarryan tcilgoenne.s showed a high
repreTsheentCaDtioRn1 ooff tGhe, HfoCllofrwoemd thbye iSm minu CnDelRib1r. aHryocwloenveesr,s hisoowleeudcianhei g(Ih)e wrraesp rperseednotamtiionnanotf siner iCnDe(RS2),,
fwolhloewreeads Rb yangdly Gci nwee(rGe) h.Higohw ine vCeDr,RC3D. LRC2 oCfDthRe1 HhaCdw aa hsigphre rdeopmreisneannttaltyioans poaf rGta, twe(itDh) ,awspiathraagrignien (inNe)
(aRn)da Dnd foarl aCnDinRe2 (aAn)da CbDunRd3a, nretsipnecCtDivRel3y.o Tfhthe eclHonCe.s Ofrnomth tehoe tihmermhuanned a,ntdh enaCïDveR l1iborafrtyh eshLoCwehda dthae
haibgsheenrcep eorfc menettahgioenoifnsee (rMin)e, (lyS)sianned (Kty),r oasnidn ecy(Yst)e.iGnely (cCin).e H(Gow)wevaesrh, gigluhteasmtianteC (DER) a2nadn dprsoelrininee (P(S)) wwearse
pardeddiotimoninalalnyt mfoisrsCinDgR fr3o.mG ltuhtea immamteu(nEe) awnads nfaoïuvned retpoebrteoairbesse, nretsfproemctivtheelya. n tibodyrepertoireofthe
immuUnnedliebrrraerpyrecsloennteesd. TahmeHinCo CaDciRdsa nwaleyrsei sfoofunnadïv etoli bbrea ryhicsltoindeinses h(oHw)e, dvaalhinigeh (rVep),r elseeunctiantieo n(Lo)f,
Gtr,yfpotlolopwhaedn b(WyS), iannCd DthRr1e.oHnionwe e(vTe).r ,Tihsoel epuoclainreity(I )dwistarsibpuretidoonm foinr aanllt CinDCRDs Rw2a,sw rhaethreears sRimainladr,G wwitehr ea
hhiigghheinr rCeDprRe3se.nLtCatiCoDn Ro1f hneaudtarahl iagnhdr espmraelsle anmtaitnioon aocfidGs ,ewxcitehpta sfpora rtahgei nCeD(RN2) aonf dthDe HfoCr,C wDhRe2rea nndo
CreDpRre3s,ernestapteioctniv oefl yt.hTish gercoluonpe osff arommintoh eacimidms wuneerea nvidsinbaleïv (eFliigburraer y6cs,hdo)w. Aedbstehnecaeb osfe npcoelaorf ammeitnhoio anciindes
(iMn )C,DlyRsi1n eof( Kth),ea HndC cfyosrt ebionteh (lCib).raHroiews ewvaesr, egvluidtaenmta, tweh(Eer)eaansd thper oCliDneR2(P o)fw theree LaCd dfritoiomn athlley immimssuinnge
flriobmrartyh eshimowmeudn tehaen adbsneanïcvee oref pneornto-piroelsa,rr easmpiencoti vaceildy.s.

Figure6.Cont.
IInntt.. JJ.. MMooll.. SSccii.. 22001177,, 1188,, 22337766 99 ooff 2211

Figure6.Analysisofaminoaciddistributionandpolarityofanti-BmR1scFvclones.CDR1,CDR2,and
Figure 6. Analysis of amino acid distribution and polarity of anti-BmR1 scFv clones. CDR1, CDR2,
CDR3showingvariationsindistributionofaminoacids.(a)Aminoaciddistributionofimmunelibrary
and CDR3 showing variations in distribution of amino acids. (a) Amino acid distribution of immune
derivedclones;(b)aminoaciddistributionofnaïvelibraryderivedclones;(c)aminoacidpolarityof
library derived clones; (b) amino acid distribution of naïve library derived clones; (c) amino acid
immunelibraryderivedclones;(d)aminoacidpolarityofnaïvelibraryderivedclones.
polarity of immune library derived clones; (d) amino acid polarity of naïve library derived clones.
2.6. SUolnudbeler rEexpprreessesniotend ofa smcFinvo Calcoindess werefoundtobehistidine(H),valine(V),leucine(L),tryptophan
(W), and threonine (T). The polarity distribution for all CDRs was rather similar, with a higher
Binding characterization was performed using the unique clones from each gene family. In
representationofneutralandsmallaminoacidsexceptfortheCDR2oftheHC,wherenorepresentation
total, there were two unique scFv clones for both immune and naïve library. Clones Ab 4 and Ab 20
ofthisgroupofaminoacidswerevisible(Figure6c,d). AbsenceofpolaraminoacidsinCDR1ofthe
were identified from the immune antibody library whereas clones Ab H4R1 and Ab G1R1 were
HCforbothlibrarieswasevident,whereastheCDR2oftheLCfromtheimmunelibraryshowedthe
isolated from the naïve library. All the soluble antibodies from both libraries were successfully
absenceofnon-polaraminoacids.
expressed and purified. Western blot analysis using anti-Cmyc was able to show the presence of a
band at approximately 37 kDa that confirms the presence of the soluble scFv clones (Figure 7a,b).
2.6. SolubleExpressionofscFvClones
Bindingcharacterizationwasperformedusingtheuniqueclonesfromeachgenefamily. Intotal,
thereweretwouniquescFvclonesforbothimmuneandnaïvelibrary. ClonesAb4andAb20were
identifiedfromtheimmuneantibodylibrarywhereasclonesAbH4R1andAbG1R1wereisolated
from the naïve library. All the soluble antibodies from both libraries were successfully expressed
and purified. Western blot analysis using anti-Cmyc was able to show the presence of a band at
approximately37kDathatconfirmsthepresenceofthesolublescFvclones(Figure7a,b).
Int. J. Mol. Sci. 2017, 18, 2376 10 of 21
Int.J.Mol.Sci.2017,18,2376 10of21
Int. J. Mol. Sci. 2017, 18, 2376 10 of 21

Figure 7. Soluble scFv protein analysis with SDS PAGE and Western blotting. (a) SDS PAGE of
iFFmiiggmuuurreen e77 ..s cSSFoovlluu bbpllreeo tssecciFFnvvs; pp(rrboo)tt eeSiinDn Saa nnPaaAllyyGssiiEss wwofii tthhn aSSïvDDeSS sPPcAFAvGG EEp raaonntdedi nWWs;e es(stcte)er rnWn beblslotoetttrtininn ggb..l o((taa )) aSSnDDalSSy sPPiAsA GGuEsEi noogff
aiimmntmmi-uCunmneey sccs FcsFvecvpo rnpodrtoeaitrneysi n;as(nb;t i)(bSboD)d SiSeDPsA Sfo GrPE aAolGlf sEnc aFïovvf e pnrsocaFtïevvienp srs.oc Ft evin ps;r(oct)eWiness; te(cr)n bWloetstaenranl ybsilsotu sainnaglyansitsi- Cumsinycg
saenctoi-nCdmaryyc asnetciobnoddaiersy faonrtaibllosdciFevs fporro atelli nssc.Fv proteins.
2.7. Binding Characterization
22..77.. BBiinnddiinngg CChhaarraacctteerriizzaattiioonn
The soluble scFv clones were subjected to immunoassay analysis against the target antigen.
This TTwhhaees ssdooolluunbebl leeto ss cccFoFvnvf cicrllmoonn eethss awwt eethrreee ssbuuinbbdjjeeiccnttgeed da bttooil iiitmmy mmofuu tnnhooeaa sscssloaanyy eaasnn waallayysss iimss aaaiggnaatiiannisnstte dtthh eein tt aasrrogglueettb laaenn fttoiiggreemnn...
TFTihhgiiussr wew a8as ss hddooownnees tttohoe c cOoonnDfif irrrmema dtthihnaagtts t thohefe abblilin nfdodiuinnrgg s caaFbbviill iicttlyyo nooeffs t thwheeh ecclnloo ninneecssu wbwaaatsse dmm waaiiinnthttaa tiihnneee ddB miinnR s1soo alluunbbtillgeee nffoo. rrAmms..
tFFhiigeg uuarrmee 8o8us shnhoto wowfs sst chtFheveO OuDsDer dera efdaoidrn igenasgcosh fo asfal lamlflop ufloeru swrc FsacvsF ckvleo cpnltoe scnoewnsh swteanhneitnn, c aiu ndbciaurtebecdatt wecdoi tmwhpitthahre itsBhomen R Bs1mhaoRnw1t isag ntehntia.gtA ecnslo. tAnhees
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Tainnh-tehi bonouedsgyea tsaihnvoteiw -ceeoGdnFtgProo aol ndstaiObmDopdlryee sas dhdioindwg sendtoo tg cosohonodfiw Orm Dan trhyee aOadDsins agryse awtdoa icnsogfnusf,n iracmtsi o etnxhipen egacstwseadeyl.l .wATlahls e ffonuuenrgc atcitloiovnneiencsgo wnwtererolell.
tsTeahsmteep dnl eeasggadatiiidnvesnt o cttohsneht ropowol sasiatnimyvepO lcDeosn rdetraioddl i nnpogrtso ,tsaehsionew x(p eaGencFytPe d)O .aDAn ldrle fadoduidirn cgnlsoo,tn aesssh weoxwepr eeacntteeysd tc.e rdAoasllsg -afroienuasrct tcitvhloietnype ost oswi ttiehvreee
bctoeasnctktergdor loapugrnaoditne asinnt d(teh cGeo FnpPtor)osailtn ipdvreod ticedoinnn.to r tosl hporwotaeniny (cerGosFsP-r)e aacntdiv idtyidt ontohte sbhaocwkg raonuyn dcraonssd-rceoancttriovlitpyr otote itnh.e
background and control protein.

FFiigguurree 88.. IImmmmuunnooaassssaayy aannaallyyssiiss ooff iissoollaatteedd ssccFFvv cclloonneess aaggaaiinnsstt BBmmRR11 aannttiiggeenn wwiitthh ccoonnttrroollss..
Figure 8. Immunoassay analysis of isolated scFv clones against BmR1 antigen with controls.