Editing Alpaca Antibody (section)

== Alpaca cDNA preparation and VHH cloning ==

RNAlater was removed from PBL and lymph node tissue (prepared as above) prior to RNA extraction. Total RNA was separately isolated from PBL and ~25 mgs of lymph node tissue using TRI REAGENT®LS (Molecular Research Center, Inc.) according to the manufacturer’s protocol. RNA was column-purified using an RNeasy Mini Kit according to the guidelines of the manufacturer (Qiagen) and the yield was calculated in a spectrophotometer at 260 and 280nm. RNA was stored at −80°C. First-strand cDNA synthesis was performed using SuperScriptTMII RNAse H− reverse transcriptase (InVitrogen) and poly(A) oligo(dT)12–18 primer to reverse transcribe up to 5μg of total RNA according to the manufacture’s protocol. cDNA was stored at −20 °C until used for PCR.

RT:&nbsp;

AL.CH2, ATGGAGAGGACGTCCTTGGGT

AL.CH2.2 TTCGGGGGGAAGAYRAAGAC

Two oligonucleotides (AL.CH2, ATGGAGAGGACGTCCTTGGGT and AL.CH2.2 TTCGGGGGGAAGAYRAAGAC) were designed to universally prime reverse transcription of mammalian immunoglobulin mRNA templates at conserved sequence motifs representing the codons that encode amino acids 11 to 15.2 and 4 to 10 (IMGT numbering system), respectively, within the CH2 domains (Lefranc et al., 2005). Reverse transcription of alpaca mRNA was performed with the CH2 primers as indicated by the manufacturer for preparing 5’-RACE-Ready cDNA (Clontech). VH and VHH cDNAs were then amplified by 5’-RACE using the SMARTTM RACE cDNA Amplification kit (Clontech) using the CH2 primers. The two-band product representing VH-CH1-hinge and VHH-CH1-hinge coding sequences was separated by electrophoresis and the lower band (VHH) was cloned into the pCR®2.1-TOPO® vector with the TOPO TA Cloning® Kit (INVITROGEN). Sequencing was performed with the T7 primer.

To prepare VHH phage display libraries, cDNA was first synthesised by reverse transcription from alpaca lymph node RNA using a combination of oligo-dT and pd(N)6 primers. Superscript II reverse transcriptase (Invitrogen) was incubated with total RNA templates according to the manufacture’s protocol. VHH coding DNA for our early libraries, including the library used to identify anti- ovTNFα VHHs, was amplified from alpaca cDNA using two primer pairs based on those successfully used on llama cDNA (VH1BACK with Lam07 or Lam08 (Harmsen et al., 2000)). The appropriate PCR products corresponding to the VHH were purified via agarose electrophoresis using QIAquick Gel Extraction kit (Qiagen) PCR products were further amplified with primers homologous to the 5’ ends of the amplified DNA from the first PCR, and that introduced appropriate restriction sites for cloning into our phage display vector. Later our alpaca VHH libraries employed primer designs based on sequences of random alpaca VHH cDNAs (see Results and Discussion). The primers used were:

AlpVh-L (GGTGGTCCTGGCTGC); based on leader sequence

'''AlpVh-F1 '''(GATCGCCGGCCAGKTGCAGCTCGTGGAGTCNGGNGG);

AlpVHH-R1 (GATCACTAGTGGGGTCTTCGCTGTGGTGCG) which primes within the short hinge coding region at the same site as Lam07;

and AlpVHH-R2 (GATCACTAGTTTGTGGTTTTGGTGTCTTGGG) which primes within the long hinge coding region at the same site as Lam08.

VH1BACK,&nbsp;AGGTSMARCTGCAGSAGTCWGG, which has often been used for PCR amplification of camel and llama VHH coding DNA, is not as good as&nbsp;AlpVh-F1

alpaca VH and VHH coding DNA could be easily amplified by PCR in combination with two hinge-specific primers (AlpVHH-R1 and AlpVHH-R2)

Amplified VHH DNA was digested with appropriate restriction enzymes and ligated into similarly digested HQ2-2 DNA. The ligated DNA was transfected by electroporation into high efficiency electroporation-competent TG1 cells (Stratagene) following the recommendation of the supplier. Transformants were scraped off the plates and recombinant phage produced according to standard methods (Li and Aitken, 2004). The total number of independent clones present in the library used in this study was 3 x 107. A quality check was made for each library in which about 40 random clones were picked and PCR amplification was performed using primers flanking the VHH cloning site. An aliquot of each PCR product was analyzed for size by agarose gel electrophoresis. Another aliquot was digested with BstN1 libraries and the “fingerprint” fragment patterns assessed by agarose gel electrophoresis. In libraries used in this study, >95% of the clones had inserts and each of the clones analyzed had unique BstN1 fingerprints (Tomlinson et al., 1992).



Amino acid sequences encoded by 42 random alpaca VHH cDNA clones separated into predicted protein domain regions



{| border="0" cellspacing="0" cellpadding="0"
|-
| <div>CLONE</div>
| LEADER
| FR1
| CDR1
| FR2
| CDR2
| FR3
|-
| A02&nbsp;
| VVLAALL&nbsp;
| QGIKARSQFVESGGGLVQPGGSLRLSCTAS&nbsp;
| G--FSLKYWA&nbsp;
| VGWFRQAPGK---EREGVLC&nbsp;
| VSQ-SGE--IT&nbsp;
| NVANSVKGRFTISRDNDK&nbsp;
|-
| A52
| VVLAALL
| QGVQAQLQLVESGGGLVQPGGSLQLACEAS
| G--VTLEEVS
| VAWFRQAPGK---EREGVSC
| INS-NGNGGST
| TYARSVKGRFTISRDSAK
|-
| A53
| VVLAALL
| QSVDAYLRIVESGGGLVQSGGSLRLSCSAS
| G--DKLARYA
| VGWFRQAPGK---QREAVAC
| IDN-SVG--NT
| TYADAVKGRFTISRDKAE
|-
| A07
| VVLAALL
| QGVQAEVQLVESGGGSVQAGGYLRLSCAVS
| G--SLASMNS
| MGWYRQSPEK---EREFVAS
| INR-SN---YT
| IYSTSVKDRFILSRDHDK
|-
| A31
| VVLAALL
| RGVQAEVRLEESGGGLVQAGGSLRLVCVAY
| G--TIFSYNA
| MGWYRQPPGK---ERELVAG
| ITA-QG---TI
| EYAEAVKGRATISRDNAK
|-
| A54
| VVLAALL
| QGVQAQVFLEESGGALVQPGDSLRLTCKVS
| G--NIADVYA
| MAWYRQAPGQ---QRKMVAG
| ISD-AG---TT
| NYGTSMRGRVTISRNSAK
|-
| A55
| VVLAALL
| QGVQAQIQLVESGGGLVQSGGSLRLSCEVS
| G--TIFSIYA
| MAWYRQAPGK---QRDLVAA
| ITT-TG---TT
| DYADSVTGRFTISRDNAK
|-
| A56
| VVLAALL
| QGVQAQEQFVETGGGLVQPGGSLTLSCSVY
| G-GAVYHRSG
| LGWYRQAPGK---ERAWVGR
| IHS-G-G--GT
| GYADFVKGRFTLSRPFGN
|-
| A57
| VVLAALL
| QGVQAQEQLVESGGGLVQPGGSLRLSCAAS
| G-FTIENYH-
| VRYYRQAPGK---ECELVAG
| IAV-GNA--WT
| SYADSVKGRFTISRDSDN
|-
| A58
| VVLAALL
| QGVRAQVQFVESGGGEVRTGESLRLSCLAS
| G--GVTSLYS
| LAWYRQTPGN---QRETVAV
| IKS-G-G--ST
| NYADSVKGRFTVSRDNTK
|-
| A59
| VVLXALX
| QGVQGQVQFVESGGDLVQPGGSXRLSCEAS
| G--IRIKLYP
| MGWYRQAPGR---QRELVAR
| VT--IRE--ST
| NYTDSVKGRFTISRDDAK
|-
| A60
| VVLAALL
| QGVQAQVQLVESGGGFVQAGGSLNLSCTAS
| G--SNLRLYG
| MGWFRQAPGS---ERELVAQ
| ISW-IGG--AA
| YYSTSVKGRFTLTRDYDQ
|-
| A61
| VVLAALL
| QGVQAQVQLKESGGGYVQPGGSLRLSCAAS
| G--STIKNYD
| MGWFRQAPGK---EREFVAV
| ISG-G-G--GD
| YYSDSVKGRFWISRDDGQ
|-
| A08
| VVLAALL
| QGVQTQLQLVESGGGLVQPGSSLLLSCSAS
| G--STFRVYK
| VGWFRTAPGK---EREKVSC
| LS---AVE-TT
| DYAAAVKGRFTTFRNKAE
|-
| A23
| VVLAALL
| QGVQAQLHFVESGGGSVQPGGSLRLTCAAS
| T--FTLNYYS
| IAWFRRAPGK---EREGVSC
| MS---GGG-VT
| VYAESVKGRFTISRDSAK
|-
| A38
| VVLAALL
| QGVQAQFQLVESGGGFVQPGGSLTLSCVGS
| G--ISLDHRG
| IGWFRQAPGK---EREGVSC
| IN---SDDRTT
| SYADSVNGRFTISKDRAK
|-
| A06&nbsp;
| VVLAALQ&nbsp;
| QGVQAQLQLAESGGGLVQPGGSLRLSCVAS&nbsp;
| G--LTLDHHN&nbsp;
| ILWFRQAPGK---DREGVSC&nbsp;
| IK---MRGGSA&nbsp;
| KYADSVKGRFTVSRDNAE
|-
| A51
| VVLAALL
| QGVQAQMHLVESGGGLVQPGDSLNLSCTAS
| G--IPLRSHV
| IGWFRQVPGK---EREAVAC
| ID---GSGGTI
| NYAGSVLDRFSISRDDAK
|-
| A11
| VVLAALL
| QGVRPQVRPVESGGGSVQPGEALRLSCTQP
| K--VNLNNFA
| IGWFRQAPGK---EREGVSC
| I----GNRGTP
| YYADFLEGRFTISRDNAK
|-
| A62
| VVLAALL
| QGVQAQFQLVESGGGLVQPGGSLRLSCTAS
| D--AAFEFYA
| IGWFRQAPGK---EREGVSC
| IS---PSRAFT
| NYTDAVKGRFTISRDNSK
|-
| A43
| VVLAALL
| QGVQAQLQLVESGGGQVQPGGSLRLSCATS
| G--GTLDVYA
| IGWFRQVPGK---DREGIAC
| IS---GSGRSS
| DYVDFVKGRFTISRDNAK
|-
| A47
| VVLAALL
| QGVQAQLQFVESGGDSVQPGGSLRLSCVAS
| G--LNFDVLA
| MGWFRQAPGK---EREAVSC
| IS---NRGGHT
| EYVDSVKGRFTISRDNAK
|-
| A39
| VVLAALL
| QGVQTQLQLVESGGGLVQPGGSLRLSCVVS
| G--IKLDHYA
| FGWFRQAPGK---EREAVGC
| IG---SGGRTT
| NYRDSVRGRATVSIDTGR
|-
| A45
| VVLAALL
| QGVQAQLQLVESGGGLVQPGGSLRLSCTVS
| G--FALDYYV
| IGWFRQAPGR---EREILSC
| IS---SGGSST
| NYADSVKGRFTISRDNDQ
|-
| A03
| VVLAALL
| QGVQPQVQLVESGGGLVQPGGSLRLSCVTS
| G--RTLSYYV
| IGWFRQAPGK---EREGVSS
| IS---STDGST
| YYSESVKGRFTISRDNAE
|-
| A12
| VVLAALL
| QGVRAQVKLVESGGGFVDAGGSLTLSCASS
| G--FVLEIFV
| IGWFRQVPGK---EREGISC
| IS---IRDGSA
| YYEDSVKGRFTVSRDNAE
|-
| A63
| VVLAALL
| QGVQPQ-QLKESGGGLVRPGGSLRLSCALS
| --ERRLEDYA
| IAWIRQAPGK---DHEAISC
| IS---VGSRST
| EYSNSVKGRFTVSRDDAR
|-
| A64
| VVLAALL
| QGVQAQPQVAESGGGLVQPGGSLTLSCVVT
| GTTSSLDYIP
| VAWFRQVPGK---EREGISC
| IGGVPHGEDHI
| FYSTPVKGRFTSSRDDAK
|-
| A30
| VVLAALL
| QGVQSQLQLVETGGGVVSPGGSLKLSCTHS
| G--FPLERRM
| ISWFRHRPGKNGDEREGLSC
| IS-TIH--GGT
| EYADSVEGRFTISRDIAK
|-
| A41
| VVLAALL
| QVVQARVQLVESGGGLVQPGGSLRLSCATS
| G--FTFANYA
| LNWVRQPPGK---GLEWVSR
| ----IHSDGDT
| KYADSVKGRFTISRDIAN
|-
| A44
| VVLAALL
| QGVQAEVQVAESGGGLVQPGGSLRLSCRAS
| G--FTFSSYS
| MGWARQIPGK---GLEWVSS
| ----IYSDGST
| YYTDSVKDRFIISRDNAK
|-
| A65
| VVLAALL
| LGVQAEVQLRESGGGSVQAGGSLRLSCEAS
| G--TTSRIDV
| LAWYRQTPGN---QRVFVAS
| IT---RDNGYT
| KYADFVNGRFDISRDNAA
|-
| A10&nbsp;
| VVLAALL&nbsp;
| QGVQAQVQLVESGGGLVQPGGSLRLSCVYS&nbsp;
| G--ILATGWP&nbsp;
| MSWVRQAPLK---GPEWLSD&nbsp;
| IN---DG--AT&nbsp;
| YYADSVKGRFTISRDDAK&nbsp;
|-
| A32
| VVLAALL
| QGVQAQLHVVESGGGLVQPGGSLRLSCAAS
| G--FTLDSHH
| IGWFRQAPGK---ERELVSC
| TG---TTGDFP
| HYADSVKGRFTISRDNAK
|-
| A66
| VVLAALL
| QGVPAQLHVVESGGGLVQPGGSLRLSCKVS
| G--LDLDYLT
| LGWFRQAPGK---EREWVSC
| VD---HSGDLE
| VYGDSVRGRFAISRDNAK
|-
| A36
| VVLAALL
| QGVQAQLQLVESGGGLVQPGGSLRLSCAAS
| G--SSLDSYA
| IGWFRQAPGK---EREGVSC
| IN---SSGGTT
| NYADSVKGRFTISRDNAK
|-
| A67
| VVLAALL
| QGVQAQLQLVESGGGLVQPGGSLRLSCAAS
| G--SSLDSYA
| IGWFRQAPGK---EREGVSC
| IN---SSGGTT
| NYADSVKGRFTISRDNAK
|-
| A48
| VVLAALL
| QGVQAQLQLVESGGGLVQPGGSLRLSCAGS
| G--FTLDSYA
| IGWFRQAPGK---EREGVSC
| IS---SSGGST
| NYADSVKGRFTISRDNAK
|-
| A68
| VVLAALL
| QGVQARLHLVESGGGSVQPGGSLRLSCVAS
| G--EPLENNA
| VGWFRQSPGN---SREGISC
| ISTLGSRGITD
| DYAGSVKGRFTVSRNDAK
|-
| A69
| VVLAALL
| QGVQAQVEPVESGGGLVQPGGSLRLSCTSH
| I--EALYHYA
| VGWFRQVPGR---KREWVAC
| IS---SSGENV
| DYHESVKGRFTISKDSTR
|-
| A14
| VVLAALL
| QGVQAQLQPARAGGNLVQPGGSLRLFCGVS
| E--RTLNGYD
| IGWFRQAYGT---EREGISC
| TS----RSGNT
| VYADSVKGRFTVARDNAK
|-
| A17
| VVLAALL
| QGVQAQLQLVESGGGLVQPGGSLRLSCVAS
| G--SSLSYYH
| IGWFRQAPGK---GREAVAC
| IS---DSGGSI
| TYADSVKGRFTISRDDAE
|-
| CLONE
| FR3
| CDR3
| FR4
| colspan="3" | HINGE
|-
| A02
| NTVYLQMNSLKPEDTSVYYC
| G-DAPTC------------SDSVGDFGS
| WGQGTQVT-VSS
| colspan="3" | -------------AHHSEDPS--SKCPKCPGPELLGGP
|-
| A52
| NAVYLQMNSLKSEDTAVYYC
| SVVEPNC------------SSGRKAFGS
| WGQGTQVT-VSS
| colspan="3" | -------------AHHSEDPS--SKCPKCPGPELLGGP
|-
| A53
| NAVYLQMNNLKPEDTAIYYC
| AADAIYC------------RSP--DFGA
| WNQGTQVT-VSS
| colspan="3" | -------------AHHSEDPS--SKCPKCPGPELLGGP
|-
| A07
| NILSLQMNGLKPEDTAMYYC
| AAEGR-------------GWG-----PV
| LGQGTQVT-VSS
| colspan="3" | -------------AHHSEDPS--SKCPKCPGPELLGGP
|-
| A31
| -KLSLQLNNLKDEDTAMYYC
| AAWDRSDY---------RGHGH----DF
| LGQGTQVT-VSS
| colspan="3" | -------------AHHSEDPS--SKCPKCPGPELLGGP
|-
| A54
| NTVWLDMNNLKPEDTAVYYC
| NALERTYS---------GGDDYGR--DY
| WGKGALVN-VSS
| colspan="3" | -------------AHHSEDPS--SKCPKCPGPELLGGP
|-
| A55
| NTVYLQMNSLKPEDTGVYYC
| AADGLRLD---------ACVFPRRTYSY
| WGQGTQVT-VSS
| colspan="3" | -------------AHHSEDPS--SKCPKCPGPELLGGP
|-
| A56
| NTVFLQMNNLEPEDTAVYYC
| NT-------------------WPMKAST
| WGQGTQVT-VSS
| colspan="3" | -------------AHHSEDPS--SKCPKCPGPELLGGP
|-
| A57
| KTVYLQMKSLQPEDTAVYHC
| NV-------------------RVFSAPC
| AGQGTQVT-VSS
| colspan="3" | -------------AHHSEDPS--SKCPKCPGPELLGGP
|-
| A58
| NTMYLQMNNLTPEDTAVYFC
| NFPAY------------GYGGQVVAQVP
| WGQGTQVT-VSS
| colspan="3" | -------------AHHSEDPS--SKCPKCPGPELLGGP
|-
| A59
| NMVYLQMTMMQPEDTAVYYC
| NADLG--------------LPSMRPFAP
| WGQGTQVT-VSS
| colspan="3" | -------------AHHSEDPS--SKCPKCPGPELLGGP
|-
| A60
| KTTLLQMNKLSPEDTAVYFC
| AAST---------------TPPIFTFNS
| YGPGTQVT-VSS
| colspan="3" | -------------AHHSEDPS--SKCPKCPGPELLGGP
|-
| A61&nbsp;
| NTVYLQMNNLKPEDTAVYYC&nbsp;
| SIESDPFDA-------IGEMIRGKHRGS&nbsp;
| WGQGTQVT-VSS&nbsp;
| colspan="3" | -------------AHHSEDPS--SKCPKCPGPELLGGP&nbsp;
|-
| A08
| TTIYLQMNNLTSEDTGVYSC
| AAYYGG------Y-HCLA----EEHYDH
| WGQGTQVT-VSA
| colspan="3" | G------------AHHSEDPS--SKCPKCPGPELLGGP
|-
| A23
| NTMYLEMNSLEPEDTAEYYC
| AADEEA------V-CSVATLKRRAKYDW
| WGQGIQVT-VSS
| colspan="3" | -------------AHHSEDPS--SKCPKCPGPELLGGP
|-
| A38
| NTVYLRMNNPKPEDTGVYRC
| AAFTWI-----CS-SAYAWANYDMDH--
| WGRGTPVN-VSS
| colspan="3" | -------------AHHSEDPS--SKCPKCPGPELLGGP
|-
| A06
| KTVYLQMDSLQPDDTAVYTC
| AAVEWR-----GS-ACPLWGN--MDS--
| WGKGTLVN-VST
| colspan="3" | -------------AHHSEDPS--SKCPKCPGPELLGGP
|-
| A51
| NTVLLQMNSLKIEDTAVYYC
| AAELWN----LSD-GELYPCSPRMEY--
| WGKGILVA-VSS
| colspan="3" | -------------AHHSEDPS--SECPKCPGPELLGGP
|-
| A11
| NTVYLQMNDLKPEDTGVYSC
| AADLGAGGCVLSP-AGTLSIARFLEV--
| MGPGTLVN-VSS
| colspan="3" | -------------AHHSEDPS--SKCPKCPGPELLGGP
|-
| A62
| NTVYLQMTSLTPEDTAVYYC
| AADRGG----------PSWCNNGMDY--
| WGKGTLVT-VSS
| colspan="3" | -------------AHHSEDPS--SKCPKCPGPELLGGP
|-
| A43
| NTVFLQMNSLDSEDTATYYC
| AATLSG------Y-SGLFWCVRPNSY--
| WGQGTQVAVVSS
| colspan="3" | -------------AHHSEDPS--SKCPKCPGPELLGGP
|-
| A47
| NTVYLQMDNLQVEDTAVYYC
| AAP-----------TALRWQCNPAYFGS
| WGQGTEVS-VSS
| colspan="3" | -------------AHHSEDPS--SKCPKCPGPELLGGP
|-
| A39
| NTVHLQMSSLELEDTGLYYC
| AA-------VLSY-GSACVISRSDVYQH
| WGQGTQVT-VSS
| colspan="3" | -------------AHHSEDPS--SKCPKCPGPELLGGP
|-
| A45
| NMAYLEMTGLKPEDTAVYYC
| AADQR----LTSI-ISTCVLDHA--YEF
| WGQGTQVT-VSS
| colspan="3" | -------------AHHSEDPS--SKCPKCPGPELLGGP
|-
| A03
| NTVYLQMNDLKSEDTAAYYC
| AAADPWK-------ISXSTMTSQEPYEY
| WGQGTQVT-VSS
| colspan="3" | -------------AHHSEDPS--SKCPKCPGPELLGGP
|-
| A12
| NTVYLQMNNLKPEDTGVYTC
| AAESFWV-------GPVQAMCNNP--HI
| WGQGTQVT-VSA
| colspan="3" | -------------AHHSEDPS--SKCPKCPGPELLGGP
|-
| A63
| NMVYLDMNALKPEDTAVYRC
| AADSGVR-------RHQLCQIDTKRYDY
| WGLGTQVT-VPS
| colspan="3" | -------------AHHSEDPS--SKCPKCPGPELLGGP
|-
| A64
| NTVYLHMNRLTPGDTAVYYC
| SPAN-----------KGFCTLDANDYKY
| WGQGTQVT-VSS
| colspan="3" | -------------AHHSEDPS--SKCPKCPGPELLGGP
|-
| A30&nbsp;
| RTVYLQMNDLETEDSGVYYC&nbsp;
| AVGGEP-------------CLYVNAMKY&nbsp;
| WGKGAEVI-VRS&nbsp;
| colspan="3" | -------------AHHSEDPS--SKCPKCPGPELLGGP&nbsp;
|-
| A41
| STLYLQMNDLKTEDTGVYYC
| VKPGS---------------------GE
| RGEGTQVT-VSS
| colspan="3" | -------------AHHSEDPS--SKCPKCPGPELLGGP
|-
| A44
| SMVYLQMNSLKPEDTAVYYC
| TTDLS-IHPFSDS-----PPLGSSQHHY
| WGQGTQVT-VSS
| colspan="3" | -EPKTPKPQPQPQPQPQPNPTTESKCPKCPAPELLGGP
|-
| A65
| NTVSLQMNSLKPEDTGTYVC
| NAHLGRIFPSRDH-----VPWDRAEDY-
| WGVGIPVT-VSA
| colspan="3" | -EPKTPKPQPQPQPQPQPNPTTESKCPKCPAPELLGGP
|-
| A10
| NTLYLQMNSLKPEDTAVYYC
| AQPRD---TQVDR-----NSWGR--YVN
| WGQGTQVT-VSS
| colspan="3" | -EPKTPKPQPQPQPQPQPNPTTESKCPKCPAPELLGGP
|-
| A32
| NTVYLQMNSLKPEDTAVYYC
| A-ADSGSRSSWSL-----CPRGSVGYDY
| WGQGTQVT-VSS
| colspan="3" | -EPKTPKPQPQPQPQPQPNPTTESKCPKCPAPELLGGP
|-
| A66
| NTVYLQMNRLEPKDQAVYYC
| A-ADP---ARFTT-----CSPD--EYDY
| WGQGTQVT-VSS
| colspan="3" | -EPKTPKPQPQPQPQPQPNPTTESKCPKCPAPELLGGP
|-
| A36
| NTVYLQMNSLKPEDTAVYYC
| A-AAPGLTTFQTL-----CLMIGG--DY
| WGQGTQVT-VSS
| colspan="3" | -EPKTPKPQPQPQPQPQPNPTTESKCPKCPAPELLGGP
|-
| A67
| NTVYLQMDSLKLEDTGIYVC
| A-AGSGSTYYDCS-----GYETGWRIDK
| WDQGTQVT-VSS
| colspan="3" | -EPKTPKPQPQPQPQPQPNPTTESKCPXCPAPELLGGP
|-
| A48
| NTVYLQMNSLKSEDTAVYYC
| A-AALGMTTVQNM-----CLMWGSG-NY
| WGQGTQVT-VSS
| colspan="3" | -EPKTPKPQPQPQPQPQPNPTTESKCPKCPAPELLGGP
|-
| A68
| NTVYLQMNNLKPEDTAVYFC
| AAVYTGAGQLLPS-----LCLDENGYDY
| WGQGTQVT-VSS
| colspan="3" | -EPKTPKPQPQPQPQPQPNPTTESKCPKCPAPELXXXX
|-
| A69
| NTAYLDIMNIEPEDTATYYC
| G--AASDLWYSGL-----YCGP--DYDY
| WGQGTRVA-VSS
| colspan="3" | -EPKTPKPQPQPQPQPQPNPTTESKCPKCPAPELLGGP
|-
| A14
| TATFLEMSDLKPEDTGTYFC
| AALGGDVCVRFGRPGAYYCIANRCYDDY
| WDQGTQVT-VSS
| colspan="3" | -EPKTPKPQPQPQPQPQPNPTTESKCPKCPAPELLGGP
|-
| A17
| NTVHLQMTSLKAEDTAIYYC
| APVTPG--YYGGS-------YSCIPDGV
| WGQGTQVT-VSV
| colspan="3" | -EPKTPKPQPQPQPQPQPNPTTESKCPKCPAPELLGGP
|}

About 30% of the random VHH cDNAs encoded the IgG3 class and about 70% the IgG2 class,&nbsp;VHH-specific PCR primers based on known camelid hinge sequences (e.g. Lam 07, Lam 08 (Harmsen et al., 2000) (van der Linden et al., 2000)) should efficiently prime the synthesis of virtually all alpaca VHH mRNAs of the IgG2 and IgG3 classes.