Alpaca Antibody

Base on https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2014515/

Preparation of alpaca lymphocytes

Alpacas were purchased locally and maintained in pasture. All animal experiments were approved by the Wallaceville Animal Ethics Committee. Blood was obtained from the jugular vein and collected into heparinised or serum collection tubes. White blood cells were isolated from about 10 mls of heparinised blood by centrifugation and peripheral blood lymphocytes (PBL) were partially purified by separation over HISTOPAQUE®-1077 (Sigma) using standard procedures and stored in RNAlater (Ambion). Serum was separated by centrifugation and stored at −20 °C until testing.

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Preparation of alpaca lymphocytes

Alpacas were purchased locally and maintained in pasture. All animal experiments were approved by the Wallaceville Animal Ethics Committee. Blood was obtained from the jugular vein and collected into heparinised or serum collection tubes. White blood cells were isolated from about 10 mls of heparinised blood by centrifugation and peripheral blood lymphocytes (PBL) were partially purified by separation over HISTOPAQUE®-1077 (Sigma) using standard procedures and stored in RNAlater (Ambion). Serum was separated by centrifugation and stored at −20 °C until testing.

The local lymph node from each animal was removed surgically under general anaesthesia, induced with sodium thiopentone (20 mg/kg intravenously) and maintained with halothane (1 – 3 % in oxygen). The pre-scapular lymph node, which drains the sites of immunizations used in these studies, was removed through a small skin incision and blunt dissection of the fat tissue and muscle overlaying the lymph node. Bleeding was controlled by ligation of the nodal artery and vein. After removal of the node, the edges of the dissected tissue and skin were re-apposed with sutures. Post surgical care included a single subcutaneous application of antibiotics (400mg procaine penicillin + 400mg dihydrostreptomycin sulphate) and an analgesic (flunixin 2 mg/kg). The excised lymph node was cut into 1 – 2 mm thick slices and either stored in RNAlater, or immediately subjected to a phenol/chloroform based RNA extraction protocol.

Alpaca immunization and serum analysis
Two adult male alpacas were given four immunizations at two week intervals, each including six 0.2 ml intra-dermal and sub-cutaneous injections of the immunogen in the pre-scapular region. The immunogen contained a total of ~400 μg of ovTNFα for each immunization prepared with 13 mg/ml aluminium hydroxide gel (Sigma) as an adjuvant. Serum samples were obtained in weekly intervals and tested for TNFα-specific antibody response by ELISA. Alpaca antibody bound to ovTNFα was detected in the ELISA using antisera from a rabbit immunized with alpaca immunoglobulins (Green et al., 1996).

Some serum samples were assayed for TNFα neutralizing activity in a standard bioassay (Flick and Gifford, 1984) which measures a reduction in TNFα cytotoxicity. Briefly, 100 μl/well of murine fibroblast L929 cells were seeded in 96-well plates at 1 × 105 cells/ml and incubated overnight. Dilutions of the serum or purified VHH were prepared in RPMI and incubated with serial twofold dilutions of ovine TNF for 30min. After removing of the supernatants of the cultured L929 cells, 100 μl of the prepared dilutions containing actinomycin D at a final concentration of 1.0 μg/ml were added to the wells. Then the plates were incubated at 37 °C for 24 h, the supernatants were removed and 0.5% crystal violet in methanol was added and incubated at room temperature for 10 min. Plates were rinsed gently with water, and the optical density (OD) of bound dye was determined at 620 nm.

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: 

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, AGGTSMARCTGCAGSAGTCWGG, which has often been used for PCR amplification of camel and llama VHH coding DNA, is not as good as 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

CLONE	LEADER	FR1	CDR1	FR2	CDR2	FR3
A02	VVLAALL	QGIKARSQFVESGGGLVQPGGSLRLSCTAS	G--FSLKYWA	VGWFRQAPGK---EREGVLC	VSQ-SGE--IT	NVANSVKGRFTISRDNDK
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	VVLAALQ	QGVQAQLQLAESGGGLVQPGGSLRLSCVAS	G--LTLDHHN	ILWFRQAPGK---DREGVSC	IK---MRGGSA	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	VVLAALL	QGVQAQVQLVESGGGLVQPGGSLRLSCVYS	G--ILATGWP	MSWVRQAPLK---GPEWLSD	IN---DG--AT	YYADSVKGRFTISRDDAK
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	HINGE
A02	NTVYLQMNSLKPEDTSVYYC	G-DAPTC------------SDSVGDFGS	WGQGTQVT-VSS	-------------AHHSEDPS--SKCPKCPGPELLGGP
A52	NAVYLQMNSLKSEDTAVYYC	SVVEPNC------------SSGRKAFGS	WGQGTQVT-VSS	-------------AHHSEDPS--SKCPKCPGPELLGGP
A53	NAVYLQMNNLKPEDTAIYYC	AADAIYC------------RSP--DFGA	WNQGTQVT-VSS	-------------AHHSEDPS--SKCPKCPGPELLGGP
A07	NILSLQMNGLKPEDTAMYYC	AAEGR-------------GWG-----PV	LGQGTQVT-VSS	-------------AHHSEDPS--SKCPKCPGPELLGGP
A31	-KLSLQLNNLKDEDTAMYYC	AAWDRSDY---------RGHGH----DF	LGQGTQVT-VSS	-------------AHHSEDPS--SKCPKCPGPELLGGP
A54	NTVWLDMNNLKPEDTAVYYC	NALERTYS---------GGDDYGR--DY	WGKGALVN-VSS	-------------AHHSEDPS--SKCPKCPGPELLGGP
A55	NTVYLQMNSLKPEDTGVYYC	AADGLRLD---------ACVFPRRTYSY	WGQGTQVT-VSS	-------------AHHSEDPS--SKCPKCPGPELLGGP
A56	NTVFLQMNNLEPEDTAVYYC	NT-------------------WPMKAST	WGQGTQVT-VSS	-------------AHHSEDPS--SKCPKCPGPELLGGP
A57	KTVYLQMKSLQPEDTAVYHC	NV-------------------RVFSAPC	AGQGTQVT-VSS	-------------AHHSEDPS--SKCPKCPGPELLGGP
A58	NTMYLQMNNLTPEDTAVYFC	NFPAY------------GYGGQVVAQVP	WGQGTQVT-VSS	-------------AHHSEDPS--SKCPKCPGPELLGGP
A59	NMVYLQMTMMQPEDTAVYYC	NADLG--------------LPSMRPFAP	WGQGTQVT-VSS	-------------AHHSEDPS--SKCPKCPGPELLGGP
A60	KTTLLQMNKLSPEDTAVYFC	AAST---------------TPPIFTFNS	YGPGTQVT-VSS	-------------AHHSEDPS--SKCPKCPGPELLGGP
A61	NTVYLQMNNLKPEDTAVYYC	SIESDPFDA-------IGEMIRGKHRGS	WGQGTQVT-VSS	-------------AHHSEDPS--SKCPKCPGPELLGGP
A08	TTIYLQMNNLTSEDTGVYSC	AAYYGG------Y-HCLA----EEHYDH	WGQGTQVT-VSA	G------------AHHSEDPS--SKCPKCPGPELLGGP
A23	NTMYLEMNSLEPEDTAEYYC	AADEEA------V-CSVATLKRRAKYDW	WGQGIQVT-VSS	-------------AHHSEDPS--SKCPKCPGPELLGGP
A38	NTVYLRMNNPKPEDTGVYRC	AAFTWI-----CS-SAYAWANYDMDH--	WGRGTPVN-VSS	-------------AHHSEDPS--SKCPKCPGPELLGGP
A06	KTVYLQMDSLQPDDTAVYTC	AAVEWR-----GS-ACPLWGN--MDS--	WGKGTLVN-VST	-------------AHHSEDPS--SKCPKCPGPELLGGP
A51	NTVLLQMNSLKIEDTAVYYC	AAELWN----LSD-GELYPCSPRMEY--	WGKGILVA-VSS	-------------AHHSEDPS--SECPKCPGPELLGGP
A11	NTVYLQMNDLKPEDTGVYSC	AADLGAGGCVLSP-AGTLSIARFLEV--	MGPGTLVN-VSS	-------------AHHSEDPS--SKCPKCPGPELLGGP
A62	NTVYLQMTSLTPEDTAVYYC	AADRGG----------PSWCNNGMDY--	WGKGTLVT-VSS	-------------AHHSEDPS--SKCPKCPGPELLGGP
A43	NTVFLQMNSLDSEDTATYYC	AATLSG------Y-SGLFWCVRPNSY--	WGQGTQVAVVSS	-------------AHHSEDPS--SKCPKCPGPELLGGP
A47	NTVYLQMDNLQVEDTAVYYC	AAP-----------TALRWQCNPAYFGS	WGQGTEVS-VSS	-------------AHHSEDPS--SKCPKCPGPELLGGP
A39	NTVHLQMSSLELEDTGLYYC	AA-------VLSY-GSACVISRSDVYQH	WGQGTQVT-VSS	-------------AHHSEDPS--SKCPKCPGPELLGGP
A45	NMAYLEMTGLKPEDTAVYYC	AADQR----LTSI-ISTCVLDHA--YEF	WGQGTQVT-VSS	-------------AHHSEDPS--SKCPKCPGPELLGGP
A03	NTVYLQMNDLKSEDTAAYYC	AAADPWK-------ISXSTMTSQEPYEY	WGQGTQVT-VSS	-------------AHHSEDPS--SKCPKCPGPELLGGP
A12	NTVYLQMNNLKPEDTGVYTC	AAESFWV-------GPVQAMCNNP--HI	WGQGTQVT-VSA	-------------AHHSEDPS--SKCPKCPGPELLGGP
A63	NMVYLDMNALKPEDTAVYRC	AADSGVR-------RHQLCQIDTKRYDY	WGLGTQVT-VPS	-------------AHHSEDPS--SKCPKCPGPELLGGP
A64	NTVYLHMNRLTPGDTAVYYC	SPAN-----------KGFCTLDANDYKY	WGQGTQVT-VSS	-------------AHHSEDPS--SKCPKCPGPELLGGP
A30	RTVYLQMNDLETEDSGVYYC	AVGGEP-------------CLYVNAMKY	WGKGAEVI-VRS	-------------AHHSEDPS--SKCPKCPGPELLGGP
A41	STLYLQMNDLKTEDTGVYYC	VKPGS---------------------GE	RGEGTQVT-VSS	-------------AHHSEDPS--SKCPKCPGPELLGGP
A44	SMVYLQMNSLKPEDTAVYYC	TTDLS-IHPFSDS-----PPLGSSQHHY	WGQGTQVT-VSS	-EPKTPKPQPQPQPQPQPNPTTESKCPKCPAPELLGGP
A65	NTVSLQMNSLKPEDTGTYVC	NAHLGRIFPSRDH-----VPWDRAEDY-	WGVGIPVT-VSA	-EPKTPKPQPQPQPQPQPNPTTESKCPKCPAPELLGGP
A10	NTLYLQMNSLKPEDTAVYYC	AQPRD---TQVDR-----NSWGR--YVN	WGQGTQVT-VSS	-EPKTPKPQPQPQPQPQPNPTTESKCPKCPAPELLGGP
A32	NTVYLQMNSLKPEDTAVYYC	A-ADSGSRSSWSL-----CPRGSVGYDY	WGQGTQVT-VSS	-EPKTPKPQPQPQPQPQPNPTTESKCPKCPAPELLGGP
A66	NTVYLQMNRLEPKDQAVYYC	A-ADP---ARFTT-----CSPD--EYDY	WGQGTQVT-VSS	-EPKTPKPQPQPQPQPQPNPTTESKCPKCPAPELLGGP
A36	NTVYLQMNSLKPEDTAVYYC	A-AAPGLTTFQTL-----CLMIGG--DY	WGQGTQVT-VSS	-EPKTPKPQPQPQPQPQPNPTTESKCPKCPAPELLGGP
A67	NTVYLQMDSLKLEDTGIYVC	A-AGSGSTYYDCS-----GYETGWRIDK	WDQGTQVT-VSS	-EPKTPKPQPQPQPQPQPNPTTESKCPXCPAPELLGGP
A48	NTVYLQMNSLKSEDTAVYYC	A-AALGMTTVQNM-----CLMWGSG-NY	WGQGTQVT-VSS	-EPKTPKPQPQPQPQPQPNPTTESKCPKCPAPELLGGP
A68	NTVYLQMNNLKPEDTAVYFC	AAVYTGAGQLLPS-----LCLDENGYDY	WGQGTQVT-VSS	-EPKTPKPQPQPQPQPQPNPTTESKCPKCPAPELXXXX
A69	NTAYLDIMNIEPEDTATYYC	G--AASDLWYSGL-----YCGP--DYDY	WGQGTRVA-VSS	-EPKTPKPQPQPQPQPQPNPTTESKCPKCPAPELLGGP
A14	TATFLEMSDLKPEDTGTYFC	AALGGDVCVRFGRPGAYYCIANRCYDDY	WDQGTQVT-VSS	-EPKTPKPQPQPQPQPQPNPTTESKCPKCPAPELLGGP
A17	NTVHLQMTSLKAEDTAIYYC	APVTPG--YYGGS-------YSCIPDGV	WGQGTQVT-VSV	-EPKTPKPQPQPQPQPQPNPTTESKCPKCPAPELLGGP

About 30% of the random VHH cDNAs encoded the IgG3 class and about 70% the IgG2 class, 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.

Screening and selection of phage antibodies

Selection was carried out by “panning” of VHH-displayed phage libraries for phage that bind to immunotubes (Nunc) coated overnight at 4° C with 5 μg/ml soluble ovTNF. The tubes were then washed three times with PBS, and blocked with 4% non-fat dried milk in PBS (MPBS) at 37° C for 2 h. A 4 ml suspension of phage in MPBS was prepared containing 5.0×1011 CFU was incubated in an immunotube at room temperature for 30 min with continuous rotation, and then for a further 90 min without rotation. The tubes were washed 20 times with PBS containing 0.1% Tween 20 (PBST) followed by 20 times with PBS. Bound phage were eluted by continuous rotation with 1 ml of 100 mM triethanolamine (Sigma) for 10 min, then, recovered and neutralised with 0.2 ml of 1 M Tris–HCl, pH 4.5. A 0.75 ml aliquot of the eluted phage was used to infect 10ml culture of log-phase E. coli TG1 cells. A small aliquot of the infected bacteria was used in serial dilutions to titrate the number of phage eluted while the remainder was processed as described above to amplify the phagemid for further selection or analysis. The binding of selected VHHs encoded by phagemid clones to ovTNFα was tested by phage ELISA using anti-M13 antibody (GE Healthcare) for detection. Positive clones were “fingerprinted” by analysis of their BstN1 digestion patterns (Tomlinson et al., 1992).

Production of soluble single domain antibodies

The VHH coding DNA was subcloned into the expression vector AB6-7 using the same restriction sites as in the HQ2-2 cloning. E. coli Rosetta-gami containing the VHH expression plasmid were grown to an optical density of 0.5 at 600 nm and then overnight in 0.1% arabinose at 28° C. Soluble protein was purified from sonicated cells and the recombinant VHH was purified by nickel affinity using Ni-NTA (Qiagen) as recommended by the manufacturer. Protein eluting in 0.2M imidazole was dialyzed against PBS. Purity of the recombinant VHH was assessed by Coomassie Blue staining of SDS-PAGE and protein concentration determined by BCA (Pierce). Western blot and ELISA detection of recombinant VHH was done using HRP anti-E-tag antibodies (GE Healthcare).

Competitive ELISA

Nunc Maxisorb plates were coated overnight at 4° C with 5 μg/ml soluble ovTNFα.in PBS, and then blocked with 4% MPBS. After washing three times with PBS, serially diluted soluble VHH antibodies were added and incubated at room temperature for 90 min to determine saturation curves. To detect bound VHH, an anti-E-Tag/HRP antibody (Amersham) was added at 1:8000 dilution in 4% MPBS for 90 min at room temperature. The wells were washed and developed with 3,3’,5,5’-tetramethyl benzidine (Applichem). Competitive ELISA was performed by preparing plates as described above and then a saturating amount of soluble expressed VHH was added and incubated for 60 min at room temperature. PBS was added to control wells containing no expressed soluble protein. Serial dilutions of bacterial supernatants containing VHH-displaying phage were added for 90 min at room temperature. Plates were then washed three times with PBST and three times with PBS. To detect bound phage, an anti-M13/HRP antibody (Amersham) was added at 1:8000 dilution in 4% MPBS for 90 min at room temperature. The wells were washed and developed as described above. Percent binding was calculated as the ODtest/ODcontrol x 100.

Alpaca serum immunoglobulin characterization

Alpaca serum was resolved by SDS-PAGE and stained for protein or transferred to PVDF membranes. Filters were probed with HRP labelled anti-llama IgG (H+L) (Bethyl) by standard western blotting methods.