rabbit monoclonal fab derived from a phage display library

12
Ž . Journal of Immunological Methods 213 1998 201–212 Rabbit monoclonal Fab derived from a phage display library Maria Foti a,b, ) , Francesca Granucci a , Paola Ricciardi-Castagnoli a , Adriano Spreafico c , Mathias Ackermann b , Mark Suter b a CNR Cellular and Molecular Pharmacology Center, Õia VanÕitelli 32, Milano 20129, Italy b Institute for Virology, UniÕersity of Zurich, Winterthurerstr. 266a, Zurich 8057, Switzerland c Dipartimento di Biologia Molecolare, UniÕersita di Siena, Policlinico Le Scotte, Siena 53100, Italy ´ Received 19 November 1997; revised 9 January 1998; accepted 30 January 1998 Abstract Ž . Rabbit monoclonal antibodies RmAb are not routinely obtained by eukaryotic cell fusion techniques. Therefore, we have applied phage display technology to produce a recombinant rabbit Fab molecule directed against the KLH model antigen. The Fab fragments selected from the rabbit phage display library were subcloned in an expression vector to permit Ž . the production of a fusion protein comprising a dimer of bacterial alkaline phosphatase phoA . This fusion protein was directly produced into the periplasmic space of Escherichia coli. We show that a crude extract containing these conjugates can be used in a direct enzyme immunoassay, as exemplified in the case of the KLH antigen. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Rabbit immunoglobulins; Phage display; Fab fragments; Alkaline phosphatase; Recombinant antibody 1. Introduction Phage display technology has been adapted to Ž . permit expression of peptides Smith, 1991 and Ž . proteins Crameri and Suter, 1993 including anti- Ž . Ž body fragments Ab , on the phage surface Kang et . al., 1991; Clackson et al., 1991 . The most powerful Abbreviations: a.a., Amino acid; mAb, monoclonal antibody; V , antibody heavy chain variable region; V , antibody light H L chain variable region; Ig, immunoglobulin; HC, heavy chain; LC, light chain; CDR, complementarity-determining region; FR, framework region; cfu, Colony-forming units; Fab, Ab-binding Ž. fragment s ; PAGE, polyacrylamide-gel electrophoresis; PCR, polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; KLH, keyhole limpet hemo- cyanin ) Corresponding author. Tel.: q39-2-70146265; fax: q39-2- 7490574; e-mail: [email protected]. aspects of this technique are the potential to generate complex libraries exceeding 10 14 phage and to screen Ž . such libraries in various ways Suter et al., 1996 to enrich for a desired ligand bound to phage-harbour- ing the genetic information for the ligand. Various vectors and approaches have been used to Ž . express immunoglobulin Ig molecules in Es- cherichia coli from different species including Ž mouse, human, chicken and rabbit Engberg et al., . 1995; Davies et al., 1995; Ridder et al., 1995 . The Ž . production of rabbit antibodies Ab by repertoire cloning is attractive for several reasons. It has been known for decades that rabbits are excellent produc- ers of polyclonal Ab against a variety of antigens, including haptens. However, monoclonal antibodies Ž . mAb from this species have not been routinely produced until very recently when a stable fusion Ž partner was made available Spieker-Polet et al., 0022-1759r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. Ž . PII S0022-1759 98 00029-5

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Page 1: Rabbit monoclonal Fab derived from a phage display library

Ž .Journal of Immunological Methods 213 1998 201–212

Rabbit monoclonal Fab derived from a phage display library

Maria Foti a,b,), Francesca Granucci a, Paola Ricciardi-Castagnoli a,Adriano Spreafico c, Mathias Ackermann b, Mark Suter b

a CNR Cellular and Molecular Pharmacology Center, Õia VanÕitelli 32, Milano 20129, Italyb Institute for Virology, UniÕersity of Zurich, Winterthurerstr. 266a, Zurich 8057, Switzerland

c Dipartimento di Biologia Molecolare, UniÕersita di Siena, Policlinico Le Scotte, Siena 53100, Italy´Received 19 November 1997; revised 9 January 1998; accepted 30 January 1998

Abstract

Ž .Rabbit monoclonal antibodies RmAb are not routinely obtained by eukaryotic cell fusion techniques. Therefore, wehave applied phage display technology to produce a recombinant rabbit Fab molecule directed against the KLH modelantigen. The Fab fragments selected from the rabbit phage display library were subcloned in an expression vector to permit

Ž .the production of a fusion protein comprising a dimer of bacterial alkaline phosphatase phoA . This fusion protein wasdirectly produced into the periplasmic space of Escherichia coli. We show that a crude extract containing these conjugatescan be used in a direct enzyme immunoassay, as exemplified in the case of the KLH antigen. q 1998 Elsevier Science B.V.All rights reserved.

Keywords: Rabbit immunoglobulins; Phage display; Fab fragments; Alkaline phosphatase; Recombinant antibody

1. Introduction

Phage display technology has been adapted toŽ .permit expression of peptides Smith, 1991 and

Ž .proteins Crameri and Suter, 1993 including anti-Ž . Žbody fragments Ab , on the phage surface Kang et

.al., 1991; Clackson et al., 1991 . The most powerful

Abbreviations: a.a., Amino acid; mAb, monoclonal antibody;V , antibody heavy chain variable region; V , antibody lightH L

chain variable region; Ig, immunoglobulin; HC, heavy chain; LC,light chain; CDR, complementarity-determining region; FR,framework region; cfu, Colony-forming units; Fab, Ab-binding

Ž .fragment s ; PAGE, polyacrylamide-gel electrophoresis; PCR,polymerase chain reaction; ELISA, enzyme-linked immunosorbentassay; BSA, bovine serum albumin; KLH, keyhole limpet hemo-cyanin

) Corresponding author. Tel.: q39-2-70146265; fax: q39-2-7490574; e-mail: [email protected].

aspects of this technique are the potential to generatecomplex libraries exceeding 1014 phage and to screen

Ž .such libraries in various ways Suter et al., 1996 toenrich for a desired ligand bound to phage-harbour-ing the genetic information for the ligand.

Various vectors and approaches have been used toŽ .express immunoglobulin Ig molecules in Es-

cherichia coli from different species includingŽmouse, human, chicken and rabbit Engberg et al.,

.1995; Davies et al., 1995; Ridder et al., 1995 . TheŽ .production of rabbit antibodies Ab by repertoire

cloning is attractive for several reasons. It has beenknown for decades that rabbits are excellent produc-ers of polyclonal Ab against a variety of antigens,including haptens. However, monoclonal antibodiesŽ .mAb from this species have not been routinelyproduced until very recently when a stable fusion

Žpartner was made available Spieker-Polet et al.,

0022-1759r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.Ž .PII S0022-1759 98 00029-5

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( )M. Foti et al.rJournal of Immunological Methods 213 1998 201–212202

.1995 . Rabbits are unusual among mammals, al-though they possess multiple germline V genes,H

they predominantly rearrange one of them, V 1. InH

addition, the V genes of rabbits generally showH

more than 80% similarity and consequently areŽmembers of one large V gene family Bernstein etH

al., 1985; McCormack et al., 1985; Currier et al.,.1988; Knight and Becker, 1990 . This is in contrast

to murine and human genes, which are grouped intoŽ10 and six V gene families, respectively BrodeurH

and Riblet, 1984; Lai et al., 1989; Pascual and. Ž .Capra, 1991 . Knight and Becker 1990 have shown

that Ab diversity in the rabbit is generated by pointmutation and somatic gene conversion like mecha-nisms of rearranged VDJ genes. The mechanismimplied that the overall structure of the Ig moleculepermits extensive alterations in the CDR regionswithout influencing the framework regions.

Among the mammals the rabbit k-chain chromo-somal region is unusual because, instead of one,

Žthere are two Ck genes, Ck1 and Ck 2 Benammarand Cazenave, 1982; Emorine and Max, 1983; Hole

.et al., 1991 . In normal rabbits, k light chainsrepresent 90–95% of total L chains and nearly all ofthem are derived from Ck1 and are designated as the

Ž .k-1 isotypes Knight and Crane, 1994 .The practical implication of the genetic informa-

tion given above is that only a limited number ofŽprimers are needed to amplify by PCR polymerase

.chain reaction both rabbit V or V in order toH L

obtain representative gene fragments for repertoirecloning.

Ž .In this report we i show the feasibility of usingŽ .PBL rather than spleen cells Ridder et al., 1995 or

Ž .bone marrow Lang et al., 1996 for library construc-tion and the production of mAb of good affinity andŽ .ii establish the production of antibodies linkeddirectly to enzyme for practical use in solid phaseassays or for the staining of tissue.

2. Materials and methods

2.1. Immunization of rabbits and cDNA synthesis

Rabbits of the V a1 and b4 allotype were origi-H

nally obtained from Andrew Kelus of the BaselInstitute of Immunology and bred in our colony.

Table 1

Oligonucleotide primers used to amplify rabbit immunoglobulinsX5 -VHA1 CAGTCGCTGCTCGAGTCCGGGGGTX3 -Hinge CACTAGTCGTGGGCTTGCTGCATGTCGX5 -VL GCCGCCGAGCTCGTGATGACCCAGACTCCAX3 -VLk1b4 CGCGGCTCTAGAACAGTCACCCCTATTGAAG

CTCTGGX3 -VLk2b4 CGCGGCTCTAGAGCAGTCACCCCTGTTGAAG

CTCTG

The restriction sites are underlined.

Rabbits were immunized by subcutaneous injec-tions of 200 mg of KLH in complete Freund’s

Ž .adjuvant Sigma and subsequently boosted twicewith 100 mg of KLH, each given on days 20 and 27,respectively.

Plasma from the immunized rabbits was assayedfor the presence of IgG antibodies to KLH after the

Ž .second boost by using a goat anti-rabbit IgG HqLpolyclonal Ab labelled with the alkaline phosphataseŽ .Southern Biotechnology Associates, USA in an

Ž .ELISA enzyme-linked immunosorbent assay -basedassay. Peripheral blood lymphocytes were harvested

Žfrom citrate-anticoagulated whole blood Coligan et.al., 1992 by centrifugation over Ficoll–Hypaque

Ž .Pharmacia . Total RNA was isolated from the nu-Ž .cleated cells B lymphocytes and Ig-specific mRNA

was selected and am plified by reverseŽ .transcription–polymerase chain reaction RT–PCR

Ž .using the BRL Superscript RT 200 Urml . Togenerate first strand cDNA, an oligo-dT was used asprimer. Ig cDNAs were amplified by PCR using theprimers specific for rabbit Ig described in Table 1.The PCR reaction mixtures containing 0.3 mMdNTPs, 50 mM KCl, 10 mM Tris–HCl, pH 8.3, 3

ŽmM MgCl and Taq polymerase 0.04 Urml2.Perkin–Elmer Cetus were then subjected to 35

rounds of amplification using a thermal cycler appa-Ž .ratus Perkin–Elmer Cetus at 948C for 15 s, 688C

for 50 s, 728C for 1.5 min for the Ig heavy chain,and 948C for 15 s, 608C for 50 s, 728C for 1.5 min,followed by a final incubation at 728C for 10 min,for the light chain.

2.2. Southern blot analysis

To verify the specificity of the amplificationbands, the PCR products were transferred on a

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Boehringer Mannheim membrane positively chargedŽ .Boehringer Mannheim in 0.4 M NaOH for 3 h andthe filter hybridized with an internal oligo. Thedifferent oligos were labelled in 3X using the DIG

X Žoligonucleotide 3 labelling kit Boehringer.Mannheim . The hybridization reaction was per-

formed according to the manufacturer’s recommen-dations.

Internal oligo sequences were as follows: Lc:TCCGTCCCAATGACATCACC, Hc: ACCT-CAAGCAGCCAGCCCGT.

2.3. Immunoglobulin gene libraries

The filamentous phage vector, pComb3, was usedŽ .as described by Barbas et al. 1991 . This vector was

derived from the phagemids excised from the l Hc2Ž .and l Lc2 vectors Huse et al., 1989 and allows the

in-frame fusion of heavy chain Fd cDNAs with theM13 gene III. The PCR products of amplified V -H

C 1 and V -C from the cDNA synthesis were gelH L L

purified, digested, and ligated into pComb3, exactlyŽ .as described by McCafferty et al. 1990 and Barbas

Ž .et al. 1991 . The size of the Fd cDNA library was1.5=106 cfu and that of the k cDNA’s was 2=106

Ž .cfu. XL1-Blue cells Stratagene transformed withthe phagemid library were infected with helper phage

Ž .VCSM13 Stratagene and phages were prepared asŽ .previously described Barbas et al., 1992 .

2.4. Biopanning

Ž .Pairs of microtiter wells Nunc, Maxisorb wereŽcoated overnight at 48C with KLH Sigma; 1

.mgrwell in 0.1 M bicarbonate, pH 8.6. The wellswere washed with water and blocked with PBScontaining 3% BSA at 378C for 1 h. The blocking

Ž 11 .solution was removed, and the library 10 cfu wasŽ .added to the microtiter wells 50 mlrwell and

incubated at 378C for 2 h. Wells were washed 10Žtimes with TBSrTween 50 mM Tris–HCl, pH 7.5,

.150 mM NaCl, 0.5% Tween 20 and once with H O2

before elution of the adherent phage with 0.1 M HCl,adjusted to pH 2.2 with glycine, containing 1 mgrmlBSA. The selected phages were then allowed to

Ž .infect E. coli XL1-Blue cultures Stratagene , whichwere used to prepare new antigen-selected phage

stocks. They were then reincubated with antigen inmicrotiter wells for a total of four rounds.

2.5. Phage ELISA

To analyse the antigen specificity of the Fabexpressed by phagemid, PEG-concentrated phage

Ž .particles Sambrook et al., 1992 were tested inŽELISA. For this test, wells of microtiter plates Max-

. Žisorb, NUNC were coated with 100 ml of KLH 10.mgrml in carbonate buffer and blocked with PBS

Ž .containing 2% skimmed milk powder Difco for 2 hat 378C. After washing, 50 ml PBSrmilk were addedto each well, followed by 20 ml of PEG-con-centrated phage. Plates were incubated for 2 h atroom temperature. Washings between the differentsteps were performed with PBS containing 0.05%Tween-20.

Binding was assayed using a sheep anti-Ž . ŽM13rHRP IgG serum Pharmacia 1r1000 dilution

.in PBSrmilk . Bound phage was indirectly deter-w X Xmined by adding the substrate, ABTS 2 ,2 -azino-

Ž .bis 3-ethylbenzthiazoline-6-sulphonic acid Di-xammonium Sigma to the washed wells. Absorbance

readings were made at 405 nm.

2.6. Preparation of soluble Fabs

Ž .The pJuFo vector Crameri and Suter, 1993 wasfirst engineered by inserting the BamHIrNheI frag-

Žment containing a stretch of six-histidines Stuber et¨.al., 1990 in BamHIrSpeI sites of pJuFo, to permit

affinity purification on a nickel–chelate column. ThepJuFo:phoA vector also contains the gene coding forthe alkaline phosphatase enzyme which was used tolabel the antibody fragment. After the six histidinesresidue, a stop codon was inserted so that the pIIIfusion protein was eliminated and the protein couldbe expressed directly into the periplasmic space ofE. coli. Fig. 3 shows the vector, now named pMF3,in which the selected heavy chain was cloned intoXhoIrSpeI sites with the six histidines at the car-boxi-terminal end, which removed the Jun gene whilethe selected light chain was inserted into SacIrXbaIsite with the alkaline phosphatase at the carboxylside replacing the Fos gene. XL1-Blue were trans-formed with the expression vector, and isolatedclones were grown overnight at 378C in super broth

wmedium SB; 30 g tryptone, 20 g yeast extract, 10 g

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Ž . Ž . Ž .Fig. 1. Heavy- and light-chain genes were amplified from oligo d T -primed cDNA. a PCR amplification of light-chain genes upper panel and map of light-chain variableŽ . Ž . Ž .region lower panel . Lane 1, V k1b4; lane 2, V k 2b4. b PCR amplification of heavy-chain genes. Lanes 1–4, V -C upper panel and map of heavy-chain variable regionL L H H

Ž . X X Xlower panel . Vk1LCFR1: light-chain 5 primer; K1b4 and K2b4: light-chain 3 primers; VHa1HCFR1: heavy-chain 5 primer.

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( )M. Foti et al.rJournal of Immunological Methods 213 1998 201–212 205

Ž . x8- N-morpholino propanesulfonic acid containing1% glucose and 50 mgrml ampicillin. This culturewas used to inoculate new SB medium, and cultureswere shaken until an OD600 of 0.9 was reached.

Ž .Next, IPTG 1 mM was added and the cultures weretransferred to 258C for 4 h. Cells were pelleted bycentrifugation and then resuspended in 20% sucrosein 50 mM Tris, pH 8.0, containing 100 mgrmllysozyme, 0.2 mM PMSF and 1 mM EDTA. The

Ž .cells were stirred using a magnetic bar at 100 rpmfor 1 h. Debris were pelleted by centrifugation for 10

Žmin at 10,000=g. The supernatant periplasmic ex-.tract was loaded at 0.5 mlrmin directly onto a 5-ml

ŽNickel–NTA metal chelating column Qiagen,. ŽHilden, Germany Stuber et al., 1990; Hochuli et¨

.al., 1987 or stored at y208C. Elution was per-formed with a gradient of imidazole from 10 mM upto 200 mM. The eluted Fab was desalted with PBSand then analysed by sodium dodecyl sulfate poly-acrylamide gel electrophoresis under nonreducingconditions. The concentration was determined with aBSA standard.

2.7. DNA sequence determination

The complete amino acid sequences of the vari-able regions of ELISA positive clones were deter-mined by the dideoxy chain termination method

Ž .using Deaza GrA sequencing mixes kit Pharmacia .Ž XThe oligonucleotides, SEQHC forward 5 TGGCC-

X. Ž XCAGGTGAAACTG3 , SEQHC backward 5 GAC-X .CAGGGTGCCTGGGCC3 , SEQLC forward

Ž X X.5 TACTCGCTGCCCAACCA3 and SEQLC back-Ž X X .ward 5 GGTCCCTCCGCCGAAAGCA3 were

used to prime sequencing reactions from the regionsimmediately adjacent to the V or V regions. Se-H L

Table 2Selective enrichment of fragments from the library as monitoredduring panning

1 Round 2 Round 3 Round 4 Round11 11 11 11Phage applied 2=10 2=10 2=10 1=105 6 7 7Phage eluted 2=10 2=10 2=10 1.5=10y4 y3 y2 y2% Yield 1=10 1=10 1=10 1=10

Ž . Ž .% Yields no. of phage eluted=100 r no. of phage applied .Enrichment as monitored by the number of phage eluted by acidfrom the plates following washings.

Fig. 2. Specificity of binding of selected phages. Phages from the4th round of selection were assayed by ELISA for binding to

Ž . Ž .KLH a or to BSA b used as negative control. Neg: clonederived from the library before panning.

Žquences were analysed with Macvector IBI, New. Ž .Haven, CT and GCG Madison, WI software pack-

ages, with comparison to GenBank.

2.8. Enzyme immunoassay

To determine antigenic specificity, plates wereŽ .coated overnight with KLH 10 mgrml . After

washings and blocking, 100 ml of a dilution ofperiplasmic extract or of the purified protein contain-ing genetically engineered conjugates were addedand the phoA activity was revealed with p-

Ž .nitrophenyl phosphate pNPP Sigma substrate. Ab-sorbance was read at 410 nm.

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2.9. Immunoblotting

Samples were subjected to SDS-PAGE and elec-troblotted onto a nitrocellulose membrane using stan-dard procedures. Proteins were detected using a goat

Ž .anti-rabbit IgG HqL polyclonal antibody labelledŽwith alkaline phosphatase Southern Biothenology,

.USA .

2.10. Nitrocellulose filter assay

To establish the presence of phagemid-encodedphoA, bacterial clones containing the appropriateconstructs were transferred to nitrocellulose. Thefilters were air-dried and developed by adding thesubstrate, bromochloroindolyl phosphaternitro blue

Ž .tetrazolium BCIPrNBT as described by the manu-Ž .facturer Boehringer .

2.11. Kinetic analysis of Fab-binding using the BI-Acorew biosensor

Kinetic binding constants of Fab anti-KLH werew Ždetermined using the BiAcore 1000 instrument Bi-

.acore, Uppsala, Sweden .Ž .Keyhole limpet hemocyanin KLH was cova-

lently bound to the dextran surface of a certifiedŽ .CM5 sensor chip BiAcore AB, Uppsala, Sweden

Ž .via primary amino groups Jonsson et al., 1991using a continuous flow rate of 5 mlrmin. Briefly,the carboxymethylated sensor chip surface was ini-tially activated by a 35-ml injection of a mixture of

X Ž .0.2 M N-ethyl-N dimethylaminopropyl carbo-Ž .diimide EDC and 0.05 M N-hydroxysuccinimide

Ž . ŽNHS . 40 ml of a KLH solution 15 mgrml in 10.mM acetate buffer pH 4.75 were then injected over

the activated surface. Following immobilization,

Fig. 3. Plasmid vector, pMF3, used for the production of the soluble molecules. The heavy chain fragment was cloned into XhoIrSpeI sites,whereas the light chain was inserted into SacIrXbaI sites. The vector contains a six-His tail and the gene for the alkaline phosphatase todirectly label the antibody fragment.

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residual unreacted N-hydroxysuccinimide estergroups were blocked by injection of 35 ml of

Ž .ethanolamine–HCL 1 M, pH 8.5 , followed by theremoval of noncovalently bound KLH by injectionof 15 ml of a 25-mM NaOH solution. At the end of

Žthe immobilization step, about 15,350 RU resonance.units of KLH were covalently bound to the matrix.

The surface was then equilibrated with HBS bufferŽ10 mM Hepes, 0.15 M NaCl, 3.4 mM EDTA and

.0.05% surfactant P20, pH 7.4 and samples of 50 mlŽof the anti-KLH Fab at different concentrations 6–12

.nM in HBS were passed over the sensor chip, one ata time, using a constant flow rate of 10 mlrmin.Regeneration of the matrix after each cycle wasachieved through the injection of 15 ml of 25 mMNaOH. The dissociation phase was followed for 300s in experiments performed for the evaluation ofkinetic constants. Kinetic rate constants were ob-tained by fitting experimental sensorgram data tokinetic models using the BIAcore BIAevaluation 2.1Software.

3. Results

3.1. Construction of a phage–Fab library

The rabbit IgH chain locus contains more than100, mostly functional V genes. However, the ma-H

Žjority of the B cells rearrange V 1 Knight andH.Becker, 1990 . Therefore, we used two specific

primers for the rabbit heavy-chain-rearranged genesand three primers for the light chain according to thesequences published in the data bank. The informa-tion was used to design the primers needed to am-plify the majority of the IgG repertoire from rabbitwith the V a1 b4 allotype. V a1 is highly conservedH H

Ž .in FR1 from a.a. 2–9 Kabat et al., 1991 and thedesign of a suitable primer was straightforward. The

Ž .restriction site, XhoI CTCGAG used for cloningwas flanked by conserved nt sequences within FR1Ž .Table 1 . For the design of the light chain primer,

Ž .the conserved a.a. 3 to 8 Kabat et al., 1991 wasused for priming. In this case, the restriction site,

Ž . XSacI GAGCTC was attached to 5 of the primer,including a 6-bp nt overhang. The nt sequence infor-mation for the primers used for the amplification ofV -C 1 and L is given in Table 1. The 3X primerH H C

Žfor C 1 anneals within the hinge region from codonH. Ž7 to 9 of the single Igg gene Knight and Crane,

. X1994 . The 3 primers for the LC were chosen toamplify the k1b4 and k 2b4 allotypes.

Rabbits were immunized with the model antigen,KLH, and peripheral blood lymphocytes were puri-fied. The successful immunization was verified byELISA using plasma obtained after preparing the

Ž .nucleated cells data not shown . RNA was extractedfrom selected nucleated cell samples and used toprepare PCR-amplified Ig-specific DNAs corre-

Ž .sponding to heavy chain Fd region and to k lightchain. Single bands of the predicted size for V -C 1H H

and V -C genes were obtained after amplificationL LŽ .of first strand cDNA Fig. 1 . The specificity of the

Fig. 4. ELISA test of serial dilution of the soluble moleculeexpressed in the periplasmic space of E. coli after the subcloningof the antibody fragments into pMF3 vector. The ELISA wasdeveloped using the phosphatase activity linked genetically to thevector. Specificity of the reaction was confirmed by analysing thebinding of the Fab fragments to BSA, as well as to KLH. ND: notdiluted sample.

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PCR products was tested by hybridization with aninternal oligonucleotide labelled using nonradioac-

Ž .tive methods Fig. 1 . The V -C 1 and V -C geneH H L L

segments were cloned into pComb3, as describedŽ . 6Barbas et al., 1991 . A library of 2=10 cfu wasobtained.

3.2. Selection of antigen binders by library panning

To select the population of rabbit Fab fragmentsthat were bound to KLH, phagemid particles wererescued from the library by superinfection with helperphage and selected for binding to antigen. Fourrounds of rescue, selection and infection were per-formed. The enrichment process was monitored bytitering the phage colony-forming units eluted fromthe antigen-coated wells at each stage. A gradualincrease of enriched phage from 2=105 cfu to2=107 cfu was monitored after each panning onantigen, as shown in Table 2. The number of phageseluted after the fourth round of panning did notincrease further, indicating that the saturation was

Ž .reached Table 2 . Panning the library against non-coated plates and elution resulted in a recovery ofsome 2=102 to 2=103 clones which did not en-rich after rescue, amplification and further pannings.The clones selected on antigen were tested for thepresence of the inserts by PCR. Two different pat-terns of migration of the heavy chain bands wereobserved, indicating that two different groups ofclones were isolated after the fourth round of selec-

Ž .tion data not shown .

3.3. Specificity of selected phage antibodies

Similar amounts of serially diluted phage wereadded to antigen-coated plates and the binding speci-ficity was determined by phage ELISA using sheepanti-M13-HRP Ab to detect phage bound to KLH.Various dilution end points were determined for thedifferent clones, indicating different affinities. A typ-

ical binding curve for two clones named Cl.2 andCl.3IV3 is shown in Fig. 2. The data show thatphage bound specifically to KLH, and thus, the datafrom phage ELISA confirmed that the gradual in-crease of enriched phage from 2=105 to 2=107

Ž .cfu was specific. A phage clone Neg, Fig. 2 pickedrandomly from the library before panning did notshow specific binding to KLH. Phage binding couldbe inhibited by coincubation of phage with the anti-

Ž .gen data not shown .

( )3.4. Production and characterization of Fab his –phoA fusion molecules

To produce a soluble Fab molecule, to purify themolecule by affinity isolation, and to have Fab frag-ments directly labelled with an enzyme which isuseful for detection, we subcloned the heavy and thelight chain of the ELISA positive clones, Cl.2 and

ŽCl.3IV3 into the modifed pJuFo vector Crameri and. Ž .Suter, 1993 named pMF3 Fig. 3 , as described in

Section 2.Some of the clones obtained after transformation

were tested for phosphatase activity in a nitrocellu-lose filter test. The clones produced a strong signalwhen compared to the control strain harbouringpJuFo. Therefore, the recombinant rabbit Fab–al-kaline phosphatase conjugate appeared to be cor-rectly folded and appeared to be expressed as solublemolecules in the periplasmic space of E. coli. How-ever, we noted different levels of phosphatase activ-ity in different clones transformed with the sameconstruct.

Proteins from individual clones expressed in theperiplasmic space and secreted in the medium wereseparated by gel electrophoresis and examined forFab molecules by immunoblot analysis using a goat

Ž .anti-rabbit IgG HqL . Under nonreducing condi-tions, a single positive band at 97 kDa was seen for

Ž .each of the clones examined data not shown . TheFab fusion protein was found in the periplasmic

Ž .Fig. 5. a cDNA sequences of the variable region gene segment of the heavy chain of the anti-KLH Fab compared with the mostŽ .homologous germline gene Genebank accession number: S76746 ; rabbHc, rabbit heavy chain. Vertical lines denote identity of the

Ž .nucleotide sequence. b Nucleotide sequence of the Vk gene of the anti-KLH antibody is compared with the most homologous germlineŽ .gene Genebank accession number: K01358 ; RabbLc, rabbit light chain. A gap has been introduced, where necessary, to align the

sequences.

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space of the E. coli, but was hardly detectable in thesupernatant, and thus, periplasmic extract was usedas the source of the Fab molecules for future experi-ments.

Ž .We tested the specificity of the soluble Fab his –phoA fusion molecules with an ELISA using KLH

Ž .or BSA as antigens Fig. 4 . As expected, a doseresponse in enzyme activity of phoA was seen withKLH as antigen, but not with BSA when unpurifiedperiplasmic extracts from clones, Cl.2 and Cl.3IV3,were diluted on solid phase-bound antigen. The neg-

Ž .ative clone Neg, Fig. 4 did not react with eitherKLH or with BSA as antigen indicating specificityof the positive clones, Cl.2 and Cl.3IV3.

( )3.5. Purification of Fab and Fab his –phoa anddetermination of binding affinity

Ž .A Fab his –phoA clone from isolate, Cl.3IV3,which showed the highest level of phosphatase activ-ity, was chosen for the purification and characteriza-tion. For this, Cl.3IV3 was grown in a large-scalepreparation in order to purify the soluble molecule

Ž .using the His tag Fig. 3 in chelate affinity purifi-6

cation. The purification was followed by analyses ofsamples for enzymatic activity of the attached phoAmolecule, either directly or in ELISA. Eventhoughwe did see an enrichment of the His -containing6

proteins, the protein was only about 60% pure, asjudged by SDS-PAGE. This purity was taken as thebasis for the calculation of the kinetic constants.

The kinetic constants for the binding of Fab toKLH were performed by surface plasmonresonance-based measurements using the BIAcore

Ž .instrument Pharmacia . The dissociation rate con-stant, k , was calculated by fitting experimental datad

to the model equation: RsR eyk d1Ž tyt0 ., where t is0

the time in seconds, R is the response expressed inRU, and R is the total response at the start time, t ,0 0

for the dissociation.The association rate constant, k , and the steadya

state response level, R , were then obtained byeq

fitting experimental data to the equation,

RsR 1yeyŽ kaCnqk d .Ž tyt0 . ,Ž .eq

where R is the steady-state response level, C is theeq

molar concentration of the analyte, n is the steric

Ž .interference factor equal to 1 , t is the start time0

for association and k is the dissociation rate con-d

stant.The calculated kinetic rate constants were: k sa

4.4=105 moly1 sy1 ; k s2.8=10y3 sy1 with ad

resulting equilibrium affinity constant, K s1.57=A

108 moly1.The nt sequence of the V and V genes fromH L

Ž .clone, Cl.3IV3, has been determined Fig. 5 . The Iggenes were of the expected allotypes and showed80% homology with the rabbit Ig gene sequencesaccessible in the GeneBank.

4. Discussion

Polyclonal rabbit antibodies have been success-fully used for decades in biology and medicine. Onlyrecently a reliable cell fusion partner for the produc-

Žtion of mAb became available Spieker-Polet et al.,.1995 .

As an alternative to cell fusion techniques, weŽhave evaluated repertoire cloning Winter et al.,

.1994; Lerner et al., 1992; Ridder et al., 1995 for theproduction of rabbit mAb. The analysis of the FR1 nt

Ž .sequences from more than 20 rearranged V V a1H HŽ .and V b4 genes confirmed that the FR region isL

Žconserved as has been shown previously Knight and.Becker, 1990 . Based on this information, two

primers for the Ig heavy chain and three for the lightchain were used for the PCR amplification of rear-ranged Ig genes. We have not amplified V se-H

Ž .quences other than V a1 Knight and Crane, 1994H

sequences because normally, they represent a smallproportion of the rearranged genes.

Ž .In contrast to earlier studies Ridder et al., 1995 ,Ž .we have used blood lymphocytes PBL rather than

spleen cells to amplify the Ig genes. Peripheral Bcells from rabbits appear to be mostly CD5 positiverecirculating cells which represent the primary im-mune repertoire in adult rabbits and therefore, areequally suited as the source of mRNA for library

Žconstruction as spleen cells Raman and Knight,.1992; Knight and Crane, 1994; Crane et al., 1996 .

In addition, because PBL and plasma can be pre-pared simultaneously, this enabled us to follow theimmune response of individual rabbits by analysing

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( )M. Foti et al.rJournal of Immunological Methods 213 1998 201–212 211

plasma for antibody reactivity to the antigen used forimmunization. After the serological analysis, thebest-suited PBL stored was taken to extract mRNAand to construct libraries from PCR-amplified Iggene segments.

From the constructed library, we have been ableto select antibodies of good affinities. Two Abs wereexpressed as Fab–enzyme conjugates using the ex-

Ž .pression vector, pJuFos Crameri and Suter, 1993 ,to allow production of a recombinant fusion proteincomprising the dimer of phoA in the periplasmicspace of E. coli. For this, the gene encoding phoAwas fused with the V -C domain, and a tail of HisL L 6

was fused with the Fd domain of the Ig fragment tofacilitate the purification by affinity chromatography.The hybrid protein maintained the affinity for theantigen, as well as the phoA activity. We haveshown that crude extracts containing these conju-gates can be used in enzyme immunoassays designedto detect binding, as exemplified for the antigen,KLH. Eventhough we did get an enrichment ofspecific antibodies by chelate affinity isolation, theFab fragments were not pure when the foldedmolecule was used. This is in contrast to fully dena-

Ž .tured proteins Dudler et al., 1992 where purifica-tion with His tails is very efficient. It is possible6

that in the folded Fab molecule, not all of the His6

a.a. could access the NTA chelator, resulting indecreased chelating activity.

5. Conclusion

The results reported here demonstrate that rabbitantibodies can be expressed in E. coli as Fab–geneIII fusion protein on the surface of filamentous phage;that in this form, they retain the capacity to bind thespecific antigen and that it is possible to detect thebinding using genetically engineered conjugates. Wepropose that this genetic approach could replace thechemical procedures currently used to label the anti-body molecules.

Acknowledgements

We would like to thank Anne Belien and AndreBernard for the antibody affinity measurement; Dr.

Malgorzata Matyszak and Dr. Mahender Singh forthe critical review of the manuscript. Rose Mage,NIH Bethesda, USA, determined the allotype ofsome rabbits and kindly provided sera against rabbitimmunoglobulins. This study was supported by theEuropean Community, TMR programme contractERB FMRXCT 960053 and by a grant from theGRD, Switzerland and from Anawa, Switzerland.

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