detection of pna/dna hybrid molecules by antibody fab fragments isolated from a phage display...

Ž .Journal of Immunological Methods 203 1997 199–207

Detection of PNArDNA hybrid molecules by antibody Fabfragments isolated from a phage display library

Margit Haahr Hansen a,c, Lise Lotte Sode a, Jens Jørgen Hyldig-Nielsen b,Jan Engberg c,)

a DAKO ArS, ProduktionsÕej 42, Glostrup, Denmarkb Boston Probes, Inc., 75E Wiggins AÕenue, Bedford, MA 01730, USA

c Royal Danish School of Pharmacy, Department of Biological Sciences, UniÕersitetsparken 2, DK-2100 Copenhagen, Denmark

Received 6 September 1996; revised 20 November 1996; accepted 11 February 1997

Abstract

ŽWe have isolated Fab fragments that specifically recognize duplexes formed between DNA and PNA peptide nucleic.acid from an immunized murine phage display library. Rearranged murine Fd- and Kappa chains were assembled by PCR

and cloned into a phagemid expression vector. Subsequently, affinity selection on immobilized PNArDNA duplexes of theFab-displaying phages resulted in the isolation of clones that uniquely recognized PNArDNA duplexes. One of these cloneswas characterized in detail, and its recognition of PNArDNA duplexes was relatively sequence independent, taking placeequally well with sticky-end and blunt end PNArDNA duplexes. Duplexes smaller than 15-mers could not be detected. Theselected clone recognized neither single-stranded DNA and PNA, nor double-stranded DNA and PNA. Binding of the Fabfragments to immobilized PNArDNA duplexes could be inhibited by PNArDNA duplex molecules in solution, with anapparent affinity in the nanomolar range. The use of this anti PNArDNA Fab-phage as an immunochemical reagent wasdemonstrated in dot blot assays.

Ž .Keywords: Phage display; Peptide nucleic acid PNA ; Recombinant antibody; Diagnostics

Abbreviations: ds: double-stranded; ELISA: enzyme-linkedimmunosorbent assay; Fd: variable region and constant domain 1

Ž .of heavy chain C 1 ; HRP: horse radish peroxidase; IPTG:H

isopropyl-b-D-thiogalactopyranoside; OPD: ortho-phenyldiamine;PCR: polymerase chain reaction; PNA: peptide nucleic acid; ss:single-stranded; V : variable region of heavy chain; V : variableH K

region of Kappa chain) Corresponding author. Tel.: q45 35 370852; Fax: q45 35

375792; e-mail: [email protected]

1. Introduction

Ž .PNA peptide nucleic acid molecules are newlydeveloped DNA analogues with the capacity to bindnucleic acids in a sequence specific manner with

Žvery high affinity Nielsen et al., 1991; Nielsen et.al., 1994 . PNArDNA duplexes are formed by Wat-

son–Crick or Hoogsteen basepairing, and it is be-lieved that they form a helical structure that is not

Žvery different from that of DNArDNA Leijon et.al., 1994 .

PNA has both anti-gene and antisense activities,

0022-1759r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved.Ž .PII S0022-1759 97 00031-8

( )M.H. Hansen et al.rJournal of Immunological Methods 203 1997 199–207200

due to the interaction with both DNA and RNAŽunder physiological conditions Hanvey et al., 1992;

.Buchardt et al., 1993 , and this feature suggests theuse of PNA molecules in various nucleic acid baseddiagnostic procedures, for example in situ hybridiza-tion to detect bacterial or viral infections. In order toavoid background problems resulting from non-hy-bridized single-stranded PNA molecules, it would beof great advantage to be able to detect uniquely thepresence of PNArDNA hybrid molecules. In thepresent work we have generated antibodies with suchspecificity using phage display technology.

Different phagemid systems have been developed,where antibody fragments are cloned in fusion with

Žthe phage coat protein pIII Barbas et al., 1991;.Clackson et al., 1991; Ørum et al., 1993 or in fusion

Žwith the phage coat protein pVIII Kang et al., 1991;.Chang et al., 1991 . The pIII system results in

Žmonovalent display of antibody fragments Garrard.et al., 1991; Johansen et al., 1995 , and thus select

antibodies by monospecific affinity and not bypolyspecific affinityravidity, as is the case in the

Ž .pVIII system Gram et al., 1992 .The preparation of the phagemid vector

pFAB5c.His that was used in the present work in-volved the use of a PCR assembly procedure fol-lowed by a one step cloning of the PCR assembledFd and Kappa chains in fusion with the gIII productŽ .Ørum et al., 1993; Engberg et al., 1995 . Theexpression of the Fab fragments was under transcrip-

Žtional control of the lac promoter, and pelB pectate.lyase from Erwinia caÕotoÕora was used as signal

sequence for export of the fragments to the periplas-mic space. The cloning strategy resulted in the fusionof the Kappa chains to a hexa-histidine tail to facili-tate the subsequent purification of the soluble Fab

Žmolecules using IMAC technology Skerra et al.,.1991; Essen and Skerra, 1993; Johansen et al., 1995 .

2. Materials and methods

2.1. PNA and DNA synthesis, labeling and hybridconstruction

The nucleotide sequences of the PNA and DNAoligomers were chosen in order to avoid self-com-plementary complex formation. The number ofpyrimidines and purines were approximately equal.

PNA oligomers were synthesized manually orusing automated equipment based on Gilson sam-pling injector 232XL according to Boc peptide

Ž .chemistry as described by Christensen et al. 1995 .Alternatively, the PNAs were synthesized employingFmoc peptide chemistry with the use of an Expedite

Ž8909 Nucleic Acid Synthesizer PerSeptive Biosys-.tems, Framingham, MA, USA . After cleavage from

the resin, the PNA oligomers were purified by re-verse-phase HPLC using a linear gradient of 0–30%acetonitrile in 0.1% trifluoracetic acid at 508C.

Biotin-labeling of PNA was performed by treatingthe resin with activated esters of biotin. After cleav-age, the labeled PNAs were purified by reverse-phaseHPLC using a linear gradient of 0–50% acetonitrilein 0.1% trifluoracetic acid at 508C.

The identity of all PNAs were confirmed byMALDI-TOF mass spectroscopy on a HewlettPackard G2025A. The experimental molecularweights were within 0.1% of the calculated molecu-lar weights. Some PNAs were purchased from

Ž .PerSeptive Biosystems, Framingham, MA, USA .DNA oligonucleotides were synthesized on an

ŽABI 381A DNA synthesizer Applied Biosystems,.Foster City, CA, USA using standard protocols and

b-cyanoethyl-N,N-diisopropylphosphoramidites. Bi-otin-labeling of DNA oligonucleotides at either the3X- or the 5X-end was carried out using 3X Biotin-ON

e ŽCPG or Biotin-ON phosphoramidites Clontech,.Palo Alto, CA, USA , and programming the DNA

synthesizer with an extended coupling time to ensurehigh coupling efficiency.

PNArDNA duplexes for immunization and char-acterization were prepared by hybridizing PNAmolecules to complementary DNA oligonucleotides.Hybridization was performed in 50 mM Tris–HCl,pH 7.5, 50 mM NaCl, with a 10 min incubation at928C and slow cooling to room temperature. Duplexformation was analyzed by Tm measurements using

Ža Lambda2S UV–VIS spectrophotometer Perkin.Elmer Corp., Norwalk, CT, USA . The PNArDNA

duplexes used in this study are listed in Table 1.

2.2. Immunization and Fab-phage library construc-tion

A female BALBrc mouse was immunized fivetimes subcutaneously over a period of approximately

( )M.H. Hansen et al.rJournal of Immunological Methods 203 1997 199–207 201

three months. According to a procedure developed atDAKO ArS, 100 mg H12 duplex including Freund’sincomplete adjuvant was used for immunization.

Two days after a booster injection of antigen notcontaining Freund’s incomplete adjuvant, RNA wasprepared from the spleen of the mouse and subjectedto cDNA synthesis and PCR amplification as previ-

Ž .ously described Engberg et al., 1995 . In brief,fragments corresponding to the complete kappachains and the Fd fragment of the IgG chains wereamplified. The 5X yprimers consist of equimolarmixtures of 25 individually synthesized V and VH L

oligonucleotides with degeneracy in two positions,and corresponding to the N-terminal ends of all

known murine IgG and kappa molecules. The 3X-primers correspond to the C-termini of the heavy

Ž .chain C 1 and kappa chain constant regions, re-H

spectively. PCR-assembled Fd- and Kappa chainsŽwere cloned in phagemid pFAB5c. His Engberg et

.al., 1995 , and the final construct was electroporatedŽinto TOP10F’ E. coli cells R&D Systems Europe,

. ŽAbingdon, UK using a BioRad E. coli pulser Bio-.Rad, Hercules, CA, USA .

Production of Fab-phages was induced by super-infecting exponentially growing cultures with R408

Ž .helper phages Stratagene, San Diego, CA, USA ata multiplicity of infection of 100 followed by induc-tion with 0.1 mM IPTG. The Fab-phages were PEG

Table 1Sequences of PNArDNA duplexes, dsPNArdsDNA duplexes and single-stranded molecules used in this investigation

PNArDNA duplexesŽ .Duplex bp DNArPNA Sequence of DNA

H2 45r15=3 GCA AAT GCT CTA GGC GCA AAT GCT CTA GGC GCA AAT GCT CTA GGC – BH3 15r15 B – GCA AAT GCT CTA GGCH7 20r20 B – CGG CCG CCG ATA TTG GCA ACH8 17r17 B – ATT GTT TCG GCA ATT GTH9 17r17 ATT GTT TCG GCA ATT GTH12 45r15=3 GCA AAT GCT CTA GGC GCA AAT GCT CTA GGC GCA AAT GCT CTA GGCH29 30r15 GCT GAC GTT CCG CAC ATG TCA ACC ATA TGT – BH29–1 30r11 GCT GAC GTT CCG CAC ATG TCA ACC ATA TGT – BH29-2 30r7 GCT GAC GTT CCG CAC ATG TCA ACC ATA TGT – BH29-3 30r15 GCT GAC GTT CCG CAC CTG TCA ACC ATA TGT – BH29-4 30r15 GCT GAC GTT CCG TAC ATG CCA ACC ATA TGT – BH29-5 30r15 GCT GAC GTT CCA CAC ATG TAA ACC ATA TGT – BH29-6 30r15 GCT GAC GTT CCG CAA CTG TCA ACC ATA TGT – BH31 45r15=3 TCC GCA CAT GTC AAC TCC GCA CAT GTC AAC TCC GCA CAT GTC AAC – B

X XAll DNA sequences are written in the 5 –3 orientation, and the complementary PNA sequences are indicated by underliningof sequences. B indicates biotin. When no B is given, the PNA moiety of the hybrid was biotinylated, except for H12- thiscomplex was not biotinylated and is identical to the duplex used as immunogen. In the H29 series, identical PNA sequenceswere used in all constructs except for H29-1 and H29-2. Introduced point mutations in DNA are indicated in bold. All constructs arehybridized in the anti-parallel orientation.

dsPNA and dsDNA duplexesŽ .H32 17r17 PNA ATT GTT TCG GCA ATT GTŽ .H20 45r15 DNA GCA AAT GCT CTA GGC GCA AAT GCT CTA GGC GCA AAT GCT CTA GGC – B

The given PNA molecule was hybridized to a biotinylated complementary PNA molecule. The 45-mer DNA sequence was hybridizedto three identical 15-mer DNA oligonucleotides.

Single-stranded moleculesŽ .L2 45 DNA GCA AAT GCT CTA GGC GCA AAT GCT CTA GGC GCA AAT GCT CTA GGC – BŽ .L8 17 PNA B – TTC AAC TCT GTG AGT TGA A


Ž .precipitated Sambrook et al., 1989 , titrated andused for the panning selection.

2.3. Panning selection

Panning selections were performed using biotin-labeled H2 PNArDNA duplexes that were immobi-lized onto Nunc Maxisorp microtiter plates coated

Ž .with streptavidin DAKO ArS . 50 ng H2 duplexeswere immobilized per well. After blocking with 4%

Žskimmed milk powder Difco Laboratories, Detroit,. ŽMI, USA in PBS 137 mM NaCl, 2.7 mM KCl, 3.4

.mM Na PO P7H O, 1.4 mM KH PO , pHs7.32 4 2 2 4Ž . 10 11PBSM , 10 –10 Fab-phages resuspended in PBSsupplemented with 1 mgr100 ml streptavidin wereadded to each well and incubated at 378C for approx-imately 2 h. Differing numbers of washes wereperformed with 300 ml 0.5% Tween 20 in PBSŽ . ŽPBST per wash 3 washes for the first round of

.panning, 5–10 washes for subsequent rounds , be-fore bound phages were eluted by a 30 min incuba-

Žtion at room temperature with 0.1% trypsin DAKO.ArS in PBS. Eluted phages were used for infection

of exponentially growing E. coli TOP10F’. Theeluted phages were titrated by spreading infectedcells on LB plates supplemented with 100 mgrmlampicillin and 0.5% glucose. Remaining cells werepropagated overnight in LB medium supplementedwith 100 mgrml ampicillin and 0.5% glucose. Analiquot of this culture was inoculated into fresh LBmedium with 100 mgrml ampicillin and 12 mgrmltetracycline, superinfected and induced with IPTG,followed by an overnight incubation and phage pre-

Žcipitation and panning the next day Engberg et al.,.1995 .

2.4. ELISA

Streptavidin coated Nunc Maxisorp ELISA platesŽ .DAKO ArS were used for ELISA assays. AllPNArDNA duplexes used for ELISA analysis ofisolated clones were biotin-labeled and bound toELISA wells by interaction with immobilizedstreptavidin. PNArDNA duplexes were diluted in

ŽTHT 50 mM Tris–HCl, 0.1 M NaCl, 0.1% Tween.20, pH 7.2 to a final concentration of 3.74 nM,

using 100 mlrwell. Microtiter wells were blockedwith 4% PBSM. All washings between incubationswere performed with THT. Fab-phages were dilutedin THT, added to the microtiter wells and incubatedfor 2 h at 378C. Detection of bound Fab-phages wasperformed with HRP conjugated rabbit anti phage

Ž .antibodies DAKO ArS , followed by detection withŽ .OPD DAKO ArS in phosphate–citrate buffer ac-

cording to the manufacturers’ instructions. Reactionswere stopped by the addition of 100 ml 1 M H SO .2 4

The extent of the reactions were measured as ab-sorbance at 490 nm.

Alternatively, free Fab fragments were preparedby mild osmotic shock according to Skerra and

Ž .Pluckthun 1988 and used in ELISA. Fab fragments¨were detected with goat anti mouse HRP conjugated

Ž .antibodies P0447, DAKO ArS , followed by detec-tion with OPD as described.

2.5. Dot blot analysis

Solutions of PNArDNA duplexes were applied toŽNY 13 N Nytran membrane Schleicher and Schuell,

.Dassel, Germany and dried at 508C. The duplexeswere crosslinked to the membrane by UV radiation

Žfor 30 s. After blocking with PBSMT PBSM sup-.plemented with 0.5% Tween 20 , the membrane was

incubated with 1011 Fab-phagesrml PBSMTovernight at 48C. Bound phages were detected withrabbit anti phage and goat anti rabbitralkaline phos-

Ž .phatase conjugated antibodies P0448, DAKO ArSand finally detected with 5-bromo-4-chloro-3-in-

Ž .dolylphosphaternitroblue tetrazolium BCIPrNBT .All washings between incubations were performed

Ž .with PBST PBS with 0.5% Tween 20 .

2.6. DNA sequencing

DNA sequencing was performed with the Sangerdideoxy method, using a Thermo Sequenase Fluores-

Žcent Labelled Primer sequencing kit Amersham,.Buckinghamshire, UK . The cycle sequencing reac-

tions were performed on a Hybaid Omn-E thermalŽ .cycler Hybaid, Teddington, UK , and sequences

were obtained from an ALFexpress automated DNAŽ .sequencer Pharmacia, Uppsala, Sweden .


3. Results

ŽUsing previously published procedures Ørum et.al., 1993; Engberg et al., 1995 , a Fab-phage library

was constructed from a BALBrc mouse hyperimmu-Ž .nized with the PNArDNA duplex H12, Table 1 .

The original library consisted of 1.5=107 indepen-dent clones, and was taken through four rounds of

Ž .panning on PNArDNA duplexes H2 immobilizedin microtiter wells. Individual colonies isolated afterthe fourth round of panning were tested for specificbinding to H2 PNArDNA duplexes in ELISA proce-dures. These assays were performed using either free

ŽFab molecules or Fab-phage fusions Kingsbury and.Junghans, 1995 . To date we have identified 24

clones as being positive using the ELISA procedure;i.e. the absorbance was at least a factor 10 higherthan background. Ten positive clones were character-ized by DNA sequencing and found to be identicalŽ . Ž .IgG1, Kappa . One such clone MHH5 was se-lected for further functional analysis.

The MHH5 clone was tested for reactivity with aŽ .variety of PNArDNA duplexes Table 1 in ELISA

based procedures. The concentration of duplexes im-mobilized onto streptavidin coated microtiter wells

Žwas 3.74 nM in all assays equivalent to 100 ngrml.of the H2 duplex . The different duplexes varied

with respect to length, sequence and the localizationŽof the biotin group within the complex i.e. which

component of the duplex that was used for immobi-lization by interaction with streptavidin in the mi-

.crotiter well .Titration curves illustrating the recognition of var-

ious duplexes by MHH5 are shown in Fig. 1. Thestrongest reactivity was observed with the H2 duplexthat is equivalent to the immunogen. Low reactivitywas seen with the H3 duplex, which is identical insequence but one third the size of the H2 duplexŽ .results not shown . This indicates that a 15-merduplex is the minimum size required for recognition

Ž .by MHH5 vide infra , since significant reactivitywas observed with the H29 duplex, that consists of a30-mer DNA and a 15-mer PNA. It is possible thatthe lack of recognition of the H3 duplex by theantibody fragment can be explained by steric hin-drance, since immobilization of H29 will permitbetter access to the epitope.

When PNArDNA duplexes of similar length but

Fig. 1. Titration of MHH5 Fab-phages. Various PNArDNA du-Ž .plexes see Table 1 were immobilized in streptavidin coated

microtiter wells. Reactivity with duplexes was followed by anincrease in absorbance at 490 nm using increasing numbers of

Ž .Fab-phagesrwell standard procedure, see Section 2 .

Žof different composition were examined cf. H2 and.H31, Fig. 1 and Table 1 , differences in reactivity

were observed, suggesting that the strength of recog-nition by MHH5 is somewhat sequence dependent.

The observed difference in reactivity with the H8and H9 duplexes was unexpected since the onlydifference between these two duplexes was the local-ization of the biotin group and thus the strand thatwas engaged in immobilization on the streptavidincoat. It is possible that the PNArDNA duplex isdisplayed differently in the two cases, thereby result-ing in a different reaction pattern.

Furthermore, the results in Fig. 1 indicate that theŽtermini of the duplexes whether A–T or C–G and.blunt-ends or sticky-end did not play any role in the

recognition. The Fab fragment reacted specificallyŽ .with both sticky-end H29 and blunt-end duplexes

Ž .H2, H7, H8, H9 and H31 .Reactivity with dsDNA, ssDNA, dsPNA and

ssPNA were also tested in order to analyze thespecificity of MHH5. As shown in Fig. 2, no signifi-


cant reaction was observed with any of these con-structs.

To demonstrate the specific interaction of the H12PNArDNA duplex with the MHH5 antibody frag-ment, we performed a competition ELISA experi-ment adding increasing amounts of the H12 duplexin solution to an ELISA procedure using immobi-

Žlized H2 duplexes H2 is a biotin-labeled version of.H12 . In the experiment described in Fig. 3, the

binding of MHH5 to immobilized H2 was inhibitedby the H12 duplex in solution with an IC of50

200–300 nM.To probe the specificity of recognition further,

two cross-competition ELISA experiments were per-formed with immobilized H9 and H31 duplexes,respectively, and with H12 as the competitor in both

Ž .cases Fig. 3 . The IC values inferred from Fig. 3,50

employing either H9 coat or H31 coat, were approxi-mately 10 nM, a value that is different from the IC50

value determined when H2 was immobilized. Thisdifference in IC indicates a stronger binding of the50

Fig. 2. Specificity of MHH5 Fab-phage clone. Reactivity withsingle- and double-stranded DNA and PNA molecules was ana-lyzed by immobilization of various duplexes to streptavidin coatedmicrotiter wells, followed by addition of increasing numbers ofFab-phagesrwell. Reactivity was measured as the absorbance at490 nm at given Fab-phage concentrations.

Ž . Ž .Fig. 3. Competitive ELISA procedure. H2 circles , H9 squaresŽ .or H31 triangles duplexes were immobilized to streptavidin

coated microtiter wells. Fab-phages were mixed with H12 du-plexes, and reacted at 378C for 1 h before transfer to ELISA wells.The % binding was determined relative to the reactivity of thesame number of Fab-phages with immobilized antigen but withoutcompeting antigen.The background reactivity with streptavidincorresponded to a relative binding of less than 10%. Processing ofthe ELISA plates was according to standard procedures.

anti PNArDNA Fab fragment to H12 compared toH9 and H31. Presumably, the stronger binding of theMHH5 Fab fragments to H12 is related to a higheroff-rate of the Fab fragment from H9 and H31 thanfrom H12.

There is good correlation between the resultspresented in Fig. 1 and the competition ELISAs,since the titration curves for MHH5 reactivity againstH9 and H31 overlapped, and the IC values in the50

cross-competitive assays were identical for the twoduplexes.

Since the H12 duplex does not contain a linker,the results from the competitive ELISA experimentsalso showed that the reactivity of the Fab fragmentswas not directed against the linker molecule betweenbiotin and PNA or DNA molecules, but was indeedspecific for PNArDNA duplexes.


The strongest reactivity of MHH5 was found withthe PNArDNA duplex used for immunization. Simi-lar, but lower reactivities with other PNArDNAduplexes were observed. Whether this observation isrelated to sequence differences among the duplexesinvestigated or to different lengths of the duplexesremains an open question. Nevertheless the observa-tions do not alter the fact that MHH5 Fab fragmentsuniquely recognize PNArDNA duplexes. It shouldbe noted that the weakest reactivity with aPNArDNA duplex was found to be 5–10-fold higherthan the reactivity with dsDNA. Thus it is possible touse the MHH5 antibody fragments as a generaldetector of PNArDNA duplexes.

The unique reactivity of the anti PNArDNA clonehas important consequences for diagnostic applica-tions, and the observed differences in reactivityshould be considered when designing PNA probes.

To investigate the minimum size of the duplexrequired for recognition, and the ability of the antiPNArDNA Fab fragments to discriminate between

Ž .Fig. 4. Variants of the H29 duplex see Table 1 were tested forreactivity with the MHH5 Fab-phage clone. The variants wereimmobilized in ELISA wells using streptavidin, and the reactivityagainst the various duplexes was monitored as the optical densityat 490 nm at increasing numbers of Fab-phagesrwell.

Fig. 5. Dot blot assay. H12 duplexes and dsDNA were applied toa nylon membrane in the same molar concentrations and subse-quently detected with anti PNArDNA Fab-phages as described inSection 2. The negative control membrane was detected withR408 helper phages.

complementary duplexes and duplexes containingbase pair mismatches, we designed different variantsof the H29 duplex. One or two base pair mismatcheswere introduced into a 30-mer DNA molecule andsubsequently hybridized to a 15-mer PNA, or a30-mer DNA was hybridized to shorter PNAs. It was

Ž .found Fig. 4 that a duplex consisting of an 11-merŽ .or smaller PNA H29-1 and H29-2 gave no reactiv-

Ž .ity, whereas a duplex made of a 15-mer PNA H29showed a specific reaction. It was also found that theintroduction of one or two point mutations into the

Ž .DNA oligonucleotide H29-3 to H29-6 had a detri-mental effect on the ability of MHH5 to bind theduplexes. This suggests that the introduced mutationsinterrupt the three dimensional structure of the du-plex necessary for specific recognition by the antiPNArDNA Fab-fragments.

The potential use of Fab-phages as immuno-Ž .chemical reagents Nissim et al., 1994 was investi-

Ž .gated by immobilizing PNArDNA duplexes H12on a nylon membrane, followed by detection of theduplexes with Fab-phages. As demonstrated in Fig.5, a specific signal was obtained when as little as 5


ng of the H12 duplex was applied to the membrane.This level of sensitivity is not appropriate for e.g. insitu hybridization procedures, but by employing invitro affinity maturation as described by Low et al.Ž . Ž .1996 and Irving et al. 1996 , the binding affinityof MHH5 may be increased, and thereby the poten-tial use of this Fab fragment.

4. Discussion

We have isolated antibody fragments from a phagedisplay library that specifically recognize duplexesformed between PNA and DNA. Antibodies withsuch specificities would become important tools inthe implementation of PNA as a diagnostic reagent.Assay formats based on a specific recognition of aPNA probe specifically hybridized to target DNAmay be developed as an alternative to existing assaysemploying DNA and RNA interaction.

All PNArDNA duplexes analyzed to date haveŽall been oriented in the antiparallel manner i.e. the

C-terminal of the PNA molecule is facing the 5X-endŽ ..of the DNA oligonucleotide Egholm et al., 1993 .

It is not known whether the opposite orientation ofthe components of the PNArDNA duplexes will berecognized by the anti PNArDNA Fab fragmentsdescribed here. Analysis of this type must be per-formed in further characterization of these Fab frag-ments, and the PNA probe design must be correlatedwith the reactivity of the antibody fragments.

Sequence analysis of the isolated anti PNArDNAFab fragments revealed that one clone had beenisolated preferentially. There may be several reasonsfor this. Firstly, the immune system may have cre-ated only one functional specificity during B-celldevelopment, and this specificity was reconstitutedduring library construction and selected in the pan-ning procedure. Secondly, the isolation of only onetype of binder may be a consequence of the panningprocedures, because the stringent washing conditions

Žare likely to enrich for the strongest binder Ander-.sen et al., 1996 .

During library construction the Fd- and Kappachains are combined randomly, and chain pairingsdiffering from parental B-cells are created. Whetherwe have isolated the parental Fd- and Kappa chainpairing remains unknown. The heavy chain promis-

Žcuity i.e. the ability of the heavy chain to combinewith different light chains and retain antigen speci-

.ficity makes it impossible to conclude anythingabout chain pairings.

We have compared the DNA sequence of theMHH5 clone to published sequences of murine VH

and V fragments in the databases. The V regionK H

showed a high degree of homology to antibodies thatreact with dsDNA and histone complexes. Out of 13clones with the highest degree of homology 10 wereDNA- or histone fraction reactive antibodies, or

Žantibodies from autoimmune mice systemic lupusŽ . .erythematosus SLE prone mice . The differences

between MHH5 and the homologous V regionsH

reside mainly in the CDR3 region. This strikinghomology could indicate that the PNArDNA duplexis similar to dsDNA and thus evokes an immuneresponse similar to that of dsDNA.

The potential of PNA as a diagnostic and thera-peutic tool is in the early phases. The antibody Fabfragment described will be of use in the futuredevelopment of PNA based assays.

Acknowledgements

We would like to thank Peter Sejer Andersen, JonAskaa, Gitte Kongsgaard Koed and Else RøngaardClausen for their help during different phases of thiswork. Jørgen Schøller is thanked for helpful discus-sions and critical reading of the manuscript. Thiswork was supported by the Danish Academy of

Ž .Technical Sciences MHH and the Danish MedicalŽ .Research Council JE .

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detection of pna/dna hybrid molecules by antibody fab fragments isolated from a phage display...

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