WO1996012397A2 - Recombinant antibody capable of binding to pna/nucleic acid complexes - Google Patents

Abstract

Recombinant antibodies or fragments thereof that are capable of binding to complexes formed between PNA (Peptide Nucleic Acid) and nucleic acids, particularly to PNA/DNA or PNA/RNA complexes, are described. The preferred antibodies are recombinant antibodies that bind to PNA/DNA OR PNA/RNA complexes, but not to single-stranded PNAs, double-stranded nucleic acids or single-stranded nucleic acids. PNAs are newly developed, not naturally occurring compounds of which some have a polyamide backbone bearing a plurality of ligands such as naturally occurring nucleobases attached to the backbone through a suitable linker. Some PNAs have been shown to possess a surprising high affinity for complementary nucleic acid, forming very stable and specific complexes. Such PNAs are thus suitable as hybridization probes for detection of nucleic acids. The antibodies now provided render the PNAs very usable as hybridization probes. The antibodies provided are useful in the capture, recognition, detection, identification or quantitation of nucleic acids in biological samples, via their ability to react with PNA-nucleic acid complexes.

Description

Recombinant antibody capable of binding to PNA/nucleic acid complexes

The present invention relates to recombinant antibodies or fragments thereof capable of binding to complexes formed between PNA (Peptide Nucleic Acid) and nucleic acids.

PNAs are newly developed, not naturally occurring compounds of which some have a polyamide backbone bearing a plurality of ligands such as naturally occurring nucleobases attached to the backbone through a suitable linker. Some PNAs have been shown to possess a surprisingly high affinity for complementary nucleic acids forming very stable and specific complexes. Such PNAs are thus suitable as hybridi¬ zation probes for detection of nucleic acids, in accordance with the present inven¬ tion, antibodies are provided which render such PNAs very usable as hybridization probes.

These antibodies are useful in the capture, recognition, detection, identification or quantitation of nucleic acids in biological samples, via their ability to bind to PNA/- nucleic acid complexes.

BACKGROUND OF THE INVENTION

The capture, recognition, detection, identification and/or quantitation of one or more chemical or biological entities is useful in the fields of recombinant DNA, human and veterinary medicine, agriculture and food science, among others. In particular, these techniques can be used to detect and identify etiologic agents such as bacteria and virus, to screen bacteria for antibiotic resistance, to aid in the diagnosis of genetic disorders and to detect cancerous cells.

The state-of-the-art nucleic acid hybridization assay techniques generally involve hybridization with a labelled form of a complementary nucleic acid probe. Hybridiza¬ tion between a particular base sequence of a nucleic acid in a sample and a labelled probe is determined by detection of the labelled complexes. The preparation of labelled probes generally involves the enzymatic incorporation of radiolabelled or modified nucleotides or chemical modification of the probe to attach or form a detec- table chemical group. Preparation of labelled probes is often time consuming and expensive and has to be carried out without destroying the ability of the probe to detectably hybridize with its complementary sequence.

Reagents for direct detecting the nucleic acid complex formed as a result of hybridi- zation between the sample and a nucleic acid probe and thereby avoid the chemical labeling of the probes, would facilitate detection.

The generation of specific polyclonal antibodies that will bind double-stranded nucleic acids but not single-stranded nucleic acids may be complicated by the fact that poly- clonal antisera raised against double-stranded nucleic acids may contain antibodies that will cross-react with single-stranded nucleic acids. Polyclonal antisera may also contain naturally occurring antibodies to single-stranded nucleic acids or antibodies to single-stranded nucleic acids arising as a result of brake down of the immunogen used for the immunization.

By use of monoclonal antibody technology, antibodies may be selected so as to possess a desired affinity and specificity.

From US 4,623,627 and US 4,833,084, monoclonal antibodies are known which bind to complexes formed between a conventional nucleic acid probe and a target nucleic acid.

An alternative to monoclonal antibodies are recombinant antibodies which may have the same type of specificities as the specificities of monoclonal antibodies.

In WO 92/20702, the term PNA is used to describe compounds having a non-cyclic backbone and bearing a plurality of ligands such as naturally occurring nucleobases attached to the backbone through a suitable linker. PNAs in which the backbone is structurally homomorphous with the deoxyribose backbone such as PNAs com- prising polymerized N-(2-aminoethyl)glycine units wherein the glycine is connected to naturally occurring nucleobases by a linker are able to hybridize to nucleic acid having a base sequence that is complementary to the base sequence of the PNA so as to form PNA-nucleic acid complexes (Egholm et al., Nature, Vol 365, 566-568 (1993)). Such PNAs have been shown to bind strongly to complementary nucleic acid sequences. The melting temperature, T_m, of the complexes formed between such PNAs and complementary nucleic acids is typically 1-2°C higher per base than the Tm value for a comparable complex formed between a DNA or RNA probe and a nucleic acid target. T_m is defined as the temperature at which half of the strands of a nucleic acid complex are dissociated or denatured.

In accordance with the present invention, novel recombinant antibodies are provided which are able to recognize, bind and detect complexes formed between PNA and nucleic acid.

SUMMARY OF THE INVENTION

One aspect of the present invention is recombinant antibodies or fragments thereof capable of binding to complexes formed between PNA and nucleic acids.

Apart from sharing the feature of base pairing, PNA/nucleic acid complexes and nucleic acid complexes, such as DNA/DNA or DNA/RNA complexes, possess sub¬ stantially different properties in that the PNA of a preferred PNA/nucleic acid com- plex comprises polymerized N-(2-aminoethyl)glycine units rendering the PNA achiral and non charged as opposed to the corresponding strands of a nucleic acid com¬ plex, wherein the backbone is a sequence of nucleotides containing one anion for each phosphate group. The ensuing steric and conformational differences between the two types of compounds makes it absolutely unpredictable whether antibodies binding specifically to PNA nucleic acid complexes could be made available.

Other aspects of the invention are recombinant antibodies or fragments thereof that are capable of binding to complexes formed between PNA and DNA or between PNA and RNA.

In preferred embodiments the recombinant antibodies or fragments thereof capable of binding to complexes formed between PNA and nucleic acids do not bind to single-stranded PNAs, double-stranded nucleic acids or single-stranded nucleic acids. In one of these embodiments, the recombinant antibody or a fragments thereof is capable of binding to a complex formed between PNA and DNA, but not to PNA RNA complexes, double-stranded DNA, DNA/RNA complexes, single-stranded PNAs or single-stranded nucleic acids.

A preferred embodiment of the present antibody is a Fab fragment comprising the Fab region of the heavy and light chain.

The present invention also provides vectors comprising recombinant DNA encoding a fragment comprising the Fab region of the heavy and light chain. Two of such vectors have been deposited at DSM (DSM 10051 and DSM 10052).

Another preferred fragment of the present recombinant antibody is a "single-chain Fv fragment" (scFv fragment) wherein the variable part of the heavy and light chains are linked by a spacer group, preferably a peptide spacer group.

Recombinant antibodies for which the specificity of the epitope(s) recognized to a higher degree is dictated by the conformation of the PNA/nucleic acid complexes than by the specific sequence of the PNA or the nucleic acid are also part of the invention.

The recombinant antibodies described herein are obtainable by immunizing a host animal, e.g. a mouse or a rabbit, with a PNA/nucleic acid complex, isolating RNA from antibody producing cells, producing single stranded cDNA from the mRNA of the isolated RNA, using specific oligonucleotide mixtures to amplify antibody encoding fragments from said cDNA and inserting it into a phagemid capable of ex¬ pressing and after superinfection displaying the antibody fragments at its surface, infecting bacteria with said phage to produce a phage library, selecting, through successive rounds of panning and reinfection of bacterial cells, the phages encoding antibody fragments of interest and using said phages for infection of bacteria for production of the antibody fragments or expressing the antibody fragment encoding DNA in another prokaryotic or eukaryotic expression system. Suitable complexes for immunization are complexes formed between PNA and DNA or between PNA and RNA wherein the PNA comprises polymerized N-(2-aminoethyl)glycine units. Recombinant antibodies of the present invention may also be obtained from large recombinatorial immunoglobulin libraries derived from non-immunized animals and if needed the affinity of the selected antibody binding sites might be increased by chain shuffling or by random mutagenesis.

Various methods for detecting a particular nucleic acid sequence in a test sample are additional aspects of the invention, whereby the present antibodies are useful in the capture, recognition, detection, identification or quantitation of one or more chemical or biological entities.

The present recombinant antibodies are very useful in the human and veterinary field. It is contemplated that the present antibodies will be very suitable to detect the presence of or the amount of infectious agents in humans such as chlamydial or gonococcal organisms or infections with Human immunodeficiency virus (HIV), Epstein Barr virus (EBV), cytomegalovirus (CMV) or papillomavirus (HPV). The present antibodies are also useful in the general field of cytogenetics such as chromosome painting.

The invention also provides a kit containing a recombinant antibody according to the invention, which antibody might be in a detectably labelled form, a PNA sequence that is complementary to all or part of the nucleic acid sequence to be detected and a visualization system.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is an overview showing the PCR primers used to generate Fab gene frag¬ ments by PCR according to ørum et al., Nucleic Acids Research, Vol 21 , No 19, 4491-4498 (1993).

Figure 2 is an illustration of the construction of combinatorial Fab libraries using a "jumping-PCR assembly" method according to ørum et al., Nucleic Acids Research, Vol 21 , No 19, 4491-4498 (1993). The diagram exemplify the primary amplification PCR (A), the linker assembly (B) and the final assembly (C), respectively. Figure 3 shows titration of Fab-phages using different complexes of PNA and DNA as test antigens. OD ₉₀ values are given for different numbers of Fab-phages per well. (1.00E+0X is 1.00 x 10^0x). The complexes tested are described in Example 1.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

As used herein, the term "recombinant antibody " is intended to describe an antibody molecule produced by any process involving the use of recombinant DNA techno¬ logy, including any analogues of natural immunoglobulins or their fragments (G. Winter and C. Milstein, Nature 349, 293-299 (1991)), such as a Fab fragments, scFv fragment, chimaeric or reshaped antibodies, or light chain or heavy chain monomers.

The term "nucleic acid" covers a nucleotide polymer composed of subunits, which are either deoxyribonucleosides or ribonucleosides joined together by phosphodie- ster bonds. They may be DNA or various types of RNA. The terms "bases" and "nucleobases" are used interchangeably for pyrimidine and purine bases of nucleic acids and PNA.

The PNAs are synthesized according to the procedure described in "Improved Synthesis, Purification and Characterization of PNA Oligomers", Presented at the 3rd Solid-Phase Symposium, Oxford UK, Aug. 31-Sept. 4, 1994, or the PNAs were obtained from PerSeptive Biosystems (Framingham, MA, USA).

The PNA-nucleic acid complex used for immunization of an animal may suitable be a complex between PNA and DNA or between PNA and RNA. Since both nucleic acid and PNA are devoid of diversed peptide sources, both nucleic acid complexes and PNA nucleic acid complexes would be expected to be essentially non-immunogenic in normal host animals (i.e. animal which are not prone to generate auto-antibodies against nucleic acid) when injected per se. Whereas under these circumstances the conventional conjugation of the non-immunogenic antigen to a carrier foreign to the host animal generally has been found to be impractical and laborious and further¬ more induce a risk of creating structural changes in the antigen, an immune response towards nucleic acid complexes has been elicited by immunizing normal host animals with non-covalent, ionic complexes formed between the polyanionic nucleic acid complex and a poly-cationic protein derivative, particular a methylated albumin or globulin species (US 4,623,627, US 4,833,084).

It has now surprisingly been found that antibodies capable of binding to PNA/DNA complexes can be produced by immunizing a normal host animal with a mixture comprising a PNA/DNA complex and a non-derivatized protein heterologous to the host animal, such as ovalbumin, and recombinant DNA technology can be used for cloning of the antibody reactivity. This technology can also be applied to immuniza¬ tion with PNA/RNA complexes.

Recombinant antibodies have a number of advantages over the conventional mono¬ clonal antibody. Firstly, recombinant antibodies, or antibody fragments, are selected within a few weeks amongst 10⁶-10⁷ different reactivities, when using the phage display technology (McCafferty, J. et al., Nature, Vol 348, 552-554 (1990)). In com- parison monoclonal antibodies are seldom selected amongst more than 2-5 x 10³ different reactivities. Secondly, the selected reactivities are physically linked to the DNA encoding the given fragments giving rise to a very high degree of flexibility during further experiments. It is relatively easy to mutate selected reactivities and select new specificities with slightly different characteristics, and the technique gives the possibility of cloning different tags for recognition, signaling, coupling or purifi¬ cation purposes to an antibody fragment. Thirdly, different types of antibody frag¬ ments, e.g. Fab-fragments or single-chain fragments consisting of the two variable regions of the heavy and light chains (scFv-fragments), may be selected. The selected antibody fragments may be expressed directly, or modified and expressed for specific applications in different multimeric combinations or in combinations with different parts of the antibody molecule.

A PNA-DNA complex can be prepared by, in a suitable buffer, mixing double- stranded or single-stranded DNA with a PNA molecule having a base sequence that is complementary to all or part of the DNA sequence, heating the mixture to form single-stranded molecules and allowing the mixture to cool slowly to room tempera¬ ture. A PNA-RNA complex can be prepared by contacting RNA with a PNA molecule having a base sequence that is complementary to all or part of the RNA sequence, heating the mixture and allowing the mixture to cool slowly to room temperature. A suitable quantity of one of the PNA-nucleic acid complexes is mixed with an adjuvant and a carrier. Examples of suitable carriers are KLH (Keyhole Limpet Hemocyanin), ovalbumin and dextrans.

Recombinant antibodies as described herein may be constructed by the antibody- phage technologies described in McCafferty, J. et al., Nature, Vol 348, 552-554 (1990) and modified by ørum et al., Nucleic Acids Research, Vol 21 , No 19, 4491- 4498 (1993). In short the antibody-phage technology involves the following steps: an animal is immunized with a PNA/nucleic acid complex, such a PNA/DNA or PNA/- RNA complex, RNA is isolated from antibody producing cells and cDNA is prepared by reverse transcription. Heavy chain and light chain Fab gene segments are speci¬ fically PCR amplified from said DNA, the DNA is inserted into a phagemid which has the capability to express and display the antibody fragment at the surface of a fd- phage and which remains intact and infectious. Following infection of bacteria, the excreted phage carrying the antibody fragment of interest are isolated in a selection procedure (panning) and used for production of the antibody fragment. The DNA encoding antibody fragments may also be transferred to another prokaryotic or eukaryotic expression system for expression of whole antibody or the antibody fragment. One advantage of expression in a eukaryotic cell line is the possibility of producing whole antibodies more efficiently than in a prokaryotic expression system, and simultaneously obtaining secondary modifications of the antibody molecule produced.

In the present case the recombinant DNA encoding a recombinant antibody or a fragment thereof was prepared by a method comprising isolation of RNA from a spleen of a mouse immunized with a PNA/DNA complex, synthesizing first-strand cDNA from mRNA by priming with oligo (dT), the four deoxyribonucleoside triphos- phates and reverse transcriptase under standard conditions for cDNA synthesis, and by amplifying heavy chain and light chain Fab gene segments in a polymerase chain reaction (PCR) using oligonucleotide primers complementary to heavy chain and light chain Fab gene segments.

The PCR technology involves repeated rounds of extension from the oligonucleotide primers added allowing specific DNA regions to be amplified. A thermostable DNA polymerase isolated and purified from Thermus brockianus was used for amplifica- tion. The enzyme has a 5'→ 3' exonuclease activity and its error frequency is twofold lower than that of the commonly used Taq DNA polymerase. The PCR amplification products were purified by gelelectrophoresis.

The oligonucleotide primers used to generate Fab fragments by PCR are described in ørum et al., Nucleic Acids Research, Vol 21 , No 19, 4491-4498 (1993). Different mixtures of oligonucleotide primers are used (Figure 1). The primer mixture denoted MVH1-25 is, in combination with the mixture MCH1-G1 , G2A, G2B, able to amplify the region from the N-terminal end of the variable domain to the C-terminal end of the first constant domain of the heavy chain (the VH and CH1 regions). MVK1-25 is, in combination with a single primer, MCK1 , able to amplify the entire light chain (the VK and CK regions).

The boxes in Figure 1 delineate the different gene-segments included in the con¬ struction of the Fab expression-cassette: Fd-chain, includes the heavy chain from the N-terminal amino acid to the cysteine residue of the hinge region which forms the disulfide bridge to the C-terminal cysteine of the light chain; Light chain, corresponds to the entire variable and constant parts of the light (Kappa) chain. LINKER, designates a DNA fragment that is used for assembly of the amplified heavy and light chains, and which DNA comprises a translational stop codon for Fd translation, a ribosome binding site for L-chain expression and the coding region corresponding to the N-terminal part of the pelB leader sequence. The pelB leader sequence encodes a signal peptide for the transportation of the expressed Fab fragment to the periplasmic space of the bacteria. Primers depicted below the boxes are forward primers and complementary to mRNA. Primers above the boxes are back-primers and complementary to first strand cDNA.

The principle of PCR based amplification and assembly of heavy and light chain fragments is illustrated in Figure 2. Firstly, the Fd and L-chain gene fragments were amplified (the primary amplification, Figure 2A). Secondly, the LINK fragment was joined to the above fragments in two separate PCR reactions by submitting the fragments to a PCR protocol in the presence of primers complementary to the opposite ends of the two fragments (the LINKER assembly, Figure 2B). A similar strategy was used for the final assembly, this time using the linker-assembled Fd and L-chain fragments (Figure 2C). The gene-fragments corresponding to the heavy chain of the Fab fragment (Fd) and the L-chains were, through the final assembly via the DNA linker as illustrated in Figure 2C, paired randomly.

The amplified and assembled DNA was digested with Notl and Sfil and ligated to Notl and Sfil-cut phagemid pFABδc.His. Phagemid pFABδc.His is a modification of pFABδc described in ørum et al., Nucleic Acids Research, Vol 21 , No 19, 4491-4498 (1993) wherein a tail of 6 histidines has been added for simplifying the subsequent purification of recombinant antibodies. The phagemid pFABδc.His has been obtained from Prof. Jan Engberg, The Royal Danish School of Pharmacy, Copenhagen, Denmark. After ligation and purification, the DNA was electrophorated into an appropriate E. coli strain using a Bio-Rad E. coli pulser. Immediately after the pulse, freshly made SOC medium ( 2% Bacto Tryptone (Difco), 0.5% Bacto Yeast extract (Difco), 10 mM NaCl, 2.5 mM KCI, 1% glucose and 10 mM MgCI₂) were added and the cells were shaken for one hour at 37°C and plated on selective plates. Trans- formed cells were superinfected with helper phage and protein synthesis was induced by adding IPTG (isopropyl-β-D-thiogalactopyranoside). Cells were pelleted leaving phage particles displaying a large repertoire of different Fab fragments as fusion proteins to gill protein in the supernatant.

The panning procedure used for selection of antigen-binding clones were performed as follows: Streptavidin coated microtiter plates were incubated with biotinylated PNA/DNA complex and a phage library consisting of 10 ⁰-10¹¹ phages were taken through 3 to 4 rounds of antigen selection (panning) before supernatants or the periplasmic fraction of individually induced clones were tested for binding to a variety of PNA/DNA complexes and single-stranded PNA and DNA as well as RNA and DNA/RNA complexes.

DNA encoding Fab fragments as described herein has been deposited in the vector pFABδc.His at "Deutsche Sammlung von Mikroorganismen und Zellkulturen" (DSM 10051 and DSM 10052).

Whereas the above described Fab fragment comprises the heavy chain from the N- terminal amino acid to the cysteine residue of the hinge region which forms the disulphide bridge to the C-terminal cysteine of the light chain and the entire variable and constant parts of the light chain another preferred fragment of an antibody as described herein comprises only the variable parts of the heavy chain and the light chain linked by a spacer group, a so-called single-chain antibody (scFv). The spacer group is preferably a peptide spacer. Also, different multimeric constructs of the Fab fragment or of the scFv (diabodies) are contemplated by the invention.

Recombinant antibodies as described herein may also be obtained from large recombinatorial immunoglobulin libraries derived from non-immunized animals, e.g. by the methods described by Marks et al., Bio/Technology, Vol 10, 779-783 (1992), Griffiths et al., The EMBO Journal, Vol 12, No 2, 725-734 (1993), Waterhouse et al., Nucleic Acid Research, Vol 21 , No 9, 2265-2266 (1993) and Gram et al., Proc. Natl. Acad. Sci. USA, Vol 89, 3576-3580 (1992).

If needed, the specificity and/or affinity of selected recombinant antibodies might be increased by chain shuffling as described in the above identified publication by Marks et al. (1992) or by random mutagenesis as described by Gram et al. (1992) and Griffiths et al. (1993) in the above identified publications.

The present recombinant antibodies have a high degree of specificity for PNA- nucleic acid complexes. They do not to any significant degree bind to double- stranded nucleic acids, single-stranded PNAs or single-stranded nucleic acids. The specificity of the epitope(s) recognized by the present antibodies appears to a high degree to be dictated by the conformation of the PNA-nucleic acid complex rather than by the specific base sequence of the PNA /nucleic acid complex.

A high specificity and affinity of the present antibodies give significant advantages when used in the isolation, detection and quantitation of complexes formed between PNA and nucleic acids to be detected in a biological sample. Thus, antibodies with a high specificity for PNA/DNA complexes are particularly valuable in PNA based assays for identifying DNA of different infectious agents in humans such as chlamydial or gonococcial organisms. These antibodies are also very useful in the general field of cytogenetics such as chromosome painting.

Antibodies according to the invention having a high specificity and affinity for PNA/RNA complexes are particularly useful in PNA based assays, for example for identifying mRNA or rRNA sequences. Depending on the particular use, the antibody may be coupled with a detectable label such as enzymatically active groups like coenzymes, enzyme inhibitors and enzymes themselves, fluorescent labels, chromophores, luminescent labels, specifically bindable ligands such as biotin or haptens.

The antibodies described herein may also be cloned directly as a fusion protein to different proteins capable of being detected, e.g. horseradish peroxidase, glucose oxidase or alkaline phosphatase. Also, DNA fragments encoding different peptide tags capable of e.g. being recognized by another antibody, or useful to facilitate a directional coupling of the present antibody to other molecules may be cloned directly to the DNA encoding the antibody reactivity.

The present antibodies are valuable tools in a number of different methods for detecting a PNA/nucleic acid complex formed between a particular nucleic acid sequence to be detected in a test sample and PNA capable of forming a complex with said particular nucleic acid sequence.

A method for detecting a particular nucleic acid sequence in a sample using the antibodies described herein may comprise the steps of

(a) forming a complex between the particular nucleic acid sequen¬ ce to be detected in the sample and a PNA having a base sequence that is sufficiently complementary to the base sequence of the nucleic acid sequence to be detected so as to form complexes, said complexes having at least one epitope for an antibody as described herein,

(b) contacting any complex that is formed between the PNA sequ- ence and the nucleic acid sequence to be detected with an antibody as described herein, and

(c) determining the presence of antibody-PNA-nucleic acid complexes. The PNA sequence may suitable be immobilized onto a solid support prior to the contact with the sample containing the nucleic acid sequence to be detected, or the antibody may be immobilized onto a solid support prior to contact with the PNA- nucleic acid complex.

If the nucleic acid sequences to be detected are present in an immobilized state in a biological specimen, a method may be used which comprises the steps of

(a) forming a complex between the particular nucleic acid sequen- ce to be detected in the specimen and a PNA having a base sequence that is sufficiently complementary to the base sequence of the nucleic acid sequence to be detected so as to form complexes, said complexes having at least one epitope for an antibody as described herein,

(b) contacting any complex that is formed between the PNA sequence and the nucleic acid sequence to be detected with an antibody as described herein, and

(c) determining the presence of antibody-PNA-nucleic acid complexes.

In a method wherein the initial step is an immobilization of the nucleic acid sequence to be detected, the method suitably comprises the steps of

(a) immobilizing the nucleic acid sequence to be detected to a solid support,

(b) forming a complex between the particular nucleic acid sequen- ce to be detected in the sample and a PNA having a base sequence that is sufficiently complementary to the base sequence of the nucleic acid sequence to be detected so as to form complexes, said complexes having at least one epitope for an antibody as described herein, (c) contacting any complex that is formed between the PNA sequence and the nucleic acid sequence to be detected with an antibody as described herein, and

(d) determining the presence of antibody-PNA-nucleic acid complexes.

Examples of applications of the present antibodies are described below.

A kit for carrying out the described methods or other methods using the present antibodies may in addition to the present antibody in labelled or unlabelled form contain a PNA sequence that is complementary to all or part of the nucleotide sequence to be detected and a visualisation system. The visualisation system may comprise an enzyme-conjugate (e.g. an enzyme conjugated antibody or an enzyme conjugated streptavidin) and a suitable substrate. The conjugate may have a reactivity to mouse immunoglobulin epitopes in cases where the unlabelled form of the present antibody is used or to hapten groups such as biotin, fluorescein or peptide in cases where the present antibody has been labelled with hapten groups. The substrate system of the kit may be selected to form a soluble coloured reaction product in cases where the PNA/nucleic acid complex is measured in an ELISA format or the substrate system may be selected to form an insoluble coloured reaction product in cases where the PNA/nucleic acid complex is measured in a biological sample or on a membrane.

Application 1 : Hybridization and detection in solution

A nucleic acid sequence of interest can be determined in solution by contacting the sample containing the nucleic acid with a PNA having a base sequence that is sufficiently complementary to the base sequence of the nucleic acid of interest so as to form complexes followed by contact with an antibody as described herein, recognising the PNA-nucleic acid complexes but not free PNA or nucleic acids. These reactions will result in a large complex which may be detected e.g. in a turbidimetric assay format.

Application 2: Solution hybridization and detection after immobilization A nucleic acid sequence of interest can be determined by contacting it with a PNA having a base sequence that is sufficiently complementary to the base sequence of the nucleic acid of interest so as to form complexes. The formed complexes are, while still in solution, contacted with a labelled antibody as described herein. The formed PNA-nucleic acid-antibody complex is then captured using an antibody as described herein which e.g. has been immobilized onto a solid support. Unbound materials are washed off and the amount of bound PNA-nucleic acid-antibody complex is determined via detection of the label on the antibody.

Alternatively, the PNAs having a base sequence that is sufficiently complementary to the base sequence of the nucleic acid of interest so as to form complexes may carry a label, e.g. biotin, a fluorescent label, or other moieties which are suitable for catching of PNA-nucleic acid complexes. Unbound materials is washed off and the amount of bound PNA-nucleic acid-antibody complex is determined either via detec- tion of the label on the antibody or by using a secondary antibody detection system.

Application 3: Capture assay

A traditional capture assay comprises the steps: recognition, capture and detection and may be composed in various ways. One example of such assay is described below.

An antibody capable of binding to a PNA-nucleic acid complex is immobilized onto a solid support, e.g. onto an ELISA plate. PNA and sample are mixed and allowed to react in solution in the wells of the ELISA-plate. If complexes between the PNA and the sample nucleic acids are formed, these complexes will be captured by the immo¬ bilized antibody. Unbound materials are washed off and the amount of bound PNA- nucleic acid-antibody complex is determined. The capture may also be based on other recognisable moieties than a PNA-nucleic acid complex. Such moieties could e.g. be biotinylated PNAs or PNAs labelled with other haptens, peptides, or poly- peptides.

In some assay formats, two or more of the steps indicated above may be performed simultaneously. Application 4: Detection of PNA/nucleic acid complexes immobilized onto a solid support

Complexes formed between PNAs and nucleic acids in which either the PNA or the nucleic acid initially was immobilized onto a solid support can be detected by the antibody described herein. This detection can be performed either directly using such an antibody conjugated to an enzyme, a fluorescent marker or another signal generating system, or indirectly using one of the secondary visualisation systems commonly used for detecting antibodies bound to a target. The solid support con¬ sidered should be understood in a very broad sense like e.g. nylon or nitrocellulose membranes (Southern or Northern blots), a tissue section, cell smears, cytospins or chromosome spreads (in situ hybridization), or a plastic surface (an ELISA format).

This system has the advantage that the normally very extensive washing procedures included in these technologies can be significantly reduced since non-specifically bound PNAs, being single-stranded, will not give rise to a signal as the antibody only recognises PNA forming complexes with nucleic acids.

Application 5: Biosensor systems

Detection and quantification of nucleic acids in a biological sample may be performed using a biosensor system such as the BIAcore biosensor system from Phamacia. The interaction of biomolecules with an immobilized ligand on a sensor chip is measured at the surface using evanescent light. The system includes a sensor chip to which the ligand can be immobilized in a hydrophilic dextran matrix, a miniaturised fluids cartridge for the transport of analytes and reagents to the sensor surface, a SPR (surface plasmon resonance) detector, an autosampler and system control and evaluation software. Specific ligands are covalently immobilized to the sensor chip through amine, thiol or aldehyde chemistry or biospecifically by e.g. biotin - avidin interaction.

The antibody as described herein may be coupled to a sensor chip of the biosensor- system used, e.g. to a dextran layer of a sensor chip in a BIAcore system. A sample is mixed with PNA and incubated so that a complex is formed between the nucleic acid in the sample and PNA having a base sequence that is sufficiently complemen¬ tary to the base sequence of the nucleic acid of interest so as to form complexes. The sample is passed through the flow system of the biosensor system and the antibody coupled to the sensor chip will bind specifically to the PNA-nucleic acid complexes if such complexes have been formed. Based on the SPR detection employed by the biosensor system, this binding will generate a signal depending on the amount of materials bound to the surface.

Application 6: Detection of bound PNA in cells

Under suitable conditions, PNAs may be able to penetrate the cell-wall of living or fixed cells, e.g. cell-lines, hemopoetic cells, and animal/human tissues (important in therapeutic applications). It may be important to be able to detect PNAs that have hybridized to different targets in the individual cells. In such cases, labelling of the PNAs with haptens or other reporter molecules may not be advantageous as this may inhibit or interfere with the penetration of the PNAs into the cells. The detection of PNAs hybridizing to a target by either immunohistochemistry (in frozen or fixed tissue biopsies) or by Flow cytometry (e.g. on cells treated with detergent, acetone or alcohol) are important. It is also advantageous to be able to detect binding and/or tissue distribution of PNA's added to a cell culture or administered to a living animal. Such detection is made possible with an antibody provided as described herein.

In the following examples, specific embodiments of the present invention are given. These examples are not intended to limit the invention in any way.

EXAMPLES

EXAMPLE 1

PNA nucleic acid complexes and single chains

PNAs comprising polymerized N-(2-aminoethyl)glycine units to which nucleobases are attached through a methylenecarbonyl linker, were synthesized and purified as described in "Improved Synthesis, Purification and characterization of PNA Oligo- mers", presented at the 3rd Solid-Phase Symposium, Oxford UK, Aug. 31 -Sept. 4, 1994, and by M. Egholm et al., J. Am. Chem. Soc. 114, 1895-1897 (1992) and M. Egholm et al., J. Chem. Soc. chem. Commun. 800-801 (1993), or such PNAs were obtained from PerSeptive Biosystems. The base sequence of the PNA used is pre¬ ferably virtually non-self-complementary in order to avoid self-hybridization in the PNA molecule. The number of purines and pyrimidines is approximately equal to allow formation of a double helix configuration rather than a triple helix configuration.

DNA sequences were synthesized on an abi 381 A DNA synthesizer from Applied Biosystems using a standard 381A cycle/procedure. The monomers used were standard β-cyanoethyl phosphoamidites for Applied Biosystems Synthesizer.

RNA sequences were purchased from "DNA Technology Aps, Science Park Aarhus, Gustav Wieds Vej 10, DK-8000 Aarhus.

The PNA and DNA sequences may be labelled or unlabelled and may optionally contain one or more linker units, preferably one or two linker units wherein the two linker units are attached end to end. Linkers are in all cases written as "-link-" independently of it being labelled PNA or DNA oligomers or the number of linker units added.

PNA oligomers can be labelled with biotin in the following way: a linker comprising one or two units of 2-(aminoethoxy)ethoxy acetic acid (AEEA) is attached to the PNA oligomer on the resin (see above), and biotin is attached in the following way. Two solutions were used. The first solution contained 0.1 M biotin in 5% s-collidin in DMF with 0.2 M of N-ethyldicyclohexylamine and the second solution contained 0.18 M HBTU (2-(1H-benzotriazol-1-yl)-1 ,1 ,3,3-tetramethyluronium hexafluorophosphate) in DMF. The two solutions were mixed in a ratio of 2 to 1 and the mixture was left for approximately one minute before it was combined with the resin to which the PNA oligomer with one or two units of AEEA were attached.

For biotin labeling of DNA the following two procedures can be used. For labeling in the 5' end of the DNA oligomers a linker (Spacer phosphoramidite, Clontech Laboratories) was connected to the 5'-OH of the oligomer and then reacted with a biotin labeling reagent (Biotin CE phosphoramidite,22-0001-35, Cruachem Limited). For labeling in the 3' end of the oligomers the DNA synthesis was started from a Biotin-CPG support (3'-Biotin-ON CPG cat # RP-5225-2 K. J. Ross Petersen, Agern Alle 3, DK-2970 Hørsholm). The first reagent was a linker (Spacer phosphoamidite, Clontech Laboratories, Inc.) and the monomer reagents were added for synthesizing the oligomer. All PNA sequences are written from the amino-terminal end which is denoted "H-" (corresponding to the 5'-end in DNA) to the C-terminal end which is denoted "CONH₂" (corresponding to the 3'-end in DNA). All DNA sequences are written from the 5'-end to the 3'-end. The following test complexes/compounds were used:

H12. An unlabelled PNA DNA complex (the immunogen) comprising a 45-mer DNA sequence (U1) and 3 units of a 15-mer PNA sequence (U2). The base sequence of the 45-mer DNA (U1) was as follows: 5'-GCA AAT GCT CTA GGC GCA AAT GCT CTA GGC GCA AAT GCT

CTA GGC-3'.

The base sequence of the 15-mer PNA (U2) was as follows: H-GCC TAG AGC ATT TGC-CONH₂

L2. A 45-mer DNA sequence (L2) with a biotin attached to the 3'-end of the

DNA.

The base sequence of the 45-mer DNA (L2) was as follows: 5'-GCA AAT GCT CTA GGC GCA AAT GCT CTA GGC GCA AAT GCT CTA GGC-link-Bio-3'.

H2. A PNA/DNA complex comprising a 45-mer DNA sequence (L2) and 3 units of a 15-mer PNA sequence (U2) wherein biotin is attached to the 3'-end of the 45-mer DNA. Apart from the biotin moiety coupled to the DNA sequence, this complex corresponds to the complex used for immunization. The base sequence of the 45-mer DNA (L2) was as follows:

5'-GCA AAT GCT CTA GGC GCA AAT GCT CTA GGC GCA AAT GCT CTA GGC-link-Bio-3'.

The base sequence of the 15-mer PNA (U2) was as follows: H-GCC TAG AGC ATT TGC-CONH₂

H3. A PNA/DNA complex comprising a 15-mer DNA sequence (L3) and a 15- mer PNA sequence (U2) wherein biotin is attached to the 5'-end of the 15- mer DNA. The base sequence of this 15-mer DNA (L3) was as follows: 5'-Bio-link-GCA AAT GCT CTA GGC-3' The base sequence of the 15-mer PNA (U2) was as follows: H-GCC TAG AGC ATT TGC-CONH₂

H20. A large DNA/DNA complex resembling the immunogen and consisting of a 45-mer DNA sequence (L2), labelled with biotin at the 3'-end, and 3 units of a 15-mer DNA sequence (U4). The sequences of the two chains in this complex is given above (in relation to the description of H2 and H4).

H7. A PNA/DNA complex comprising a 20-mer PNA sequence and a 20-mer DNA sequence having a base sequence that are different from the sequen¬ ce of the complex used for immunization and wherein the PNA sequence is labelled with biotin at the amino-terminal end. The base sequence of the 20-mer PNA (L5) was as follows: Bio-link-CGG CCG CCG ATA TTG GCA AC-CONH₂ The base sequence of this 20-mer DNA (U6) was as follows:

5'-GTT GCC AAT ATC GGC GGC CG-3'

H8. A PNA/DNA complex comprising a 17-mer PNA sequence and a 17-mer

DNA sequence wherein the base sequence is different from the complexes previous described, but related to the complex H9 described below. The

DNA strand of this complex is labelled with biotin at the 5'-end The base sequence of the 17-mer DNA (L6) was as follows 5'-Bio-link-ATT GTT TCG GCA ATT GT-3' The base sequence of the 17-mer PNA (U7) was as follows. H-link-ACA ATT GCC GAA ACA AT-CONH₂

H9. A PNA/DNA complex comprising a 17-mer PNA sequence and a 17-mer

DNA sequence wherein the base sequence is different from the complexes previous described, but related to the complex H8 described above. The PNA strand of this complex is labelled with biotin at the amino-terminal end.

The base sequence of the 17-mer DNA (U8) was as follows: 5'-ATT GTT TCG GCA ATT GT-3'

The base sequence of the 17-mer PNA (L7) was as follows: Bio-link-ACA ATT GCC GAA ACA AT-CONH₂ L8. A 19-mer PNA sequence labelled with biotin at the amino-terminal end. The base sequence of the 19-mer PNA (L8) was as follows: Bio-link-TTC AAC TCT GTG AGT TGA A-CONH₂

H10. A PNA/RNA complex comprising a 19-mer PNA sequence (L8) and a 19- mer RNA sequence (U9) with a base sequence complementary to the PNA base sequence. The PNA strand of this complex is labelled with biotin at the amino-terminal end.

The base sequence of the 19-mer PNA (L8) was as follows: Bio-link-TTC AAC TCT GTG AGT TGA A-CONH₂

The base sequence of the 19-mer RNA (U9) was as follows: 5'-UUC AAC UCA CAG AGU UGA A-3'

H22. A PNA/DNA complex comprising a 19-mer PNA sequence (L8) and a 19- mer DNA sequence (U26) with a base sequence complementary to the PNA base sequence. The PNA strand of this complex is labelled with biotin at the amino-terminal end.

The base sequence of the 19-mer PNA (L8) was as follows:

Bio-link-TTC AAC TCT GTG AGT TGA A-CONH₂ The base sequence of the 19-mer DNA (U26) was as follows:

5'-TTC AAC TCA CAG AGT TGA A-3'

H29. A PNA/DNA complex comprising a 15-mer PNA sequence (U27) and a 30- mer DNA sequence (L11). The 30-mer DNA sequence is labelled with biotin in the 3'-end. The PNA sequence is complementary to the central part of the

DNA resulting in single stranded DNA overhangs both 5'- and 3'- to the PNA/DNA complex.

The base sequence of the 30-mer DNA (L11 ) was as follows: 5'-GCT GAC GTT CCG CAC ATG TCA ACC ATA TGT-link-Bio-3^* The base sequence of the 15-mer PNA (U27) was as follows:

H-link-GTT GAC ATG TGC GGA-CONH₂

H30. A PNA/DNA complex comprising a 45-mer DNA sequence (L12) and 3 units of a 15-mer PNA sequence (U13) wherein biotin is attached to the 5'-end of the 45-mer DNA sequence. The base sequence of the 45-mer DNA (L12) was as follows: Bio-link-TCC GCA CAT GTC AAC TCC GCA CAT GTC AAC TCC GCA CAT GTC AAC-3'.

The base sequence of the 15-mer PNA (U13) was as follows: H-GTT GAC ATG TGC GGA-CONH₂.

H31. A PNA/DNA complex comprising a 45-mer DNA sequence (L13) and 3 units of a 15-mer PNA sequence (U13) wherein biotin is attached to the 3'-end of the 45-mer DNA sequence. The base sequence of L13 is identical to the base sequence of L12 above.

Thus the sequence of L13 is as follows:

5-TCCGCACATGTCAACTCCGCACATGTCAACTCCGCACAT

GTCAAC-link-Bio-3'.

The sequence of the 15-mer PNA (U13) was as follows: H-GTT GAC ATG TGC GGA-CONH₂.

H32 A PNA/PNA complex (dsPNA) comprising two 17-mer PNA sequences, L7 and U28, wherein biotin is attached to the 5'-end of L7. The base sequence of L7 is as follows: Bio-link-ACA ATT GCC GAA ACA AT-CONH₂

The base sequence of U28 is as follows: H-ATT GTT TCG GCA ATT GT- CONH₂

The PNA/nucleic acid complexes are prepared by, in a suitable buffer (e.g. 50 mM Tris-HCI, pH 7.6, 50 M NaCl), mixing the nucleic acid with PNA having a base sequence that is complementary to all or a part of the nucleic acid sequence, heating the mixture to form single-stranded molecules and allowing the mixture to cool slowly to room temperature.

Complex formation was characterized as described below.

T_rn determinations: T_m measurements of PNA/DNA and PNA/RNA complexes were performed in a Lambda 2S UV/VIS spectrometer (Perkin Elmer) equipped with a "cell holder" with a heating facility (Peltier heating element). For the preparation of a final amount of 2 nmol complex in 3 mL 50 mM Tris-HCI, pH 7.6, 50 mM NaCl buffer, a suitable amount of each strand and the buffer are mixed in a 3.6 L NUNC CryoTube. The mixture is heated to 95°C for 10 minutes and the allowed to cool slowly to room temperature (3 to 4 hours). Approximately 2.8 mL is transferred to a 3 mL quartz cuvette with a lid and a stirring magnet. The tempera¬ ture of the cuvette is increased at a speed of 0.2 °C/minute, starting at 20°C and ending at 95°C. Absorbance at 260 nm is measured continuously. The T_m value is determined as the top point of the first derivative of the melting curve.

Acrylamide gel electrophoresis: The complex formation was also tested by running the complexes in a 20 % polyacrylamide gel in TBE buffer (89 mM Tris-borate, 2 mM EDTA). The complexes were transferred to Nytran 13N filter paper (Schleicher & Schuell). Complexes were visualised in accordance with the label of the complex. Complexes containing either a biotin or a fluorescein label were visualised using alkaline phosphatase (AP) conjugated streptavidin or anti fluorescein antibody, respectively. Unlabelled complexes were visualised either directly in the poly¬ acrylamide gel by staining with ethidium bromide or by use of a polyclonal PNA - nucleic acid antibody as described in WO 95/17430 followed by a secondary antibody, e.g. swine anti-rabbit/AP. Bound AP conjugates were visualised using the chromagen mixture NBT/BCIP.

Dot blot: A dilution row of a complex (from 20 ng to 2 ng per dot) was spotted onto a Nytran 13N filter paper (Schleicher & Schuell). Visualisation were performed as described above.

EXAMPLE 2

Immunization

The antigen used for immunization (PNA/DNA complex H12) was prepared by mixing the following in a total volume of 2 mL:

0.939 mg 45-mer polydeoxyribonucleotide (DNA) 1.145 mg 15-mer PNA oligomer 50 mM Tris-HCI, pH 7.5 50 mM NaCl This mixture was heated to 92°C in a heating block and allowed to cool slowly to room temperature. Final concentration of PNA/DNA complex was 1.04 mg/mL.

Complex formation was tested as described in Example 1.

To the PNA/DNA complex at a concentration of 1.04 mg/mL Govalbumin was added to a concentration of 250 mg/mL. This mixture was further mixed with Freunds incomplete adjuvant at a ratio of 1 :1 v/v and was used for immunizing female BALB/c mice intraperitoneally or subcutaneously five times with an interval of approximately three weeks.

EXAMPLE 3

Isolation of RNA. amplification and cloning of Fab fragment encoding DNA Isolation of RNA

The spleen from the above mentioned immunized mouse was isolated, and immediately transferred to 10 mL 4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarcosyl, 0.1 M β-mercapto ethanol (all reagents from Sigma). All solutions were kept on ice at all times. Ten minutes later the spleen was homogenized in a Polytron homogenizer at full speed for about 10 seconds.

Subsequently, 1 mL 2 M sodium acetate, pH 4.0, and 10 mL phenol/chloroform/- isoamylalcoho! (125:24:1), pH 4.7 was added to the homogenate, and mixed vigorously. The mixture was centrifuged at 10000 rpm (JA20 rotor, Beckman centrifuge) for 20 minutes at 4°C.

The supernatant containing RNA was transferred to a fresh tube, and one volume of phenol/chloroform/isoamylalcohol (125:24:1), pH 4.7, was added, mixed vigorously and centrifuged as before. The supernatant was transferred to a fresh tube, and the remaining organic phase was back-extracted with 7.5 mL 4 M guanidinium thio- cyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarcosyl, 0.1 M β-mercapto ethanol, 0.75 mL 2 M sodium acetate, pH 4.0. The sample was mixed and centrifuged as previously mentioned. The RNA containing supernatants were pooled. The total volume was 12.5 mL, and RNA was precipitated with 1 volume isopropyl alcohol at -20°C for a minimum of 30 minutes. RNA was collected by centrifugation at 12000 rpm for 30 minutes at 4°C.

The pellet was resuspended in 0.5 mL 4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarcosyl, 0.1 M β-mercapto ethanol to dissolve the RNA, and the solution was transferred to an eppendorf tube, and subsequently added 50 μl 2 M sodium acetate, pH 4.0 and 0.5 mL phenol/chloroform/isoamylalcohol (125:24:1 ), pH 4.7. After vigorously mixing the sample was centrifuged in a microfuge at full speed for 5 minutes. The supernatant was transferred to a fresh tube, and the RNA was precipitated with 1 volume isopropyl alcohol for 30 minutes at -20°C. RNA was collected by centrifugation in a microfuge for 30 minutes at full speed at 4°C. The RNA was extracted and precipitated once more as described above.

The pellet was resuspended in 0.7 mL DEPC (diethylpyrocarbonate) treated H₂0, and 70 μl 2 M sodium acetate, pH 4.0 and 0.7 mL phenol/chloroform/isoamylalcohol (125:24:1), pH 4.7 was added. Vigorously mixing was followed by 5 minutes centri¬ fugation in a microfuge. The supernatant was transferred to a fresh tube, and RNA precipitated with 1 volume isopropyl alcohol for 30 minutes at -20°C, followed by a 30 minutes centrifugation at full speed at 4°C. The pellet was washed with 80% ethanol, and the RNA pellet was subsequently dried briefly in a SpeedVac. RNA was dissolved in DEPC-treated H₂O, and stored frozen at -70°C. The yield was app. 187 μg total cellular RNA as estimated by the optical density at 260 nm.

cDNA synthesis 50 μg total RNA and 1.25 μg oligo(dT)₁₈ were mixed and denatured by a 10 minutes incubation at 70°C, followed by a brief cooling on ice. Reverse transcription was performed with SuperScript™ll (Gibco BRL) under the following assay conditions:

50 mM Tris-HCI, pH 8.3 at room temperature 75 mM KCI

10 mM DTT 3 mM MgCI₂ 0.5 mM each dATP, dCTP, dGTP, dTTP Denatured RNA was added to the reaction tube, and the 50 μl reaction was incu¬ bated 3 minutes at 45°C before adding 600 U of SuperScript™ll RNaseH^" Reverse Transcriptase. Incubation at 45°C was continued for 1 hour. cDNA was then preci¬ pitated according to standard procedures, and finally resuspended in 15 μl H₂O, and stored frozen at -20°C.

PCR amplification

Fd and Kappa chains were initially amplified by PCR in separate reactions, followed by an assembly PCR reaction essentially as described by ørum et al. (Nucleic Acids Reseach, Vol 21 , No 19, 4491-4498 (1993).

DyNAzyme thermostable DNA polymerase (Finnzymes Oy, Finland) was used under the following assay conditions:

10 mM Tris-HCI (pH 8.8 at 25°C)

1.5 mM MgCI₂

50 mM KCI

0.1% Triton X-100

0.1 mM each dATP, dCTP, dGTP, dTTP 0.2 μM forward primers (see figure 1)

0.2 μM back primers (see figure 1)

Half of the prepared cDNA was used as template for amplification of Fd chains, the other half was used for amplification of Kappa chains. A total of three 50 μl reactions was performed per chain. cDNA was added to the reaction tubes, and after an initial incubation (denaturation) at 94°C, 1 U DyNAzyme was added per 50 μl reaction. The reactions were cycled 25 times (denaturation at 94°C - 1 minute; annealing at 55°C - 1 minute; and elongation at 72°C - 1 minute) using a DNA Thermal Cycler (Perkin Elmer Cetus).

Amplified material was precipitated and gelpurified using JetSorp (Genomed) according to the manufacturers instructions.

The Kappa and Fd chain libraries were in separate PCR reactions (step B, figure 2) linked to a specific fragment (LINK in figure 1 and 2) in order to facilitate a final assembly of the two libraries in a final assembly PCR reaction and cloning of the complete repertoire in one step. The reactions were performed under the same conditions as mentioned above, except the primer concentrations in these reactions were 0.25 μM for both primers. Template DNA was for the Kappa reaction 5 ng purified primary Kappa PCR product and 7 ng link fragment per 100 μl PCR reaction. To the Fd reaction was added 10 ng purified primary Fd PCR product and 14 ng link fragment per 100 μl PCR reaction. After initial denaturation at 94°C for 5 minutes, 1.5 U DyNAzyme/100 μl reaction was added, and subsequently cycled 25 times (Fd) or 21 times (Kappa) at (94°C - 1 minute; 65°C - 1 minute; 72°C - 1 minute).

Amplified material was precipitated and gelpurified using JetSorp (Genomed) according to the manufacturer's instructions.

The two separate, extended chains were then assembled by PCR in a final reaction (step C, figure 2), employing identical assay conditions as in the primary reactions, except the primer concentration was 5 μM, and as template DNA was added approx. 4 ng of purified, extended Kappa chain and Fd chain, respectively, per 100 μl reaction. After an initial denaturation at 94°C for 5 minutes, 1 U DyNAzyme was added/100 μl reaction, and cycling was performed 25 times at (94°C - 1.5 minutes; 69°C - 1 minute; 72°C - 2 minutes.

Amplified material was precipitated and gelpurified using JetSorp (Genomed) according to the manufacturer's instructions.

Cloning

Amplified material was restricted with Sfil and Notl restriction enzymes according to the manufacturers instructions (Boehringer Mannheim), and subsequently gelpurified using JetSorp (Genomed).

A suitable vector for cloning should comprise an origin of replication, such as f1 , marker sequence(s) for selection, such as ampicillin resistance, expression control sequence(s), such as promotors etc, and signal sequence(s) to guide the expressed protein to the periplasmic space of the bacteria, such as a pelB leader. Furthermore numerous restrictions sites for flexible incorporation of sequences are required. A suitable vector is pFAB5c.His. Phagemid pFABδc.His is a modification of pFAB5c described in ørum et al., Nucleic Acids Research, Vol 21 , No 19, 4491-4498 (1993) wherein a tail of 6 histidines has been added for simplifying the subsequent purification of recombinant antibodies. The phagemid pFABδc.His has been obtained from Prof. Jan Engberg, The Royal Danish School of Pharmacy, Copenhagen, Denmark.

The expression vector used can be reconstructed by an Eagl digestion. With this treatment, Δglll is removed from the vector, and the Fab fragment can be expressed as a free Fab; i.e. not fused to the phage gill protein. By this treatment, the ability to display the Fab fragment on the surface of a phage is also lost.

The vector pFABδc.His used here was treated with the same enzymes as the amplified material, Sfil and Notl, and gelpurified using JetSorp.

Approx. 400 ng digested and purified vector was ligated to 400 ng digested and purified PCR amplified material using 4 units of T4 DNA ligase form Boehringer Mannheim in a total of 100 μl for 3 hours at room temperature under the following conditions:

66 mM Tris-HCI 5 mM MgCI₂ 1 mM DTT 1 mM ATP pH 7.5 (20°C)

This reaction was performed in duplicate.

Post ligation, the mixture was extracted once with phenol/chloroform/isoamylalcohol (25:24:1), followed by a chloroform extraction of the aqueous phase. Finally, the

DNA was precipitated with ethanol using standard conditions. The pellet was washed twice with 70% ethanol, and after brief drying in SpeedVac, the DNA was resuspended in 15 μl H₂O.

EXAMPLE 4 Transformation of E. coli

The DNA obtained in example 3 was introduced to electrocompetent E.coli cells (TOPIOF'/lnvitrogen) in 1μl portions using BioRad E. coli Pulser, and 0.1 cm electro- poration cuvettes (BioRad) employing a field strength of 18 kV/cm. Immediately after pulsing, the cuvette was flushed twice with freshly prepared 1 mL SOC medium, and the cells were shaken for 1 hour at 37°C. The size of the library was estimated by plating appropriate dilutions on selective plates, before transferring the electro- porated cells to LB medium supplemented with 100 μg/mL ampicillin and 0.5% glucose. Shaking at 37°C was continued for app. 8 hours (OD₆₀o approx. 1.5), before a culture for superinfection with helper phage was started, and plasmid DNA was prepared from the remaining part of the culture (Qiagen plasmid preparation).

Features of host cell, E. coli: Top10F':

F' {/acl^q 7n10 (Tet^R)} mcrA A(mrr-hsdRMS-mcrBC) θ80/acZΔM15 Δ/acX74 deoR recA1 araD139 Δ(ara-/eu)7697 ga/U galK rps endA1 nupG.

EXAMPLE 5

Phage display of Fab fragment and selection of antigen binding phages (panning) 50 mL LB supplemented with 100 μg/mL ampicillin and 12 μg/mL tetracycline was inoculated with 1-5 x 10⁸ transformed cells, and shaken at 37°C to an OD₆₀o of approx. 0.5. R408 helper phage (Stratagene) was added at a multiplicity of δO-100, and infection proceeded at slow shaking for 20 minutes at 37°C. Subsequently, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 100 μM, and incubation was performed overnight at room temperature with shaking at approx. 250 rpm.

Following pelleting of the cells, phages were precipitated for 1 hour on ice with 4% PEG₆ooo and 0.5 M NaCl, and finally the phages were collected by centrifugation for 30 minutes at 4°C in a JA20 rotor/Beckman centrifuge at 20,000 rpm. The pellet was resuspended in approx. 1 mL supernatant and recentrifuged at full speed in a micro¬ fuge for 30 minutes at 4°C . Finally, the phages were resuspended in 400 μl PBS (137 mM NaCl, 2.7 mM KCI, 4.3 mM Na₂HPO₄-7H₂O, 1.4 mM KH₂PO₄, pH 7.3), followed by a clearing spin before transferring the phages to a fresh tube. The freshly prepared phages were used immediately for subsequent procedures: titration and panning.

Titration was done by infecting 50 μl of exponentially growing Eco//TOP10F' with 1 μl phages from appropriate dilutions, and after 20 minutes incubation at 37°C, the bacteria were transferred to selective agar plates. Phage titers were calculated from colony numbers the following day.

Panning: Microtiter plates (Maxisorp, NUNC) coated with streptavidin were incubated for a minimum of 2 hours with 50 ng of biotinylated PNA/DNA complexes (H2) at 37°C or overnight at 4°C. PNA/DNA complexes were diluted in THT (50 mM Tris- HCI, pH 7.2, 0.1 M NaCl, 0.1% Tween 20). Following binding of PNA/DNA com¬ plexes, excess binding sites in the microtiter wells were blocked with 4% dehydrated skim milk (Difco Laboratories) in PBS (PBSM) for approx. 2 hours at 37°C. Finally, the wells were washed 3 - 5 times with 0.5% Tween 20 in PBS (PBST). 10¹⁰-10¹¹ phages (50-7δ μl of the prepared phages) were supplemented with 1 μg/100 μl streptavidin and subsequently added to the microtiter well, and incubated at 37°C on a rocking table for 1 /4 - 2 hours. A varying number of washes were performed with 300 μl PBST (3 times for first round of panning; 5 - 10 times for subsequent rounds). Bound phages were eluted with 100 μl 0.1% trypsin (DAKO S2012) in PBS for 30 minutes at room temperature. Eluates were added to 2 mL exponentially growing E.coli, TOP10F', and after a 20 minute incubation at 37°C, eluted phages were titrated by spreading appropriate dilutions on selective agar plates. The remaining cells were propagated by an overnight incubation with shaking at 37°C in 50 mL LB medium, supplemented with 100 μg/mL ampicillin and 0.5% glucose. Next day, the titers of eluted phages were estimated, and a 50 mL culture for superinfection were started as described in this example, and phages for the next round of panning were prepared as previously described.

The panning procedure was performed 4 times, followed by a colony screening in order to isolate single colonies with the desired binding specificities. Single colonies were picked and inoculated in 5 mL LB medium supplemented with 100 μg/mL ampicillin, and shaken at 37°C to an OD₆₀₀ of 0.8-1. Then IPTG was added to a final concentration of 1 mM, and incubation was continued at room temperature over- night. Cells were then pelleted, resuspended in PBS, and taken through 4 cycles of freezing in dry ice/ethanol and thawing in a waterbath at room temperature. After centrifugation, the supernatants was analyzed in ELISA.

The following clones were obtained : Mouse anti PNA/DNA clone 5, mouse anti PNA/DNA clone 6 and mouse anti PNA/DNA clone 16.

EXAMPLE 6

Analysis of selected positive clones ELISA results with Fab fragments

Microtiter plates (Maxisorp, Nunc) coated with streptavidin were incubated with biotinylated PNA/DNA complexes, at a concentration of 100 ng/mL in THT, for a minimum of 2 hours at 37°C or overnight at 4°C. Following binding of PNA DNA complexes, the plates were washed in THT buffer and non-specific binding to the wells was blocked with 4% dehydrated skim milk in PBS (PBSM) for approx. 1 /4-2 hours. After washing with THT (Denley WeWash 4) recombinant Fab fragments were added to separate wells, and incubated as a standard for 2 hours on a rocking table at 37°C. Detection of binding fragments was performed with HRP conjugated goat- anti-mouse IgG (DAKO) and OPD (DAKO) according to manufactures instructions. Detection reactions were stopped after 1 δ-30 minutes with 1 M H₂SO₄, and finally plates were read at OD₄₉₀ (Molecular Devices). All washings between different steps were done with THT (Denley WeWash 4).

Fab fragments produced from the initially isolated clones were further analyzed against a variety of PNA/nucleic acid complexes according to Table 1. The results are shown as OD ₉₀ values and the background, which as an average was below 0.1 , has not been subtracted. TABLE 1

Test complex Number of bases Clone 5 Clone 6 Clone 16 in the complex

H2 (PNA/DNA)¹ 3x15/45 - 1.28 1.24

H7 (PNA/DNA)¹ 20/20 - 0.34 0.33

H9 (PNADNA)¹ 17/17 - 0.63 0.62

H22 (PNA/DNA)' 19/19 - 0.17 0.22

H29 (PNA/DNA)¹ 15/30 - 0.42 0.44

H2 (PNA/DNA)' 3x15/45 - 1.70 1.28

H3 (PNA/DNA)' 15/15 0.26 0.22 -

H8 (PNA/DNA)' 17/17 0.51 0.38 -

L8 (ssPNA)' 19 0.13 0.12 -

L2 (ssDNA)' 45 - 0.18 -

H2 (PNA/DNA)^J 3x15/45 2.32 2.69 1.36

H3 (PNA/DNA)^J 3x15/45 0.55 0.45 0.92

H8 (PNA/DNA)^J 17/17 0.56 0.46 1.09

H9 (PNA/DNA)^J 17/17 1.46 1.55 1.07

Streptavidin" 0.10 0.12 0.15

H2 (PNADNA)⁴ 3x15/45 3.31 3.47 3.66

H7 (PNADNA)⁴ 20/20 0.55 0.40 0.57

H9 (PNA/DNA)⁴ 17/17 1.94 1.83 2.05

H10 (PNA/RNA)⁴ 19/19 0.14 0.14 0.18

H20 (DNA/DNA)⁴ 3x15/45 0.094 0.13 0.11

The numbers 1 to 4 given as superscripts indicate that the results were obtained on different streptavidin coated microtiter plates. As different preparations of Fab fragments have been used, the absolute values obtained on the individual plates cannot be compared.

As shown in Table 1 , the Fab fragment according to the invention reacted with most of PNA/DNA complexes tested, although with varying intensities. The base sequence of the PNA/DNA complexes tested varied as described in Example 1 and the results obtained showed that the antibody response appears not to be dependent on the base sequence. Only insignificant reaction was seen with single-stranded PNA or DNA or double-stranded DNA or PNA/RNA.

Thus, the present recombinant antibodies have a high degree of specificity for PNA- nucleic acid complexes. The specificity of the epitope(s) recognized by the present antibodies appears to a high degree to be dictated by the conformation of the PNA- nucleic acid complex rather than by the specific base sequence of the PNA /nucleic acid complex.

ELISA results with Fab-phage fusions

Microtiter plates were coated with streptavidin as described above, but in this case the plates were incubated with equal molar amounts of the different PNA/DNA complexes, i.e. 100 μL of a 3.74 nM solution of each complex were used for coating each well instead of 100 μL of a solution of 100 ng/mL. Non-specific binding was blocked with 4% skim milk in PBS for 2 hours. After washing with THT, different numbers of single clone Fab-phages produced as described in Example δ were added to separate wells. The plates were incubated on a rocking table at 37°C for 2 hours. Detection of binding Fab-phages was performed with HRP conjugated rabbit anti phage (DAKO) and OPD/citrate (DAKO) according to manufactures instructions. Detection reactions were stopped as described above and OD₄₉o values were read. Washings between different steps were carried out with THT.

The reactivity of Fab-phages from the three different clones obtained were very similar. Titrations of Fab-phages, clone 6, using a variety of PNA/DNA complexes as test antigens are shown in Figure 3. The curves illustrate OD₄₉₀ values for different numbers of Fab-phages per well. (1.00E+0X denotes 1.00 x 10^ox).

Figure 3 shows a dose-response curve of the antibody reactivity towards the individual PNA/DNA complexes. These results confirm the variations in the antibody response towards the different PNA/DNA complexes.

The reactivity of Fab-phages towards double-stranded PNA was also tested. The PNA/PNA complex (H32) is given in Example 1. The OD₄₉₀ values obtained with H32 and Fab-phages from three clones were similar to the OD₄₉₀ values obtained for single-stranded PNA (L8) and single-stranded DNA (L2). Detection with goat anti mouse Kappa secondary antibody

An ELISA assay was performed using an anti mouse antibody specific for the Kappa chain in the detection step. The antibody was an alkaline phosphatase conjugate, and detection was performed with PNPP (p-nitrophenyl phosphatase; Sigma) in 1 M diethanolamin/O.δ mM MgCI. OD₄₀₅ readings were as indicated in Table 2:

TABLE 2

Antibody conjugate Clone 5 Clone 6 Clone 16 goat anti mouse/AP 0.48 0.64 0.53 goat anti mouse Kappa/AP 1.03 0.39 0.38

These results show that the Kappa chain is present in the expressed Fab fragment.

Competitive ELISA assay:

The reactivities of the recombinant Fab fragments have been tested in the presence of immunogen (PNA DNA complex H12; a non-biotinylated version of H2 PNA/DNA complex). Periplasmic fractions containing recombinant antibody fragments were incubated with increasing amounts of H12 PNA/DNA complex, ending at a concentration of 2 μg/mL. The mixture was preincubated for approx. 1 hour at 37°C, before transfer to ELISA wells containing bound H2 PNA/DNA complexes. The assay was completed as described in the general ELISA procedure. Two negative controls included were a periplasmic extract without expressed Fab fragment, and an inert mouse monoclonal antibody (DAKO code no X0931). The OD readings increased by a factor 2-2. δ when going from 2 μg H12/mL to no H12 complex, whereas no signal beyond the background was obtained when testing the two negative controls.

Subtype determination

The antibody subtype of the recombinant fragments were determined in an ELISA assay according to the general description, incubated with subtype specific antibodies (IgG 1 , IgG 2a, IgG 2b, IgG 3) and detected with HRP conjugated goat anti rabbit antibody. OD₄₉₀ readings are listed in Table 3. TABLE 3

Clone anti IgG 1 anti IgG 2a anti IgG 2b anti IgG 3

5 1.18 0.13 0.31 0.28

6 0.97 0.07 0.11 0.13

16 0.80 0.05 0.07 0.14

Negative 0.06 0.06 0.06 0.13

The negative clone is a periplasmic extract prepared from a culture of E.coli transformed with pFABδc.His (the original plasmid), i.e. no Fab fragments are expressed. The results show that the Fab fragments are all of IgG 1 subtype.

Dot blot analysis

NYTRAN NY 13 N 0.4δ μ membrane (Schleicher & Schuell) was used in the dot blot assay. Two fold dilutions of H2 PNA/DNA complex were dotted onto the membrane, starting at 20 ng/dot, ending at 0.625 ng/dot, including a negative dot only containing dilution buffer (10 mM Tris-HCI, 0.1 mM EDTA, pH 8.0). The membrane was treated as follows:

1. After airdrying the dots, complexes were UV fixed for 2 minutes.

2. Blocking of membrane with 0.5% casein prepared in TS at 65°C. Incubation was continued with shaking for approx. 1 hour at room temperature.

3. Membranes were incubated with periplasmic fractions overnight at room temperature.

4. Washing of membrane: 3 x 10 minutes in TST.

5. Incubation for VA hours with detecting antibody: goat anti mouse/AP conjugate (DAKO), diluted in 0.δ% casein in TS.

6. Washing performed as in step 4.

7. Staining with NBT/BCIP.

Positive reactions with Fab fragments from all three clones were seen down to 10 ng per dot. Negative control was the periplasmic extract from E.coli transformed with pFABδc.His. No reaction was seen in the negative control.

Solutions: TS: 0.05 M Tris-HCI, pH 9.0, 0.5 M NaCl TST: 0.5% Tween in TS NBT: Nitro Blue Tetrazolium (Sigma)

BCIP: 5-bromo-4-chloro-3- indolylphosphate, p-toluidine salt (Sigma). NBT/BCIP: 75 mg/mL NBT, 50 mg/mL BCIP. Staining is performed using a

1 :400 dilution in 0.1 M Tris-HCI, pH 9.0, 0.05 M MgCI₂, 0.1 M NaCl.

DNA analysis of clones

Plasmid DNA prepared from the three selected clones (mouse anti PNA/DNA clone 5, clone 6, and clone 16, respectively) has been prepared and analyzed by restriction enzyme analysis according to the manufacturers instructions (Boehringer

Mannheim).

Sfil + Notl digest yielded two fragments of approx. 1700 bp and 4600 bp respec¬ tively. Esll digest yielded three fragments of approx. 400 bp, 1600 bp and 42-4300 bp, respectively.

No differences between the three clones have been detected so far by restriction enzyme analysis. From partial sequence analysis, the three clones also appeared to be very similar.

Hybrid vectors comprising clone 6 or clone 16 sequences inserted into pFABδc.His sequences have been deposited at DSM on June 19, 1995 (DSM 10051 and DSM 10062, respectively).

Although PNA comprising a N-(2-aminoethyl)glycine backbone has been used in the present work, this should not be taken as a limitation. It is expected that PNA with other types of backbone can be used in a similar way as long as the PNA is capable of forming stable complexes with nucleic acids.

The disclosure in Danish patent application No 717/95 from which this application claims priority are incorporated by reference.

Claims

37CLAIMS

1. A recombinant antibody or fragment thereof, characterized in that it is capable of binding to complexes formed between PNA (Peptide Nucleic Δcid) and nucleic acids.

2. A recombinant antibody according to claim 1 , characterized in that it does not bind to single-stranded PNAs, double-stranded nucleic acids, or single- stranded nucleic acids.

3. A recombinant antibody according to claim 2, characterized in that it binds to a complex formed between PNA and DNA, but not to PNA RNA complexes, single-stranded or double-stranded DNA, DNA/RNA-complexes, RNA or single-stranded PNAs.

4. A recombinant antibody according to any of the claims 1 to 3, characterized in that it is a fragment comprising the Fab region of the heavy chain and light chain.

5. A Fab fragment according to claim 4, characterized in that it is obtainable by immunizing a host animal with a complex formed between PNA having a N- (2-aminoethyl)glycine backbone and DNA, isolating RNA from antibody pro¬ ducing cells, producing single stranded cDNA from the mRNA of the isolated RNA, using specific oligonucleotide mixtures to amplify antibody encoding fragments from said cDNA and inserting it into a phagemid capable of ex¬ pressing and after superinfection displaying the antibody fragments at its surface, infecting bacteria with said phage to produce a phage library, se¬ lecting, through successive rounds of panning and reinfection of bacterial cells, the phages encoding antibody fragments of interest and using said phages for infection of bacteria for production of the antibody fragments or expressing the antibody fragment encoding DNA in another prokaryotic or eukaryotic expression system.

6. A recombinant antibody according to claim 2, characterized in that it is a fragment comprising the Fab region of the heavy chain and light chain and that it is obtainable by immunizing a host animal with a complex formed between PNA having a N-(2-aminoethyl)glycine backbone and RNA, isolating RNA from antibody producing cells, producing single stranded cDNA from the mRNA of the isolated RNA, using specific oligonucleotide mixtures to amplify antibody encoding fragments from said cDNA and inserting it into a phagemid capable of expressing and after superinfection displaying the antibody frag¬ ments at its surface, infecting bacteria with said phage to produce a phage library, selecting, through successive rounds of panning and reinfection of bacterial cells, the phages encoding antibody fragments of interest and using said phages for infection of bacteria for production of the antibody fragments or expressing the antibody fragment encoding DNA in another prokaryotic or eukaryotic expression system.

7. A recombinant antibody according to any of the claims 1 to 6 having attached a tag for recognition, signaling, coupling or purification purposes.

8. A recombinant DNA comprising an insert coding for a recombinant antibody or a fragment thereof according to any of the claims 1 to 7.

9. A recombinant DNA according to claim 8, characterized in that it is a hybrid vector further comprising an origin of replication, marker sequence(s) for selection, expression control sequences, signal sequence(s) and restriction sites.

10. A hybrid vector according to claim 9, characterized in that it comprises DNA encoding a recombinant antibody according to any of the claims 1 to 7 inserted into the vector pFABδc.His.

11. A hybrid vector according to claim 10, wherein the hybrid vector is DSM 10061.

12. A hybrid vector according to claim 10, wherein the hybrid vector is DSM 10062.

13. Use of a recombinant antibody or a fragment thereof according to any of the claims 1 to 7 for detecting a PNA/nucleic acid complex formed between a particular nucleic acid sequence to be detected in a test sample and PNA capable of forming a complex with said particular nucleic acid sequence.

14. A kit for detecting a particular nucleic acid sequence in a sample, said kit containing a recombinant antibody or fragment thereof according to any of the claims 1 to 7, a PNA sequence that is capable of forming a complex with the nucleic acid sequence to be detected and a visualization system.