WO1996014341A1 - Anticorps monoclonal pouvant se fixer a des complexes de pna/acides nucleiques - Google Patents

Anticorps monoclonal pouvant se fixer a des complexes de pna/acides nucleiques Download PDF

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Publication number
WO1996014341A1
WO1996014341A1 PCT/DK1995/000485 DK9500485W WO9614341A1 WO 1996014341 A1 WO1996014341 A1 WO 1996014341A1 DK 9500485 W DK9500485 W DK 9500485W WO 9614341 A1 WO9614341 A1 WO 9614341A1
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pna
dna
nucleic acid
complexes
complex
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PCT/DK1995/000485
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English (en)
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Jens Jørgen HYLDIG-NIELSEN
Karl-Johan Pluzek
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Dako A/S
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Application filed by Dako A/S filed Critical Dako A/S
Priority to AU43271/96A priority Critical patent/AU4327196A/en
Priority to EP95942055A priority patent/EP0840751A1/fr
Priority to JP8514968A priority patent/JPH11507202A/ja
Publication of WO1996014341A1 publication Critical patent/WO1996014341A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids

Definitions

  • the present invention relates to monoclonal antibodies capable of binding to com ⁇ plexes formed between PNA (Eeptide nucleic Acid) and nucleic acids. 5
  • 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 10. forming very stable and specific complexes. Such PNAs are thus suitable as hybri ⁇ dization 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.
  • the capture, recognition, detection, identification 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.
  • these techniques can be used to detect and identify etiologic agents such as bacteria and 5 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- 0 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 label ⁇ led probes generally involves the enzymatic incorporation of radiolabelled or modi ⁇ fied nucleotides or chemical modification of the probe to attach or form a detectable chemical group. Preparation of labelled probes is often time consuming and expen- sive and has to be carried out without destroying the ability of the probe to detectably hybridize with its complementary sequence.
  • Reagents for direct detecting a nucleic acid duplex formed as a result of hybridiza- tion between the sample and a nucleic acid probe and thereby avoid the chemical labelling of the used probes, would facilitate detection.
  • polyclonal 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.
  • antibodies may be selected so as to possess a desired affinity and specificity.
  • 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 stable PNA-nucleic acid complexes (Egholm et al., Nature, Vol 365, 566- 568 (1993)).
  • T- melting temperature
  • T m melting temperature
  • novel monoclonal antibodies are provided which are able to recognize, bind and detect complexes formed between PNA and nucleic acid.
  • One aspect of the present invention is monoclonal antibodies that are capable of binding to complexes formed between PNAs and nucleic acids.
  • PNA/nucleic acid complexes and nucleic acid duplexes possess substanti ⁇ ally different properties in that the PNA of a preferred PNA/nucleic acid complex comprises polymerized N-(2-aminoethyl)glycine units rendering the PNA achiral and non charged as opposed to the corresponding strands of a nucleic acid duplex, wherein the backbone is a sequence of nucleotides containing one anion for each phosphate group.
  • the monoclonal antibodies capable of binding to com ⁇ plexes formed between PNAs and nucleic acids do not bind to single-stranded PNAs, double-stranded nucleic acids or single-stranded nucleic acids.
  • the monoclonal antibody is capable of binding to a complex formed between PNA and DNA, but not to PNA/RNA complexes, double- stranded DNA, DNA/RNA duplexes, single-stranded PNA or single-stranded nucleic acids. In another of these embodiments, the monoclonal antibody is capable of binding to a complex formed between PNA and RNA, but not to PNA DNA complexes, double- stranded DNA, DNA/RNA duplexes, single-stranded PNAs or single-stranded nucleic acids.
  • Monoclonal antibodies for which the specificity of the epitope(s) recognised to a higher degree is dictated by the conformation of the PNA/nucleic acid complexes than by the specific sequence of the PNA/nucleic acid complex are also part of the invention.
  • the monoclonal antibodies described herein are obtainable from a hybridoma using generally known techniques.
  • a selected host animal is immunized with a complex formed by contacting a PNA with a nucleic acid.
  • Lymphocytes that secretes anti ⁇ bodies are taken form the immunized animal and are fused with myeloma cells to produce hybridomas.
  • Hybridoma cells producing antibodies which bind to PNA- nucleic acid complexes are selected. These selected hybridomas are subcloned to assure monoclonality of the secreted antibody.
  • Suitable complexes for immunization are complexes formed between PNA having a backbone of N-(2-aminoethyl)glycine units and DNA or RNA.
  • 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 antibodies may be very useful in the human and veterinary field. It is contempla ⁇ ted 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 Epstein Barr virus or papillomavirus.
  • the present antibodies are also useful in the general field of cytogenetics such as chromosome painting.
  • the invention also provides a kit containing a monoclonal antibody according to the invention, which antibody is optionally labelled, a PNA sequence capable of forming a complex with the nucleic acid sequence to be detected and a visualisation system. DESCRIPTION OF THE SPECIFIC EMBODIMENTS
  • the term "monoclonal antibody” is intended to include whole, intact antibodies, antibody fragments, or in general any antibody-derived substance that comprises at least one antibody combining site having the characteristics described herein.
  • Antibodies of any of the known classes and subclasses of immunoglobulins are encompassed, e.g., IgG, IgM, and so forth, as well as active fragments such as the Ig fragments conventionally known as Fab, F(ab'), and F(ab') 2 .
  • nucleic acid covers a nucleotide polymer composed of subunits, which are either deoxyribonucleosides or ribonucleosides joined together by phosphodie- ster bridges between the 5'-position of one nucleoside and the 3'-position of another nucleoside. They may be DNA or various types of RNA.
  • bases and “nucleobases” are used interchangeably for pyrimidine and purine bases of nucleic acids and PNAs.
  • 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 may suitably 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.
  • antibodies directed against PNA/DNA com ⁇ plexes can be raised 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.
  • This technology can also be applied to immunization with PNA/- RNA complexes.
  • a PNA-DNA complex can be prepared by contacting double-stranded or single-stran ⁇ ded 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 temperature.
  • a PNA-RNA complex might 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.
  • Complex formation may be characterized by determining T m of the complex or by performing electrophoresis in polyacrylamide gels.
  • a suitable quantity of one of the PNA-nucleic acid complexes is mixed with an adju ⁇ vant and a carrier.
  • suitable carriers are KLH (Keyhole Limpet Hemo- cyanin), ovalbumin and dextrans.
  • the monoclonal antibodies can be harvested from the secretions of hybridoma cells produced by somatic cell hybridization techniques originated from the work of K ⁇ hler and Milstein, Nature, 256, 495 (1975). This technique is well-known and has undergone various refinements and improvements. Details are described in referen- ces such as: Waldmann, M. C. H. "Production of murine monoclonal antibodies” in Monoclonal Antibodies, Beverly, P. C. L., ed., Churchhill Livingstone, London, 1986; Melchers, F. et al.
  • Lymphocytes producing antibodies against PNA-nucleic acid complexes may be obtained from various sites, e.g. the lymph nodes, spleen or peripheral blood.
  • the selected lymphocytes are preferably spleen cells from a host animal, e.g. a mouse or a rat, preferably a mouse, which has been immunized with a PNA-nucleic acid complex such as complexes between PNA and DNA or between PNA and RNA.
  • a mouse may be immunized intraperitoneally, subcutaneously or intravenously with a mixture of a PNA/DNA complex, wherein the PNA in preferred embodiments com ⁇ prises polymerized N-(2-aminoethyl)glycine units, ovalbumin and a suitable adjuvant.
  • myeloma cells of various animal origin can be used, for example, mye ⁇ loma cells from mice, rats or humans. However, for reasons of genetic stability, it may be preferred to fuse lymphocytes and myeloma cells derived from the same animal species and most preferably from the same strain of such animal species. Murine lymphocytes and myeloma cells are most commonly used.
  • the myeloma cells are preferably from the BALB/c strain, most preferred is myeloma cell line P3- X63-Ag.8.
  • Fusion of the lymphocyte and myeloma cells to form hybridomas and selection of antibodies are accomplished by methods known per se.
  • the selected hybridomas are cultured in vitro for an appropriate time period and aliquots of the culture fluid are drawn off to provide monoclonal antibody-rich fractions.
  • Monoclonal antibodies as described herein may be recovered from these fractions by purification using methods known per se for purification of monoclonal antibodies.
  • the present antibodies have a high degree of specificity for PNA-nucleic acid complexes. No significant degree of binding to double-stranded nucleic acids, double-stranded PNAs, single-stranded PNAs or single-stranded nucleic acids is observed.
  • the specificity of the epitope(s) recognised by the present antibodies appears to be dictated by the conformation of the PNA-nucleic acid complex rather than by the specific sequence of the PNAs or the nucleic acids.
  • the clones obtained produced antibodies capable of binding specifically to PNA/DNA complexes. Only insignificant reaction with single-stranded PNAs or DNA or double- stranded DNA or double-stranded PNA or PNA/RNA was observed with these clones.
  • a high specificity and affinity of the present antibodies give significant advantages when used in the isolation, detection and quantitation of complexes formed between a PNA and a nucleic acid to be detected in a biological sample.
  • antibodies having a high specificity for PNA/DNA complexes are particularly valuable in PNA based analysis for identifying infectious agents in humans such as chlamydial or gonococcial organisms or in animals.
  • These antibodies are also very useful in the general field of cytogenetics such as specific detection of chromosomes.
  • Antibodies according to the invention having a high specificity and affinity for PNA/- RNA complexes are particularly useful in PNA based analysis, for example for identi ⁇ fying mRNA or rRNA sequences.
  • 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, speci ⁇ fically bindable ligands such as biotin or haptens.
  • a detectable label such as enzymatically active groups like coenzymes, enzyme inhibitors and enzymes themselves, fluorescent labels, chromophores, luminescent labels, speci ⁇ fically bindable ligands such as biotin or haptens.
  • a method for detecting a particular nucleic acid sequence in a sample using the antibodies described herein may comprise the steps of
  • the PNA sequence may suitably 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
  • the method suitably comprises the steps of
  • 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 reac ⁇ tivity 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 sub- strate 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.
  • 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 turbi- dimetric assay format.
  • 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.
  • 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 detection of the label on the antibody or by using a secondary antibody detection sy ⁇ stem.
  • 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 detection of the label on the antibody or by using a secondary antibody detection sy ⁇ stem.
  • a traditional capture assay comprises the steps: recognition, capture and detection and may be composed in various ways.
  • recognition recognition, capture and detection and may be composed in various ways.
  • capture and detection 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 step 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 polypeptides.
  • two or more of the steps indicated above may be performed simultaneously.
  • 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 detection systems com ⁇ monly used for detecting antibodies bound to a target.
  • 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.
  • Detection and quantification of nucleic acids in a biological sample may be per- formed 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
  • PNAs may be able to penetrate the cell-wall of living or fixed cells, e.g. cell-lines, hemopoetic ceils, 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.
  • 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 Inc using a standard 381A cycle/procedure. The monomers used were standard ⁇ -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 sequences or the number of linker units added.
  • PNA 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 on the resin (see above), and biotin is attached in the following way.
  • AEEA 2-(aminoethoxy)ethoxy acetic acid
  • 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 with one or two units of AEEA were attached.
  • 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 "CONH 2 " (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-CONH 2
  • ssDNA single stranded 45-mer DNA sequence with a biotin attached to the 5'-end of the DNA sequence.
  • the base sequence of the 45-mer DNA (L1) was as follows: 5'-Bio-link-GCA AAT GCT CTA GGC GCA AAT GCT CTA GGC GCA AAT GCT CTA GGC- 3'.
  • L2 A 45-mer DNA sequence (L2) with a biotin attached to the 3'-end of the
  • the base sequence of the 45-mer DNA (L2) was as follows:
  • 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 sequence. 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:
  • the base sequence of the 15-mer PNA (U2) was as follows:
  • a single stranded 15-mer PNA sequence (ssPNA) with a base sequence corresponding to the 15-mer PNA in the complex used for immunization, and labelled with biotin in the 5' end.
  • the base sequence of this 15-mer is a single stranded 15-mer PNA sequence (ssPNA) with a base sequence corresponding to the 15-mer PNA in the complex used for immunization, and labelled with biotin in the 5' end.
  • PNA PNA
  • L4 Bio-link-GCC TAG AGC ATT TGC-CONH 2 L3.
  • the base sequence of this 15-mer DNA (L3) was as follows: 5'-Bio-link-GCA AAT GCT CTA GGC-3'
  • 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 sequence.
  • the base sequence of this 15-mer DNA (L3) was as follows: ⁇ '-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-CONH 2
  • a DNA/DNA complex comprising a 15-mer DNA sequence (L3) and a complementary 15-mer DNA sequence (U4).
  • the biotin is attached to the 5'- end of the L3 15-mer DNA sequence.
  • 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).
  • L2 45-mer DNA sequence
  • U4 15-mer DNA sequence
  • L5 A 20-mer PNA sequence wherein the PNA is labelled with biotin at the amino-terminal end.
  • the base sequence of the 20-mer PNA (L5) was as follows:
  • a PNA/DNA complex comprising a 20-mer PNA sequence and a 20-mer DNA having a base sequence that is different from the sequence of the complex used for immunization and wherein the PNA is labelled with biotin at the amino-terminal end.
  • the base sequence of the 20-mer PNA (L5) was as follows:
  • L6 A 17-mer DNA sequence with a biotin attached 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'
  • 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 was as follows: ⁇ '-Bio-link-ATT GTT TCG GCA ATT GT-3'
  • the base sequence of the 17-mer PNA was as follows: H-link-ACA ATT GCC GAA ACA AT-CONH 2
  • the base sequence of the 17-mer PNA (L7) was as follows: Bio-link-ACA ATT GCC GAA ACA AT-CONH 2
  • a PNA/DNA complex comprising a 17-mer PNA sequence and a 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-CONH 2
  • 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-CONH 2
  • 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 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-CONH 2
  • the base sequence of the 19-mer RNA (U9) was as follows: 5'-UUC AAC UCA CAG AGU UGA A-3'
  • 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-CONH 2
  • the base sequence of the 19-mer DNA (U26) was as follows: 5'-TTC AAC TCA CAG AGT TGA A-3'
  • 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 sequence resulting in single-stranded DNA overhangs both 5'- and 3'- to the PNA/DNA complex.
  • the base sequence of the 30-mer DNA 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 was as follows: H-link-GTT GAC ATG TGC GGA-CONH 2 .
  • 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-TCCGCACATGTCAACTCCGCACATGTCAACTCCGCA
  • the base sequence of the 15-mer PNA (U13) was as follows:
  • 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.
  • sequence of L13 is as follows:
  • the sequence of the 15-mer PNA (U13) was as follows:
  • a PNA PNA complex 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:
  • dsDNA double-stranded DNA
  • dsDNA double-stranded DNA
  • Sigma D-1501 converted to fragments comprising from 200 to 1000 bp
  • the PNA/nucleic acid complexes are prepared by, in a suitable buffer (e.g. 50 mM Tris-HCI, pH 7.6, 50 mM NaCI), 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.
  • a suitable buffer e.g. 50 mM Tris-HCI, pH 7.6, 50 mM NaCI
  • 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).
  • 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.
  • AP alkaline phosphatase
  • Unlabelled complexes were visualised either directly in the polyacrylamide 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.
  • the antigen used for immunization was prepared by mixing the following in a total volume of 2 mL: 0.939 mg the 45-mer polydeoxyribonucleot.de (DNA)
  • the mouse was immunized intraperitone ⁇ ally without adding adjuvant to the mixture of the PNA/DNA complex and ovalbumin.
  • the fusion was carried out using standard fusion procedures with spleen cells from the immunized mouse and the myeloma cell line P3-X63-Ag.8 as fusion partner.
  • H2 is a biotinylated version of the PNA/DNA complex used for immunization. Positive clones were cultivated and supernatants were re- tested in the ELISA systems described below. Clonality was ensured by limited dilu ⁇ tion of the cells followed by screening of the supernatants in ELISA. Hybridomas were grown under standard conditions in RPMI 1640 medium supplemented with 10% foetal calf serum.
  • Detection of binding antibodies were performed with HRP conjugated rabbit anti mouse IgG (DAKO) and OPD (o-phenylenediamine) (DAKO) according to the manufactures instructions. Detection reactions were stopped after 10-15 minutes with 1 M H 2 S0 4 , and finally the plates were read at OD 492 (Molecular Devices). All washings between different steps were done with THT. The complexes/compounds used for testing the specificity are described in Example 1.
  • the supernatants were tested in the test systems described above.
  • the supernatant from two of the clones of fusion F86 (1 B11 and 1G12) showed preferential binding to PNA/DNA complexes as shown in Table 1.
  • the optical density at 492 nm obtained after diluting the supernatant 1 :2 is shown in tests of supernatant using the test complexes/compounds shown and which are described in Example 1.
  • the background values (measured by omitting the antibody) was below 0.150 and has not been subtracted from the OD values given in Table 1. (nd denotes not determined).
  • the present monoclonal 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.
  • the immunoglobulin class of the supernatants were tested in standard ELISA system for determination of the Ig class.
  • the antibodies from both clones were found to be of the IgM type.
  • Clone F86 1 B11 has been deposited with ECACC on May 31 , 1995 (deposit number ECACC 95053111.)
  • PNA comprising a N-(2-aminoethyl)glycine backbone

Abstract

On décrit des anticorps monoclonaux pouvant se fixer à des complexes formés entre des PNA (acides nucléiques de peptides) et des acides nucléiques, notamment des complexes de PNA/ADN ou PNA/ARN. Les anticorps monoclonaux préférés peuvent se fixer à des complexes de PNA/ADN ou PNA/ARN, mais pas à des PNA simple-brin ni à des acides nucléiques double-brin ou simple-brin. Les PNA sont des composés nouvellement développés, non naturels, parmi lesquels certains possèdent un squelette polyamide portant une pluralité de ligands tels que des nucléobases naturelles fixées sur le squelette par l'intermédiaire d'une liaison appropriée. Certains PNA se sont révélés posséder de façon inattendue une affinité élevée pour l'acide nucléique complémentaire formant des complexes très stables et spécifiques. De tels PNA sont alors appropriés en tant que sondes d'hybridation destinées à la détection d'acides nucléiques. Les anticorps décrits rendent ces PNA très appropriées en tant que sondes d'hybridation, et ils sont utiles dans la capture, la reconnaissance, la détection, l'identification ou la quantification d'acides nucléiques dans des échantillons biologiques, grâce à leur capacité à réagir avec des complexes de PNA/acides nucléiques.
PCT/DK1995/000485 1995-06-22 1995-12-01 Anticorps monoclonal pouvant se fixer a des complexes de pna/acides nucleiques WO1996014341A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU43271/96A AU4327196A (en) 1995-06-22 1995-12-01 Monoclonal antibody capable of binding to pna/nucleic acid complexes
EP95942055A EP0840751A1 (fr) 1995-06-22 1995-12-01 Anticorps monoclonal pouvant se fixer a des complexes de pna/acides nucleiques
JP8514968A JPH11507202A (ja) 1995-06-22 1995-12-01 Ｐｎａ／核酸複合体に結合することができるモノクローナル抗体

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DK0718/95 1995-06-22

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EP0135159A2 (fr) * 1983-08-19 1985-03-27 Cetus Corporation Anticorps monoclonal spécifique pour la structure à double hélice de l'ADN naissant et méthodes de diagnostic utilisant cet anticorps
WO1992020703A1 (fr) * 1991-05-24 1992-11-26 Ole Buchardt Utilisation d'analogues d'acides nucleiques dans des procedures diagnostiques et analytiques
WO1995015974A1 (fr) * 1993-12-06 1995-06-15 Pna Diagnostics A/S Acides nucleiques de protection et procedes d'analyse
WO1995017430A1 (fr) * 1993-12-23 1995-06-29 Dako A/S Anticorps polyclonal contre des complexes anp/acide nucleique

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EP0135159A2 (fr) * 1983-08-19 1985-03-27 Cetus Corporation Anticorps monoclonal spécifique pour la structure à double hélice de l'ADN naissant et méthodes de diagnostic utilisant cet anticorps
WO1992020703A1 (fr) * 1991-05-24 1992-11-26 Ole Buchardt Utilisation d'analogues d'acides nucleiques dans des procedures diagnostiques et analytiques
WO1995015974A1 (fr) * 1993-12-06 1995-06-15 Pna Diagnostics A/S Acides nucleiques de protection et procedes d'analyse
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6287772B1 (en) 1998-04-29 2001-09-11 Boston Probes, Inc. Methods, kits and compositions for detecting and quantitating target sequences
US7135563B2 (en) 1998-04-29 2006-11-14 Boston Probes, Inc. Compositions for detecting target sequences

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EP0840751A1 (fr) 1998-05-13
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