WO2014168242A1 - Anticorps monoclonal contre un peptide spécifique des maladies périodontiques, et son utilisation - Google Patents

Anticorps monoclonal contre un peptide spécifique des maladies périodontiques, et son utilisation Download PDF

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WO2014168242A1
WO2014168242A1 PCT/JP2014/060531 JP2014060531W WO2014168242A1 WO 2014168242 A1 WO2014168242 A1 WO 2014168242A1 JP 2014060531 W JP2014060531 W JP 2014060531W WO 2014168242 A1 WO2014168242 A1 WO 2014168242A1
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periodontal disease
antibody
antigen
hybridoma
monoclonal antibody
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Japanese (ja)
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友宏 吉村
若本　裕晶
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Jnc株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/18—Dental and oral disorders

Definitions

  • the present invention relates to a monoclonal antibody that recognizes a periodontal disease-specific peptide, a method for quantifying periodontal disease-specific peptide using the monoclonal antibody, an immunocytochemistry or histochemical examination method for periodontal disease-specific peptide, periodontal It relates to diagnostic agents for diseases.
  • Periodontal disease is a condition in which any of the gingiva, alveolar bone, cementum, and periodontal ligament, which are the periodontal tissues that support the teeth, is impaired. Chronic marginal periodontitis is a typical disease. It is done. Periodontal disease is a lifestyle-related disease found in about 80% of adults and is caused by bacterial infection.
  • a variety of undesirable effects have been suggested. For example, diabetic patients are not only prone to periodontal disease, but periodontal disease may exacerbate diabetes. It has also been confirmed that periodontal disease is a high risk factor for heart disease caused by arteriosclerosis, and that pregnant women with periodontal disease are likely to have premature birth of low-weight infants.
  • periodontal disease is not only about missing teeth, but also deeply involved in the onset and exacerbation of serious diseases that can lead to death, so it is effective for early diagnosis and treatment and prevention. Development of new means is desired.
  • Non-patent Document 1 Porphyromonas gingivalis
  • P. gingivalis is an anaerobic gram-negative bacillus that enters the plaque and releases enzymes such as proteases for its own survival. This enzyme causes gingival inflammation and develops gingivitis, the beginning of periodontal disease.
  • Non-Patent Documents 2 and 3 trypsin-like cysteine proteases
  • Keratin is a protein constituting an intermediate filament that is the cytoskeleton of epithelial cells.
  • epithelial cells are filled with an intermediate filament made of special keratin called hard keratin, and die and harden.
  • keratin plays an important role as a constituent protein of intermediate filaments, and the sheet-like structure of epithelial tissue maintains mechanical strength by keratin fibers.
  • Patent Document 5 discloses that keratin and its degradation products migrate into the blood to stimulate T cell proliferation and induce an autoimmune response thereto, and that keratin and its degradation products are RANKL in T cells. This activated T cell induces differentiation from osteoclast progenitor cells to osteoclasts and promotes bone resorption. It has been shown to be deeply involved in the onset and progression, as well as the development of systemic complications.
  • Patent Document 5 also describes an antibody that recognizes a periodontal disease-specific peptide.
  • Patent Document 5 discloses a monoclonal antibody that can detect periodontal disease-specific peptides in a test sample with high sensitivity and quantitatively, a hybridoma that produces the monoclonal antibody, and an analysis that uses the monoclonal antibody. The method is not described.
  • JP 2004-143127 A JP 2005-35909 JP JP2003-335648 Japanese Patent Laid-Open No. 2007-16002 WO2011 / 115225
  • the present inventors have found that periodontal disease bound to a carrier protein.
  • a monoclonal antibody having excellent specificity was obtained by using a specific peptide as an antigen, and the present invention was completed. That is, the present invention includes the following monoclonal antibody or antigen-binding fragment thereof; hybridoma; periodontal disease-specific peptide quantification method, immunocytochemistry or histochemical examination method, periodontal disease diagnostic agent, etc. provide.
  • a periodontal disease-specific peptide contained in a test sample, characterized in that the antibody or antigen-binding fragment thereof according to any one of [1] to [3] above is used.
  • Quantitation method [7] The method according to [6] above, wherein the quantification method is performed in an ELISA system.
  • a diagnostic agent for periodontal disease comprising the antibody or antigen-binding fragment thereof according to any one of [1] to [3] above.
  • [10] Use of the antibody or antigen-binding fragment thereof according to any one of [1] to [3] in the manufacture of a diagnostic agent for periodontal disease.
  • [10A] The antibody or antigen-binding fragment thereof according to any one of the above [1] to [3] for diagnosis of periodontal disease.
  • [10B] A method for diagnosing periodontal disease, wherein periodontal disease is diagnosed using the antibody or antigen-binding fragment thereof according to any one of [1] to [3] above.
  • the present invention provides a monoclonal antibody or an antigen-binding fragment thereof capable of highly sensitively and quantitatively detecting a periodontal disease-specific peptide in a test sample.
  • the monoclonal antibody and antigen-binding fragment thereof of the present invention can be used for a periodontal disease-specific peptide quantification method, immunocytochemistry or histochemical examination method, periodontal disease diagnostic agent, and the like.
  • the antibody of the present invention is a monoclonal antibody that recognizes a periodontal disease-specific peptide or an antigen-binding fragment thereof.
  • Periodontal disease specific peptide is a peptide produced by degrading a parent protein by periodontal disease enzyme using keratin present in gingival epithelial cells as a parent protein.
  • the keratin in the present invention is specifically Kelatin® 6 or Kelatin® 5, preferably Kelatin® 6.
  • the existence of subtypes is known in Kelatin 6 or Kelatin 5, but any subtype is included in keratin in the present invention as long as it exists in gingival epithelial tissue.
  • Periodontal disease enzyme is an enzyme that is produced and released by the causative bacteria of periodontal disease for their own survival.
  • the periodontal disease enzyme in the present invention is specifically Kgp released by Porphyromonas gingivalis.
  • Kgp is one of three types of gingipain (HRgpA, RgpB, Kgp) produced by P. gingivalis.
  • the periodontal disease-specific peptide is a Kgp degradation product of human Keratin 6B (UniprotKB database Accession No.P04259; SEQ ID NO: 4), that is, a partial amino acid sequence at positions 360 to 378 of human Keratin 6B. It is a peptide (peptide 1) consisting of (SEQ ID NO: 1).
  • Periodontal disease-specific peptides in a preferred embodiment of the present invention include human Keratin B paralogs (eg, human keratin 6A (UniprotKB / Swiss-protproP02538), human keratin 6C (UniprotKB / Swiss-prot P48668), human keratin 5 (UniprotKB / Swiss-prot P13647)), or its orthologs (eg, rat Keratin 6A (UniprotKB / Swiss-prot Q4FZU2), mouse Keratin 6A (UniprotKB / Swiss-prot P50446), mouse Keratin 6B (UniprotKBt ), Rat Keratin 5 (UniprotKB / Swiss-prot Q6P6Q2), Mouse Keratin 5 (UniprotKB / Swiss-prot Q922U2), Chimpanzee Keratin 5 (UniprotKB / Swiss-prot A5A6M8), or Bo
  • the N-terminus and / or C-terminus of peptide 1 is about 1 to 3 residues from the partial amino acid sequence, and the parent protein N-terminus or C-terminus. Those that are shifted to the above are also included in the periodontal disease-specific peptide in the preferred embodiment of the present invention.
  • the periodontal disease-specific peptide in the most preferred embodiment of the present invention is peptide 1.
  • the antibody in a preferred embodiment of the present invention is a monoclonal antibody that specifically recognizes peptide 1.
  • an “antigen-binding fragment” is a fragment having an antigen-binding or variable region of an intact antibody, such as a Fab fragment, Fab ′ fragment, F (ab ′) 2 fragment, Fv, or ScFv fragment of the antibody of the present invention. Can be mentioned.
  • a preferred example of the antibody of the present invention is a monoclonal antibody produced by hybridoma A17-7-8 or an antigen-binding fragment thereof.
  • Another preferred example of the antibody of the present invention is a monoclonal antibody produced by the hybridoma JB29-3-7 or an antigen-binding fragment thereof. “A17-7-8” and “JP29-3-7” are described in detail later.
  • the isotypes of the monoclonal antibodies produced by these two hybridomas A17-7-8 and JB29-3-7 are both IgG1 for the H chain and ⁇ for the L chain.
  • the present invention also encompasses class switch variants of the above antibodies, for example, variants belonging to isotype IgG3, IgG1, IgG2b, IgG2a and other immunoglobulin subclasses, such variants can be obtained by the method of Martin et al. (J Immunol Methods. 1991 Dec 15; 145 (1-2): 11-8.).
  • Some embodiments of the present invention include chimeric antibodies, humanized antibodies, or human antibodies, which can also be used.
  • the antibody of the present invention may be labeled.
  • the label is, for example, an enzyme, an enzyme substrate, a coenzyme, an enzyme precursor, an apoenzyme, a fluorescent substance, a dye substance, a chemiluminescent compound, a luminescent substance, a coloring substance, a magnetic substance, a metal particle, or a radioactive substance.
  • Hybridoma of the present invention provides a hybridoma that produces a monoclonal antibody that recognizes a periodontal disease-specific peptide or an antigen-binding fragment thereof.
  • the hybridoma according to a preferred embodiment of the present invention is referred to as “A17-7-8” and, as of February 6, 2013, is an independent administrative agency, Product Evaluation Technology Infrastructure Organization, Patent Microorganism Depositary Center (Kazusakami, Kisarazu City, Chiba Prefecture 292-0818). Deposited at NITE Biotechnology Headquarters (patent microorganism deposit center) as deposit number NITE BP-1530. The depositor is JNC Corporation (2-1-1 Otemachi, Chiyoda-ku, Tokyo 100-8105, Japan). The hybridoma produces the monoclonal antibody used in the examples described below.
  • hybridoma of the present invention is referred to as “JB29-3-7” and dated February 6, 2013, the National Institute of Technology and Evaluation, Patent Microorganism Depositary Center (Kisarazu City, Chiba Prefecture 292-0818, Japan). Deposited with NITE BP-1531 at the NITE Biotechnology Headquarters Patent Microorganism Depositary Center). The depositor is JNC Corporation (2-1-1, Otemachi, Chiyoda-ku, Tokyo 100-8105, Japan). The hybridoma produces the monoclonal antibody used in the examples described below.
  • the antibody of the present invention comprises a step of cell fusion between myeloma cells and antibody-producing cells, selection of a hybridoma, and cloning after immunization of an animal using a periodontal disease-specific peptide as an immunogen. It can be produced through a step of collecting a monoclonal antibody.
  • Preparation of antigen requires a periodontal disease-specific peptide or a partial peptide thereof that can be used as an immunogenic antigen.
  • a periodontal disease-specific peptide or a partial peptide thereof as an antigen can be prepared by synthesis or degradation of keratin by a periodontal disease bacterial enzyme.
  • Periodontal disease-specific peptides or partial peptides thereof can be directly immunized if they are immunogenic, but these antigenic peptides are usually used when low molecular weight antigens are used. Since the hapten molecule has low immunogenicity (hereinafter sometimes referred to as hapten), it can be immunized as a complex bound or adsorbed to an appropriate carrier (hereinafter sometimes referred to as carrier). A natural or synthetic polymer can be used as the carrier.
  • hemoglobin of mammals such as bovine, rabbit, human, and sheep, bovine serum albumin (BSA), or keyhole lymph hemocyanin (KLH) is used.
  • BSA bovine serum albumin
  • KLH keyhole lymph hemocyanin
  • Examples of the synthetic polymer include various latexes such as polymers or copolymers of polyamino acids, polystyrenes, polyacryls, polyvinyls, polypropylenes, and the like.
  • any substance may be bound or adsorbed at any ratio as long as an antibody against the antigen bound or adsorbed to the carrier is efficiently produced.
  • the above-mentioned natural or synthetic polymer carrier which is commonly used for the production of an antibody against a hapten, can be used which is bound or adsorbed at a ratio of 0.1 to 100 with respect to hapten 1 by weight.
  • Various coupling agents can be used for coupling the hapten and the carrier.
  • diazonium compounds such as bisdiazotized benzidine that crosslinks tyrosine, histidine, and tryptophan; dialdehyde compounds such as glutaraldehyde that crosslink amino groups; diisocyanate compounds such as toluene-2,4-diisocyanate; Dimaleimide compounds such as N, N′-o-phenylene dimaleimide; maleimide active ester compounds that crosslink amino groups and thiol groups; or carbodiimide compounds that crosslink amino groups and carboxyl groups.
  • diazonium compounds such as bisdiazotized benzidine that crosslinks tyrosine, histidine, and tryptophan
  • dialdehyde compounds such as glutaraldehyde that crosslink amino groups
  • diisocyanate compounds such as toluene-2,4-diisocyanate
  • Dimaleimide compounds such as
  • amide groups are introduced into the other amino group by introducing a thiol group by reducing the cross-linking between amino groups by reacting an active ester reagent (for example, SPDP) having a dithiopyridyl group with one amino group.
  • an active ester reagent for example, SPDP
  • the reaction can be carried out.
  • coupling of the hapten and the carrier is performed by adding a cysteine residue to the N-terminus or C-terminus of the hapten (peptide), introducing a maleimide group into the amino group of the carrier protein with a maleimide active ester reagent, and then reacting both. Can also be performed.
  • the antigen prepared as described above is administered to mammals such as rats, mice (for example, BALB / cAJcl mice or FI mice) or rabbits for immunization.
  • the dose of the antigen per animal can be appropriately set depending on the presence or absence of an adjuvant.
  • the adjuvant include Freund's incomplete adjuvant, Libi adjuvant, pertussis vaccine, BCG, liposome, aluminum hydroxide, or silica gel.
  • Immunization is performed mainly by injecting intravenously, footpads, subcutaneously, intraperitoneally, and the like. Further, the immunization interval is not particularly limited, and immunization is performed 1 to 10 times at intervals of several days to several weeks. Then, antibody-producing cells are collected 1 to 60 days after the final immunization day. Examples of antibody-producing cells include spleen cells, lymph node cells, and peripheral blood cells, with spleen cells being preferred.
  • Cell fusion Cell fusion between antibody-producing cells and myeloma cells is performed to obtain hybridomas.
  • myeloma cells to be fused with antibody-producing cells generally available cell lines of animals such as mice that do not produce immunoglobulins can be used.
  • Cell fusion is performed in animal cell culture media such as serum-free DMEM and RPMI-1640 media, and 1 ⁇ 10 6 to 1 ⁇ 10 10 antibody-producing cells and 1 ⁇ 10 6 to 1 ⁇ 10 10 cells Mix with / mL myeloma cells.
  • a fusion reaction is performed in the presence of a cell fusion promoter (eg, polyethylene glycol). If necessary, a small amount of dimethyl sulfoxide can be added to further promote cell fusion.
  • antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device utilizing electrical stimulation (for example, electroporation).
  • the target hybridoma is selected from the cells after cell fusion treatment.
  • the cell suspension is appropriately diluted in IMEM medium or RPMI-1640 medium containing hypoxanthine, aminopterin, thymidine and fetal calf serum, or IMDM medium containing hypoxanthine, aminopterin and thymidine, and then microtiter. Seed on plate and add selective medium to each well. As a result, cells growing from about 7 days after the start of culture in the selective medium can be obtained as hybridomas.
  • Hybridoma screening is not particularly limited, and may be performed according to ordinary methods. For example, a part of the culture supernatant contained in a well grown as a hybridoma can be collected and screened by ELISA, EIA, RIA, FIA or the like.
  • Fusion cells are cloned by limiting dilution.
  • An antibody showing strong reactivity with a periodontal disease-specific peptide is determined by flow cytometry or the like, and a hybridoma producing the antibody is selected and established.
  • culturing means growing the hybridoma in a culture dish or culture bottle, or growing the hybridoma in the abdominal cavity of an animal.
  • the hybridoma is cultured in an animal cell culture medium such as RPMI-1640 medium containing 10% (v / v) fetal bovine serum, MEM medium, and serum-free medium under normal culture conditions (for example, 37 ° C). And 5% (v / v) CO 2 concentration) for 7 to 14 days, and antibodies are obtained from the culture supernatant.
  • an animal cell culture medium such as RPMI-1640 medium containing 10% (v / v) fetal bovine serum, MEM medium, and serum-free medium under normal culture conditions (for example, 37 ° C). And 5% (v / v) CO 2 concentration) for 7 to 14 days, and antibodies are obtained from the culture supernatant.
  • hybridomas are administered into the abdominal cavity of a myeloma cell-derived mammal or a homologous animal or an immunodeficient rat, and the hybridoma is proliferated in large quantities. Ascites is collected after 1-2 weeks.
  • Antigen-binding fragment is also included in the antibody of the present invention.
  • Antigen-binding fragments can be produced by proteolysis of intact antibodies, for example by papain degradation (see eg WO 94/29348). Alternatively, it can be produced directly from a transformed host cell by genetic recombination according to a conventional method.
  • Chimeric antibody, humanized antibody, or human antibody Some embodiments of the present invention include chimeric antibodies, humanized antibodies, or human antibodies.
  • the variable region (V region) of a mouse antibody is linked to a human constant region. Methods for producing chimeric antibodies are well known in the art.
  • CDRs complementarity determining regions
  • FR framework regions
  • the antibody of the present invention may be labeled.
  • the label is, for example, an enzyme, an enzyme substrate, a coenzyme, an enzyme precursor, an apoenzyme, a fluorescent substance, a dye substance, a chemiluminescent compound, a luminescent substance, a coloring substance, a magnetic substance, a metal particle, or a radioactive substance.
  • the antibody of the present invention can be labeled using a reaction between a thiol group and a maleimide group, a reaction between a pyridyl disulfide group and a thiol group, a reaction between an amino group and an aldehyde group, or the like.
  • the antibody of the present invention can be used for a periodontal disease-specific peptide quantification method, immunohistochemical or cytochemical examination method, periodontal disease diagnostic agent, and the like.
  • the antibody of the present invention can also be used for protein purification, affinity columns and the like.
  • the present invention provides a method for quantifying periodontal disease specific peptide contained in a test sample, which uses the antibody of the present invention. Periodontal disease-specific peptides can be quantified with high sensitivity by using the antibody of the present invention.
  • the quantification method of the present invention is not particularly limited, and the amount of antibody, antigen or antibody-antigen complex corresponding to the amount of antigen in the test sample (for example, the amount of periodontal disease specific peptide) is chemically determined. Any quantitative method may be used as long as it is a quantitative method in which it is detected by physical or physical means and is prepared and calculated using a standard sample containing a known amount of antigen.
  • the quantitative method is specifically an immunoassay (for example, a competitive type or a non-competitive type immunoassay).
  • the immunoassay include RIA, IRMA, EIA, ELISA, latex agglutination method, immunocytochemistry or histochemical method, a method using an immunoblot, or an immunoprecipitation method.
  • the immunoassay is preferably a method using an ELISA system, more preferably a sandwich type assay.
  • test sample is preferably a biological sample such as blood (eg, whole blood, serum, plasma), saliva, interdental fluid, urine, other body fluids, cell culture fluid, tissue culture fluid, tissue homogenate, or live A test sample, more preferably plasma.
  • blood eg, whole blood, serum, plasma
  • saliva interdental fluid
  • urine other body fluids
  • cell culture fluid cell culture fluid
  • tissue culture fluid tissue homogenate
  • live A test sample more preferably plasma.
  • the cell or tissue used in the inspection method of the present invention is a cell or tissue derived from a living body, or a cultured cell or tissue, but is preferably a cell or tissue derived from a living body.
  • the cell or tissue derived from a living body is preferably a cell or tissue derived from the oral cavity (eg, gingiva), more preferably a gingival epithelial cell.
  • the staining of cells or tissues and the confirmation of the presence of periodontal disease specific peptides in cells or tissues can be performed according to conventional methods.
  • the present invention provides a diagnostic agent for periodontal disease containing the antibody of the present invention.
  • the present invention also includes (a) the use of the antibody of the present invention in the production of a diagnostic agent for periodontal disease, (b) the antibody of the present invention for the diagnosis of periodontal disease, and (c) the antibody of the present invention.
  • a method for diagnosing periodontal disease including the above is provided.
  • Periodontal disease-specific peptide in a subject-derived test sample is detected or measured using the antibody of the present invention.
  • Periodontal disease-specific peptides can be detected or measured by the same method as the quantitative method of the present invention and the test method of the present invention.
  • an increase in the amount of periodontal disease-specific peptide in the test sample indicates that the subject has periodontal disease.
  • “Increase in the amount of periodontal disease-specific peptide in the test sample” means, for example, a periodontal disease-specific peptide in the test sample as compared to a sample derived from a subject not suffering from periodontal disease Means that the amount of is increasing.
  • Kit of the present invention The present invention further provides a kit containing the antibody of the present invention.
  • the kit of the present invention can be used for the above-described quantification method of the present invention, the inspection method of the present invention, the diagnostic method of periodontal disease, and the like.
  • Example 1 Preparation of anti-periodontal disease-specific peptide monoclonal antibody An anti-periodontal disease-specific peptide monoclonal antibody was prepared according to the following procedure.
  • mice with high antibody titers intraperitoneally administer a solution of antigen solution for immunization in physiological saline 2 weeks after the second booster so that the periodontal disease-specific peptide is about 100 ⁇ g / mouse.
  • spleen cells were prepared from the immunized mice and used for the following cell fusion.
  • the plate was washed 4 times with PBS-T, HRP-labeled mouse IgG antibody (manufactured by Invitrogen) diluted 5000 times was added at 100 ⁇ L / well, and reacted at 37 ° C. for 30 minutes. After the plate was washed 4 times with PBS-T, 3,3 ′, 5,5′-tetramethylbenzidine solution was added at 100 ⁇ L / well and allowed to react at room temperature for 12 minutes. Thereafter, 50 ⁇ L of 1N H 2 SO 4 was added to stop the reaction, and the absorbance at 450 nm was measured.
  • the fused cells were suspended in a medium (HAT medium) in which hypoxanthine, aminopterin, and thymidine were added to an IMDM medium, and dispensed at 100 ⁇ L / well into a 96-well microplate (Sumitomo Bakelite).
  • the fused cells were cultured in a CO 2 incubator (37 ° C, 7% (v / v) CO 2 ) while changing the medium by half every 3 to 5 days. Only hybridomas that can be cultured in HAT medium were selectively cultured.
  • Hybridomas that produce antibodies that bind to periodontal disease-specific peptides were cloned twice by limiting dilution to obtain antibodies that specifically bind to periodontal disease-specific peptides.
  • Two types of hybridoma A17-7-8 cell line (NITE BP-1530) and hybridoma JB29-3-7 cell line (NITE BP-1531) which were produced and had stable growth ability were obtained.
  • Example 2 Development of ELISA system using anti-periodontal disease specific peptide antibody (a) ELISA method (competitive method) A periodontal disease-specific peptide solution prepared by the same method as that for the antigen for immunization was adjusted to 1 ⁇ g / mL with PBS and adsorbed to an ELISA plate at 50 ⁇ l / well for 2 hours at 37 ° C. After washing 3 times with PBS-T, blocking was performed at 37 ° C. for 2 hours using 50 mM carbonate buffer (pH 9.6) containing 10 mM glycine. Thereafter, the sample washed 3 times with PBS-T was used for evaluation.
  • a ELISA method (competitive method) A periodontal disease-specific peptide solution prepared by the same method as that for the antigen for immunization was adjusted to 1 ⁇ g / mL with PBS and adsorbed to an ELISA plate at 50 ⁇ l / well for 2 hours at 37 ° C. After washing
  • Periodontal disease-specific peptide was diluted with PBS to an appropriate concentration (0 to 1,000 ng / mL), and 100 ⁇ L of this was added. Subsequently, 100 ⁇ L each of the HRP-labeled monoclonal antibody obtained in Example 1 (i) was added and mixed well, followed by reaction at 37 ° C. for 1 hour. After the plate was washed 4 times with PBS-T, 3,3 ′, 5,5′-tetramethylbenzidine solution was added by 100 ⁇ L / well and allowed to react at room temperature for 12 minutes. Thereafter, 50 ⁇ L of 1N H 2 SO 4 was added to stop the reaction, and the absorbance at 450 nm was measured.
  • FIG. 1 shows the results when using the antibody produced by hybridoma A17-7-8
  • FIG. 2 shows the results when using the antibody produced by hybridoma JB29-3-7.
  • the horizontal axis represents the periodontal disease-specific peptide concentration (ng / mL), and the vertical axis represents the inhibition rate (%).
  • the inhibition rate (%) was determined from the measured optical density (450 nm) as follows.
  • Inhibition rate (%) 100-S / N x 100
  • S Optical density of sample well (450 nm)
  • N Optical density (450 nm) of blank well (P1 peptide 0 ng / mL)
  • FIG. 3 shows the results when using the antibody produced by hybridoma A17-7-8
  • FIG. 4 shows the results when using the antibody produced by hybridoma JB29-3-7.
  • Example 6 of Patent Document 5 describes that the concentration of periodontal disease-specific peptide in the plasma of periodontal disease patients and healthy individuals was measured by ELISA. From FIG. 6 of Patent Document 5, it can be estimated that the lower limit of the detection concentration of periodontal disease-specific peptide is 500 ng / mL to 1000 ng / mL.
  • the antibody of the present invention can detect periodontal disease-specific peptides in the blood with higher sensitivity than conventional antibodies.
  • the present invention provides a monoclonal antibody that specifically binds to a periodontal disease-specific peptide.
  • the antibody of the present invention it became possible to detect a periodontal disease-specific peptide in a sample with high sensitivity and quantitative.
  • [SEQ ID NO: 1] Amino acid sequence of a periodontal disease-specific peptide (peptide 1) (partial amino acid sequence at positions 360 to 378 of human Keratin 6B).
  • [SEQ ID NO: 2] is an amino acid sequence of partial peptide A (a 12-residue peptide on the N-terminal side of a periodontal disease-specific peptide).
  • [SEQ ID NO: 3] This is the amino acid sequence of partial peptide B (11-residue peptide on the C-terminal side of periodontal disease-specific peptide).
  • [SEQ ID NO: 4] This is the amino acid sequence of human Keratin 6B (UniprotKB database Accession No. P04259).

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Abstract

L'invention concerne un anticorps monoclonal apte à détecter, avec une sensibilité élevée et d'une manière quantitative, un peptide spécifique des maladies périodontiques dans un échantillon d'intérêt. La présente invention décrit : un anticorps monoclonal qui est produit grâce à un hybridome A17-7-8 représenté par le numéro d'enregistrement NITE BP-1530 ou un fragment de liaison à l'antigène de l'anticorps monoclonal ; et un anticorps monoclonal qui est produit par un hybridome JB29-3-7 représenté par le numéro d'enregistrement NITE BP-1531 ou un fragment de liaison à l'antigène de l'anticorps monoclonal.
PCT/JP2014/060531 2013-04-12 2014-04-11 Anticorps monoclonal contre un peptide spécifique des maladies périodontiques, et son utilisation WO2014168242A1 (fr)

Applications Claiming Priority (2)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3988563A3 (fr) * 2016-08-26 2022-07-27 Immatics Biotechnologies GmbH Nouveaux peptides et échafaudages destinés à être utilisés en immunothérapie contre le carcinome à cellules squameuses de la tête et du cou et d'autres cancers

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011115225A1 (fr) * 2010-03-17 2011-09-22 国立大学法人 鹿児島大学 Peptide spécifique d'une pathologie parodontale et traitement et diagnostic d'une pathologie parodontale l'incluant

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011115225A1 (fr) * 2010-03-17 2011-09-22 国立大学法人 鹿児島大学 Peptide spécifique d'une pathologie parodontale et traitement et diagnostic d'une pathologie parodontale l'incluant

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
EIJI ISHIKAWA ET AL., SOKUTEI KANDO, KOSO MEN'EKI SOKUTEIHO, 15 December 1982 (1982-12-15), pages 50 - 66 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3988563A3 (fr) * 2016-08-26 2022-07-27 Immatics Biotechnologies GmbH Nouveaux peptides et échafaudages destinés à être utilisés en immunothérapie contre le carcinome à cellules squameuses de la tête et du cou et d'autres cancers

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