WO2014168242A1

WO2014168242A1 - Monoclonal antibody against peptide specific to periodontal diseases, and use thereof

Info

Publication number: WO2014168242A1
Application number: PCT/JP2014/060531
Authority: WO
Inventors: 友宏吉村; 若本　裕晶
Original assignee: Jnc株式会社
Priority date: 2013-04-12
Filing date: 2014-04-11
Publication date: 2014-10-16

Abstract

A monoclonal antibody that can detect a peptide specific to periodontal diseases in a sample of interest with high sensitivity and quantitatively has been demanded. The present invention provides: a monoclonal antibody which is produced by a hybridoma A17-7-8 represented by Accession No. NITE BP-1530 or an antigen-binding fragment of the monoclonal antibody; and a monoclonal antibody which is produced by a hybridoma JB29-3-7 represented by Accession No. NITE BP-1531 or an antigen-binding fragment of the monoclonal antibody.

Description

Monoclonal antibody against periodontal disease specific peptide and use thereof

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.

Periodontal bacteria themselves and the toxic substances they produce, or leukocytes that resist periodontal bacteria, cytokines released from white blood cells, prostaglandins, and other substances enter the body and are transported to the body. 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.

In this way, 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.

There are more than 10 types of pathogens causing periodontal disease, but the most important pathogen is Porphyromonas gingivalis (Non-patent Document 1). 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.

There are several molecular species in the protease produced by P. gingivalis, but trypsin-like cysteine proteases (gingipain; HRgpA, RgpB, Kgp) are the main enzymes produced by this bacterium, and various studies have been conducted. (Non-Patent Documents 2 and 3). Through these studies, it is suggested that gingipain plays a major role in the maintenance and growth of P. gingivalis and the infection process, and we are trying to develop these protease inhibitors for the treatment and prevention of periodontal disease. Attempts to do this have been made vigorously (Patent Documents 1 to 4, Non-Patent Documents 4 to 7).

Keratin is a protein constituting an intermediate filament that is the cytoskeleton of epithelial cells. In keratinous tissues such as nails and hair, epithelial cells are filled with an intermediate filament made of special keratin called hard keratin, and die and harden. Even in non-keratinized epithelial cells such as mucous membranes, keratin (cytokeratin) 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.

Regarding the relationship between periodontal disease and periodontal bacterial enzymes and keratin, the existence of periodontal disease-specific peptides produced by digestion of keratin in the gingival epithelial tissue by periodontal bacterial enzymes has been clarified. It has been confirmed that the periodontal disease-specific peptide is contained in the interdental fluid and serum of patients more than in healthy subjects (Patent Document 5). Furthermore, 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. However, 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

In the above situation, a monoclonal antibody capable of detecting periodontal disease-specific peptide in a test sample with high sensitivity and quantitatively has been demanded.

As a result of intensive studies to provide a monoclonal antibody capable of detecting a periodontal disease-specific peptide in a test sample with high sensitivity and quantitatively, 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.

[1] A monoclonal antibody or an antigen-binding fragment thereof produced by a hybridoma A17-7-8 whose accession number is NITE BP-1530.
[2] A monoclonal antibody or an antigen-binding fragment thereof produced by the hybridoma JB29-3-7 whose accession number is NITE BP-1531.
[3] The antibody or antigen-binding fragment thereof according to [1] or [2] above, wherein the antibody or antigen-binding fragment thereof recognizes a periodontal disease-specific peptide.
[4] Hybridoma A17-7-8 whose accession number is represented by NITE BP-1530.
[5] Hybridoma JB29-3-7 whose accession number is represented by NITE BP-1531.
[6] 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.
[8] Confirmation of the presence of periodontal disease-specific peptides in cells or tissues using the antibody or antigen-binding fragment thereof according to any one of [1] to [3] above For immunocytochemistry or histochemical examination.
[9] 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.

It is a figure which shows the result of ELISA method (competition method) when the monoclonal antibody produced by hybridoma A17-7-8 is used (Example 2 (a)). It is a figure which shows the result of ELISA method (competition method) when the monoclonal antibody produced by hybridoma JB29-3-7 is used (Example 2 (a)). It is a figure which shows the result of ELISA method (competition method) when the monoclonal antibody produced by hybridoma A17-7-8 is used (Example 2 (b)). It is a figure which shows the result of ELISA method (competition method) when the monoclonal antibody produced by hybridoma JB29-3-7 is used (Example 2 (b)).

Hereinafter, the present invention will be described in detail.

1. Antibody of the Present Invention 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.

In a preferred embodiment of the present invention, 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 Bovine Keratin 5 (Q ), A peptide consisting of a partial amino acid sequence corresponding to peptide 1, or a variant or polymorphism of those peptides existing in nature (substitution, deletion, substitution, insertion and / or addition of 1-2 amino acids; example , Polymorphism 365 Ile is substituted with Val in peptide 1 (NCBI SNP database Accession No.rs437014)) are also included. In addition, since the cleavage site by Kgp may not always be exact, 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 κ 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.

2. Hybridoma of the present invention 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.

Another preferred embodiment of the 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.

3. Production of the Antibody of the Present Invention 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.

(1) Preparation of antigen Preparation of the antibody of the present invention 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.

As the natural polymer, for example, hemoglobin of mammals such as bovine, rabbit, human, and sheep, bovine serum albumin (BSA), or keyhole lymph hemocyanin (KLH) is used.

Examples of the synthetic polymer include various latexes such as polymers or copolymers of polyamino acids, polystyrenes, polyacryls, polyvinyls, polypropylenes, and the like.

As for the mixing ratio of the carrier and the hapten, 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. Usually, 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. For example, 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. In addition, 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. After introducing a maleimide group with a reagent, the reaction can be carried out. In addition, 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.

(2) Immunization of animals and preparation of antibody-producing cells 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. Examples of 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.

(3) Cell fusion Cell fusion between antibody-producing cells and myeloma cells is performed to obtain hybridomas. As 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. Next, 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. In addition, antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device utilizing electrical stimulation (for example, electroporation).

(4) Hybridoma selection and cloning process The target hybridoma is selected from the cells after cell fusion treatment. As a method, 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.

Next, the culture supernatant of the grown hybridoma is screened for the presence of antibodies that react with periodontal disease-specific peptides. 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.

(5) Collection of monoclonal antibody As a method for culturing the established hybridoma and collecting the monoclonal antibody from the obtained culture, a normal cell culture method, ascites formation method, or the like can be employed. “Culturing” means growing the hybridoma in a culture dish or culture bottle, or growing the hybridoma in the abdominal cavity of an animal.

For example, in the cell culture method, 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.

In the case of the ascites formation method, about 1 × 10 ⁷ 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.

In the above antibody collection method, when purification of the antibody is required, a known method such as ammonium sulfate salting out method, caprylic acid precipitation method, ion exchange chromatography, gel filtration, affinity chromatography is appropriately selected, Or it can refine | purify by combining these.

(6) Production of antigen-binding fragment An 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.

(7) Chimeric antibody, humanized antibody, or human antibody Some embodiments of the present invention include chimeric antibodies, humanized antibodies, or human antibodies.
When producing a chimeric antibody, 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.

When preparing humanized antibodies, complementarity determining regions (CDRs) are transplanted from mouse antibody V regions to human V regions, and framework regions (FR) are derived from human, Creates a reconstructed V region consisting of mouse-derived material. These humanized reshaped human V regions are then ligated to human constant regions. Methods for producing humanized antibodies are well known in the art.
A human antibody generally has the same structure as that of a human antibody in the hypervariable region (Hyper Variable region), which is an antigen-binding site of the V region, the rest of the V region, and the constant region. However, the hypervariable site may be derived from another animal. Techniques for producing human antibodies are also known, and methods for producing gene sequences common to humans by genetic engineering techniques have been established.

(8) Labeling of the antibody 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.

4. Use of the Antibody of the Present Invention 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.

(1) Method for Quantifying Periodontal Disease Specific Peptide 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). Examples of 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.

Any type of solution, colloidal solution, non-fluid sample, etc. can be used as the test sample. The 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.

(2) Immunohistochemical or cytochemical examination method of periodontal disease specific peptide The present invention confirms the presence of a periodontal disease specific peptide in a cell or tissue, using the antibody of the present invention. An immunocytochemical or histochemical test method is also provided.

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.

(3) Diagnostic Agent for Periodontal Disease 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.

When diagnosing periodontal disease, specifically, a 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.

And 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.

(4) 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.

It should be noted that all documents and publications described in this specification are incorporated herein by reference in their entirety regardless of their purposes. In addition, this specification is a disclosure of claims, description, and drawings of Japanese Patent Application No. 2013-84027 (filed on April 12, 2013), which is a Japanese patent application that is the basis for claiming priority of the present application. Includes content.

Hereinafter, the present invention will be described more specifically by way of examples. However, the present invention is not limited to these examples.

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.

(a) Preparation of immunogen Partial peptide A (12 peptide sequence YEELQITAGRHG (SEQ ID NO: 2) on the N-terminal side) and partial peptide B (11 peptide sequence GRHGDDLRNTK (C-terminal side) of the periodontal disease-specific peptide (SEQ ID NO: The BSA conjugate of 3)) was commissioned by Nippon Bioservice Co., Ltd. (purity 80% or more).

(b) Immunization For four 6-week-old female BALB / cAJcl mice, the antigen solution for immunization prepared in (a) above was mixed 1: 1 with Freund's incomplete adjuvant (Wako Pure Chemical Industries). The emulsion was injected intraperitoneally so that the periodontal disease-specific peptide was about 100 μg / animal. After that, booster immunization is performed twice every 2 weeks, and the antigen solution for immunization is mixed with ALUM (made by LSL) in 1: 1 and emulsified, resulting in a periodontal disease-specific peptide of about 100 μg / mouse. Prepared and injected intraperitoneally. Three days after the second boost, blood was collected from the tail vein, and the antibody titer in the serum was measured by the following ELISA method. For 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 μg / mouse. Three days later, spleen cells were prepared from the immunized mice and used for the following cell fusion.

(c) ELISA method (direct solid phase method)
A periodontal disease-specific peptide solution was prepared at 1 μg / ml in PBS and adsorbed to an ELISA plate at 50 μL / 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. After washing with PBS-T three times, the serum obtained in (b) above was diluted 5-fold with a serum dilution solution (phosphate buffered saline), added 100 μL / well, and incubated at 37 ° C for 1 hour. Reacted. The plate was washed 4 times with PBS-T, HRP-labeled mouse IgG antibody (manufactured by Invitrogen) diluted 5000 times was added at 100 μL / 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 μL / well and allowed to react at room temperature for 12 minutes. Thereafter, 50 μL of 1N H ₂ SO ₄ was added to stop the reaction, and the absorbance at 450 nm was measured.

(d) Cell fusion, hybridoma preparation The spleen was removed 3 days after the final immunization from 2 mice that showed an increase in antibody titer against periodontal disease-specific peptide as a result of the above ELISA method, and splenocytes were obtained by a conventional method. Was prepared. Myeloma SP2 cells derived from BALB / cAJcl mice confirmed to be hypoxanthine / guanine / phosphoribosyltransferase (HGPRT) -deficient strains, which were selected in advance in a medium containing 20 μg / mL 8-azaguanine. The strain was used. SP2 cells 1 × 10 combined ⁸ splenocytes 1 × 10 ^8, and polyethylene glycol 4000; the (PEG 4000 Merck Co.) was used as a cell fusion promoter, were performed cell fusion according to a conventional method. 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 μL / 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.

(e) Screening for hybridomas Wells confirmed to form colonies were screened by the same ELISA method as shown in (c) above to confirm the presence or absence of antibodies against periodontal disease-specific peptides in the culture supernatant. And colonies that strongly reacted with the periodontal disease-specific peptide were selected and subjected to cloning.

(f) Cloning of hybridomas 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.

(g) Monoclonal antibody typing Using 0.5 mL of the culture supernatant of the two hybridoma A17-7-8 cell lines and the hybridoma JB29-3-7 cell line obtained above, Pierce Rapid Isotyping Kit (Thermo Fisher Scientific The isotype was examined using the same product. The isotypes of the monoclonal antibodies produced by the two hybridoma A17-7-8 cell lines and JB29-3-7 cell line were both IgG for the H chain and κ for the L chain.

(h) Preparation and purification of monoclonal antibody The two hybridoma A17-7-8 cell lines and hybridoma JB29-3-7 cell line obtained by cloning in (f) above were cultured in 100 mL of RPMI-1640 medium, respectively. . After recovering the culture solution, it was centrifuged at 1,400 rpm for 5 minutes, and the supernatant was recovered. Next, each monoclonal antibody was purified from the obtained supernatant using rProtein A Sepharose Fast Flow 2 (GE). The absorbance at 280 nm of this antibody solution was measured to measure the antibody concentration.

(i) HRP labeling of monoclonal antibody 200 μg of each monoclonal antibody prepared in (h) above was labeled with HRP using Peroxidase Labeling Kit-NH2 (manufactured by Dojindo Laboratories).

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 μg / mL with PBS and adsorbed to an ELISA plate at 50 μl / 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.

Periodontal disease-specific peptide was diluted with PBS to an appropriate concentration (0 to 1,000 ng / mL), and 100 μL of this was added. Subsequently, 100 μL 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 μL / well and allowed to react at room temperature for 12 minutes. Thereafter, 50 μL 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, and FIG. 2 shows the results when using the antibody produced by hybridoma JB29-3-7.

In the figure, 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)

As is clear from this result, in FIGS. 1 and 2, linearity was obtained in the periodontal disease-specific peptide concentration range of 5 to 1000 ng / mL. That is, if the periodontal disease-specific peptide concentration is within this range, the sample can be quantified.

(B) Measurement of periodontal disease-specific peptides in blood (competitive method)
Periodontal disease-specific peptides were diluted to an appropriate concentration (0 to 2,000 ng / mL) with normal human serum (manufactured by Innovative Reserch), and further diluted 2-fold with a serum dilution solution (phosphate buffered saline). 100 μL of each of these was added to the ELISA plate, and then 100 μL 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 μL / well and allowed to react at room temperature for 12 minutes. Thereafter, 50 μL of 1N H ₂ SO ₄ was added to stop the reaction, and the absorbance at 450 nm was measured.

FIG. 3 shows the results when using the antibody produced by hybridoma A17-7-8, and FIG. 4 shows the results when using the antibody produced by hybridoma JB29-3-7.

As is clear from FIGS. 3 and 4, linearity was obtained when the periodontal disease-specific peptide concentration was in the range of 10 to 1000 ng / mL. That is, since the serum is diluted 2-fold at the time of measurement, the specimen can be quantified if the periodontal disease-specific peptide concentration in the blood is in the range of 20 to 2000 ng / mL.

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.

Therefore, it can be seen that the antibody of the present invention can detect periodontal disease-specific peptides in the blood with higher sensitivity than conventional antibodies.

As shown in the above examples, the present invention provides a monoclonal antibody that specifically binds to a periodontal disease-specific peptide. By using 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).

Claims

A monoclonal antibody produced by hybridoma A17-7-8 represented by the deposit number NITE BP-1530 or an antigen-binding fragment thereof.
A monoclonal antibody produced by the hybridoma JB29-3-7 whose accession number is NITE 受 BP-1531 or an antigen-binding fragment thereof.
3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof recognizes a periodontal disease-specific peptide.
Hybridoma A17-7-8 whose accession number is represented by NITE BP-1530.
* Hybridoma JB29-3-7 whose accession number is represented by NITE BP-1531.
A method for quantifying periodontal disease-specific peptides contained in a test sample, comprising using the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.
The method according to claim 6, wherein the quantification method is performed in an ELISA system.
An immunocytochemistry for confirming the presence of a periodontal disease-specific peptide in a cell or tissue, characterized by using the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3. Histochemical examination method.
A diagnostic agent for periodontal disease comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.
Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 in the manufacture of a diagnostic agent for periodontal disease.