WO2022172901A1 - Anti-myoglobin monoclonal antibody or antigen-binding fragement thereof, method for detecting myoglobin, kit, and polypeptide - Google Patents

Anti-myoglobin monoclonal antibody or antigen-binding fragement thereof, method for detecting myoglobin, kit, and polypeptide Download PDF

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WO2022172901A1
WO2022172901A1 PCT/JP2022/004739 JP2022004739W WO2022172901A1 WO 2022172901 A1 WO2022172901 A1 WO 2022172901A1 JP 2022004739 W JP2022004739 W JP 2022004739W WO 2022172901 A1 WO2022172901 A1 WO 2022172901A1
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amino acid
myoglobin
seq
monoclonal antibody
acid sequence
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French (fr)
Japanese (ja)
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ゆき 若林
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デンカ株式会社
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies

Definitions

  • the present invention provides anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof, methods and kits for detecting myoglobin using the anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof, and anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof. It relates to recognized polypeptides.
  • Myoglobin is a heme protein with a molecular weight of about 17 kDa that is mainly present in cardiac and skeletal muscles.
  • myoglobin In muscle tissue, myoglobin has the function of receiving oxygen carried by hemoglobin in red blood cells, transporting and storing it, and supplying it to the energy production system.
  • Myoglobin is also present at basal levels in the blood of healthy individuals. When muscle cells are damaged, myoglobin in the muscle cells escapes outside the cells, flows into the blood within several hours, and then is excreted in the urine. When muscle cells are damaged, the myoglobin concentration in the blood rises several times to several tens of times higher than the basal level (concentration) of a healthy person.
  • the concentration of myoglobin in blood is used as a diagnostic marker for damage in the very early stages (0.5 to 10 hours after injury) after skeletal muscle injury.
  • Diseases for which blood myoglobin concentration can be used as a diagnostic marker include myocardial disorders (myocardial infarction or myocarditis), skeletal muscle diseases (muscular dystrophy, dermatomyositis, polymyositis, or rhabdomyolysis), thyroid Hypofunction, malignant hyperthermia, renal failure and the like. Blood myoglobin concentration is also used as a prognostic monitor after cardiac surgery.
  • the blood concentration of myoglobin is measured by an immunoassay method that utilizes an antigen-antibody reaction.
  • immunosorbent test method ELISA method
  • CLIA method chemiluminescence enzyme immunoassay method
  • homogeneous methods that do not involve B/F separation in the measurement process (eg, latex agglutination method).
  • Anti-human myoglobin polyclonal antibodies obtained from immune hosts other than humans have been used to measure myoglobin blood levels by these immunoassays.
  • the anti-human myoglobin polyclonal antibody has a problem of low reproducibility between lots (Non-Patent Document 1).
  • the present invention has been made in view of the above problems, and includes an anti-myoglobin monoclonal antibody or an antigen-binding fragment thereof suitable for immunoassay, and detection of myoglobin using the anti-myoglobin monoclonal antibody or antigen-binding fragment thereof. It is an object of the present invention to provide methods and kits for the detection and polypeptides recognized by said anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof.
  • epitopes 1 to 5 in FIG. 1 As a result, it was found that a specific region in the amino acid sequence of human myoglobin has strong antigenicity and that this region is an epitope (indicated as epitopes 1 to 5 in FIG. 1) effective for the formation of immune complexes. I completed the present invention.
  • the anti-myoglobin monoclonal antibody or antigen-binding fragment thereof comprises (a) positions 78 to 104, (b) positions 112 to 130, and (c) in the myoglobin amino acid sequence shown in SEQ ID NO: 1. Binds to the 141st to 154th amino acid region, (d) 2nd to 18th amino acid region, or (e) 35th to 64th amino acid region.
  • the anti-myoglobin monoclonal antibody or antigen-binding fragment thereof comprises (a) positions 79 to 103, (b) positions 117 to 127, (c) positions 144 to 154, (d) in the myoglobin amino acid sequence shown in SEQ ID NO: 1.
  • a method for detecting myoglobin in a sample comprises detecting myoglobin in a sample by immunoassay using two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof. and the two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof are (a) an anti-myoglobin monoclonal antibody that binds to the amino acid region from 78 to 104 in the amino acid sequence shown in SEQ ID NO: 1, (b) SEQ ID NO: 1, (c) an anti-myoglobin monoclonal antibody that binds to the 141st to 154th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1, ( d) an anti-myoglobin monoclonal antibody that binds to the 2nd to 18th amino acid regions in the amino acid sequence shown by SEQ ID NO: 1; Two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof selected from the two or more anti-myo
  • a kit for detecting myoglobin comprises two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof, and the two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof are ( a) an anti-myoglobin monoclonal antibody that binds to the 78th to 104th amino acid region in the amino acid sequence shown by SEQ ID NO: 1, (b) an anti-myoglobin that binds to the 112th to 130th amino acid region in the amino acid sequence shown by SEQ ID NO: 1 A monoclonal antibody, (c) an anti-myoglobin monoclonal antibody that binds to the 141st to 154th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1, (d) to the 2nd to 18th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1 Two or more anti-myoglobin monoclonal antibodies selected from the group consisting of binding anti-myo
  • the two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof (a) bind to the 79th to 103rd amino acid regions in the amino acid sequence shown in SEQ ID NO: 1.
  • an anti-myoglobin monoclonal antibody (b) an anti-myoglobin monoclonal antibody that binds to the 117th to 127th amino acid region in the amino acid sequence shown in SEQ ID NO: 1, c) the 144th to 154th amino acid region in the amino acid sequence shown in SEQ ID NO: 1 (d) an anti-myoglobin monoclonal antibody that binds to the 2nd to 12th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1, and (e) 38 to 38 in the amino acid sequence shown in SEQ ID NO: 1
  • Two or more anti-myoglobin monoclonal antibodies selected from the group consisting of anti-myoglobin monoclonal antibodies that bind to the 60th amino acid region or antigen-binding fragments thereof, (a) in the amino acid sequence shown in SEQ ID NO: 1
  • An anti-myoglobin monoclonal antibody that binds to the 79th to 85th and 92nd to 99th amino acid regions (b)
  • an anti-myoglobin monoclonal antibody that binds to the 78th to 104th amino acid region in the amino acid sequence shown in SEQ ID NO: 1
  • the 112th to 130th amino acid region in the amino acid sequence shown in SEQ ID NO: 1 and
  • two or more anti-myoglobin monoclonal antibodies selected from the group consisting of an anti-myoglobin monoclonal antibody that binds to the 141st to 154th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1, or Even if it is an antigen-binding fragment thereof good.
  • a polypeptide according to one aspect of the present invention consists of an amino acid sequence represented by any one of SEQ ID NOs: 2-6.
  • the polypeptide may consist of the amino acid sequence shown in any one of SEQ ID NOS:4-6.
  • the anti-myoglobin monoclonal antibody or antigen-binding fragment thereof recognizes an epitope that is effective in forming immune complexes. Therefore, according to the present invention, an anti-myoglobin monoclonal antibody or antigen-binding fragment thereof suitable for immunological assays is provided.
  • the present invention also provides a method and kit for detecting myoglobin using the anti-myoglobin monoclonal antibody or antigen-binding fragment thereof, and a polypeptide recognized by the anti-myoglobin monoclonal antibody or antigen-binding fragment thereof. be done.
  • FIG. 2 is a graph showing the antigenicity score of human myoglobin.
  • FIG. FIG. 10 is a graph showing that when mice were immunized with peptides A to C, the blood antibody titers of mice increased.
  • FIG. 4 is a sensorgram showing that anti-myoglobin monoclonal antibodies generated using peptides A to C as immunogens can bind to the same myoglobin molecule and form immune complexes without structurally competing with each other.
  • An anti-myoglobin monoclonal antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof that specifically binds to myoglobin.
  • the anti-myoglobin monoclonal antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof that binds to any of epitopes 1 to 5 shown in FIG. 1 (that is, recognizes any of epitopes 1 to 5). be.
  • FIG. 1 is a graph showing the antigenicity score of human myoglobin shown in SEQ ID NO:1. Epitopes 1 to 5 shown in FIG. 1 are highly antigenic regions in human myoglobin and are effective epitopes for the formation of immune complexes.
  • the anti-myoglobin monoclonal antibody or antigen-binding fragment thereof has, in the myoglobin amino acid sequence shown in SEQ ID NO: 1, (a) amino acid regions 78-104 (corresponding to epitope 3), preferably 79-103, more preferably 79-85 and 92-99, (b) amino acid regions 112-130 (corresponding to epitope 4), preferably 117-127, more preferably 118-126, (c) 141st to 154th amino acid region (corresponding to epitope 5), preferably 144th to 154th, more preferably 145th to 154th amino acid region, (d) 2nd to 18th (corresponding to epitope 1), preferably 2nd to 12th, more preferably 2nd to 7th amino acid region, or (e) 35th to 64th (corresponding to epitope 2), preferably 38 It binds to an amino acid region of ⁇ 60th, more preferably 42nd to 55th.
  • the anti-myoglobin monoclonal antibody or antigen-binding fragment thereof is, for example, (a) 78th to 104th, (b) 112th to 130th, or (c) 141st to 154th in the myoglobin amino acid sequence shown in SEQ ID NO: 1. may bind to the amino acid region of
  • the term “specifically” means that in a liquid system containing myoglobin, a protein component other than myoglobin, and an anti-myoglobin monoclonal antibody according to this aspect, the anti-myoglobin monoclonal antibody is combined with a protein component other than myoglobin. It means that no antigen-antibody reaction occurs at a detectable level, or that even if some binding reaction or association reaction does occur, it only causes a reaction that is clearly weaker than the antigen-antibody reaction of the anti-myoglobin monoclonal antibody with myoglobin. .
  • myoglobin specifically refers to human myoglobin.
  • the anti-myoglobin monoclonal antibody "binds to the mn-th region in the amino acid sequence" means that the anti-myoglobin monoclonal antibody binds to one or more arbitrary positions in the mn-th region of the amino acid sequence. It means to bind, but does not necessarily mean to bind throughout the m to n th region of the amino acid sequence.
  • an anti-myoglobin monoclonal antibody "binds to the 78th to 104th amino acid regions in the myoglobin amino acid sequence shown in SEQ ID NO: 1" means that the anti-myoglobin monoclonal antibody binds to any one or more (eg, 79th to 85th and 92nd to 99th amino acids), but does not necessarily mean binding to all of the 78th to 104th amino acids.
  • the class of anti-myoglobin monoclonal antibodies is not limited to IgG, but may be IgY, IgM, camelid Ig, or Ig NAR.
  • the antigen-binding fragment thereof is not particularly limited, and may be Fab, Fab', F(ab') 2 or a single chain antibody (scFv).
  • a method for producing an anti-myoglobin monoclonal antibody is not particularly limited.
  • a hybridoma produced by immunizing an animal with a full-length myoglobin molecule or a partial peptide thereof using a known immunological technique and using cells of the immunized animal. can be obtained from Alternatively, anti-myoglobin monoclonal antibodies can be produced as recombinant antibodies using gene recombination technology.
  • the length of the peptide used for immunization is not particularly limited, but is preferably 5 amino acids or more, more preferably 10 amino acids or more, and still more preferably 13 amino acids or more.
  • Peptides are degraded in vivo by antigen-presenting cells and only a portion thereof is presented as an antigen. Therefore, peptides used for immunization are epitopes 1 to 5 shown in FIG. preferably a peptide containing amino acids in the immediately preceding and/or immediately following region).
  • a peptide used for immunization may be, for example, a polypeptide according to another aspect of the present invention, which will be described later.
  • Myoglobin used as an immunogen may be obtained, for example, from a human biological sample derived from blood or urine, or may be obtained by introducing a plasmid vector incorporating DNA encoding a myoglobin protein into a host cell for expression. good.
  • the myoglobin molecule or its partial peptide used as an immunogen may be fused with other proteins. That is, a myoglobin molecule or a partial peptide thereof may be expressed as a fusion protein with another protein and used as an immunogen after or without purification.
  • a carrier protein such as keyhole limpet hemocyanin (KLH) may be conjugated to the peptide.
  • GST glutathione S-transferase
  • MBP maltose binding protein
  • TRX thioredoxin
  • Nus tag S tag, HSV tag, FLRAG tag, polyhistidine tag, Strep tag, Strep-II tag
  • Proteins commonly used as protein expression and/or purification tags such as Myc tag, HA tag, V5 tag, E tag, T7 tag, VSV-G tag, Glu-Glu tag, and Avi tag may be used, These tags are preferably cleaved using digestive enzymes after expression of the fusion protein.
  • An anti-myoglobin monoclonal antibody can be easily prepared from an immunized animal by the well-known method of Keller et al. (Kohler, et al. Nature. 1975; 256: 495-497). That is, antibody-producing cells such as splenocytes and lymphocytes are recovered from the immunized animal, the antibody-producing cells are fused with mouse myeloma cells to prepare hybridomas, and the hybridomas are cloned by limiting dilution or the like.
  • An anti-myoglobin monoclonal antibody can be prepared by selecting a monoclonal antibody that causes an antigen-antibody reaction with myoglobin from the monoclonal antibodies produced by each hybridoma.
  • Cultivation of hybridomas can be carried out in the ascites fluid or culture medium of animals such as mice.
  • a known immunoglobulin purification method can be used to purify the anti-myoglobin monoclonal antibody from ascites fluid or culture supernatant. For example, fractionation by salting out using ammonium sulfate or sodium sulfate, polyethylene glycol (PEG) fractionation, ethanol fractionation, DEAE ion exchange chromatography, gel filtration and the like can be mentioned.
  • the anti-myoglobin monoclonal antibody is purified by affinity chromatography using a carrier to which protein A, protein G, or protein L is bound, depending on the animal species to be immunized and the class of the anti-myoglobin monoclonal antibody. good too.
  • a method for detecting myoglobin in a sample according to one aspect of the present invention comprises two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof according to the above aspect of the present invention, ie epitopes 1 to 5 shown in FIG. detecting myoglobin in a sample by an immunoassay using two or more of the anti-myoglobin monoclonal antibodies that bind to any of
  • the method for detecting myoglobin in a sample is an immunological assay using two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof.
  • detecting myoglobin wherein two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof are (a) in the amino acid sequence shown by SEQ ID NO: 1, anti-binding to the 78th to 104th (corresponding to epitope 3), preferably 79th to 103rd, more preferably 79th to 85th and 92nd to 99th amino acid regions myoglobin monoclonal antibody, (b) an anti-myoglobin monoclonal antibody that binds to the 112th to 130th (corresponding to epitope 4), preferably 117th to 127th, more preferably 118th to 126th amino acid region in the amino acid sequence shown by SEQ ID NO: 1; (c) an anti-myoglobin monoclonal antibody that bind
  • the two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof are, for example, (a) an anti-myoglobin monoclonal antibody that binds to the 78th to 104th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1, (b) SEQ ID NO: from an anti-myoglobin monoclonal antibody that binds to the 112th to 130th amino acid regions in the amino acid sequence shown by SEQ ID NO: 1, and (c) an anti-myoglobin monoclonal antibody that binds to the 141st to 154th amino acid regions in the amino acid sequence shown by SEQ ID NO: Two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof selected from the group consisting of:
  • anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof For detection of myoglobin, 2 or more, 3 or more, 4 or more, or 5 anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof can be used, preferably 2 to 5, more preferably 2 ⁇ 3 of the above anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof are used.
  • Samples include, for example, human or non-human animal blood, serum, plasma, urine, semen, cerebrospinal fluid, saliva, sweat, tears, ascites, amniotic fluid and other bodily fluids; mucus; feces; organs such as blood vessels and liver; Examples include biological samples that may contain myoglobin, such as tissues; cells; or extracts thereof.
  • the sample is preferably blood (whole blood, plasma, or serum) or urine, which is easy to collect.
  • a method for collecting a sample is not particularly limited, and a known method can be adopted.
  • the immunological measurement methods include known immunological measurement methods such as the competitive method, agglutination method, Western blot method, immunostaining method, and sandwich method, with the sandwich method being preferred.
  • the sandwich method can be performed by techniques such as immunochromatography, ELISA, latex agglutination, and CLEIA.
  • a kit for detecting myoglobin according to one aspect of the present invention comprises two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof used in the myoglobin detection method according to the above aspect of the present invention. Details of the two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof are as described above.
  • the kit can further include known reagents, materials, instruments, etc. used in techniques such as immunochromatography, ELISA, latex agglutination, and CLEIA.
  • Polypeptide recognized by anti-myoglobin monoclonal antibody provides a polypeptide recognized by an anti-myoglobin monoclonal antibody or antigen-binding fragment thereof according to the above aspects of the invention.
  • Polypeptides include part or all of any of epitopes 1-5 shown in FIG. More specifically, the polypeptide comprises at least (a) positions 79 to 85 and 92 to 99, preferably 79 to 103, more preferably 78 to 104 in the myoglobin amino acid sequence shown in SEQ ID NO: 1.
  • the polypeptide is, for example, in the myoglobin amino acid sequence shown in SEQ ID NO: 1, (a) 75th to 100th (SEQ ID NO: 4), (b) 112-131 (SEQ ID NO: 5), (c) 133-154 (SEQ ID NO: 6), (d) 2nd to 18th (SEQ ID NO: 2), or (e) 35th to 64th (SEQ ID NO: 3) may be a peptide consisting of an amino acid region, and is represented by any one of SEQ ID NOS: 4 to 6 It may be a peptide consisting of an amino acid sequence.
  • a polypeptide according to this aspect can be used, for example, as an immunogen for producing anti-human myoglobin monoclonal antibodies by immunological techniques.
  • the polypeptide is degraded by antigen-presenting cells in vivo and only a portion thereof is presented as an antigen. Therefore, the polypeptide is a peptide obtained by adding one or more extra amino acids to epitopes 1 to 5 shown in FIG. 1 (for example, in addition to epitopes 1 to 5 in FIG. /or a peptide containing the amino acids in the immediately following region).
  • Polypeptides may be conjugated to carrier proteins such as KLH.
  • polypeptide according to this aspect can be used together with the anti-myoglobin monoclonal antibody or antigen-binding fragment thereof according to the above aspect to detect myoglobin by an immunological competition method.
  • one or more polypeptides according to the present aspect are immobilized on a substrate, and a sample that may contain myoglobin and a labeled anti-myoglobin monoclonal antibody according to the aspect or an antigen-binding fragment thereof are prepared as described above. By contacting the substrate, myoglobin can be competitively detected.
  • another aspect of the present invention is to detect myoglobin in a sample by an immunological competition method using the polypeptide and the anti-myoglobin monoclonal antibody or antigen-binding fragment thereof. It can also be said to provide a method for detecting myoglobin and a kit for detecting myoglobin comprising the polypeptide and the anti-myoglobin monoclonal antibody or antigen-binding fragment thereof.
  • Peptides A to C shown in Table 1 were synthesized and administered to BALB/c mice as antigens, respectively.
  • Peptides AC are conjugates of peptides having amino acid sequences shown in SEQ ID NOs: 4-6 and KLH, respectively.
  • the antigen was administered 5 times at 2-week intervals, and blood was collected in the week following the administration.
  • Antibody titers in the resulting plasma were measured by ELISA. Specifically, first, a plate on which human myoglobin having the amino acid sequence shown by SEQ ID NO: 1 was immobilized was blocked by a known method, and washed with a wash buffer (0.05% Tween 20-containing PBS) at 6000 times.
  • 20,000-fold diluted plasma was added and allowed to react at room temperature for 2 hours.
  • an enzyme-labeled anti-mouse IgG antibody appropriately diluted with a wash buffer was added and allowed to react at room temperature for 1 hour, followed by color development reaction using a substrate solution.
  • the absorbance at 450 nm and 620 nm was measured with a plate reader, and the antibody titer was obtained from the difference between the absorbance at 450 nm and the absorbance at 620 nm.
  • FIG. 2 Graphs (A), (B), and (C) in FIG. 2 show plasma antibody titers in mice to which peptides A, B, and C were administered, respectively. As shown in FIG. 2, it was confirmed that the antibody titer against human myoglobin increased according to the administration frequency of peptides A to C.
  • Antibody-producing B cells were obtained from mice immunized with any of peptides AC in Example 1. This antibody-producing B was fused with myeloma cells to obtain an immortal antibody-producing hybridoma. The hybridoma was administered intraperitoneally to another mouse, and the anti-myoglobin monoclonal antibody produced by the hybridoma was obtained from the ascites fluid of the mouse.
  • the anti-myoglobin monoclonal antibodies obtained using peptides A to C as immunogens are referred to as antibodies A to C, respectively. It was confirmed by the surface plasmon resonance (SPR) method that antibodies A to C bind to the same myoglobin molecule to form immune complexes without structurally competing with each other.
  • SPR surface plasmon resonance
  • a sensor chip (Series S Sensor Chip CM5, manufactured by Cytiva) on which about 7000 RU of antibody A is immobilized by an amine coupling method using Biacore (registered trademark) 8K (manufactured by Cytiva), 500 nM myoglobin (analyte 1) and 500 nM antibody B (analyte 2) were added until the sensorgram was saturated, and finally, as analyte 3, any of antibodies A to C (500 nM), buffer (0. 01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, and 0.05% Tween 20) or normal mouse IgG were added.
  • FIG. 3 The resulting sensorgram is shown in Figure 3.
  • (A) is the entire sensorgram
  • (B) is an enlarged view of the sensorgram when analyte 3 was added.
  • (B) only when antibody C was added as analyte 3, a response greater than the signal when analyte 2 was added was shown. This result confirms that antibodies A, B, and C bind to the same myoglobin molecule without structurally competing with each other.

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Abstract

The present invention provides: an anti-myoglobin monoclonal antibody or antigen-binding fragment thereof suitable for immunological measurement; a method and kit for detecting myoglobin by using said anti-myoglobin monoclonal antibody or antigen-binding fragment thereof; and a polypeptide recognized via said anti-myoglobin monoclonal antibody or antigen-binding fragment thereof. An anti-myoglobin monoclonal antibody or antigen-binding fragment thereof according to one aspect of the present invention binds to the (a) 78th to 104th, (b) 112th to 130th, (c) 141th to 154th, (d) 2nd to 18th, or (e) 35th to 64th amino acid region.

Description

抗ミオグロビンモノクローナル抗体又はその抗原結合性断片、ミオグロビンを検出する方法、キット及びポリペプチドAnti-myoglobin monoclonal antibody or antigen-binding fragment thereof, method, kit and polypeptide for detecting myoglobin
　本発明は、抗ミオグロビンモノクローナル抗体又はその抗原結合性断片、該抗ミオグロビンモノクローナル抗体又はその抗原結合性断片を用いてミオグロビンを検出する方法及びキット、並びに該抗ミオグロビンモノクローナル抗体又はその抗原結合性断片によって認識されるポリペプチドに関する。 The present invention provides anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof, methods and kits for detecting myoglobin using the anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof, and anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof. It relates to recognized polypeptides.
　ミオグロビン（Ｍｂ）は、主に心筋や骨格筋に存在する、分子量約１７ｋＤａのヘムタンパク質である。筋組織において、ミオグロビンは、赤血球中のヘモグロビンにより運ばれてきた酸素を受けとって運搬及び貯蔵し、エネルギー産生系に供給する機能を担っている。また、ミオグロビンは、健常人の血中にも基底レベルの濃度で存在している。筋細胞が損傷を受けると、筋細胞内のミオグロビンは細胞外へと逸脱し、数時間で血中へ流入した後、尿中に排泄される。筋細胞が損傷を受けたときの血中のミオグロビン濃度は、健常人の基底レベル（濃度）の数倍～数十倍に上昇する。このため、血中のミオグロビン濃度は、骨格筋損傷後の極早期（損傷から０．５～１０時間後）における損傷の診断マーカーとして用いられている。血中のミオグロビン濃度を診断マーカーとして用いることができる疾患としては、心筋障害（心筋梗塞又は心筋炎）、骨格筋疾患（筋ジストロフィー症、皮膚筋炎、多発性筋炎、又は横紋筋融解症）、甲状腺機能低下症、悪性高熱症、腎不全等が挙げられる。血中のミオグロビン濃度は、心臓手術後の予後モニターとしても用いられている。 Myoglobin (Mb) is a heme protein with a molecular weight of about 17 kDa that is mainly present in cardiac and skeletal muscles. In muscle tissue, myoglobin has the function of receiving oxygen carried by hemoglobin in red blood cells, transporting and storing it, and supplying it to the energy production system. Myoglobin is also present at basal levels in the blood of healthy individuals. When muscle cells are damaged, myoglobin in the muscle cells escapes outside the cells, flows into the blood within several hours, and then is excreted in the urine. When muscle cells are damaged, the myoglobin concentration in the blood rises several times to several tens of times higher than the basal level (concentration) of a healthy person. Therefore, the concentration of myoglobin in blood is used as a diagnostic marker for damage in the very early stages (0.5 to 10 hours after injury) after skeletal muscle injury. Diseases for which blood myoglobin concentration can be used as a diagnostic marker include myocardial disorders (myocardial infarction or myocarditis), skeletal muscle diseases (muscular dystrophy, dermatomyositis, polymyositis, or rhabdomyolysis), thyroid Hypofunction, malignant hyperthermia, renal failure and the like. Blood myoglobin concentration is also used as a prognostic monitor after cardiac surgery.
　ミオグロビンの血中濃度は、抗原抗体反応を利用した免疫学的測定法により測定され、免疫学的測定法としては、測定工程にＢ／Ｆ分離を含む方法（例えば、放射免疫測定法、酵素結合免疫吸着検査法（ＥＬＩＳＡ法）、又は化学発光酵素免疫測定法（ＣＬＥＩＡ法））、及び測定工程にＢ／Ｆ分離を含まないホモジニアスな方法（例えば、ラテックス凝集法）が挙げられる。これら免疫学的測定法によるミオグロビンの血中濃度の測定には、ヒト以外の免疫宿主から得た抗ヒトミオグロビンポリクローナル抗体が用いられてきた。しかしながら、抗ヒトミオグロビンポリクローナル抗体は、ロット間の再現性が低いという問題があった（非特許文献１）。 The blood concentration of myoglobin is measured by an immunoassay method that utilizes an antigen-antibody reaction. immunosorbent test method (ELISA method), or chemiluminescence enzyme immunoassay method (CLEIA method)), and homogeneous methods that do not involve B/F separation in the measurement process (eg, latex agglutination method). Anti-human myoglobin polyclonal antibodies obtained from immune hosts other than humans have been used to measure myoglobin blood levels by these immunoassays. However, the anti-human myoglobin polyclonal antibody has a problem of low reproducibility between lots (Non-Patent Document 1).
　上記ロット間の再現性の問題は、抗ミオグロビンモノクローナル抗体を用いることで解決される。しかしながら、免疫複合体の形成に効果的なミオグロビン分子上のエピトープは、従来解明されておらず、免疫学的測定法に適した抗ミオグロビンモノクローナル抗体は、存在していなかった。 The above lot-to-lot reproducibility issue is resolved by using an anti-myoglobin monoclonal antibody. However, an epitope on the myoglobin molecule that is effective for immune complex formation has not been elucidated, and an anti-myoglobin monoclonal antibody suitable for immunoassays has not existed.
　本発明は上記課題に鑑みてなされたものであり、免疫学的測定法に適した抗ミオグロビンモノクローナル抗体又はその抗原結合性断片、該抗ミオグロビンモノクローナル抗体又はその抗原結合性断片を用いてミオグロビンを検出する方法及びキット、並びに該抗ミオグロビンモノクローナル抗体又はその抗原結合性断片によって認識されるポリペプチドを提供することを目的とする。 The present invention has been made in view of the above problems, and includes an anti-myoglobin monoclonal antibody or an antigen-binding fragment thereof suitable for immunoassay, and detection of myoglobin using the anti-myoglobin monoclonal antibody or antigen-binding fragment thereof. It is an object of the present invention to provide methods and kits for the detection and polypeptides recognized by said anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof.
　これまでに抗原性を予測するアルゴリズムが複数報告されている（例えば、Chou PY, Fasman GD. 1978. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol 47:45-148）。しかしながら、これらのアルゴリズムは、互いに相反する指標を含んでいることから、個々のアルゴリズムでは抗原性予測の精度に乏しいという問題点があった。そこで、本発明者らは、これらのアルゴリズムの統合及び改良を行い、抗原性予測の精度を向上し得るアルゴリズムを見いだした。そして、本発明者らは、このアルゴリズムを用いて、配列番号１で示されるヒトミオグロビンのアミノ酸配列における各アミノ酸の抗原性の強さをスコア化した。この抗原性スコアを図１に示す。その結果、ヒトミオグロビンのアミノ酸配列における特定の領域において抗原性が強く、当該領域が免疫複合体の形成に効果的なエピトープ（図１中、エピトープ１～５として示す。）であることを見いだし、本発明を完成させた。 Several algorithms for predicting antigenicity have been reported so far (e.g., Chou PY, Fasman GD. 1978. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol 47:45-148 ). However, since these algorithms contain mutually contradictory indicators, there is a problem that individual algorithms have poor accuracy in predicting antigenicity. Therefore, the present inventors integrated and improved these algorithms and found an algorithm capable of improving the accuracy of antigenicity prediction. Using this algorithm, the present inventors scored the strength of antigenicity of each amino acid in the human myoglobin amino acid sequence shown in SEQ ID NO:1. This antigenicity score is shown in FIG. As a result, it was found that a specific region in the amino acid sequence of human myoglobin has strong antigenicity and that this region is an epitope (indicated as epitopes 1 to 5 in FIG. 1) effective for the formation of immune complexes. I completed the present invention.
　本発明の一側面に係る抗ミオグロビンモノクローナル抗体又はその抗原結合性断片は、配列番号１で示されるミオグロビンのアミノ酸配列における、（ａ）７８～１０４番目、（ｂ）１１２～１３０番目、（ｃ）１４１～１５４番目、（ｄ）２～１８番目、又は（ｅ）３５～６４番目のアミノ酸領域に結合する。抗ミオグロビンモノクローナル抗体又はその抗原結合性断片は、配列番号１で示されるミオグロビンのアミノ酸配列における、（ａ）７９～１０３番目、（ｂ）１１７～１２７番目、（ｃ）１４４～１５４番目、（ｄ）２～１２番目、又は（ｅ）３８～６０番目のアミノ酸領域に結合してもよく、配列番号１で示されるミオグロビンのアミノ酸配列における、（ａ）７９～８５番目及び９２～９９番目、（ｂ）１１８～１２６番目、（ｃ）１４５～１５４番目、（ｄ）２～７番目、又は（ｅ）４２～５５番目のアミノ酸領域に結合してもよく、配列番号１で示されるミオグロビンのアミノ酸配列における、（ａ）７８～１０４番目、（ｂ）１１２～１３０番目、又は（ｃ）１４１～１５４番目のアミノ酸領域に結合してもよい。 The anti-myoglobin monoclonal antibody or antigen-binding fragment thereof according to one aspect of the present invention comprises (a) positions 78 to 104, (b) positions 112 to 130, and (c) in the myoglobin amino acid sequence shown in SEQ ID NO: 1. Binds to the 141st to 154th amino acid region, (d) 2nd to 18th amino acid region, or (e) 35th to 64th amino acid region. The anti-myoglobin monoclonal antibody or antigen-binding fragment thereof comprises (a) positions 79 to 103, (b) positions 117 to 127, (c) positions 144 to 154, (d) in the myoglobin amino acid sequence shown in SEQ ID NO: 1. ) 2nd to 12th or (e) 38th to 60th amino acid regions, and (a) 79th to 85th and 92nd to 99th in the myoglobin amino acid sequence shown in SEQ ID NO: 1, ( b) 118th to 126th, (c) 145th to 154th, (d) 2nd to 7th, or (e) 42nd to 55th amino acid region, and the amino acid of myoglobin shown in SEQ ID NO: 1 It may bind to the amino acid region (a) 78th to 104th, (b) 112th to 130th, or (c) 141st to 154th in the sequence.
　本発明の一側面に係る試料中のミオグロビンを検出する方法は、２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片を用いて、免疫学的測定法により試料中のミオグロビンを検出することを含み、２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片は、（ａ）配列番号１で示されるアミノ酸配列における７８～１０４番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、（ｂ）配列番号１で示されるアミノ酸配列における１１２～１３０番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、（ｃ）配列番号１で示されるアミノ酸配列における１４１～１５４番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、（ｄ）配列番号１で示されるアミノ酸配列における２～１８番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、並びに（ｅ）配列番号１で示されるアミノ酸配列における３５～６４番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体からなる群より選ばれる２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片である。 A method for detecting myoglobin in a sample according to one aspect of the present invention comprises detecting myoglobin in a sample by immunoassay using two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof. and the two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof are (a) an anti-myoglobin monoclonal antibody that binds to the amino acid region from 78 to 104 in the amino acid sequence shown in SEQ ID NO: 1, (b) SEQ ID NO: 1, (c) an anti-myoglobin monoclonal antibody that binds to the 141st to 154th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1, ( d) an anti-myoglobin monoclonal antibody that binds to the 2nd to 18th amino acid regions in the amino acid sequence shown by SEQ ID NO: 1; Two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof selected from the group consisting of myoglobin monoclonal antibodies.
　本発明の一側面に係るミオグロビンを検出するためのキットは、２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片を含み、２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片は、（ａ）配列番号１で示されるアミノ酸配列における７８～１０４番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、（ｂ）配列番号１で示されるアミノ酸配列における１１２～１３０番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、（ｃ）配列番号１で示されるアミノ酸配列における１４１～１５４番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、（ｄ）配列番号１で示されるアミノ酸配列における２～１８番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、並びに（ｅ）配列番号１で示されるアミノ酸配列における３５～６４番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体からなる群より選ばれる２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片である。 A kit for detecting myoglobin according to one aspect of the present invention comprises two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof, and the two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof are ( a) an anti-myoglobin monoclonal antibody that binds to the 78th to 104th amino acid region in the amino acid sequence shown by SEQ ID NO: 1, (b) an anti-myoglobin that binds to the 112th to 130th amino acid region in the amino acid sequence shown by SEQ ID NO: 1 A monoclonal antibody, (c) an anti-myoglobin monoclonal antibody that binds to the 141st to 154th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1, (d) to the 2nd to 18th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1 Two or more anti-myoglobin monoclonal antibodies selected from the group consisting of binding anti-myoglobin monoclonal antibodies and (e) anti-myoglobin monoclonal antibodies that bind to the 35th to 64th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1, or It is an antigen-binding fragment.
　本発明の上記側面に係る方法及びキットにおいて、２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片は、（ａ）配列番号１で示されるアミノ酸配列における７９～１０３番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、（ｂ）配列番号１で示されるアミノ酸配列における１１７～１２７番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、ｃ）配列番号１で示されるアミノ酸配列における１４４～１５４番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、（ｄ）配列番号１で示されるアミノ酸配列における２～１２番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、並びに（ｅ）配列番号１で示されるアミノ酸配列における３８～６０番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体からなる群より選ばれる２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片であってもよく、（ａ）配列番号１で示されるアミノ酸配列における７９～８５番目及び９２～９９番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、（ｂ）配列番号１で示されるアミノ酸配列における１１８～１２６番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、（ｃ）配列番号１で示されるアミノ酸配列における１４５～１５４番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、（ｄ）配列番号１で示されるアミノ酸配列における２～７番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、並びに（ｅ）配列番号１で示されるアミノ酸配列における４２～５５番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体からなる群より選ばれる２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片であってもよく、（ａ）配列番号１で示されるアミノ酸配列における７８～１０４番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、（ｂ）配列番号１で示されるアミノ酸配列における１１２～１３０番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、並びに（ｃ）配列番号１で示されるアミノ酸配列における１４１～１５４番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体からなる群より選ばれる２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片であってもよい。 In the methods and kits according to the above aspects of the present invention, the two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof (a) bind to the 79th to 103rd amino acid regions in the amino acid sequence shown in SEQ ID NO: 1. An anti-myoglobin monoclonal antibody, (b) an anti-myoglobin monoclonal antibody that binds to the 117th to 127th amino acid region in the amino acid sequence shown in SEQ ID NO: 1, c) the 144th to 154th amino acid region in the amino acid sequence shown in SEQ ID NO: 1 (d) an anti-myoglobin monoclonal antibody that binds to the 2nd to 12th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1, and (e) 38 to 38 in the amino acid sequence shown in SEQ ID NO: 1 Two or more anti-myoglobin monoclonal antibodies selected from the group consisting of anti-myoglobin monoclonal antibodies that bind to the 60th amino acid region or antigen-binding fragments thereof, (a) in the amino acid sequence shown in SEQ ID NO: 1 An anti-myoglobin monoclonal antibody that binds to the 79th to 85th and 92nd to 99th amino acid regions, (b) an anti-myoglobin monoclonal antibody that binds to the 118th to 126th amino acid region in the amino acid sequence shown in SEQ ID NO: 1, (c) (d) an anti-myoglobin monoclonal antibody that binds to the 145th to 154th amino acid region in the amino acid sequence shown in SEQ ID NO: 1, (d) an anti-myoglobin monoclonal antibody that binds to the 2nd to 7th amino acid region in the amino acid sequence shown in SEQ ID NO: 1 and (e) two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof selected from the group consisting of anti-myoglobin monoclonal antibodies that bind to the 42nd to 55th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1. (a) an anti-myoglobin monoclonal antibody that binds to the 78th to 104th amino acid region in the amino acid sequence shown in SEQ ID NO: 1, (b) the 112th to 130th amino acid region in the amino acid sequence shown in SEQ ID NO: 1 and (c) two or more anti-myoglobin monoclonal antibodies selected from the group consisting of an anti-myoglobin monoclonal antibody that binds to the 141st to 154th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1, or Even if it is an antigen-binding fragment thereof good.
　本発明の一側面に係るポリペプチドは、配列番号２～６のいずれか一つで示されるアミノ酸配列からなる。ポリペプチドは、配列番号４～６のいずれか一つで示されるアミノ酸配列からなってもよい。 A polypeptide according to one aspect of the present invention consists of an amino acid sequence represented by any one of SEQ ID NOs: 2-6. The polypeptide may consist of the amino acid sequence shown in any one of SEQ ID NOS:4-6.
　上記抗ミオグロビンモノクローナル抗体又はその抗原結合性断片は、免疫複合体の形成に効果的なエピトープを認識する。このため、本発明によれば、免疫学的測定法に適した抗ミオグロビンモノクローナル抗体又はその抗原結合性断片が提供される。また、本発明によれば、該抗ミオグロビンモノクローナル抗体又はその抗原結合性断片を用いてミオグロビンを検出する方法及びキット、並びに該抗ミオグロビンモノクローナル抗体又はその抗原結合性断片によって認識されるポリペプチドも提供される。 The anti-myoglobin monoclonal antibody or antigen-binding fragment thereof recognizes an epitope that is effective in forming immune complexes. Therefore, according to the present invention, an anti-myoglobin monoclonal antibody or antigen-binding fragment thereof suitable for immunological assays is provided. The present invention also provides a method and kit for detecting myoglobin using the anti-myoglobin monoclonal antibody or antigen-binding fragment thereof, and a polypeptide recognized by the anti-myoglobin monoclonal antibody or antigen-binding fragment thereof. be done.
ヒトミオグロビンの抗原性スコアを示すグラフである。FIG. 2 is a graph showing the antigenicity score of human myoglobin. FIG. ペプチドＡ～Ｃをマウスに免疫した際に、マウスの血中抗体価が上昇したことを示すグラフである。FIG. 10 is a graph showing that when mice were immunized with peptides A to C, the blood antibody titers of mice increased. ペプチドＡ～Ｃを免疫原として作製した抗ミオグロビンモノクローナル抗体が、互いに構造的に競合せずに、同一のミオグロビン分子に結合して免疫複合体を形成し得ることを示すセンサーグラムである。FIG. 4 is a sensorgram showing that anti-myoglobin monoclonal antibodies generated using peptides A to C as immunogens can bind to the same myoglobin molecule and form immune complexes without structurally competing with each other.
＜抗ミオグロビンモノクローナル抗体又はその抗原結合性断片＞
　本発明の一側面に係る抗ミオグロビンモノクローナル抗体又はその抗原結合性断片は、ミオグロビンに特異的に結合するモノクローナル抗体又はその抗原結合性断片である。該抗ミオグロビンモノクローナル抗体又はその抗原結合性断片は、図１に示すエピトープ１～５のいずれかに結合する（すなわち、エピトープ１～５のいずれかを認識する）モノクローナル抗体又はその抗原結合性断片である。上述のとおり、図１は、配列番号１で示されるヒトミオグロビンの抗原性スコアを示すグラフである。この図１に示すエピトープ１～５は、ヒトミオグロビンにおける抗原性の強い領域であって、免疫複合体の形成に効果的なエピトープである。 <Anti-myoglobin monoclonal antibody or antigen-binding fragment thereof>
An anti-myoglobin monoclonal antibody or antigen-binding fragment thereof according to one aspect of the present invention is a monoclonal antibody or antigen-binding fragment thereof that specifically binds to myoglobin. The anti-myoglobin monoclonal antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof that binds to any of epitopes 1 to 5 shown in FIG. 1 (that is, recognizes any of epitopes 1 to 5). be. As described above, FIG. 1 is a graph showing the antigenicity score of human myoglobin shown in SEQ ID NO:1. Epitopes 1 to 5 shown in FIG. 1 are highly antigenic regions in human myoglobin and are effective epitopes for the formation of immune complexes.
　すなわち、本側面に係る抗ミオグロビンモノクローナル抗体又はその抗原結合性断片は、配列番号１で示されるミオグロビンのアミノ酸配列における、
（ａ）７８～１０４番目（エピトープ３に対応）、好ましくは７９～１０３番目、より好ましくは７９～８５番目及び９２～９９番目のアミノ酸領域、
（ｂ）１１２～１３０番目（エピトープ４に対応）、好ましくは１１７～１２７番目、より好ましくは１１８～１２６番目のアミノ酸領域、
（ｃ）１４１～１５４番目（エピトープ５に対応）、好ましくは１４４～１５４番目、より好ましくは１４５～１５４番目のアミノ酸領域、
（ｄ）２～１８番目（エピトープ１に対応）、好ましくは２～１２番目、より好ましくは２～７番目のアミノ酸領域、又は
（ｅ）３５～６４番目（エピトープ２に対応）、好ましくは３８～６０番目、より好ましくは４２～５５番目のアミノ酸領域
に結合する。 That is, the anti-myoglobin monoclonal antibody or antigen-binding fragment thereof according to this aspect has, in the myoglobin amino acid sequence shown in SEQ ID NO: 1,
(a) amino acid regions 78-104 (corresponding to epitope 3), preferably 79-103, more preferably 79-85 and 92-99,
(b) amino acid regions 112-130 (corresponding to epitope 4), preferably 117-127, more preferably 118-126,
(c) 141st to 154th amino acid region (corresponding to epitope 5), preferably 144th to 154th, more preferably 145th to 154th amino acid region,
(d) 2nd to 18th (corresponding to epitope 1), preferably 2nd to 12th, more preferably 2nd to 7th amino acid region, or (e) 35th to 64th (corresponding to epitope 2), preferably 38 It binds to an amino acid region of ˜60th, more preferably 42nd to 55th.
　抗ミオグロビンモノクローナル抗体又はその抗原結合性断片は、例えば、配列番号１で示されるミオグロビンのアミノ酸配列における、（ａ）７８～１０４番目、（ｂ）１１２～１３０番目、又は（ｃ）１４１～１５４番目のアミノ酸領域に結合してよい。 The anti-myoglobin monoclonal antibody or antigen-binding fragment thereof is, for example, (a) 78th to 104th, (b) 112th to 130th, or (c) 141st to 154th in the myoglobin amino acid sequence shown in SEQ ID NO: 1. may bind to the amino acid region of
　本明細書において、「特異的に」とは、ミオグロビン、とミオグロビン以外のタンパク質成分と、本側面に係る抗ミオグロビンモノクローナル抗体とを含む液系において、該抗ミオグロビンモノクローナル抗体がミオグロビン以外のタンパク質成分と検出可能なレベルで抗原抗体反応を起こさないか、何らかの結合反応又は会合反応を起こしたとしても、該抗ミオグロビンモノクローナル抗体のミオグロビンとの抗原抗体反応よりも明らかに弱い反応しか起こさないことを意味する。本明細書において、ミオグロビンとはヒトミオグロビンを特に意味する。 As used herein, the term “specifically” means that in a liquid system containing myoglobin, a protein component other than myoglobin, and an anti-myoglobin monoclonal antibody according to this aspect, the anti-myoglobin monoclonal antibody is combined with a protein component other than myoglobin. It means that no antigen-antibody reaction occurs at a detectable level, or that even if some binding reaction or association reaction does occur, it only causes a reaction that is clearly weaker than the antigen-antibody reaction of the anti-myoglobin monoclonal antibody with myoglobin. . As used herein, myoglobin specifically refers to human myoglobin.
　本明細書において、抗ミオグロビンモノクローナル抗体が「アミノ酸配列におけるｍ～ｎ番目の領域に結合する」とは、抗ミオグロビンモノクローナル抗体がアミノ酸配列のｍ～ｎ番目の領域中の１以上の任意の位置に結合することを意味し、必ずしもアミノ酸配列のｍ～ｎ番目の領域の全体にわたって結合することを意味するものではない。例えば、抗ミオグロビンモノクローナル抗体が「配列番号１で示されるミオグロビンのアミノ酸配列における７８～１０４番目のアミノ酸領域に結合する」とは、抗ミオグロビンモノクローナル抗体が７８～１０４番目のアミノ酸領域における１以上の任意のアミノ酸（例えば、７９～８５番目及び９２～９９番目のアミノ酸）に結合することを意味し、必ずしも７８～１０４番目のアミノ酸の全てと結合することを意味しない。 As used herein, the anti-myoglobin monoclonal antibody "binds to the mn-th region in the amino acid sequence" means that the anti-myoglobin monoclonal antibody binds to one or more arbitrary positions in the mn-th region of the amino acid sequence. It means to bind, but does not necessarily mean to bind throughout the m to n th region of the amino acid sequence. For example, the expression that an anti-myoglobin monoclonal antibody "binds to the 78th to 104th amino acid regions in the myoglobin amino acid sequence shown in SEQ ID NO: 1" means that the anti-myoglobin monoclonal antibody binds to any one or more (eg, 79th to 85th and 92nd to 99th amino acids), but does not necessarily mean binding to all of the 78th to 104th amino acids.
　抗ミオグロビンモノクローナル抗体のクラスは、ＩｇＧに限定されず、ＩｇＹ、ＩｇＭ、ラクダＩｇ、又はＩｇ　ＮＡＲであってもよい。また、その抗原結合性断片は、特に限定されず、Ｆａｂ、Ｆａｂ’、Ｆ（ａｂ’）２、又は一本鎖抗体（ｓｃＦｖ）であってよい。 The class of anti-myoglobin monoclonal antibodies is not limited to IgG, but may be IgY, IgM, camelid Ig, or Ig NAR. Moreover, the antigen-binding fragment thereof is not particularly limited, and may be Fab, Fab', F(ab') 2 or a single chain antibody (scFv).
　抗ミオグロビンモノクローナル抗体の作製方法は、特に限定されず、例えば、公知の免疫学的手法を用いてミオグロビン分子全長又はその部分ペプチドを動物に免疫し、免疫された動物の細胞を用いて作製したハイブリドーマから得ることができる。あるいは、抗ミオグロビンモノクローナル抗体は、遺伝子組換え技術を用いて、組換え抗体として作製することもできる。 A method for producing an anti-myoglobin monoclonal antibody is not particularly limited. For example, a hybridoma produced by immunizing an animal with a full-length myoglobin molecule or a partial peptide thereof using a known immunological technique and using cells of the immunized animal. can be obtained from Alternatively, anti-myoglobin monoclonal antibodies can be produced as recombinant antibodies using gene recombination technology.
　免疫に用いるペプチドの長さは、特に限定されないが、好ましくは５アミノ酸以上、より好ましくは１０アミノ酸以上、さらに好ましくは１３アミノ酸以上である。ペプチドは生体内で抗原提示細胞によって分解されて、その一部のみが抗原として提示される。このため、免疫に用いるペプチドは、図１に示すエピトープ１～５に、１つ以上の余分なアミノ酸が付加されたペプチド（例えば、図１のエピトープ１～５に加えて、エピトープ１～５の直前及び／又は直後の領域のアミノ酸を含むペプチド）であることが好ましい。免疫に用いるペプチドは、例えば、後述する本発明の別の側面に係るポリペプチドであってよい。 The length of the peptide used for immunization is not particularly limited, but is preferably 5 amino acids or more, more preferably 10 amino acids or more, and still more preferably 13 amino acids or more. Peptides are degraded in vivo by antigen-presenting cells and only a portion thereof is presented as an antigen. Therefore, peptides used for immunization are epitopes 1 to 5 shown in FIG. preferably a peptide containing amino acids in the immediately preceding and/or immediately following region). A peptide used for immunization may be, for example, a polypeptide according to another aspect of the present invention, which will be described later.
　免疫原とするミオグロビンは、例えば、血液又は尿に由来するヒト生体試料から得てもよく、ミオグロビンのタンパク質をコードするＤＮＡを組み込んだプラスミドベクターを宿主細胞に導入して発現させることにより得てもよい。 Myoglobin used as an immunogen may be obtained, for example, from a human biological sample derived from blood or urine, or may be obtained by introducing a plasmid vector incorporating DNA encoding a myoglobin protein into a host cell for expression. good.
　免疫原として用いるミオグロビン分子又はその部分ペプチドには、他のタンパク質が融合されていてもよい。すなわち、ミオグロビン分子又はその部分ペプチドを他のタンパク質との融合タンパク質として発現させ、精製後又は精製せずに、免疫原として用いてもよい。例えば、免疫原としてペプチドを用いる場合は、キーホールリンペットヘモシアニン（ＫＬＨ）等のキャリアタンパク質をペプチドにコンジュゲートさせてもよい。上記他のタンパク質として、グルタチオンＳ－トランスフェラーゼ（ＧＳＴ）、マルトース結合タンパク質（ＭＢＰ）、チオレドキシン（ＴＲＸ）、Ｎｕｓタグ、Ｓタグ、ＨＳＶタグ、ＦＬＲＡＧタグ、ポリヒスチジンタグ、Ｓｔｒｅｐタグ、Ｓｔｒｅｐ－ＩＩタグ、Ｍｙｃタグ、ＨＡタグ、Ｖ５タグ、Ｅタグ、Ｔ７タグ、ＶＳＶ－Ｇタグ、Ｇｌｕ‐Ｇｌｕタグ、Ａｖｉタグ等、タンパク質発現及び／又は精製タグとして一般的に用いられるタンパク質を用いてもよいが、これらのタグは、融合タンパク質発現後に消化酵素を用いて切断することが好ましい。 The myoglobin molecule or its partial peptide used as an immunogen may be fused with other proteins. That is, a myoglobin molecule or a partial peptide thereof may be expressed as a fusion protein with another protein and used as an immunogen after or without purification. For example, when using a peptide as an immunogen, a carrier protein such as keyhole limpet hemocyanin (KLH) may be conjugated to the peptide. Examples of the other proteins include glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (TRX), Nus tag, S tag, HSV tag, FLRAG tag, polyhistidine tag, Strep tag, Strep-II tag, Proteins commonly used as protein expression and/or purification tags such as Myc tag, HA tag, V5 tag, E tag, T7 tag, VSV-G tag, Glu-Glu tag, and Avi tag may be used, These tags are preferably cleaved using digestive enzymes after expression of the fusion protein.
　免疫した動物からの抗ミオグロビンモノクローナル抗体の調製は、ケラーらの周知の方法（Ｋｏｈｌｅｒ，ｅｔ　ａｌ．Ｎａｔｕｒｅ．１９７５；２５６：４９５－４９７）により容易に行うことができる。すなわち、免疫した動物から、脾細胞、リンパ球等の抗体産生細胞を回収し、抗体産生細胞をマウスミエローマ細胞と融合させてハイブリドーマを作製し、ハイブリドーマを限界希釈法等によりクローニングし、クローニングされた各ハイブリドーマが産生するモノクローナル抗体のうち、ミオグロビンと抗原抗体反応を起こすモノクローナル抗体を選択することにより、抗ミオグロビンモノクローナル抗体を調製することができる。 An anti-myoglobin monoclonal antibody can be easily prepared from an immunized animal by the well-known method of Keller et al. (Kohler, et al. Nature. 1975; 256: 495-497). That is, antibody-producing cells such as splenocytes and lymphocytes are recovered from the immunized animal, the antibody-producing cells are fused with mouse myeloma cells to prepare hybridomas, and the hybridomas are cloned by limiting dilution or the like. An anti-myoglobin monoclonal antibody can be prepared by selecting a monoclonal antibody that causes an antigen-antibody reaction with myoglobin from the monoclonal antibodies produced by each hybridoma.
　ハイブリドーマの培養は、マウス等動物の腹水又は培地中で行うことができる。腹水又は培養上清からの抗ミオグロビンモノクローナル抗体の精製には、公知のイムノグロブリン精製法を用いることができる。例えば、硫酸アンモニウム又は硫酸ナトリウムを用いた塩析による分画法、ポリエチレングリコール（ＰＥＧ）分画法、エタノール分画法、ＤＥＡＥイオン交換クロマトグラフィー法、ゲルろ過法などが挙げられる。あるいは、免疫動物種と抗ミオグロビンモノクローナル抗体のクラスに応じて、プロテインＡ、プロテインＧ、及びプロテインＬのいずれかを結合させた担体を用いて、アフィニティクロマトグラフィー法により抗ミオグロビンモノクローナル抗体を精製してもよい。 Cultivation of hybridomas can be carried out in the ascites fluid or culture medium of animals such as mice. A known immunoglobulin purification method can be used to purify the anti-myoglobin monoclonal antibody from ascites fluid or culture supernatant. For example, fractionation by salting out using ammonium sulfate or sodium sulfate, polyethylene glycol (PEG) fractionation, ethanol fractionation, DEAE ion exchange chromatography, gel filtration and the like can be mentioned. Alternatively, the anti-myoglobin monoclonal antibody is purified by affinity chromatography using a carrier to which protein A, protein G, or protein L is bound, depending on the animal species to be immunized and the class of the anti-myoglobin monoclonal antibody. good too.
＜ミオグロビンの検出方法＞
　本発明の一側面に係る試料中のミオグロビンを検出する方法は、本発明の上記側面に係る抗ミオグロビンモノクローナル抗体又はその抗原結合性断片のうち２種以上、すなわち、図１に示すエピトープ１～５のいずれかに結合する抗ミオグロビンモノクローナル抗体のうち２種以上を用いて免疫学的測定法により試料中のミオグロビンを検出することを含む。 <Method for detecting myoglobin>
A method for detecting myoglobin in a sample according to one aspect of the present invention comprises two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof according to the above aspect of the present invention, ie epitopes 1 to 5 shown in FIG. detecting myoglobin in a sample by an immunoassay using two or more of the anti-myoglobin monoclonal antibodies that bind to any of
　より具体的には、本発明の一側面に係る試料中のミオグロビンを検出する方法は、２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片を用いて、免疫学的測定法により試料中のミオグロビンを検出することを含み、２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片は、
　（ａ）配列番号１で示されるアミノ酸配列における、７８～１０４番目（エピトープ３に対応）、好ましくは７９～１０３番目、より好ましくは７９～８５番目及び９２～９９番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、
　（ｂ）配列番号１で示されるアミノ酸配列における、１１２～１３０番目（エピトープ４に対応）、好ましくは１１７～１２７番目、より好ましくは１１８～１２６番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、
　（ｃ）配列番号１で示されるアミノ酸配列における、１４１～１５４番目（エピトープ５に対応）、好ましくは１４４～１５４番目、より好ましくは１４５～１５４番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、
　（ｄ）配列番号１で示されるアミノ酸配列における、２～１８番目（エピトープ１に対応）、好ましくは２～１２番目、より好ましくは２～７番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、並びに
　（ｅ）配列番号１で示されるアミノ酸配列における、３５～６４番目（エピトープ２に対応）、好ましくは３８～６０番目、より好ましくは４２～５５番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体
からなる群より選ばれる２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片である。これら抗ミオグロビンモノクローナル抗体又はその抗原結合性断片は、互いに競合しない。 More specifically, the method for detecting myoglobin in a sample according to one aspect of the present invention is an immunological assay using two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof. detecting myoglobin, wherein two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof are
(a) in the amino acid sequence shown by SEQ ID NO: 1, anti-binding to the 78th to 104th (corresponding to epitope 3), preferably 79th to 103rd, more preferably 79th to 85th and 92nd to 99th amino acid regions myoglobin monoclonal antibody,
(b) an anti-myoglobin monoclonal antibody that binds to the 112th to 130th (corresponding to epitope 4), preferably 117th to 127th, more preferably 118th to 126th amino acid region in the amino acid sequence shown by SEQ ID NO: 1;
(c) an anti-myoglobin monoclonal antibody that binds to the 141st to 154th (corresponding to epitope 5), preferably 144th to 154th, more preferably 145th to 154th amino acid region in the amino acid sequence shown by SEQ ID NO: 1;
(d) an anti-myoglobin monoclonal antibody that binds to the 2nd to 18th (corresponding to epitope 1), preferably 2nd to 12th, more preferably 2nd to 7th amino acid regions in the amino acid sequence shown by SEQ ID NO: 1, and (e) consisting of an anti-myoglobin monoclonal antibody that binds to the 35th to 64th (corresponding to epitope 2), preferably 38th to 60th, more preferably 42nd to 55th amino acid regions in the amino acid sequence shown by SEQ ID NO: 1 Two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof selected from the group. These anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof do not compete with each other.
　２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片は、例えば、（ａ）配列番号１で示されるアミノ酸配列における７８～１０４番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、（ｂ）配列番号１で示されるアミノ酸配列における１１２～１３０番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、及び（ｃ）配列番号１で示されるアミノ酸配列における１４１～１５４番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体からなる群より選ばれる２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片であってよい。 The two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof are, for example, (a) an anti-myoglobin monoclonal antibody that binds to the 78th to 104th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1, (b) SEQ ID NO: from an anti-myoglobin monoclonal antibody that binds to the 112th to 130th amino acid regions in the amino acid sequence shown by SEQ ID NO: 1, and (c) an anti-myoglobin monoclonal antibody that binds to the 141st to 154th amino acid regions in the amino acid sequence shown by SEQ ID NO: Two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof selected from the group consisting of:
　ミオグロビンの検出には、２種以上、３種以上、４種以上、又は５種の上記抗ミオグロビンモノクローナル抗体又はその抗原結合性断片を用いることができ、好ましくは２～５種、より好ましくは２～３種の上記抗ミオグロビンモノクローナル抗体又はその抗原結合性断片を用いる。 For detection of myoglobin, 2 or more, 3 or more, 4 or more, or 5 anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof can be used, preferably 2 to 5, more preferably 2 ˜3 of the above anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof are used.
　試料としては、例えば、ヒト又はヒト以外の動物の血液、血清、血漿、尿、精液、髄液、唾液、汗、涙、腹水、羊水などの体液；粘液；糞便；血管、肝臓などの臓器；組織；細胞；又はそれらの抽出液など、ミオグロビンが含まれる可能性のある生体試料が挙げられる。試料は、好ましくは採取が容易である、血液（全血、血漿、若しくは血清）又は尿である。試料を採取する方法は特に限定されず、公知の方法を採用することができる。 Samples include, for example, human or non-human animal blood, serum, plasma, urine, semen, cerebrospinal fluid, saliva, sweat, tears, ascites, amniotic fluid and other bodily fluids; mucus; feces; organs such as blood vessels and liver; Examples include biological samples that may contain myoglobin, such as tissues; cells; or extracts thereof. The sample is preferably blood (whole blood, plasma, or serum) or urine, which is easy to collect. A method for collecting a sample is not particularly limited, and a known method can be adopted.
　免疫学的測定法としては、競合法、凝集法、ウェスタンブロット法、免疫染色法、サンドイッチ法等、公知の免疫学的測定法が挙げられ、サンドイッチ法が好ましい。サンドイッチ法は、例えば、イムノクロマト法、ＥＬＩＳＡ法、ラテックス凝集法、ＣＬＥＩＡ法などの手法により行うことができる。 The immunological measurement methods include known immunological measurement methods such as the competitive method, agglutination method, Western blot method, immunostaining method, and sandwich method, with the sandwich method being preferred. The sandwich method can be performed by techniques such as immunochromatography, ELISA, latex agglutination, and CLEIA.
＜ミオグロビンの検出キット＞
　本発明の一側面に係るミオグロビンを検出するためのキットは、本発明の上記側面に係るミオグロビンの検出方法において用いられる、２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片を含む。２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片の詳細は、上述したとおりある。キットは、例えば、イムノクロマト法、ＥＬＩＳＡ法、ラテックス凝集法、ＣＬＥＩＡ法などの手法において用いられる公知の試薬、材料、器具等をさらに含むことができる。 <Detection kit for myoglobin>
A kit for detecting myoglobin according to one aspect of the present invention comprises two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof used in the myoglobin detection method according to the above aspect of the present invention. Details of the two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof are as described above. The kit can further include known reagents, materials, instruments, etc. used in techniques such as immunochromatography, ELISA, latex agglutination, and CLEIA.
＜抗ミオグロビンモノクローナル抗体によって認識されるポリペプチド＞
　本発明の一側面は、本発明の上記側面に係る抗ミオグロビンモノクローナル抗体又はその抗原結合性断片によって認識されるポリペプチドを提供する。ポリペプチドは、図１に示すエピトープ１～５のいずれかの一部又は全体を含む。より具体的には、ポリペプチドは、配列番号１で示されるミオグロビンのアミノ酸配列における、少なくとも
　（ａ）７９～８５番目及び９２～９９番目、好ましくは７９～１０３番目、より好ましくは７８～１０４番目（エピトープ３に対応）のアミノ酸領域、
　（ｂ）１１８～１２６番目、好ましくは１１７～１２７番目、より好ましくは１１２～１３０番目（エピトープ４に対応）のアミノ酸領域、
　（ｃ）１４５～１５４番目、好ましくは１４４～１５４番目、より好ましくは１４１～１５４番目（エピトープ５に対応）のアミノ酸領域、
　（ｄ）２～７番目、好ましくは２～１２番目、より好ましくは２～１８番目（エピトープ１に対応）のアミノ酸領域、又は
　（ｅ）４２～５５番目、好ましくは３８～６０番目、より好ましくは３５～６４番目（エピトープ２に対応）のアミノ酸領域
を含む。 <Polypeptide recognized by anti-myoglobin monoclonal antibody>
One aspect of the invention provides a polypeptide recognized by an anti-myoglobin monoclonal antibody or antigen-binding fragment thereof according to the above aspects of the invention. Polypeptides include part or all of any of epitopes 1-5 shown in FIG. More specifically, the polypeptide comprises at least (a) positions 79 to 85 and 92 to 99, preferably 79 to 103, more preferably 78 to 104 in the myoglobin amino acid sequence shown in SEQ ID NO: 1. the amino acid region of (corresponding to epitope 3),
(b) an amino acid region at positions 118-126, preferably 117-127, more preferably 112-130 (corresponding to epitope 4);
(c) an amino acid region from 145 to 154, preferably from 144 to 154, more preferably from 141 to 154 (corresponding to epitope 5);
(d) 2nd to 7th, preferably 2nd to 12th, more preferably 2nd to 18th amino acid region (corresponding to epitope 1), or (e) 42nd to 55th, preferably 38th to 60th, more preferably contains a region of amino acids 35-64 (corresponding to epitope 2).
　ポリペプチドは、例えば、配列番号１で示されるミオグロビンのアミノ酸配列における、
　（ａ）７５～１００番目（配列番号４）、
　（ｂ）１１２～１３１番目（配列番号５）、
　（ｃ）１３３～１５４番目（配列番号６）、
　（ｄ）２～１８番目（配列番号２）、又は
　（ｅ）３５～６４番目（配列番号３）のアミノ酸領域からなるペプチドであってよく、配列番号４～６のいずれか一つで示されるアミノ酸配列からなるペプチドであってよい。 The polypeptide is, for example, in the myoglobin amino acid sequence shown in SEQ ID NO: 1,
(a) 75th to 100th (SEQ ID NO: 4),
(b) 112-131 (SEQ ID NO: 5),
(c) 133-154 (SEQ ID NO: 6),
(d) 2nd to 18th (SEQ ID NO: 2), or (e) 35th to 64th (SEQ ID NO: 3) may be a peptide consisting of an amino acid region, and is represented by any one of SEQ ID NOS: 4 to 6 It may be a peptide consisting of an amino acid sequence.
　本側面に係るポリペプチドは、例えば、免疫学的手法により抗ヒトミオグロビンモノクローナル抗体を作製するための免疫原として用いることができる。ポリペプチドを免疫原として用いる場合、ポリペプチドは、生体内で抗原提示細胞によって分解されて、その一部のみが抗原として提示される。このため、ポリペプチドは、図１に示すエピトープ１～５に、１つ以上の余分なアミノ酸が付加されたペプチド（例えば、図１のエピトープ１～５に加えて、エピトープ１～５の直前及び／又は直後の領域のアミノ酸を含むペプチド）であることが好ましい。ポリペプチドは、ＫＬＨ等のキャリアタンパク質にコンジュゲートさせてもよい。 A polypeptide according to this aspect can be used, for example, as an immunogen for producing anti-human myoglobin monoclonal antibodies by immunological techniques. When a polypeptide is used as an immunogen, the polypeptide is degraded by antigen-presenting cells in vivo and only a portion thereof is presented as an antigen. Therefore, the polypeptide is a peptide obtained by adding one or more extra amino acids to epitopes 1 to 5 shown in FIG. 1 (for example, in addition to epitopes 1 to 5 in FIG. /or a peptide containing the amino acids in the immediately following region). Polypeptides may be conjugated to carrier proteins such as KLH.
　また、本側面に係るポリペプチドは、上記側面に係る抗ミオグロビンモノクローナル抗体又はその抗原結合性断片とともに、免疫学的競合法によるミオグロビンの検出にも用いることができる。具体的には、本側面に係る１種以上のポリペプチドを基板上に固相化し、ミオグロビンを含み得る試料と、標識化した上記側面に係る抗ミオグロビンモノクローナル抗体又はその抗原結合性断片とを上記基板に接触させることで、競合的にミオグロビンを検出することができる。したがって、本発明の別の側面は、上記ポリペプチドと、上記抗ミオグロビンモノクローナル抗体又はその抗原結合性断片とを用いて免疫学的競合法により試料中のミオグロビンを検出することを含む、試料中のミオグロビンを検出する方法、並びに、上記ポリペプチドと、上記抗ミオグロビンモノクローナル抗体又はその抗原結合性断片とを含む、ミオグロビンを検出するためのキットを提供するともいえる。 In addition, the polypeptide according to this aspect can be used together with the anti-myoglobin monoclonal antibody or antigen-binding fragment thereof according to the above aspect to detect myoglobin by an immunological competition method. Specifically, one or more polypeptides according to the present aspect are immobilized on a substrate, and a sample that may contain myoglobin and a labeled anti-myoglobin monoclonal antibody according to the aspect or an antigen-binding fragment thereof are prepared as described above. By contacting the substrate, myoglobin can be competitively detected. Therefore, another aspect of the present invention is to detect myoglobin in a sample by an immunological competition method using the polypeptide and the anti-myoglobin monoclonal antibody or antigen-binding fragment thereof. It can also be said to provide a method for detecting myoglobin and a kit for detecting myoglobin comprising the polypeptide and the anti-myoglobin monoclonal antibody or antigen-binding fragment thereof.
　以下、本発明を実施例に基づきより詳細に説明する。ただし、本発明は下記実施例に限定されるものではない。 Hereinafter, the present invention will be described in more detail based on examples. However, the present invention is not limited to the following examples.
＜実施例１＞
　表１に示すペプチドＡ～Ｃを合成し、ＢＡＬＢ／ｃマウスに抗原としてそれぞれ投与した。ペプチドＡ～Ｃは、それぞれ配列番号４～６で示すアミノ酸配列を有するペプチドとＫＬＨとのコンジュゲートである。抗原の投与は２週間おきに５回行い、投与の翌週に採血を行った。得られた血漿中の抗体価をＥＬＩＳＡ法にて測定した。具体的には、まず、配列番号１で示されるアミノ酸配列を有するヒトミオグロビンを固相化したプレートを公知の方法にてブロッキングし、そこにウォッシュバッファー（０．０５％Ｔｗｅｅｎ２０含有ＰＢＳ）で６０００倍又は２００００倍希釈した血漿を加えて室温で２時間反応させた。プレートを洗浄後、ウォッシュバッファーで適宜希釈した酵素標識抗マウスＩｇＧ抗体を添加して室温で１時間反応させ、基質溶液を用いて発色反応を行った。反応停止後、プレートリーダーにて４５０ｎｍ及び６２０ｎｍの吸光度を測定し、４５０ｎｍの吸光度と６２０ｎｍの吸光度の差から抗体価を求めた。 <Example 1>
Peptides A to C shown in Table 1 were synthesized and administered to BALB/c mice as antigens, respectively. Peptides AC are conjugates of peptides having amino acid sequences shown in SEQ ID NOs: 4-6 and KLH, respectively. The antigen was administered 5 times at 2-week intervals, and blood was collected in the week following the administration. Antibody titers in the resulting plasma were measured by ELISA. Specifically, first, a plate on which human myoglobin having the amino acid sequence shown by SEQ ID NO: 1 was immobilized was blocked by a known method, and washed with a wash buffer (0.05% Tween 20-containing PBS) at 6000 times. Alternatively, 20,000-fold diluted plasma was added and allowed to react at room temperature for 2 hours. After washing the plate, an enzyme-labeled anti-mouse IgG antibody appropriately diluted with a wash buffer was added and allowed to react at room temperature for 1 hour, followed by color development reaction using a substrate solution. After stopping the reaction, the absorbance at 450 nm and 620 nm was measured with a plate reader, and the antibody titer was obtained from the difference between the absorbance at 450 nm and the absorbance at 620 nm.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　結果を図２に示す。図２のグラフ（Ａ）、（Ｂ）、（Ｃ）は、それぞれペプチドＡ、Ｂ、Ｃを投与したマウスの血漿中抗体価を示す。図２に示されるように、ペプチドＡ～Ｃの投与回数に応じて、ヒトミオグロビンに対する抗体価が上昇することが確認された。 The results are shown in Figure 2. Graphs (A), (B), and (C) in FIG. 2 show plasma antibody titers in mice to which peptides A, B, and C were administered, respectively. As shown in FIG. 2, it was confirmed that the antibody titer against human myoglobin increased according to the administration frequency of peptides A to C.
＜実施例２＞
　実施例１においてペプチドＡ～Ｃのいずれかを免疫したマウスから、抗体産生Ｂ細胞を得た。この抗体産生Ｂをミエローマ細胞と融合して、不死化した抗体産生ハイブリドーマを得た。ハイブリドーマを別のマウスの腹腔へ投与し、マウスの腹水から、ハイブリドーマが産生する抗ミオグロビンモノクローナル抗体を得た。ここで、ペプチドＡ～Ｃを免疫原として得られた抗ミオグロビンモノクローナル抗体を、それぞれ抗体Ａ～Ｃと呼ぶ。抗体Ａ～Ｃが、互いに構造的に競合せずに、同一のミオグロビン分子に結合して免疫複合体を形成することを、表面プラズモン共鳴（ＳＰＲ）法により確認した。具体的には、まず、Ｂｉａｃｏｒｅ（登録商標）　８Ｋ（サイティバ社製）を用いてアミンカップリング法により抗体Ａを約７０００ＲＵ固相化したセンサーチップ（Ｓｅｒｉｅｓ　Ｓ　Ｓｅｎｓｏｒ　Ｃｈｉｐ　ＣＭ５、サイティバ社製）に、５００ｎＭのミオグロビン（アナライト１）及び５００ｎＭの抗体Ｂ（アナライト２）をセンサーグラムが飽和するまで添加し、最後にアナライト３として、抗体Ａ～Ｃのいずれか（５００ｎＭ）、バッファー（０．０１Ｍ　ＨＥＰＥＳ、ｐＨ７．４、０．１５Ｍ　ＮａＣｌ、３ｍＭ　ＥＤＴＡ、及び０．０５％Ｔｗｅｅｎ２０）、又は正常マウスＩｇＧを添加した。 <Example 2>
Antibody-producing B cells were obtained from mice immunized with any of peptides AC in Example 1. This antibody-producing B was fused with myeloma cells to obtain an immortal antibody-producing hybridoma. The hybridoma was administered intraperitoneally to another mouse, and the anti-myoglobin monoclonal antibody produced by the hybridoma was obtained from the ascites fluid of the mouse. Here, the anti-myoglobin monoclonal antibodies obtained using peptides A to C as immunogens are referred to as antibodies A to C, respectively. It was confirmed by the surface plasmon resonance (SPR) method that antibodies A to C bind to the same myoglobin molecule to form immune complexes without structurally competing with each other. Specifically, first, a sensor chip (Series S Sensor Chip CM5, manufactured by Cytiva) on which about 7000 RU of antibody A is immobilized by an amine coupling method using Biacore (registered trademark) 8K (manufactured by Cytiva), 500 nM myoglobin (analyte 1) and 500 nM antibody B (analyte 2) were added until the sensorgram was saturated, and finally, as analyte 3, any of antibodies A to C (500 nM), buffer (0. 01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, and 0.05% Tween 20) or normal mouse IgG were added.
　得られたセンサーグラムを図３に示す。図３中、（Ａ）はセンサーグラム全体であり、（Ｂ）はアナライト３添加時のセンサーグラムの拡大図である。（Ｂ）に示されるように、アナライト３として抗体Ｃを添加したときにのみ、アナライト２添加時のシグナル以上のレスポンスを示した。この結果から、抗体Ａ、Ｂ、及びＣは、互いに構造的に競合せずに同一のミオグロビン分子に結合することが確認された。 The resulting sensorgram is shown in Figure 3. In FIG. 3, (A) is the entire sensorgram, and (B) is an enlarged view of the sensorgram when analyte 3 was added. As shown in (B), only when antibody C was added as analyte 3, a response greater than the signal when analyte 2 was added was shown. This result confirms that antibodies A, B, and C bind to the same myoglobin molecule without structurally competing with each other.

Claims (14)

  1. 　配列番号１で示されるミオグロビンのアミノ酸配列における、
    　（ａ）７８～１０４番目、
    　（ｂ）１１２～１３０番目、
    　（ｃ）１４１～１５４番目、
    　（ｄ）２～１８番目、又は
    　（ｅ）３５～６４番目
    のアミノ酸領域に結合する、抗ミオグロビンモノクローナル抗体又はその抗原結合性断片。     In the myoglobin amino acid sequence shown in SEQ ID NO: 1,
    (a) 78th to 104th,
    (b) 112th to 130th,
    (c) 141st to 154th,
    (d) an anti-myoglobin monoclonal antibody or an antigen-binding fragment thereof that binds to the 2nd to 18th amino acid region, or (e) the 35th to 64th amino acid region;
  2. 　配列番号１で示されるミオグロビンのアミノ酸配列における、
    　（ａ）７９～１０３番目、
    　（ｂ）１１７～１２７番目、
    　（ｃ）１４４～１５４番目、
    　（ｄ）２～１２番目、又は
    　（ｅ）３８～６０番目
    のアミノ酸領域に結合する、請求項１に記載の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片。     In the myoglobin amino acid sequence shown in SEQ ID NO: 1,
    (a) 79th to 103rd,
    (b) 117th to 127th,
    (c) 144th to 154th,
    The anti-myoglobin monoclonal antibody or antigen-binding fragment thereof according to claim 1, which binds to (d) the 2nd to 12th amino acid regions, or (e) the 38th to 60th amino acid regions.
  3. 　配列番号１で示されるミオグロビンのアミノ酸配列における、
    　（ａ）７９～８５番目及び９２～９９番目、
    　（ｂ）１１８～１２６番目、
    　（ｃ）１４５～１５４番目、
    　（ｄ）２～７番目、又は
    　（ｅ）４２～５５番目
    のアミノ酸領域に結合する、請求項２に記載の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片。     In the myoglobin amino acid sequence shown in SEQ ID NO: 1,
    (a) 79th to 85th and 92nd to 99th,
    (b) 118th to 126th,
    (c) 145th to 154th,
    The anti-myoglobin monoclonal antibody or antigen-binding fragment thereof according to claim 2, which binds to the (d) 2nd to 7th amino acid region, or (e) the 42nd to 55th amino acid region.
  4. 　配列番号１で示されるミオグロビンのアミノ酸配列における、
    　（ａ）７８～１０４番目、
    　（ｂ）１１２～１３０番目、又は
    　（ｃ）１４１～１５４番目
    のアミノ酸領域に結合する、請求項１に記載の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片。     In the myoglobin amino acid sequence shown in SEQ ID NO: 1,
    (a) 78th to 104th,
    The anti-myoglobin monoclonal antibody or antigen-binding fragment thereof according to claim 1, which binds to (b) the 112th to 130th amino acid region, or (c) the 141st to 154th amino acid region.
  5. 　２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片を用いて、免疫学的測定法により試料中のミオグロビンを検出することを含み、
    　２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片は、
    　（ａ）配列番号１で示されるアミノ酸配列における７８～１０４番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、
    　（ｂ）配列番号１で示されるアミノ酸配列における１１２～１３０番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、
    　（ｃ）配列番号１で示されるアミノ酸配列における１４１～１５４番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、
    　（ｄ）配列番号１で示されるアミノ酸配列における２～１８番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、並びに
    　（ｅ）配列番号１で示されるアミノ酸配列における３５～６４番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体
    からなる群より選ばれる２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片である、試料中のミオグロビンを検出する方法。     detecting myoglobin in a sample by immunoassay using two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof;
    Two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof,
    (a) an anti-myoglobin monoclonal antibody that binds to the 78th to 104th amino acid region in the amino acid sequence shown in SEQ ID NO: 1;
    (b) an anti-myoglobin monoclonal antibody that binds to the 112th to 130th amino acid region in the amino acid sequence shown in SEQ ID NO: 1;
    (c) an anti-myoglobin monoclonal antibody that binds to the 141st to 154th amino acid region in the amino acid sequence shown in SEQ ID NO: 1;
    (d) an anti-myoglobin monoclonal antibody that binds to the 2nd to 18th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1, and (e) binds to the 35th to 64th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1 A method for detecting myoglobin in a sample, which is two or more anti-myoglobin monoclonal antibodies selected from the group consisting of anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof.
  6. 　２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片が、
    　（ａ）配列番号１で示されるアミノ酸配列における７９～１０３番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、
    　（ｂ）配列番号１で示されるアミノ酸配列における１１７～１２７番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、
    　（ｃ）配列番号１で示されるアミノ酸配列における１４４～１５４番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、
    　（ｄ）配列番号１で示されるアミノ酸配列における２～１２番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、並びに
    　（ｅ）配列番号１で示されるアミノ酸配列における３８～６０番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体
    からなる群より選ばれる２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片である、請求項５に記載の方法。     Two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof,
    (a) an anti-myoglobin monoclonal antibody that binds to the 79th to 103rd amino acid region in the amino acid sequence shown in SEQ ID NO: 1;
    (b) an anti-myoglobin monoclonal antibody that binds to the 117th to 127th amino acid region in the amino acid sequence shown in SEQ ID NO: 1;
    (c) an anti-myoglobin monoclonal antibody that binds to the 144th to 154th amino acid region in the amino acid sequence shown in SEQ ID NO: 1;
    (d) an anti-myoglobin monoclonal antibody that binds to the 2nd to 12th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1, and (e) binds to the 38th to 60th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1 The method according to claim 5, wherein two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof are selected from the group consisting of anti-myoglobin monoclonal antibodies.
  7. 　２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片が、
    　（ａ）配列番号１で示されるアミノ酸配列における７９～８５番目及び９２～９９番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、
    　（ｂ）配列番号１で示されるアミノ酸配列における１１８～１２６番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、
    　（ｃ）配列番号１で示されるアミノ酸配列における１４５～１５４番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、
    　（ｄ）配列番号１で示されるアミノ酸配列における２～７番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、並びに
    　（ｅ）配列番号１で示されるアミノ酸配列における４２～５５番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体
    からなる群より選ばれる２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片である、請求項６に記載の方法。     Two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof,
    (a) an anti-myoglobin monoclonal antibody that binds to the 79th to 85th and 92nd to 99th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1;
    (b) an anti-myoglobin monoclonal antibody that binds to the 118th to 126th amino acid region in the amino acid sequence shown in SEQ ID NO: 1;
    (c) an anti-myoglobin monoclonal antibody that binds to the 145th to 154th amino acid region in the amino acid sequence shown in SEQ ID NO: 1;
    (d) an anti-myoglobin monoclonal antibody that binds to the 2nd to 7th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1, and (e) binds to the 42nd to 55th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1 7. The method according to claim 6, which is two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof selected from the group consisting of anti-myoglobin monoclonal antibodies.
  8. 　２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片が、
    　（ａ）配列番号１で示されるアミノ酸配列における７８～１０４番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、
    　（ｂ）配列番号１で示されるアミノ酸配列における１１２～１３０番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、並びに
    　（ｃ）配列番号１で示されるアミノ酸配列における１４１～１５４番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体
    からなる群より選ばれる２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片である、請求項５に記載の方法。     Two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof,
    (a) an anti-myoglobin monoclonal antibody that binds to the 78th to 104th amino acid region in the amino acid sequence shown in SEQ ID NO: 1;
    (b) an anti-myoglobin monoclonal antibody that binds to the 112th to 130th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1; and (c) binds to the 141st to 154th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1 The method according to claim 5, wherein two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof are selected from the group consisting of anti-myoglobin monoclonal antibodies.
  9. 　２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片を含み、
    　２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片は、
    　（ａ）配列番号１で示されるアミノ酸配列における７８～１０４番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、
    　（ｂ）配列番号１で示されるアミノ酸配列における１１２～１３０番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、
    　（ｃ）配列番号１で示されるアミノ酸配列における１４１～１５４番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、
    　（ｄ）配列番号１で示されるアミノ酸配列における２～１８番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、並びに
    　（ｅ）配列番号１で示されるアミノ酸配列における３５～６４番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体
    からなる群より選ばれる２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片である、ミオグロビンを検出するためのキット。     comprising two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof,
    Two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof,
    (a) an anti-myoglobin monoclonal antibody that binds to the 78th to 104th amino acid region in the amino acid sequence shown in SEQ ID NO: 1;
    (b) an anti-myoglobin monoclonal antibody that binds to the 112th to 130th amino acid region in the amino acid sequence shown in SEQ ID NO: 1;
    (c) an anti-myoglobin monoclonal antibody that binds to the 141st to 154th amino acid region in the amino acid sequence shown in SEQ ID NO: 1;
    (d) an anti-myoglobin monoclonal antibody that binds to the 2nd to 18th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1, and (e) binds to the 35th to 64th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1 A kit for detecting myoglobin, which is two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof selected from the group consisting of anti-myoglobin monoclonal antibodies.
  10. 　２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片が、
    　（ａ）配列番号１で示されるアミノ酸配列における７９～１０３番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、
    　（ｂ）配列番号１で示されるアミノ酸配列における１１７～１２７番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、
    　（ｃ）配列番号１で示されるアミノ酸配列における１４４～１５４番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、
    　（ｄ）配列番号１で示されるアミノ酸配列における２～１２番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、並びに
    　（ｅ）配列番号１で示されるアミノ酸配列における３８～６０番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体
    からなる群より選ばれる２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片である、請求項９に記載のキット。     Two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof,
    (a) an anti-myoglobin monoclonal antibody that binds to the 79th to 103rd amino acid region in the amino acid sequence shown in SEQ ID NO: 1;
    (b) an anti-myoglobin monoclonal antibody that binds to the 117th to 127th amino acid region in the amino acid sequence shown in SEQ ID NO: 1;
    (c) an anti-myoglobin monoclonal antibody that binds to the 144th to 154th amino acid region in the amino acid sequence shown in SEQ ID NO: 1;
    (d) an anti-myoglobin monoclonal antibody that binds to the 2nd to 12th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1, and (e) binds to the 38th to 60th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1 The kit according to claim 9, which is two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof selected from the group consisting of anti-myoglobin monoclonal antibodies.
  11. 　２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片が、
    　（ａ）配列番号１で示されるアミノ酸配列における７９～８５番目及び９２～９９番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、
    　（ｂ）配列番号１で示されるアミノ酸配列における１１８～１２６番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、
    　（ｃ）配列番号１で示されるアミノ酸配列における１４５～１５４番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、
    　（ｄ）配列番号１で示されるアミノ酸配列における２～７番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、並びに
    　（ｅ）配列番号１で示されるアミノ酸配列における４２～５５番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体
    からなる群より選ばれる２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片である、請求項１０に記載のキット。     Two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof,
    (a) an anti-myoglobin monoclonal antibody that binds to the 79th to 85th and 92nd to 99th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1;
    (b) an anti-myoglobin monoclonal antibody that binds to the 118th to 126th amino acid region in the amino acid sequence shown in SEQ ID NO: 1;
    (c) an anti-myoglobin monoclonal antibody that binds to the 145th to 154th amino acid region in the amino acid sequence shown in SEQ ID NO: 1;
    (d) an anti-myoglobin monoclonal antibody that binds to the 2nd to 7th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1, and (e) binds to the 42nd to 55th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1 The kit according to claim 10, which is two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof selected from the group consisting of anti-myoglobin monoclonal antibodies.
  12. 　２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片が、
    　（ａ）配列番号１で示されるアミノ酸配列における７８～１０４番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、
    　（ｂ）配列番号１で示されるアミノ酸配列における１１２～１３０番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体、並びに
    　（ｃ）配列番号１で示されるアミノ酸配列における１４１～１５４番目のアミノ酸領域に結合する抗ミオグロビンモノクローナル抗体
    からなる群より選ばれる２種以上の抗ミオグロビンモノクローナル抗体又はその抗原結合性断片である、請求項９に記載のキット。     Two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof,
    (a) an anti-myoglobin monoclonal antibody that binds to the 78th to 104th amino acid region in the amino acid sequence shown in SEQ ID NO: 1;
    (b) an anti-myoglobin monoclonal antibody that binds to the 112th to 130th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1; and (c) binds to the 141st to 154th amino acid regions in the amino acid sequence shown in SEQ ID NO: 1 The kit according to claim 9, which is two or more anti-myoglobin monoclonal antibodies or antigen-binding fragments thereof selected from the group consisting of anti-myoglobin monoclonal antibodies.
  13. 　配列番号２～６のいずれか一つで示されるアミノ酸配列からなる、ポリペプチド。 A polypeptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 2-6.
  14. 　配列番号４～６のいずれか一つで示されるアミノ酸配列からなる、請求項１３に記載のポリペプチド。 The polypeptide according to claim 13, which consists of the amino acid sequence shown in any one of SEQ ID NOS: 4-6.
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