WO2022172901A1 - Anticorps monoclonal anti-myoglobine ou fragment de liaison à l'antigène de celui-ci, procédé de détection de myoglobine, kit et polypeptide - Google Patents

Anticorps monoclonal anti-myoglobine ou fragment de liaison à l'antigène de celui-ci, procédé de détection de myoglobine, kit et 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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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

La présente invention concerne : un anticorps monoclonal anti-myoglobine ou un fragment de liaison à l'antigène de celui-ci approprié pour une mesure immunologique ; un procédé et un kit de détection de la myoglobine à l'aide dudit anticorps monoclonal anti-myoglobine ou d'un fragment de liaison à l'antigène de celui-ci ; et un polypeptide reconnu par l'intermédiaire dudit anticorps monoclonal anti-myoglobine ou du fragment de liaison à l'antigène de celui-ci. Un anticorps monoclonal anti-myoglobine ou un fragment de liaison à l'antigène de celui-ci selon un aspect de la présente invention se lie aux (a) 78e à 104e, (b) 112e à 130e, (c) 141e à 154e, (d) 2e à 18e, ou (e) 35e à 64e régions d'acides aminés.
PCT/JP2022/004739 2021-02-12 2022-02-07 Anticorps monoclonal anti-myoglobine ou fragment de liaison à l'antigène de celui-ci, procédé de détection de myoglobine, kit et polypeptide WO2022172901A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023238821A1 (fr) * 2022-06-09 2023-12-14 デンカ株式会社 Anticorps monoclonal antimyoglobine

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013145139A (ja) * 2012-01-13 2013-07-25 Konica Minolta Inc Ｓｐｆｓ（表面プラズモン励起増強蛍光分光法）を用いたミオグロビンの免疫学的測定法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013145139A (ja) * 2012-01-13 2013-07-25 Konica Minolta Inc Ｓｐｆｓ（表面プラズモン励起増強蛍光分光法）を用いたミオグロビンの免疫学的測定法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BERIT JOHNE , KARL HANSEN , EINAR MORK , JOSTEIN HOLTLUND: "Colloidal gold conjugated monoclonal antibodies, studied in the BIAcore biosensor and in the Nycocard immunoassay format", JOURNAL OF IMMUNOLOGICAL METHODS, vol. 183, no. 1, 14 June 1995 (1995-06-14), NL , pages 167 - 174, XP004021036, ISSN: 0022-1759, DOI: 10.1016/0022-1759(95)00047-E *
MIHOC DELIA, LUPU LOREDANA-MIRELA, WIEGAND PASCAL, KLEINEKOFORT WOLFGANG, MÜLLER OLIVER, VÖLKLEIN FRIEDEMANN, GLOCKER MICHAEL O., : "Antibody Epitope and Affinity Determination of the Myocardial Infarction Marker Myoglobin by SPR-Biosensor Mass Spectrometry", JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY, vol. 32, no. 1, 6 January 2021 (2021-01-06), US , pages 106 - 113, XP055957710, ISSN: 1044-0305, DOI: 10.1021/jasms.0c00234 *

Cited By (1)

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