WO2023238821A1 - Anticorps monoclonal antimyoglobine - Google Patents

Anticorps monoclonal antimyoglobine Download PDF

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Publication number
WO2023238821A1
WO2023238821A1 PCT/JP2023/020804 JP2023020804W WO2023238821A1 WO 2023238821 A1 WO2023238821 A1 WO 2023238821A1 JP 2023020804 W JP2023020804 W JP 2023020804W WO 2023238821 A1 WO2023238821 A1 WO 2023238821A1
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antigen
monoclonal antibody
antibody
seq
human myoglobin
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PCT/JP2023/020804
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Japanese (ja)
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建人 坂下
昌弘 冨田
幹太 湊元
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デンカ株式会社
国立大学法人三重大学
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Publication of WO2023238821A1 publication Critical patent/WO2023238821A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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

Definitions

  • the present invention relates to a monoclonal antibody that specifically binds to human myoglobin.
  • Myoglobin is one of the major proteins in skeletal and cardiac muscles, and is a heme protein that plays a role in storing oxygen.
  • This oxygen-binding hemoprotein consists of a single polypeptide chain with a reported molecular weight of 17.8 kDa.
  • the tertiary structure of myoglobin has been extensively studied, and it has been reported that 75% of the main chain is folded into an alpha-helical structure.
  • myoglobin detection methods have been established, but one with high affinity for human myoglobin is used as an immune system reagent to measure myoglobin levels in blood, serum or plasma in patients with myocardial injury (e.g. myocardial infarction).
  • myocardial injury e.g. myocardial infarction
  • monoclonal antibodies that demonstrate.
  • Patent Document 1 a method has been reported in which a target high-performance monoclonal antibody-producing hybridoma can be obtained with high efficiency.
  • An object of the present invention is to provide an antibody that has a high ability to detect human myoglobin.
  • the present inventors have discovered that the performance of detecting human myoglobin can be improved by using an anti-human myoglobin antibody that reacts with the human myoglobin peptide sequence ALGGILKKKG (SEQ ID NO: 2) or IPGHGQEVLI (SEQ ID NO: 3). Completed the invention.
  • the present invention is as follows. [1] An anti-human myoglobin monoclonal antibody or an antigen-binding fragment thereof, which reacts with the partial peptide sequence ALGGILKKKG (SEQ ID NO: 2) of human myoglobin or a partial sequence thereof. [2] An anti-human myoglobin monoclonal antibody or an antigen-binding fragment thereof, which is characterized by reacting with the partial peptide sequence IPGHGQEVLI (SEQ ID NO: 3) of human myoglobin or a partial sequence thereof.
  • the antigen-binding fragment of [1] is a peptide fragment selected from the group consisting of Fab, Fab', F(ab') 2 , single chain antibody (scFv), dsFv, diabody, and minibody.
  • Anti-human myoglobin monoclonal antibody or antigen-binding fragment thereof is a human myoglobin detection reagent comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [3].
  • the human myoglobin detection reagent of [4] which is for sandwich method.
  • [6] Detection of human myoglobin in [5], where the combination of two antibodies in the sandwich method is a combination of the monoclonal antibody or antigen-binding fragment thereof in [1] and the monoclonal antibody or antigen-binding fragment thereof in [2] reagent.
  • a human myoglobin detection kit comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [3].
  • the method of [7] which is a sandwich method.
  • the method of the present invention has high sensitivity because it uses an antibody that reacts with the human myoglobin peptide sequence ALGGILKKKG (SEQ ID NO: 2) or IPGHGQEVLI (SEQ ID NO: 3) for immunoassay.
  • the present invention also provides an immunoassay instrument and a monoclonal antibody for use in the novel detection method of the present invention.
  • FIG. 3 shows the peptide sequence of hMyo antigen.
  • FIG. 2 is a diagram showing a schematic diagram and measurement pattern of an epitope binning assay.
  • FIG. 2A shows a schematic diagram of the measurement, and
  • FIG. 2B shows a typical measurement pattern.
  • FIG. 3 is a diagram showing measurement results of an epitope binning assay.
  • the subject to be detected by the method of the present invention is human myoglobin, and a monoclonal antibody that recognizes the peptide sequence ALGGILKKKG (SEQ ID NO: 2) or IPGHGQEVLI (SEQ ID NO: 3) as an antigen is used.
  • the peptide sequence ALGGILKKKG (SEQ ID NO: 2) is a peptide sequence consisting of amino acids 71 to 80 of the full-length amino acid sequence of human myoglobin (SEQ ID NO: 1)
  • the peptide sequence IPGHGQEVLI (SEQ ID NO: 3) is the full-length amino acid sequence of human myoglobin. This is a peptide sequence consisting of the 21st to 30th amino acids of the sequence (SEQ ID NO: 1).
  • an immunoassay is performed using a monoclonal antibody or an antigen-binding fragment thereof that recognizes the human myoglobin peptide sequence ALGGILKKKG (SEQ ID NO: 2) or IPGHGQEVLI (SEQ ID NO: 3) as an antigen.
  • “recognize” means reacting specifically, that is, causing an antigen-antibody reaction.
  • “Specific” means that the antibody does not cause an antigen-antibody reaction with the protein components of other antigens at a detectable level in a liquid system where the protein and the antibody are mixed, or that some binding reaction or association reaction occurs. Even if it is, it means that the reaction between the antibody and the antigen is clearly weaker than the antigen-antibody reaction.
  • Antigen-binding fragments based on the monoclonal antibodies of the present invention in which only the antigen-binding site has been isolated can also be used in the method of the present invention. That is, Fab, Fab', F(ab') 2 , single chain antibody (scFv) that binds to the human myoglobin peptide sequence ALGGILKKKG (SEQ ID NO: 2) or IPGHGQEVLI (SEQ ID NO: 3), produced by a known method.
  • recombinant or modified antibodies e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted antibodies, primatized antibodies, deimmunized antibodies, synhumanized antibodies, dsFv, diabodies, minibodies, etc.
  • fragments having a specific antigen-binding property e.g., antigen-binding fragments
  • Fab is a fragment obtained by treating an antibody with papain, a proteolytic enzyme, and has an antigen-binding activity with a molecular weight of approximately 50,000, in which approximately half of the amino terminal side of the H chain and the entire L chain are bonded by disulfide bonds.
  • an antibody fragment with F(ab') 2 is an antibody fragment with a molecular weight of approximately 100,000, which is obtained by treating IgG with pepsin, a proteolytic enzyme, in which Fab is bonded via a disulfide bond in the hinge region.
  • Fab' is an antibody fragment with a molecular weight of approximately 50,000, obtained by cleaving the disulfide bond in the hinge region of F(ab') 2 mentioned above.
  • An scFv which is one type of Fv fragment, is an antibody fragment in which one heavy chain variable region (VH) and one light chain variable region (VL) are linked via a peptide linker.
  • a diabody is an antibody fragment obtained by dimerizing scFv and has bivalent antigen-binding activity.
  • the class of monoclonal antibodies is not limited to IgG, but may be IgM or IgY.
  • the monoclonal antibodies used in the method of the present invention can be obtained by immunizing an immunized animal with human myoglobin or a partial peptide thereof using known immunological techniques and producing hybridomas using the cells of the immunized animal.
  • the length of the peptide used for immunization is not particularly limited, but a peptide of preferably 3 or more amino acids, more preferably 5 or more amino acids, even more preferably 10 or more amino acids can be used as an immunogen.
  • a peptide consisting of the human myoglobin peptide sequence ALGGILKKKG (SEQ ID NO: 2) or IPGHGQEVLI (SEQ ID NO: 3) may be used as an immunogen.
  • the immunogen can also be obtained by incorporating DNA encoding human myoglobin into a plasmid vector, introducing the vector into a host cell, and expressing the vector.
  • Human myoglobin or a partial peptide thereof as an immunogen can be expressed as a fusion protein with proteins such as those exemplified below, and can be used as an immunogen after purification or as an unpurified state.
  • GST glutathione S-transferase
  • MBP maltose binding protein
  • TRX thioredoxin
  • Nus tag, S tag, HSV which are commonly used as "protein expression/purification tags" by those skilled in the art are used.
  • Tags, FRAG tags, polyhistidine tags, etc. can be used. It is preferable to use a fusion protein with these as an immunogen after cleaving human myoglobin or its partial peptide portion and other tag portions using a digestive enzyme, separating and purifying the protein.
  • Monoclonal antibodies can be easily prepared from immunized animals by the well-known method of Kohler et al. (Kohler and Milstein, Nature, vol, 256, p495-497 (1975)). That is, antibody-producing cells such as splenocytes and lymphocytes are collected from the immunized animal, fused with mouse myeloma cells using a conventional method to create a hybridoma, and the resulting hybridoma is cloned using a limiting dilution method or the like.
  • a monoclonal antibody that reacts with an antigen-antibody reaction with a peptide consisting of the human myoglobin peptide sequence ALGGILKKKG (SEQ ID NO: 2) or IPGHGQEVLI (SEQ ID NO: 3) is selected.
  • Patent Document 1 a technique such as a method in which the target B cells and myeloma cells are cross-linked in advance with biotin/avidin and then subjected to an electric pulse to obtain the target hybridoma (Patent Document 1) can also be used.
  • a known immunoglobulin purification method can be used to purify monoclonal antibodies from ascites or culture supernatant. Examples include fractionation by salting out using ammonium sulfate or sodium sulfate, PEG fractionation, ethanol fractionation, DEAE ion exchange chromatography, and gel filtration. Purification can also be carried out by affinity chromatography using a carrier to which protein A, protein G, or protein L is bound, depending on the species of the immunized animal and the class of the monoclonal antibody.
  • the monoclonal antibodies used in the present invention can be produced using genetically modified plants. Such antibodies can be produced using plant transient expression systems. Furthermore, the monoclonal antibodies used in the present invention can also be obtained as recombinant antibodies using mammalian cells as expression hosts. Examples of mammalian cells in this case include, but are not limited to, CHO (Chinese Hamster Ovary) cells and HEK293 (Human Embryonic Kidney cells 293) cells. Furthermore, regarding methods for obtaining genetic recombinants, there are transient expression systems using plasmid vectors or viral vectors that lack autonomous replication ability, and semi-transient expression systems that use episomal vectors that are endowed with nuclear import signals and have autonomous replication ability. -stable expression systems, stable expression systems in which a gene of interest is inserted into the genome of an expression host, etc., and are not particularly limited.
  • the antibody that recognizes the human myoglobin peptide sequence ALGGILKKKG (SEQ ID NO: 2) or IPGHGQEVLI (SEQ ID NO: 3) of the present invention has flexibility in reactivity, for example, by recognizing the three-dimensional structure formed by these peptides. There is a possibility that Furthermore, the antibody of the present invention that recognizes the human myoglobin peptide sequence ALGGILKKKG (SEQ ID NO: 2) can recognize a peptide sequence consisting of a part of the peptide sequence ALGGILKKKG (SEQ ID NO: 2).
  • a partial sequence in the peptide sequence ALGGILKKKG is a continuous amino acid sequence in the peptide sequence ALGGILKKKG (SEQ ID NO: 2), and includes 3, 4, 5, 6, and 7 amino acids. , 8 or 9 amino acids, preferably 3 or more amino acids, more preferably 5 or more amino acids.
  • the antibody that recognizes the human myoglobin peptide sequence IPGHGQEVLI (SEQ ID NO: 3) of the present invention can recognize a peptide sequence consisting of a part of the peptide sequence IPGHGQEVLI (SEQ ID NO: 3).
  • the partial sequence in the peptide sequence IPGHGQEVLI is a continuous amino acid sequence in the peptide sequence IPGHGQEVLI (SEQ ID NO: 3), and includes 4, 5, 6, 7, and 8 amino acids. Or an amino acid sequence consisting of 9 amino acids, preferably an amino acid sequence consisting of 5 or more.
  • the monoclonal antibody or its antigen-binding fragment produced as described above (hereinafter, unless it is clear from the context, “antibody” refers to “antibody or its antigen-binding fragment”) It is measured by immunoassay that utilizes the antigen-antibody reaction between the antigen in the specimen and the antigen in the specimen.
  • immunoassay method any method well known to those skilled in the art can be used, such as a competitive method, an agglutination method, a Western blotting method, an immunostaining method, and a sandwich method.
  • “measurement” includes all of quantitative determination, semi-quantitative determination, and detection.
  • a sandwich method is preferred for immunoassay.
  • a complex is formed by sandwiching an antigen between two antibodies, and the complex is detected.
  • the sandwich method itself is well known in the field of immunoassay, and can be performed using, for example, immunochromatography or ELISA. All of these sandwich methods are well known, and the method of the present invention uses a monoclonal antibody that recognizes the human myoglobin peptide sequence ALGGILKKKG (SEQ ID NO: 2) or IPGHGQEVLI (SEQ ID NO: 3) as an antigen. , can be carried out by the well-known sandwich method.
  • the sandwich method uses one type of antibody that recognizes the antigen (if the antigen forms a multimer) or two or more types of antibodies (an antibody immobilized on a solid phase and a labeled antibody).
  • two or more types of antibodies are used, at least one of these two types of antibodies is a monoclonal antibody that recognizes the above-mentioned human myoglobin peptide sequence ALGGILKKKG (SEQ ID NO: 2) or IPGHGQEVLI (SEQ ID NO: 3) as an antigen. It is an antibody.
  • it is sufficient to form a complex by sandwiching the antigen between the same two antibodies, and when the antigen forms a multimer, one type of antibody binds to one antigen.
  • One type of antibody can be used when there are multiple sites to be obtained.
  • one or two types of antibodies that recognize the above-mentioned peptides are used.
  • any antibody that recognizes a portion other than the peptide consisting of the human myoglobin peptide sequence ALGGILKKKG (SEQ ID NO: 2) or IPGHGQEVLI (SEQ ID NO: 3) can be used.
  • a monoclonal antibody that recognizes the human myoglobin peptide sequence ALGGILKKKG (SEQ ID NO: 2) as an antigen
  • a monoclonal antibody that recognizes the human myoglobin peptide sequence IPGHGQEVLI (SEQ ID NO: 3) as an antigen.
  • the antibody immobilized on the solid phase is a monoclonal antibody that recognizes the peptide sequence ALGGILKKKG (SEQ ID NO: 2) of human myoglobin as an antigen
  • the labeled antibody recognizes the peptide sequence IPGHGQEVLI (SEQ ID NO: 3) of human myoglobin as an antigen.
  • the antibody immobilized on the solid phase may be a monoclonal antibody that recognizes the human myoglobin peptide sequence IPGHGQEVLI (SEQ ID NO: 3) as an antigen
  • the labeled antibody may be a monoclonal antibody that recognizes the human myoglobin peptide sequence ALGGILKKKG (SEQ ID NO: 2).
  • ) may be used as a monoclonal antibody that recognizes it as an antigen.
  • any solid phase on which antibodies can be immobilized can be used using any known technique, such as a porous thin film (membrane) with capillary action. , particulate matter, test tubes, resin flat plates, and other known materials can be selected arbitrarily. Furthermore, as substances for labeling antibodies, enzymes, radioactive isotopes, fluorescent substances, luminescent substances, colored particles, colloid particles, etc. can be used.
  • the above immunoassay method can be used for the myoglobin detection reagent containing a monoclonal antibody that recognizes the human myoglobin peptide sequence ALGGILKKKG (SEQ ID NO: 2) or IPGHGQEVLI (SEQ ID NO: 3) of the present invention.
  • the myoglobin detection kit containing a monoclonal antibody that recognizes the human myoglobin peptide sequence ALGGILKKKG (SEQ ID NO: 2) or IPGHGQEVLI (SEQ ID NO: 3) of the present invention can use the above immunoassay method.
  • the kit may also include a brochure, a sample collection device, and the like.
  • Specimen samples include, for example, human or animal blood, serum, plasma, urine, semen, spinal fluid, saliva, sweat, tears, body fluids such as ascites or amniotic fluid; mucus; feces; organs such as blood vessels or liver. ;
  • biological samples that may contain human myoglobin or a part thereof, such as tissues; cells, or their extracts; the method for collecting these samples is not particularly limited, and any known method may be used. be able to.
  • hMyo Human myoglobin
  • N-hydroxysuccinimide (NHS)-biotin and polyethylene glycol (PEG) 4000 were purchased from Sigma-Aldrich.
  • Streptavidin (StAv), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), complete Freund's adjuvant (CFA), and incomplete Freund's adjuvant (IFA) were purchased from Fuji Film Wako Pure Chemical Industries, Ltd.
  • Anti-mouse IgG (H+L) antibody conjugated with HRP was purchased from BioSource International.
  • Goat anti-mouse IgG (H+L) antibody labeled with Alexa Fluor 488 was purchased from Thermo Fisher Scientific.
  • BALB/cAJcl mice were purchased from Claire Japan.
  • mice from which heart blood was collected were sterilized by immersing them in a beaker containing 70% ethanol, and placed on aluminum foil placed in a clean bench.
  • a cut was made in the integument on the left flank of the mouse using dissecting scissors, and the integument was expanded until the spleen was visible.
  • the endothelium was cut to expose the spleen.
  • the fat near the spleen was grabbed with forceps, lifted up along with the spleen, and the fat around the spleen was cut with scissors and removed from the mouse.
  • a series of operations for splenectomy were all performed aseptically.
  • the removed spleen was placed in a 15 ml conical tube containing 10 ml of DMEM + [Dulbecco's Modified Eagle's medium (DMEM, Nissui Pharmaceutical Co., Ltd.) + 100 ⁇ g/ml kanamycin sulfate (Meiji Seika Co., Ltd.]] and transported to a clean bench. .
  • DMEM Dulbecco's Modified Eagle's medium
  • kanamycin sulfate Meiji Seika Co., Ltd.
  • a stainless steel mesh was set in the final Petri dish, and the spleen was placed there and gently crushed with a rubber policeman.
  • the splenocyte suspension was then transferred to a 50 ml conical tube.
  • the Petri dish containing the stainless steel mesh was washed with 10 ml of DMEM + , and the liquid was collected and added to the 50 ml conical tube. Repeat this operation until the volume in the tube reaches 40 ml, centrifuge the suspension at 2,000 rpm (800 g) for 5 minutes, and transfer the cell pellet to 5 ml of red blood cell lysing buffer (SIGMA).
  • SIGMA red blood cell lysing buffer
  • the red blood cells were hemolyzed by suspending them in water and leaving them on ice for 5 minutes. Then, quickly add 45 ml of DMEM + to make a total of 50 ml, mix, and centrifuge at 2,000 rpm (800 g) for 5 minutes. Cells were prepared.
  • BCT method (1) Optimized BCT method Spleen cells (5.0 ⁇ 10 7 to 1.0 ⁇ 10 8 ) containing sensitized B lymphocytes were suspended in 2.5 mL DMEM + . 50 ⁇ g of optimized biotinylated antigen (3.75-7.5 times molar equivalent) dissolved in another 2.5 mL of DMEM + was mixed with 2.5 mL of the above splenocyte suspension and gently rotated for 2 hours at 4°C. The cell suspension was then centrifuged at 2,000 rpm (800 g) for 5 min and washed with 10 mL DMEM + . The splenocyte pellet was resuspended in 2.5 mL DMEM + .
  • Myeloma cell pellets were suspended in 5 mL PBS + , mixed with 5 mL PBS containing 10 ⁇ L NHS biotin (1 mg per 30 ⁇ L DMF), and gently rotated for 30 min at 37 °C.
  • Biotinylated myeloma cells were pelleted by centrifugation at 800 rpm (130 g) for 5 min and washed with 10 mL DMEM + .
  • Splenocyte suspension containing B cell-antigen-biotin-streptavidin complexes was mixed with biotinylated myeloma cells at a ratio of 4:1 (spleen cell myeloma cells) and centrifuged at 1,000 rpm (200 g) for 10 min.
  • B lymphocytes bound to biotinylated myeloma cells due to the strong and specific interaction of biotin and streptavidin.
  • B cell-myeloma cell complexes were selectively fused by electrical pulses (10 ⁇ sec, 2.0 kV/cm to 3.0 kV/cm, 4 cycles at 1 second intervals).
  • the fused hybridoma cells were cultured in HAT selection medium for 2 weeks and HT medium for an additional 2 weeks.
  • (2) Conventional BCT method The conventional BCT method was basically performed using the same protocol as the optimized BCT technique, except that a 30-fold molar equivalent of biotinylated antigen was used.
  • PEG method Mix spleen cells (5.0x10 7 to 1.0x10 8 ) containing sensitized B lymphocytes suspended in DMEM + and mouse myeloma cells at a ratio of 10:1 (spleen cells: myeloma cells), and add 1,000 Centrifugation was performed at rpm (200 g) for 10 minutes. After removing the supernatant, 1 mL 50% (wt/vol) polyethylene glycol 4000 was added dropwise to the cell pellet over 1 min to fuse the cells. After adding 10 mL DMEM + , centrifugation was performed at 1,000 rpm (200 g) for 10 min, and the supernatant was completely removed. Thereafter, the cells were cultured for 2 weeks in HAT selection medium and for an additional 2 weeks in HT medium.
  • hybridoma cells were diluted to 9, 3, 1, and 0.5 cells/well, seeded in a 96-well plate, and cultured in a 5% carbon dioxide incubator at 37°C. .
  • ELISA enzyme-linked immunosorbent assay
  • Epitope binning assay was performed using Octet 384 Red system (Sartorius). All steps were performed in 384-well plates containing 100 ⁇ L of solution in each well at 30°C with shaking at 1,000 rpm. PBS was used for diluting and washing antibodies and antigens. The primary monoclonal antibody (1st Ab 200 nM) was loaded onto the anti-mouse IgG Fc capture (AMC) biosensor for 200 seconds, followed by a 30 second wash. Then, a mouse IgG antibody (333 nM) irrelevant to the reaction was added to the AMC biosensor to block the AMC biosensor for 300 seconds, followed by a 60 second wash.
  • AMC anti-mouse IgG Fc capture
  • hMyo antigen 200 nM
  • 2nd Ab 200 nM secondary monoclonal antibody
  • Evaluation result 2 Epitope binning assay results A combination that can sandwich the hMyo antigen using the monoclonal antibodies obtained from the optimized BCT method and the PEG method was measured using an epitope binning assay.
  • a schematic diagram of measurement by epitope binning assay is shown in FIG. 2A. Briefly, after capturing the primary monoclonal antibody with an anti-mouse IgG Fc Capture (AMC) biosensor (step 1), the sensorgram was washed (step 2). Thereafter, a mouse IgG antibody unrelated to the reaction was added to the AMC sensor for blocking (Step 3), followed by washing (Step 4).
  • AMC anti-mouse IgG Fc Capture
  • Step 5 a secondary monoclonal antibody premixed with hMyo antigen was added and reacted.
  • a typical measurement pattern of the epitope binning assay is shown in FIG. 2B.
  • step 5 it can be seen that when the combination of monoclonal antibodies was able to sandwich the hMyo antigen, the sensorgram increased quantitatively depending on the affinity of the monoclonal antibodies.
  • the strength of the reaction was defined as 0.50 nm ⁇ sss, 0.45 nm ⁇ ss ⁇ 0.50 nm, 0.40 nm ⁇ s ⁇ 0.45 nm, 0.15 nm ⁇ w ⁇ 0.40 nm, and No response ⁇ 0.15 nm. .
  • the measurement results of the epitope binning assay are shown in a matrix in FIG. In FIG. 3, "5", “9”, “15”, and “17” in the "1st mAb” and “2nd mAb” columns indicate the above-mentioned peptide numbers.
  • Antibody pairs carrying the same epitope are indicated with “No response”, while antibody pairs carrying different epitopes can sandwich antigens and are marked with “w”, “s", “ss”, “ sss”. Additionally, pairs of antibodies directed against unknown epitopes may or may not be able to sandwich the antigen. Although not all combinations are shown here, a large number of monoclonal antibodies were produced using the PEG method, but most of them possessed the same epitope (peptide 9 or 8-9), so there were only a few combinations that could be sandwiched. There wasn't many.
  • the monoclonal antibody created using the optimized BCT method has an epitope that binds to the rare peptide 5 and 15 that could not be obtained using the PEG method, so it is necessary to use the monoclonal antibody created using the optimized BCT method as the primary antibody or both.
  • strong reactivity of sss and ss was confirmed.
  • the combination of primary antibody 4F-1-5M and secondary antibody H4-2-B2 both obtained by the optimized BCT method, showed the strongest reactivity.
  • the monoclonal antibodies obtained by the optimized BCT method were found to have high affinity.
  • the anti-myoglobin monoclonal antibody of the present invention can be used for highly sensitive myoglobin measurement.

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Abstract

L'invention concerne un anticorps ayant une capacité élevée à détecter la myoglobine humaine.　Un anticorps monoclonal anti-myoglobine humaine caractérisé par la réaction avec une séquence peptidique partielle ALGGILKKKG (SEQ ID NO : 2) de la myoglobine humaine ou d'une séquence partielle de celle-ci, ou caractérisé par la réaction avec une séquence peptidique partielle IPGHGQEVLI (SEQ ID NO : 3) de la myoglobine humaine ou d'une séquence partielle de celle-ci ; ou un fragment de liaison à l'antigène de l'anticorps monoclonal anti-myoglobine humaine.
PCT/JP2023/020804 2022-06-09 2023-06-05 Anticorps monoclonal antimyoglobine WO2023238821A1 (fr)

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Citations (5)

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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
CN104862283A (zh) * 2015-04-03 2015-08-26 暨南大学 一对高特异性高亲和力结合人肌红蛋白的单克隆抗体及其应用
US20190086424A1 (en) * 2017-09-15 2019-03-21 Derek James Wilson Methods for epitope mapping
CN109721655A (zh) * 2018-12-28 2019-05-07 江苏众红生物工程创药研究院有限公司 抗人肌红蛋白抗体及其在检测试剂盒中的应用
WO2022172901A1 (fr) * 2021-02-12 2022-08-18 デンカ株式会社 Anticorps monoclonal anti-myoglobine ou fragment de liaison à l'antigène de celui-ci, procédé de détection de myoglobine, kit et polypeptide

Patent Citations (5)

* 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
CN104862283A (zh) * 2015-04-03 2015-08-26 暨南大学 一对高特异性高亲和力结合人肌红蛋白的单克隆抗体及其应用
US20190086424A1 (en) * 2017-09-15 2019-03-21 Derek James Wilson Methods for epitope mapping
CN109721655A (zh) * 2018-12-28 2019-05-07 江苏众红生物工程创药研究院有限公司 抗人肌红蛋白抗体及其在检测试剂盒中的应用
WO2022172901A1 (fr) * 2021-02-12 2022-08-18 デンカ株式会社 Anticorps monoclonal anti-myoglobine ou fragment de liaison à l'antigène de celui-ci, procédé de détection de myoglobine, kit et polypeptide

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