Anti-myoglobin monoclonal antibody

Abstract

Provided is an antibody having a high ability to detect human myoglobin.　An anti-human-myoglobin monoclonal antibody characterized by reacting with a partial peptide sequence ALGGILKKKG (SEQ ID NO: 2) of human myoglobin or a partial sequence thereof, or characterized by reacting with a partial peptide sequence IPGHGQEVLI (SEQ ID NO: 3) of human myoglobin or a partial sequence thereof; or an antigen-binding fragment of the anti-human myoglobin monoclonal antibody.

Description

[Technical Field]

[0001] This invention relates to a monoclonal antibody that specifically binds to human myoglobin. [Background technology]

[0002] Myoglobin is one of the major proteins in skeletal and cardiac muscle, and is a heme protein that plays a role in oxygen storage. This oxygen-binding heme protein 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 within an alpha helix structure.

[0003] When muscle tissue is damaged, such as in a myocardial infarction, myoglobin levels in the blood and urine rise significantly within a few hours. In recent years, it has also been pointed out that elevated myoglobin levels associated with muscle tissue damage can lead to kidney damage.

[0004] Therefore, accurately measuring myoglobin concentration is of extremely important significance in diagnosing heart disease and kidney disease.

[0005] While several myoglobin detection methods have been established, there is a need for monoclonal antibodies that exhibit high affinity for human myoglobin, which can be used as reagents in the immune system to measure myoglobin levels in the blood, serum, or plasma of patients with myocardial damage (e.g., myocardial infarction).

[0006] Furthermore, a method for obtaining high-performance monoclonal antibody-producing hybridomas with high efficiency has been reported (Patent Document 1). [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Patent No. 6311959 [Overview of the project] [Problems that the invention aims to solve]

[0008] Currently, various test reagents using anti-human myoglobin antibodies are available on the market. However, the performance of these conventional test reagents in detecting human myoglobin has been insufficient. The present invention aims to provide an antibody with high detection capability for human myoglobin. [Means for solving the problem]

[0009] The inventors of the present invention have discovered that the performance of detecting human myoglobin can be improved by using an anti-human myoglobin antibody that reacts to the human myoglobin peptide sequence ALGGILKKKG (SEQ ID NO: 2) or IPGHGQEVLI (SEQ ID NO: 3), and have completed the present invention.

[0010] In other words, the present invention is as follows. [1] An anti-human myoglobin monoclonal antibody or an antigen-binding fragment thereof, characterized by reacting with the partial peptide sequence ALGGILKKKG (SEQ ID NO: 2) of human myoglobin or a part thereof. [2] An anti-human myoglobin monoclonal antibody or an antigen-binding fragment thereof, characterized by reacting with the partial peptide sequence IPGHGQEVLI (SEQ ID NO: 3) of human myoglobin or a part thereof. [3] The anti-human myoglobin monoclonal antibody of [1] or its antigen-binding fragment, wherein the antigen-binding fragment is a peptide fragment selected from the group consisting of Fab, Fab', F(ab')2, single-chain antibody (scFv), dsFv, diabody, and minibody. [4] A reagent for detecting human myoglobin containing one of the monoclonal antibodies or antigen-binding fragments thereof from [1] to [3]. [5] A reagent for detecting human myoglobin of [4], for use in the sandwich method. [6] The human myoglobin detection reagent of [5], wherein the combination of two antibodies in the sandwich method is a combination of the monoclonal antibody of [1] or its antigen-binding fragment and the monoclonal antibody of [2] or its antigen-binding fragment. [7] A kit for detecting human myoglobin, comprising the monoclonal antibody of any one of [1] to [3] or its antigen-binding fragment. [8] A method for detecting human myoglobin by an immunological detection method using the monoclonal antibody of any one of [1] to [3] or its antigen-binding fragment. [9] The method of [7], which is a sandwich method.

[10] The method of [9], wherein the combination of two antibodies in the sandwich method is a combination of the monoclonal antibody of [1] or its antigen-binding fragment and the monoclonal antibody of [2] or its antigen-binding fragment. This specification incorporates the disclosure of Japanese Patent Application No. 2022-093483, which is the basis of the priority of this application. [Advantages of the Invention]

[0011] The method of the present invention uses an antibody that reacts with the peptide sequence ALGGILKKKG (SEQ ID NO: 2) or IPGHGQEVLI (SEQ ID NO: 3) of human myoglobin in immunoassay, so it has high sensitivity. Further, according to the present invention, an immunoassay instrument and a monoclonal antibody used in the novel detection method of the present invention are provided. [Brief Description of the Drawings]

[0012] [Figure 1] It is a diagram showing the peptide sequence of the hMyo antigen. [Figure 2] It is a diagram showing a schematic diagram of an epitope binning assay and a measurement pattern. FIG. 2A shows a schematic diagram of the measurement, and FIG. 2B shows a typical measurement pattern. [Figure 3] It is a diagram showing the measurement result of the epitope binning assay. [Embodiments for Carrying Out the Invention]

[0013] Next, embodiments of the present invention will be described. The technical scope of the present invention is not limited by these embodiments, and it can be implemented in various forms without changing the gist of the invention.

[0014] The test subject 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), and the peptide sequence IPGHGQEVLI (SEQ ID NO: 3) is a peptide sequence consisting of amino acids 21 to 30 of the full-length amino acid sequence of human myoglobin (SEQ ID NO: 1).

[0015] The method of the present invention performs immunoassay using a monoclonal antibody or its antigen-binding fragment that recognizes the human myoglobin peptide sequence ALGGILKKKG (SEQ ID NO: 2) or IPGHGQEVLI (SEQ ID NO: 3) as an antigen. Here, "recognize" means to react specifically, that is, to undergo an antigen-antibody reaction. "Specific" means that in a liquid system in which the protein and the antibody are mixed, the antibody does not undergo an antigen-antibody reaction with the protein component of the other antigen at a detectable level, or even if it undergoes some binding or association reaction, the reaction is significantly weaker than the antigen-antibody reaction of the antibody with the antigen.

[0016] Antigen-binding fragments obtained by isolating only the antigen-binding site from the monoclonal antibody of the present invention can also be used in the method of the present invention. That is, even when using Fab, Fab', F(ab')2, single-chain antibodies (scFv), recombinant antibodies, or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR transplant antibodies, primate-like antibodies, deimmunized antibodies, synhumanized antibodies, dsFv, diabody, minibody, etc., which have specific antigen-binding properties (antigen-binding fragments)) prepared by known methods that bind to the human myoglobin peptide sequence ALGGILKKKG (SEQ ID NO: 2) or IPGHGQEVLI (SEQ ID NO: 3), these fragments are also included in the monoclonal antibody and are within the scope of the present invention. Fab is a fragment obtained by treating an antibody with papain, a proteolytic enzyme, and is an antibody fragment having binding activity to an antigen 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 linked by disulfide bonds. F(ab')2 is an antibody fragment with a molecular weight of approximately 100,000, obtained by treating IgG with the protease pepsin, with Fab linked 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. scFv, one of the Fv fragments, 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. Diabody is an antibody fragment obtained by dimerizing scFv, and is an antibody fragment with bivalent antigen-binding activity. Furthermore, the class of monoclonal antibody is not limited to IgG, but may also be IgM or IgY.

[0017] The monoclonal antibody 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 methods, and by producing a hybridoma using the cells of the immunized animal. The length of the peptide used for immunization is not particularly limited, but preferably a peptide of 3 amino acids or more, more preferably 5 amino acids or more, and even more preferably 10 amino acids or more can be used as an immunogen. Furthermore, it is preferable to use a peptide consisting of the human myoglobin peptide sequence ALGGILKKKG (SEQ ID NO: 2) or IPGHGQEVLI (SEQ ID NO: 3) as the immunogen. The immunogen can also be obtained by incorporating DNA encoding human myoglobin into a plasmid vector and introducing it into host cells for expression. The human myoglobin or partial peptide thereof used as an immunogen can be expressed as a fusion protein with a protein as exemplified below, and after purification, or unpurified, it can be used as an immunogen. For the preparation of fusion proteins, glutathione S-transferase (GST), maltose-binding protein (MBP), thioredoxin (TRX), Nus tag, S tag, HSV tag, FRAG tag, polyhistidine tag, etc., which are commonly used by those skilled in the art as "protein expression and purification tags," can be used. It is preferable to use the fusion proteins with these tags as immunogens after cleaving the human myoglobin or partial peptide portion from the other tag portion using digestive enzymes, separating and purifying them.

[0018] The preparation of monoclonal antibodies from immunized animals can be easily carried out using the well-known method of Kohler et al. (Kohler and Milstein, Nature, vol, 256, pp. 495-497 (1975)). Specifically, antibody-producing cells such as spleen cells and lymphocytes are collected from immunized animals, and these are fused with mouse myeloma cells using a conventional method to create hybridomas. The resulting hybridomas are then cloned using methods such as limiting dilution, and from the monoclonal antibodies produced by each cloned hybridoma, a monoclonal antibody that reacts with a peptide consisting of the human myoglobin peptide sequence ALGGILKKKG (SEQ ID NO: 2) or IPGHGQEVLI (SEQ ID NO: 3) is selected.

[0019] Furthermore, techniques such as the method of obtaining the desired hybridoma by cross-linking the target B cells and myeloma cells with biotin / avidin beforehand and then applying an electrical pulse can also be used (Patent Document 1).

[0020] Monoclonal antibodies can be purified from ascites fluid or culture supernatant using known immunoglobulin purification methods. Examples include fractionation by salting out with ammonium sulfate or sodium sulfate, PEG fractionation, ethanol fractionation, DEAE ion exchange chromatography, and gel filtration. Furthermore, depending on the immunizing animal species and the class of the monoclonal antibody, purification is also possible using affinity chromatography with a carrier conjugated to either protein A, protein G, or protein L.

[0021] Furthermore, the monoclonal antibodies used in the present invention can be produced from genetically modified plants. Such antibodies can be produced using a plant transient expression system. In addition, the monoclonal antibodies used in the present invention can also be obtained as genetically modified organisms using mammalian cells as the expression host. 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, the methods for obtaining genetically modified organisms are not particularly limited and include transient expression systems using plasmid vectors or viral vectors lacking autologous replication ability, semi-stable expression systems using episomal vectors that confer nuclear localization signals and possess autologous replication ability, and stable expression systems in which the target gene is inserted into the genome of the expression host.

[0022] The antibodies that recognize the human myoglobin peptide sequences ALGGILKKKG (SEQ ID NO: 2) or IPGHGQEVLI (SEQ ID NO: 3) of the present invention exhibit reactivity flexibility and may, for example, recognize the three-dimensional structure formed by these peptides. Furthermore, the antibodies that recognize the human myoglobin peptide sequence ALGGILKKKG (SEQ ID NO: 2) of the present invention may recognize a peptide sequence consisting of a portion of the peptide sequence ALGGILKKKG (SEQ ID NO: 2). The portion of the peptide sequence ALGGILKKKG (SEQ ID NO: 2) refers to a continuous amino acid sequence within the peptide sequence ALGGILKKKG (SEQ ID NO: 2) consisting of 3, 4, 5, 6, 7, 8, or 9 amino acids, preferably 3 or more, more preferably 5 or more amino acids. The antibodies that recognize the human myoglobin peptide sequence IPGHGQEVLI (SEQ ID NO: 3) of the present invention may recognize a peptide sequence consisting of a portion of the peptide sequence IPGHGQEVLI (SEQ ID NO: 3). The part of the peptide sequence IPGHGQEVLI (SEQ ID NO: 3) refers to a continuous amino acid sequence within the peptide sequence IPGHGQEVLI (SEQ ID NO: 3) that consists of 4, 5, 6, 7, 8, or 9 amino acids, preferably 5 or more amino acids.

[0023] The immunoassay method of the present invention uses an antigen-antibody reaction between a monoclonal antibody or its antigen-binding fragment (hereinafter, unless otherwise clearly stated in the context, "antibody" means "antibody or its antigen-binding fragment") prepared as described above and an antigen in the sample for measurement. Any method well known to those skilled in the art can be used as the immunoassay method for this purpose, such as competitive assay, agglutination assay, Western blotting assay, immunostaining assay, or sandwich assay. In this invention, "measurement" encompasses quantitative, semi-quantitative, and detection assays.

[0024] For immunoassays, the sandwich method is preferred. In the sandwich method, a complex is formed by sandwiching an antigen between two antibodies, and this complex is detected. The sandwich method itself is well known in the field of immunoassays and can be performed, for example, by immunochromatography or ELISA. All of these sandwich methods are well known, and the method of the present invention can be performed by known sandwich methods, except that it 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.

[0025] The sandwich method uses one type of antibody that recognizes the antigen (when the antigen forms a multimer) or two or more types of antibodies (an antibody immobilized on the solid phase and a labeled antibody). When two or more types of antibodies are used, at least one of these two antibodies is a monoclonal antibody that recognizes the human myoglobin peptide sequence ALGGILKKKG (SEQ ID NO: 2) or IPGHGQEVLI (SEQ ID NO: 3) as the antigen. When using one type of antibody, a complex can be formed by sandwiching the antigen between the two identical antibodies. One type of antibody can be used when there are multiple sites on a single antigen to which one type of antibody can bind, such as when the antigen forms a multimer. Preferably, one or two types of antibodies that recognize the above peptide are used. The other antibody can be any antibody that recognizes a portion of the human myoglobin other than the peptide sequence ALGGILKKKG (SEQ ID NO: 2) or IPGHGQEVLI (SEQ ID NO: 3). Furthermore, as the combination of two antibodies used in the sandwich method, a combination of a monoclonal antibody that recognizes the human myoglobin peptide sequence ALGGILKKKG (SEQ ID NO: 2) as an antigen and a monoclonal antibody that recognizes the human myoglobin peptide sequence IPGHGQEVLI (SEQ ID NO: 3) as an antigen may be used. In this case, the antibody immobilized on the solid phase may be a monoclonal antibody that recognizes the human myoglobin peptide sequence ALGGILKKKG (SEQ ID NO: 2) as an antigen, and the labeled antibody may be a monoclonal antibody that recognizes the human myoglobin peptide sequence IPGHGQEVLI (SEQ ID NO: 3) as an antigen, or 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, and the labeled antibody may be a monoclonal antibody that recognizes the human myoglobin peptide sequence ALGGILKKKG (SEQ ID NO: 2) as an antigen.

[0026] In immunoassays using the sandwich method as the detection principle, any known material on which antibodies can be immobilized can be used as the solid phase to which the antibodies are immobilized. For example, porous thin films (membranes) with capillary action, particulate matter, test tubes, resin plates, and other known materials can be arbitrarily selected. Furthermore, substances such as enzymes, radioisotopes, fluorescent substances, luminescent substances, colored particles, and colloidal particles can be used to label the antibodies.

[0027] The myoglobin detection reagent of the present invention, which includes a monoclonal antibody that recognizes the human myoglobin peptide sequence ALGGILKKKG (SEQ ID NO: 2) or IPGHGQEVLI (SEQ ID NO: 3), can be used with the immunoassay method described above. Furthermore, the myoglobin detection kit of the present invention, which includes a monoclonal antibody that recognizes the human myoglobin peptide sequence ALGGILKKKG (SEQ ID NO: 2) or IPGHGQEVLI (SEQ ID NO: 3), can be used with the immunoassay method described above. The kit may also include a bronchograph, a sample collection device, and the like.

[0028] Examples of specimens include human or animal bodily fluids such as blood, serum, plasma, urine, semen, cerebrospinal fluid, saliva, sweat, tears, ascites, or amniotic fluid; mucus; feces; organs such as blood vessels or the liver; tissues; cells; or extracts thereof, which may contain human myoglobin or a part thereof. The method of collecting these specimens is not particularly limited, and known methods can be employed. [Examples]

[0029] <Material> Human myoglobin (hMyo) was purchased from Oriental Yeast. 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 Fujifilm Wako Pure Chemical Industries, Ltd. HRP-conjugated anti-mouse IgG(H+L) antibody was purchased from BioSource International. Alexa Fluor 488-labeled goat anti-mouse IgG(H+L) antibody was purchased from Thermo Fisher Scientific. BALB / cAJcl mice were purchased from CREA Japan.

[0030] <Method> [1] Culture of myeloma cells Mouse myeloma cells of PAI, P3X63 Ag8.653 (X63), and SP2 / 0 were cultured in complete RPMI1640 medium supplemented with 10% FBS, 2 mM L-glutamine (Nissui Pharmaceutical Co., Ltd.), and 100 μg / mL kanamycin sulfate (Meiji Seika Co., Ltd.) in a 37°C, 5% carbon dioxide incubator.

[0031] [2] Immunization method BALB / c mice aged 5-7 weeks were used for immunization. hMyo antigen and CFA were mixed in a 1:1 (v / v) ratio to prepare a water-in-oil (w / o) emulsion. The water-in-oil (w / o) emulsion containing 50 μg of antigen was injected intraperitoneally. Two weeks later, hMyo antigen and IFA were mixed in a 1:1 (v / v) ratio, and the antigen in the IFA was used for a second immunization. Final immunization was performed 3-5 days before fusion.

[0032] [3] Preparation of spleen cells Mice that had undergone final immunization were placed in a glass bottle filled with isoflurane and anesthetized by inhalation. After confirming that the mice were anesthetized, they were fixed to the dissection table with a hypodermic needle. The abdomen of the mouse was disinfected with cotton wool soaked in 70% ethanol, and then the outer skin near the center of the abdomen was grasped with forceps, an incision was made in the outer skin with dissection scissors, and the incision was widened towards the heart. Next, the endothelium near the ribs was grasped with forceps, the thoracic cavity was opened until the heart was visible, and blood was drawn from the heart using a 5 ml syringe. After that, the mouse from which the heart blood was drawn was sterilized by immersing it in a beaker containing 70% ethanol and placed on aluminum foil laid in a clean bench. To remove the spleen, an incision was made in the outer skin on the left flank of the mouse with dissection scissors, and the outer skin was pushed open until the spleen was visible. Then, the endothelium was cut to expose the spleen. Next, the fat around the spleen was grasped with forceps, lifted together with the spleen, and the fat surrounding the spleen was cut with scissors to remove it from the mouse. The entire procedure for splenectomy was performed aseptically. The removed spleen was treated with DMEM. + The spleen was placed in a 15 ml conical tube containing 10 ml of [Dulbecco's Modified Eagle's Medium (DMEM, Nissui Pharmaceutical Co.) + 100 μg / ml Kanamycin Sulfate (Meiji Seika Co.)] and transported to a clean bench. The excised spleen was immediately placed in 10 ml of DMEM. + Transfer to an empty petri dish, rinse briefly, and then immediately hydrate. + The spleen was transferred to another petri dish containing 10 ml of DMEM, and the surrounding fat was removed using scissors and tweezers while washing. This washing was repeated three more times. A stainless steel mesh was placed in the last petri dish, the spleen was placed on it, and gently crushed with a rubber policeman. Next, the spleen cell suspension was transferred to a 50 ml conical tube. Furthermore, the petri dish with the stainless steel mesh was placed in 10 ml of DMEM. +Wash with it, collect the liquid, and add it to the 50 ml conical tube used earlier. Repeat this operation until the liquid volume in the tube reaches 40 ml. After centrifuging the suspension at 2,000 rpm (800 g) for 5 minutes, suspend the cell pellet in 5 ml of red blood cell lysing buffer (SIGMA) and let it stand in ice for 5 minutes to lyse red blood cells. Then, quickly add DMEM + 45 ml, make a total of 50 ml, mix well, centrifuge at 2,000 rpm (800 g) for 5 minutes, and then suspend the obtained cell pellet in 2.5 ml of DMEM + to prepare spleen cells.

[0033] [4] Preparation of Biotinylated Antigen Dissolve human myoglobin (hMyo) in PBS pH 7.2 to a final concentration of 1 mg / mL. Add N-hydroxysuccinimide (NHS) biotin (1 mg / 1 mL DMSO) to the hMyo solution at 0.9, 3.75, 4, 7.5, 30-fold molar equivalents, and gently rotate at room temperature for 30 minutes for biotinylation. After biotinylation, add glycine to a final concentration of 20 mM to stop the reaction.

[0034] [5] BCT Method (1) Optimized BCT Method Spleen cells (5.0×10 7 to 1.0×10 8 ) carrying sensitized B lymphocytes were suspended in 2.5 mL of DMEM + . 50 μg (3.75 - 7.5-fold molar equivalent) of the optimized biotinylated antigen dissolved in another 2.5 mL of DMEM + was mixed with 2.5 mL of the above spleen cell suspension and gently rotated at 4°C for 2 hours. Then, centrifuge the cell suspension at 2,000 rpm (800 g) for 5 minutes and wash it with 10 mL of DMEM + . Resuspend the spleen cell pellet in 2.5 mL of DMEM + . Another 2.5 mL of DMEM + containing 200 μg of streptavidinThe spleen cell suspension was added and gently rotated at 4°C for 1 hour. Finally, the cell suspension was centrifuged at 2,000 rpm (800 g) for 5 minutes and condensed in 10 mL of DMEM. + Washed with 5 mL of DMEM. Spleen cell pellet. + The cells were resuspended. Here, the B cell-antigen-biotin-streptavidin complex was formed. Myeloma cells (2.0 × 10⁻⁶) 7 From 4.0 x 10 7 ) is collected by centrifugation at 800 rpm (130 g) for 5 minutes, and 10 mL of sterile PBS (PBS + Washed with ) Myeloma cell pellet in 5 mL PBS + The cells were suspended in and mixed with 5 mL of PBS containing 10 μL of NHS biotin (1 mg per 30 μL of DMF), and gently rotated at 37 °C for 30 minutes. The biotinylated myeloma cells were centrifuged at 800 rpm (130 g) for 5 minutes to form a pellet, and then immersed in 10 mL of DMEM. + Washed with [method]. A spleen cell suspension containing B cell-antigen-biotin-streptavidin complex was mixed with biotinylated myeloma cells in a 4:1 ratio (spleen cells to myeloma cells), centrifuged at 1,000 rpm (200 g) for 10 minutes, and dispensed into 5 mL of DMEM. + The cells were resuspended in [a specific medium]. This cell mixture was gently rotated at room temperature for 0.5–1 hour. Due to the strong and specific interaction of biotin and streptavidin, B lymphocytes bound to biotinylated myeloma cells. The B cell-myeloma cell complexes were resuspended in 1–2 mL of isotonic sucrose buffer consisting of 0.25 M sucrose, 2 mM NaH2PO4 / Na2HPO4 (pH = 7.2), 0.1 mM MgCl2, and 0.1 mM CaCl2. The B cell-myeloma cell complexes were selectively fused by electrical pulses (10 μsec, 2.0 kV / cm to 3.0 kV / cm, 4 cycles at 1-second intervals). The fused hybridoma cells were cultured in HAT selective medium for 2 weeks and then in HT medium for a further 2 weeks. (2) Conventional BCT method The conventional BCT method was performed using essentially the same protocol as the optimized BCT technique, except that it used a 30-fold molar equivalent of biotinylated antigen.

[0035] [6] PEG method D-Receive + Spleen cells containing sensitized B lymphocytes suspended in (5.0 x 10) 7 From 1.0x10 8 The spleen cells and mouse myeloma cells were mixed in a 10:1 ratio (spleen cells:myeloma cells) and centrifuged at 1,000 rpm (200 g) for 10 minutes. After removing the supernatant, 1 mL of 50% (wt / vol) polyethylene glycol 4000 was added to the cell pellet over 1 minute to fuse the cells. 10 mL DMEM + After adding the reagent, the mixture was centrifuged at 1,000 rpm (200 g) for 10 minutes, and the supernatant was completely removed. The mixture was then cultured in HAT selective medium for 2 weeks, and then in HT medium for another 2 weeks.

[0036] [7] Cloning of hybridoma cells Using the limiting dilution method, hybridoma cells were diluted to 9, 3, 1, and 0.5 cells / well, seeded in 96-well plates, and cultured at 37°C in a 5% carbon dioxide incubator.

[0037] [8]ELISA (enzyme-linked immunosorbent assay) method The antigen was diluted to 1-10 μg / ml with 0.1 M NaHCO3, added to 50 μl / well of a 96-well plate, and allowed to stand overnight at 4 °C to adsorb the antigen onto the plate. Next, the plate was washed three times with PBS, and 350 μl / well of 1% gelatin (1 g / 100 ml PBS) was added and incubated at 37 °C for 2 hours to block the plate. Subsequently, the plate was washed three times with PBST (PBS + 0.05% Triton X-100), and 50 μl / well of hybridoma cell culture supernatant was added as the primary antibody and incubated at 37 °C for 1 hour. After washing three times with PBST, 50 μl / well of a secondary antibody (anti-mouse IgG(H+L) antibody conjugated with HRP), diluted 10,000-fold with PBST, was added and incubated at 37 °C for 1 hour. Finally, after washing five times with PBST, 100 μl / well of the chromogenic agent [0.1M sodium citrate buffer (pH 5.2) + o-phenylene diamine (1 mg / ml) + 0.02% H2O2] was added and incubated at 37°C for 10 minutes. The reaction was then stopped by adding 50 μl / well of 1M H2SO4. The OD490nm was measured using a plate reader.

[0038] [9] Epitope mapping To identify the epitopes of the anti-hMyo antibody, epitope mapping was performed using the hMyo antigen peptide. Based on the hMyo sequence, 10-amino acid peptides, numbered 1 through 30, were prepared with 5-amino acid overlaps, and their N-terminuses were biotinylated. The biotinylated peptides were immobilized on a streptavidin-coated 96-well plate. ELISA was performed using anti-hMyo antibodies prepared by PEG and optimized BCT methods as primary antibodies. Figure 1 shows the peptide sequences of the hMyo antigen. Amino acids that differ between mouse and human are indicated with thick black borders and bold text.

[0039]

[10] Epitope binning assay The epitope binning assay was performed using the Octet 384 Red system (Sartorius). All steps were performed in a 384-well plate containing 100 μL of solution per well, at 30°C and shaken at 1,000 rpm. PBS was used for antibody and antigen dilution and washing. The primary monoclonal antibody (1st Ab 200 nM) was loaded onto an anti-mouse IgG Fc capture (AMC) biosensor for 200 seconds, followed by washing for 30 seconds. Then, mouse IgG antibody (333 nM), unrelated to the reaction, was added to the AMC biosensor, the AMC biosensor was blocked for 300 seconds, and washed for 60 seconds. The hMyo antigen (200 nM) and secondary monoclonal antibody (2nd Ab 200 nM) were premixed at room temperature for 4 hours. After the hMyo antigen was completely saturated with 2nd Ab, the reaction was allowed to proceed for 200 seconds in the final step.

[0040] <Result> Evaluation Result 1: Epitope Mapping Results Epitope mapping was performed on anti-hMyo monoclonal antibodies, and the epitopes of each monoclonal antibody were analyzed. The results are shown in Table 1. Most of the epitopes of anti-hMyo monoclonal antibodies produced by the PEG method were peptide numbers 9 or 8-9, while the monoclonal antibodies produced by the optimized BCT method had peptide numbers 5 (IPGHGQEVLI (SEQ ID NO: 3)) and 15 (ALGGILKKKG (SEQ ID NO: 2)). Thus, the epitopes of the two monoclonal antibodies were completely different. However, when the epitopes of mouse antiserum obtained by PEG and optimized BCT immunization were similarly examined, the antiserum reacted strongly to the peptide number 8-9 region (Table 2). For monoclonal antibodies obtained by random fusion using the PEG method, it is thought that the same site as the site where the antiserum reacts strongly was preferentially established because there are many antibody-producing B lymphocytes in the peptide number 8-9 region. Conversely, the optimized BCT method selectively produced monoclonal antibodies that reacted with peptides other than 9 or 8-9. As a result, we were able to establish a monoclonal antibody that reacted with peptide number 15, which could never be obtained by the PEG method. This factor is related to the biotinylated antigen used for B cell selection in the optimized BCT method. LC-MS analysis of the biotinylated hMyo antigen revealed that mainly the N-terminal amino acid and lysine residues at positions 42 and 63 were biotinylated (Figure 1). The lysine residue at position 42 is a site included in peptides 8 and 9, and this biotinylation inhibited the selection of antibody-producing B cells that bind to peptides 8-9 in the optimized BCT method. As a result, monoclonal antibodies that bind to sequences other than peptides 8 and 9 could be preferentially produced in the optimized BCT method.

[0041] [Table 1]

[0042] [Table 2]

[0043] Evaluation result 2: Epitope binning assay results The combinations of monoclonal antibodies obtained from the optimized BCT method and the PEG method that can sandwich the hMyo antigen were measured using an epitope binning assay. A schematic diagram of the measurement by the epitope binning assay is shown in Figure 2A. Briefly, the primary monoclonal antibody was captured with an anti-mouse IgG Fc Capture (AMC) biosensor (Step 1), and the sensorgram was washed (Step 2). Then, mouse IgG antibody unrelated to the reaction was added to the AMC sensor to block it (Step 3), and then washed again (Step 4). Finally, a secondary monoclonal antibody premixed with the hMyo antigen was added and the reaction was allowed to proceed (Step 5). A typical measurement pattern of the epitope binning assay is shown in Figure 2B. In Step 5, it can be seen that when the combination of monoclonal antibodies was able to sandwich the hMyo antigen, the sensorgram increased quantitatively in proportion to the affinity of the monoclonal antibody. The strength of the reaction was defined from the binding rate (nm) of the measurement results as follows: 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 Figure 3. In Figure 3, "5", "9", "15", and "17" in the "1st mAb" and "2nd mAb" columns indicate the peptide numbers mentioned above. Antibody pairs possessing the same epitope are indicated as "No response", while antibody pairs possessing different epitopes were able to sandwich the antigen and are indicated as "w", "s", "ss", and "sss". Furthermore, antibody pairs against unknown epitopes sometimes could sandwich the antigen and sometimes could not. While not all combinations are shown here, numerous monoclonal antibodies could be produced using the PEG method. However, since most of them possessed the same epitope (peptide 9 or 8-9), there were few combinations that could be used for sandwiching.On the other hand, monoclonal antibodies produced by the optimized BCT method possess epitopes that bind to rare peptide 5 and peptide 15, which could not be obtained by the PEG method. When monoclonal antibodies produced by the optimized BCT method were used as the primary antibody or both, strong reactivity of sss and ss was observed. Among these, 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. It was found that monoclonal antibodies obtained by the optimized BCT method have high affinity. [Industrial applicability]

[0044] The anti-myoglobin monoclonal antibody of the present invention can be used for highly sensitive myoglobin measurement. [Sequence Listing Free Text]

[0045] Sequence ID 2 Synthesis Sequence ID 3 Synthesis All publications, patents, and patent applications cited herein shall be incorporated herein by direct reference.

Claims

1. An anti-human myoglobin monoclonal antibody or its antigen-binding fragment, characterized by comprising at least five consecutive amino acids from the 2nd to the 9th position in the partial peptide sequence ALGGILKKKG (SEQ ID NO: 2) of human myoglobin, and reacting to any epitope consisting of five to eight consecutive amino acids in the amino acid sequence from the 69th to the 82nd position of SEQ ID NO:

1.

2. An anti-human myoglobin monoclonal antibody or its antigen-binding fragment, characterized by comprising at least five consecutive amino acids from the 2nd to the 9th position in the partial peptide sequence IPGHGQEVLI (SEQ ID NO: 3) of human myoglobin, and reacting to any epitope consisting of five to eight consecutive amino acids in the amino acid sequence from the 19th to the 32nd position in SEQ ID NO:

1.

3. The antigen-binding fragments are Fab, Fab', and F(ab'). ２ The anti-human myoglobin monoclonal antibody or antigen-binding fragment thereof according to claim 1, which is a peptide fragment selected from the group consisting of single-chain antibody (scFv), dsFv, diabody, and minibody.

4. A reagent for detecting human myoglobin comprising a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3.

5. A reagent for detecting human myoglobin according to claim 4, for use in the sandwich method.

6. The human myoglobin detection reagent according to claim 5, wherein the combination of two antibodies in the sandwich method is a combination of the monoclonal antibody or its antigen-binding fragment according to claim 1 and the monoclonal antibody or its antigen-binding fragment according to claim 2.

7. A kit for detecting human myoglobin comprising a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3.

8. A method for detecting human myoglobin by an immunological detection method using a monoclonal antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 3.

9. The method according to claim 7, which is a sandwich method.

10. The method according to claim 9, wherein the combination of two antibodies in the sandwich method is a combination of the monoclonal antibody or its antigen-binding fragment according to claim 1 and the monoclonal antibody or its antigen-binding fragment according to claim 2.

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