WO2019167874A1 - Ａｐｏａ４に対するモノクローナル抗体、免疫学的測定方法及び測定用キット - Google Patents

Ａｐｏａ４に対するモノクローナル抗体、免疫学的測定方法及び測定用キット Download PDF

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WO2019167874A1
WO2019167874A1 PCT/JP2019/007033 JP2019007033W WO2019167874A1 WO 2019167874 A1 WO2019167874 A1 WO 2019167874A1 JP 2019007033 W JP2019007033 W JP 2019007033W WO 2019167874 A1 WO2019167874 A1 WO 2019167874A1
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antibody
apoa4
amino acid
acid sequence
seq
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French (fr)
Japanese (ja)
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曜子 井田
小原　隆
英美子 湯本
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Eisai R&D Management Co Ltd
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Eisai R&D Management Co Ltd
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Priority to JP2020503487A priority Critical patent/JPWO2019167874A1/ja
Priority to EP19760446.5A priority patent/EP3760641A4/en
Priority to US16/965,161 priority patent/US11327078B2/en
Publication of WO2019167874A1 publication Critical patent/WO2019167874A1/ja
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    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies
    • 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
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2470/00—Immunochemical assays or immunoassays characterised by the reaction format or reaction type
    • G01N2470/04—Sandwich assay format

Definitions

  • the present invention relates to a monoclonal antibody that binds to apolipoprotein A-IV (hereinafter referred to as APOA4), a method for immunoassay of APOA4 using the monoclonal antibody, and a measurement kit comprising the monoclonal antibody .
  • APOA4 a monoclonal antibody that binds to apolipoprotein A-IV
  • APOA4 apolipoprotein A-IV
  • APOA4 is a glycoprotein with a molecular weight of 46 kDa. Most of APOA4 in plasma is considered to be derived from the small intestine (Non-Patent Documents 1 to 3). APOA4 is known to have many functions such as antioxidant effect or appetite reduction effect in addition to being involved in lipid absorption, transport and metabolism (Non-patent Document 4). APOA4 is also known as a biomarker in the diagnosis of early renal dysfunction, liver disease, and tumor (Patent Documents 1 to 3).
  • APOA4 exists in blood in a lipid-bound state bound to free monomer, dimer, chylomicrons, or high density lipoproteins (HDL) (Non-patent Documents 2 and 5).
  • Non-patent Document 6 the APOA4 monomer and the APOA4 dimer are interconverted due to storage temperature, concentration, freeze-thawing, etc., and the ratio is not constant.
  • Non-Patent Documents 7 and 8 There are also reports of measuring APOA4 by ELISA using an anti-APOA4 polyclonal antibody without using a denaturing agent.
  • Patent Documents 1 to 3 There are also reports of measuring APOA4 by ELISA using an anti-APOA4 polyclonal antibody without using a denaturing agent.
  • Patent Documents 1 to 3 There are also reports of measuring APOA4 by ELISA using an anti-APOA4 polyclonal antibody without using a denaturing agent.
  • Omori et al Measured APOA4 in human serum by ELISA using an anti-APOA4 monoclonal antibody without treatment with a serum denaturing agent.
  • the present invention relates to an anti-APOA4 monoclonal antibody for accurately measuring APOA4 present in various forms such as monomers and dimers in a specimen, an immunoassay method for APOA4 using the antibody, and APOA4 measurement including the antibody
  • the purpose is to provide a kit for use.
  • the present inventors prepared an anti-APOA4 monoclonal antibody that recognizes and binds to APOA4, from which the APOA4-His monomer once denatured with a denaturant and then diluted and refolded is used as an antigen.
  • the anti-APOA4 monoclonal antibody was screened to find an anti-APOA4 monoclonal antibody that binds to the APOA4 monomer and dimer, and an APOA4 immunoassay method using the antibody and an APOA4 measurement kit using the antibody were constructed.
  • Heavy chain complementarity determining region (CDR) 1 comprising the amino acid sequence represented by SEQ ID NO: 3; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 4; A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 5; A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 15; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 16; and A light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 17; An anti-APOA4 monoclonal antibody or an antibody fragment thereof that specifically binds to APOA4 comprising the amino acid sequence represented by SEQ ID NO: 2.
  • a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 27; and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 31;
  • An anti-APOA4 monoclonal antibody that specifically binds to the 122st to 144th amino acid sequence, the 156th to 177th amino acid sequence, and the 242nd to 252nd amino acid sequence of APOA4 represented by SEQ ID NO: 2 or Its antibody fragment.
  • Heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 6; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 7; A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 8; A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 18; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 19; and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 20
  • a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 28; and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 32;
  • Heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 9; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 10; Heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 11; A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 21; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 22; and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 23; An anti-APOA4 monoclonal antibody or an antibody fragment thereof that specifically binds to APOA4 comprising the amino acid sequence represented by SEQ ID NO: 2.
  • a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 29; and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 33;
  • a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 12; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 13; Heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 14; A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 24; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 25; and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 26; An anti-APOA4 monoclonal antibody or an antibody fragment thereof that specifically binds to APOA4 comprising the amino acid sequence represented by SEQ ID NO: 2.
  • a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 30; and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 34;
  • the detection antibody that specifically binds to the APOA4 is labeled.
  • a conjugated labeled detection antibody is added to generate an immune complex comprising the APOA4, a capture antibody, and a labeled detection antibody, and the amount of the label in the generated immune complex is measured.
  • the capture antibody and the detection antibody are selected from the anti-APOA4 monoclonal antibody or the antibody fragment thereof according to any one of [1] to [14], and the capture antibody, the detection antibody, Immunoassay of APOA4 in a specimen, which is different from each other.
  • the capture antibody is the anti-APOA4 monoclonal antibody or antibody fragment thereof according to any one of [1] to [4], and the detection antibody is any one of [5] to [8].
  • the capture antibody is the anti-APOA4 monoclonal antibody or antibody fragment thereof according to any one of [1] to [4], and the detection antibody is any one of [9] to [11].
  • [15] The measurement method according to any one of [15] to [17], which is the anti-APOA4 monoclonal antibody or the antibody fragment thereof according to item.
  • the capture antibody is the anti-APOA4 monoclonal antibody or antibody fragment thereof according to any one of [1] to [4], and the detection antibody is any one of [12] to [14].
  • [21] The measurement method according to any one of [15] to [20], wherein the label is an enzyme.
  • a specimen comprising a capture antibody that specifically binds to APOA4 comprising the amino acid sequence represented by SEQ ID NO: 2, and a labeled detection antibody having a label bound to the detection antibody that specifically binds to the APOA4
  • the capture antibody and the detection antibody are selected from the anti-APOA4 monoclonal antibody or antibody fragment thereof according to any one of [1] to [14], and the capture antibody and A kit for measuring APOA4 in a specimen, which is different from the detection antibody.
  • the kit according to [26] wherein the capture antibody is the anti-APOA4 monoclonal antibody or antibody fragment thereof according to any one of [1] to [4].
  • the kit according to [26] or [27], wherein the detection antibody is the anti-APOA4 monoclonal antibody or antibody fragment thereof according to any one of [5] to [14].
  • the capture antibody is the anti-APOA4 monoclonal antibody or antibody fragment thereof according to any one of [1] to [4], and the detection antibody is any one of [5] to [8].
  • the capture antibody is the anti-APOA4 monoclonal antibody or the antibody fragment thereof according to any one of [1] to [4], and the detection antibody is any one of [9] to [11].
  • the capture antibody is the anti-APOA4 monoclonal antibody or the antibody fragment thereof according to any one of [1] to [4]
  • the detection antibody is any one of [12] to [14].
  • an anti-APOA4 monoclonal antibody capable of accurately measuring APOA4 in a specimen, an APOA4 immunoassay method using the antibody, and an APOA4 measurement kit containing the antibody are provided.
  • FIG. 3 is a graph showing the reactivity of the anti-APOA4 monoclonal antibody of the present invention to rhAPOA4-His monomer and dimer. It is the figure which showed the analysis result by the western blotting of the monomer and dimer of APOA4 in human serum. It is the figure which showed the measurement result of APOA4 in human serum by ELISA using the anti-APOA4 monoclonal antibody of this invention. It is the figure which showed the measurement result of APOA4 in the sample for specimen dilution by ELISA using the anti-APOA4 monoclonal antibody of this invention. It is the figure which showed the correlation of the measured value by ELISA using the anti- APOA4 monoclonal antibody of this invention, and the measured value by LC / MS of APOA4 in human serum.
  • the “APOA4 gene” is a gene encoding apolipoprotein A-IV, and is known to be expressed not only in humans but also in rodents such as mice and rats.
  • GenBank Accession No. Examples include the base sequence registered in NM_000482 or the base sequence represented by SEQ ID NO: 1.
  • amino acid sequence of human APOA4 GenBank Accession No. Examples include the amino acid sequence registered under NP_000473 or the amino acid sequence represented by SEQ ID NO: 2.
  • APOA4 refers to human APOA4 unless otherwise specified.
  • APOA4 is represented by a peptide comprising the amino acid sequence represented by SEQ ID NO: 2, or an amino acid sequence in which one or more amino acids are substituted, added, or deleted in the amino acid sequence. Contains peptides.
  • the “plurality” used for APOA4 is not limited as long as it retains the functional characteristics equivalent to the peptide represented by the amino acid sequence that forms the group, but 2 to 40, for example, 2 to 35 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10, or 2 to 5.
  • APOA4 comprises at least 90% of the amino acid sequence set forth in SEQ ID NO: 2, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Or a peptide represented by an amino acid sequence having 99% homology.
  • “homology” of amino acid sequences means homology when a comparison between two sequences (Pairwise Alignment) is performed using the CLUSTALW algorithm under the following parameter settings.
  • K-tuple (word) size 1 Window size: 5 Gap Penalty: 3 Number of Top Diagonals: 5 Scoring Method: PERCENT
  • the APOA 4 used in the present invention may be commercially available, extracted / generated from blood, or manufactured by genetic recombination technology.
  • Production of APOA4 by gene recombination technology includes, for example, cloning a gene encoding APOA4, preparing a vector containing the gene, introducing it into a host cell, and transforming the cell expressing APOA4. It can be obtained and obtained by cell culture.
  • the cells, vector types, cell types, culture conditions, and the like used in this preparation are within the technical scope of those skilled in the art, and appropriate conditions can be set as appropriate.
  • C of human APOA4 Recombinant human APOA4 (hereinafter also referred to as rhAPOA4-His) having a histidine tag attached to the end can be used.
  • the cells, vector types, cell types, culture conditions, etc. used for the preparation of rhAPOA4-His are within the technical scope of those skilled in the art, and appropriate conditions can be set as appropriate.
  • antibody refers to a full-length immunoglobulin molecule that is naturally occurring or produced by genetic engineering techniques
  • antibody fragment refers to an antigen-binding fragment of such an immunoglobulin molecule.
  • Antibody fragments include F (ab ′) 2 , F (ab) 2 , Fab ′, Fab, Fv, scFv, variants thereof, fusion proteins or fusion peptides containing antibody portions, and APOA4 binding sites, Examples include modified structures other than immunoglobulin molecules.
  • “monoclonal antibody” means an antibody obtained from a population of substantially homogeneous antibodies, and each individual antibody contained in the population is a natural abrupt that may be slightly present. Identical except for variants.
  • a monoclonal antibody is an antibody that exhibits one binding specificity and affinity for a particular epitope of an antigen.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous antibody population, and is not to be construed as limiting the production of the antibody by any particular method.
  • an antibody “heavy chain” of an antibody refers to the larger of the two types of polypeptide chains present in all antibody molecules in its naturally occurring conformation.
  • an antibody “light chain” refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformation.
  • anti-APOA4 antibody refers to an antibody that binds to APOA4.
  • the anti-APOA4 antibody or antibody fragment thereof of the present invention is preferably an antibody or antibody fragment thereof that specifically binds to APOA4.
  • the anti-APOA4 antibody or antibody fragment thereof of the present invention is preferably an anti-APOA4 monoclonal antibody or antibody fragment thereof.
  • the anti-APOA4 antibody may be any class such as IgG, IgA or IgM, or a subclass thereof, and is not limited to a specific class.
  • immunoglobulins are classified into different classes.
  • the five main immunoglobulin classes are IgA, IgD, IgE, IgG and IgM, some of which are, for example, IgG1, IgG2, IgG3, IgG4 (in the case of mice, IgG1, IgG2a, IgG2b, IgG2c, IgG3), IgA1 and IgA2 (in the case of mice, IgA) can be further subdivided into subclasses (isotypes).
  • the corresponding heavy chain constant regions of the different classes of immunoglobulins are called ⁇ chain, ⁇ chain, ⁇ chain, ⁇ chain, and ⁇ chain, respectively.
  • ⁇ chains and ⁇ chains as types of antibody light chains (sometimes referred to as L chains).
  • the antibody variable region means the variable region of the antibody light chain, the variable region of the antibody heavy chain, or both.
  • the constant region of an antibody means the constant region of an antibody light chain, the constant region of an antibody heavy chain, or both.
  • the variable regions of the heavy chain and the light chain each consist of three complementarity determining regions (CDRs), also known as hypervariable regions, and four framework regions (FR) linked by CDRs.
  • CDRs complementarity determining regions
  • FR framework regions linked by CDRs.
  • the CDR in each chain is held in the vicinity by the FR and contributes to the formation of the antigen-binding site of the antibody together with the CDR in the other chain.
  • Techniques for determining CDRs are not limited. For example, (1) an approach based on heterogeneous sequence variability (for example, Kabat et al. Sequences of Proteins of Immunological Institute, 5th ed., 1991, National Institutes). of Health, Bethesda MD); and (2) An approach based on crystal structural studies of antigen-antibody complexes (Al-lazikani et al., J. Molec. Biol. 273, 927-948, 1997). Can do. A combination of these approaches and other approaches may be used.
  • the anti-APOA4 antibody or antibody fragment thereof of the present invention can be prepared according to a known method.
  • An anti-APOA4 monoclonal antibody or an antibody fragment thereof is produced by, for example, isolating antibody-producing cells from a non-human mammal immunized with APOA4 or APOA4 fragments, and fusing them with myeloma cells to produce a hybridoma.
  • the purified antibody can be purified.
  • the APOA4 fragment is a partial peptide of APOA4, and a monoclonal antibody against the APOA4 fragment or an antibody fragment thereof binds to APOA4.
  • immunogens examples include APOA4 or APOA4 fragments such as primates such as humans and monkeys, and rodents such as rats and mice, preferably human APOA4 or APOA4 fragments.
  • the anti-APOA4 antibody or antibody fragment thereof of the present invention can also be produced using a known gene recombination technique. Specifically, a monoclonal antibody produced by the hybridoma prepared above, a gene encoding the heavy chain variable region, light chain variable region, heavy chain CDR, light chain CDR, etc.
  • a vector containing the gene can be produced by introducing a cell into a host cell and transforming it to obtain a cell expressing the anti-APOA4 antibody or antibody fragment thereof of the present invention and culturing it.
  • the cells, vector types, cell types, culture conditions, and the like used in this preparation are within the technical scope of those skilled in the art, and appropriate conditions can be set as appropriate.
  • the anti-APOA4 antibody or antibody fragment thereof of the present invention specifically binds to the 89th to 110th amino acid sequences of APOA4 containing the amino acid sequence represented by SEQ ID NO: 2 and the 200th to 224th amino acid sequences.
  • Anti-APOA4 monoclonal antibody or an antibody fragment thereof specifically binds to the 122nd to 144th amino acid sequence of APOA4 represented by SEQ ID NO: 2, the 156th to 177th amino acid sequence, and the 242nd to 252nd amino acid sequence And anti-APOA4 monoclonal antibody or antibody fragment thereof.
  • Examples of the anti-APOA4 monoclonal antibody or an antibody fragment thereof that specifically binds to the 89th to 110th amino acid sequence of APOA4 including the amino acid sequence represented by SEQ ID NO: 2 and the 200th to 224th amino acid sequence include: A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 3; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 4; A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 5; A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 15; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 16; and A light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 17; Anti-APOA4 monoclonal antibody No. 2 which specifically binds to APOA4 comprising the amino acid sequence represented by SEQ ID NO: 2.
  • 30 hereinafter abbreviated as antibody No. 30 or an antibody fragment thereof.
  • a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 6
  • a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 7
  • a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 8
  • a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 18
  • a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 19
  • a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 20
  • Anti-APOA4 monoclonal antibody No. 2 which specifically binds to APOA4 comprising the amino acid sequence represented by SEQ ID NO: 2.
  • 33 hereinafter abbreviated as antibody No
  • the anti-APOA4 antibody or antibody fragment thereof of the present invention includes the antibody No. 30 and antibody no. 33, as well as antibody fragments thereof, for example, A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 9; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 10; Heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 11; A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 21; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 22; and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 23; Anti-APOA4 monoclonal antibody No.
  • a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 12; A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 13; Heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 14; A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 24; A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 25; and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 26; Anti-APOA4 monoclonal antibody No. 2 which specifically binds to APOA4 comprising the amino acid sequence represented by SEQ ID NO: 2.
  • 37 hereinafter abbreviated as antibody No. 37 or an antibody fragment thereof.
  • An anti-APOA4 monoclonal antibody or an antibody fragment thereof that specifically binds to APOA4 containing the amino acid sequence represented by SEQ ID NO: 2, including these heavy chain CDR1 to CDR3 and light chain CDR1 to CDR3, is a known gene recombination technique.
  • the genes encoding heavy chain CDR1-CDR3 and light chain CDR1-CDR3 are incorporated into vectors containing genes encoding the antibody FR and the antibody constant region, respectively, and introduced into host cells.
  • the cells expressing the antibody can be obtained by transformation and cultured by culturing the cells.
  • the cells, vector types, cell types, culture conditions, and the like used in this preparation are within the technical scope of those skilled in the art, and appropriate conditions can be set as appropriate.
  • the antibody No. 30 includes a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 27 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 31.
  • the antibody No. 33 includes a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 28 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 32.
  • the antibody No. 36 includes a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 29 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 33.
  • the antibody No. 37 includes a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 30 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 34.
  • An anti-APOA4 monoclonal antibody or an antibody fragment thereof that specifically binds to APOA4 containing the amino acid sequence represented by SEQ ID NO: 2, including these heavy chain variable region and light chain variable region, is prepared using a known gene recombination technique. Can be produced. Specifically, a gene encoding each of the heavy chain variable region and the light chain variable region is incorporated into a vector containing a gene encoding the constant region of the antibody, and introduced into a host cell for transformation, It can be produced by obtaining cells that express the antibody and culturing the cells. The cells, vector types, cell types, culture conditions, and the like used in this preparation are within the technical scope of those skilled in the art, and appropriate conditions can be set as appropriate.
  • the APOA4 measurement method comprises the step of binding APOA4 in a sample with a capture antibody that specifically binds to the APOA4, and then labeling the detection antibody that specifically binds to the APOA4 with a label.
  • a conjugated labeled detection antibody is added to generate an immune complex comprising the APOA4, a capture antibody, and a labeled detection antibody, and the amount of the label in the generated immune complex is measured.
  • the “capture antibody” and the “detection antibody” are respectively an anti-APOA4 antibody or an antibody fragment thereof that specifically binds to APOA4, and preferably an anti-APOA4 monoclonal antibody or an antibody fragment thereof.
  • examples of the specimen include body fluids, and examples of the body fluid include, but are not limited to, blood such as serum, plasma or whole blood, lymph fluid, tissue fluid, spinal fluid, body cavity fluid. , Digestive fluid, runny nose, tears, sweat, urine, and the like. From the viewpoint of easy acquisition and processing, it is preferable to use serum or plasma as the specimen.
  • the bodily fluid may be a bodily fluid collected from a subject, or may be one obtained by subjecting the collected bodily fluid to a process such as dilution or concentration that is normally performed.
  • the specimen used in the present invention may be collected or prepared at the time of carrying out the present invention, or may be collected or prepared and stored in advance.
  • immunological measurement method examples include enzyme immunoassay (EIA or ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), fluorescence polarization immunoassay (FPIA), chemiluminescence immunoassay (CLIA) depending on the label of the labeled detection antibody. ), Electrochemiluminescence immunoassay, etc., and any of these can be used in the measurement method of the present invention, but ELISA is preferable because it can easily and rapidly measure the detection target.
  • the capture antibody is preferably immobilized on a solid support. After appropriately reacting a biological sample that has been treated with the capture antibody immobilized on the solid support, the label is bound to a detection antibody that specifically binds to APOA4, which is different from the capture antibody. A labeled detection antibody is added to generate an immune complex composed of the APOA4, the capture antibody, and the labeled detection antibody.
  • the solid support is not particularly limited as long as it can stably hold an antibody or antibody fragment.
  • Preferred materials for the solid support include polymer materials such as polystyrene, polycarbonate, polyvinyl toluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, gelatin, agarose, cellulose, nitrocellulose, cellulose acetate, cellulose acetate, and polyethylene terephthalate. , Glass, ceramics, magnetic particles and metals.
  • Preferable shapes of the solid support include tubes, beads, plates, fine particles such as latex, sticks and the like.
  • APOA4 in the sample is measured by measuring the label in the immune complex.
  • APOA4 in a sample can be measured by reacting an enzyme as a label with a substrate of the enzyme and measuring the absorbance of the colored product.
  • an unlabeled anti-APOA4 antibody or antibody fragment is added, and the antibody against the primary antibody labeled with an enzyme or the like.
  • APOA4 in the sample can be measured by further adding an antibody fragment (secondary antibody) and measuring the label of the secondary antibody.
  • the label examples include an enzyme such as peroxidase and alkaline phosphatase in ELISA, a radioactive substance such as 125 I, 131 I, 35 S, and 3 H in RIA method, and a fluorescein isothiocyanate, rhodamine, dansyl chloride in FPIA method, Fluorescent substances such as phycoerythrin, tetramethylrhodamine isothiocyanate, near-infrared fluorescent materials, and the CLIA method, luciferase, ⁇ -galactosidase and other enzymes and luminescent substrates that change to luminescent substances with each enzyme, luciferin, aequorin, etc.
  • a luminescent material can be used.
  • nanoparticles such as gold colloids and quantum dots can be used as labels.
  • the substrate of the enzyme to be labeled is 3, 3′-diaminobenzidine (DAB), 3,3 ′, 5,5′-tetramethylbenzidinein (TMB), O-phenylenediamine (OPD), etc.
  • DAB 3, 3′-diaminobenzidine
  • TMB 3,3 ′, 5,5′-tetramethylbenzidinein
  • OPD O-phenylenediamine
  • pNPP p-nitropheny phosphatase or the like can be used.
  • the capture antibody that specifically binds to APOA4 specifically binds to the 89th to 110th amino acid sequences of APOA4 including the amino acid sequence represented by SEQ ID NO: 2 and the 200th to 224th amino acid sequences.
  • Examples thereof include an anti-APOA4 monoclonal antibody or an antibody fragment thereof.
  • the capture antibody is preferably an anti-APOA4 antibody or an antibody fragment thereof that specifically binds to the monomer and dimer of APOA4. 30, Antibody No. 33, Antibody No. 36, Antibody No. 37, antibody fragments thereof, and the like. 30 or antibody fragments thereof are preferred.
  • the detection antibody that specifically binds to APOA4, which is different from the capture antibody includes an anti-APOA4 antibody or an antibody fragment thereof that binds to an epitope different from the capture antibody.
  • the detection antibody is represented by SEQ ID NO: 2.
  • the detection antibody is preferably an anti-APOA4 antibody or an antibody fragment thereof that specifically binds to the monomer of APOA4. 30, Antibody No. 33, Antibody No. 36, Antibody No. 37, antibody fragments thereof, and the like. 33, Antibody No. 36, Antibody No. 37, or an antibody fragment thereof, is preferred. More preferred is 33 or an antibody fragment thereof.
  • the combination of the capture antibody and the detection antibody is not particularly limited as long as the capture antibody and the detection antibody are different. 30 or an antibody fragment thereof, and the detection antibody is antibody No. No. 33 or a combination thereof as an antibody fragment, and the capture antibody is antibody No. 30 or an antibody fragment thereof, and the detection antibody is antibody No. 36 or a combination thereof as an antibody fragment, and the capture antibody was designated as antibody No. 30 or an antibody fragment thereof, and the detection antibody is antibody No. 37 or a combination thereof as an antibody fragment, and the like. 30 or an antibody fragment thereof, and the detection antibody is antibody No. A combination of 33 or an antibody fragment thereof is preferred.
  • the kit of the present invention is a kit used in the method for measuring APOA4 of the present invention, and specifically binds to a capture antibody that specifically binds to APOA4 comprising the amino acid sequence represented by SEQ ID NO: 2 and the APOA4 And a labeled detection antibody having a label bound to the detection antibody.
  • the capture antibody and the detection antibody used in the kit of the present invention are different.
  • the capture antibody and the detection antibody bind to different epitopes on APOA4, respectively.
  • Examples of the capture antibody and the detection antibody used in the kit of the present invention include the capture antibody and the detection antibody mentioned in the above ⁇ Measurement method> section.
  • Examples of the combination of the capture antibody and the detection antibody used in the kit of the present invention include the combination of the capture antibody and the detection antibody mentioned in the above section ⁇ Measurement Method>.
  • the kit of the present invention may further include a reagent and a device necessary for measuring APOA4 in the sample by the immunological measurement method of APOA4 in the sample of the present invention.
  • the kit of the present invention includes a solid support such as a microtiter plate and a reagent for measuring the label in addition to the capture antibody and the labeled detection antibody.
  • a reagent for measuring the label for example, when the label is an enzyme, a substrate of alkaline phosphatase such as paranitrophenyl phosphate (pNPP), or 3,3′-diaminobenzidine tetrahydrochloride (DAB), 3, Examples include substrates of peroxidase such as 3 ′, 5,5′-tetramethylbenzidine (TMB) and o-phenylenediamine dihydrochloride (OPD).
  • pNPP paranitrophenyl phosphate
  • DAB 3,3′-diaminobenzidine tetrahydrochloride
  • peroxidase such as 3 ′, 5,5′-tetramethylbenzidine (TMB) and o-phenylenediamine dihydrochloride (OPD).
  • a capture antibody is immobilized on a solid support such as a microtiter plate, and then an appropriately treated and diluted sample is added thereto, followed by incubation, and a sample unbound to the capture antibody is removed by washing. To do.
  • the labeled detection antibody is added and incubated, and the label on the solid support is measured.
  • the label is an enzyme
  • it is possible to measure APOA4 in a sample by adding a substrate of the enzyme to cause color development and measuring the color development using a microtiter plate reader or the like.
  • the kit of the present invention may contain a secondary antibody that is an antibody or an antibody fragment thereof that binds to an anti-APOA4 antibody or an antibody fragment thereof.
  • a secondary antibody that is an antibody or an antibody fragment thereof that binds to an anti-APOA4 antibody or an antibody fragment thereof.
  • a kit include a solid support such as a microtiter plate, a capture antibody, an anti-APOA4 antibody or an antibody fragment thereof as a primary antibody, an alkaline phosphatase, a peroxidase for an anti-APOA4 antibody or an antibody fragment thereof as a secondary antibody.
  • kits containing a labeled secondary antibody labeled with an enzyme such as, and a substrate of alkaline phosphatase such as pNPP, or a substrate of peroxidase such as DAB, TMB, or OPD.
  • a capture antibody is immobilized on a solid support such as a microtiter plate, and a sample that has been appropriately treated and diluted is added thereto, followed by incubation. Remove. Subsequently, after the primary antibody is added and incubated, washing is performed, and further, an enzyme-labeled secondary antibody that recognizes the primary antibody is added and incubated. Thereafter, the labeling enzyme substrate is added to cause color development, and the color development is measured using a microtiter plate reader or the like, whereby APOA4 in the sample can be measured.
  • a secondary antibody the reaction is amplified and the detection sensitivity can be increased.
  • the kit of the present invention may further contain necessary buffers, enzyme reaction stop solutions, microplate readers, product instructions, and the like.
  • the label is not particularly limited, and enzymes such as peroxidase and alkaline phosphatase, radioactive materials such as 125 I, 131 I, 35 S, and 3 H, fluorescein isothiocyanate, rhodamine, dansyl chloride, phycoerythrin, tetramethyl Examples thereof include rhodamine isothiocyanate, fluorescent materials such as near-infrared fluorescent materials, luminescent materials such as luciferase, luciferin, and aequorin, nanoparticles such as gold colloids and quantum dots.
  • biotinylated anti-APOA4 antibody or an antibody fragment thereof can be used as a detection antibody, and avidin or streptavidin labeled in the kit can be included.
  • Example 1 Preparation of Recombinant Human APOA4 with Histidine Tag Binding
  • a recombinant protein in which a histidine tag is bound to the C-terminus of human APOA4 is prepared by the following steps. It was prepared by.
  • the gene encoding human APOA4 was amplified from human liver cDNA by PCR. This gene was inserted into the HindIII / NotI site of a pcDNA3.4 vector (manufactured by ThermoFisher Scientific) into which a gene encoding a histidine tag was inserted.
  • the prepared vector was transformed into Expi293F cells according to the standard method of Expi293 Expression System (ThermoFisher Scientific), and a recombinant protein in which a histidine tag was bound to the C-terminus of human APOA4 (rhAPOA4-His in a supernatant of culture medium). Secreted.
  • the rhAPOA4-His secreted into the culture supernatant was purified from the culture supernatant with TALON Superflow Metal Affinity Resin (CLONTECH) and dialyzed against D-PBS (Wako Pure Chemical Industries).
  • Example 2 Preparation of rhAPOA4-His monomer and dimer
  • the rhAPOA4-His purified in Example 1 was subjected to size fractionation by size exclusion chromatography (Size Exclusion Chromatography; SEC).
  • Size Exclusion Chromatography SEC
  • SEC Size Exclusion Chromatography
  • Superdex200 10/300 GL manufactured by GE Healthcare
  • AKTA explorer 10s chromatography system was used and carried out at 4 ° C.
  • the solvent used was D-PBS ( ⁇ ) (manufactured by Wako Pure Chemical Industries, Ltd.), and the flow rate was 0.5 ml / min.
  • the protein elution profile was detected by absorbance at 280 nm, and the elution fraction was fractionated into 0.3-well plates in 96-well plates.
  • the elution fraction was collected using the peak of Retention 11.8 ml as a dimer and the peak of Retention 14.2 ml as a monomer.
  • the protein amount of the dimer and monomer fractions was calculated from a standard curve using bovine serum albumin (BSA) of known concentration as a standard substance using Micro BCA Protein Assay Kit (manufactured by ThermoFisher Scientific).
  • BSA bovine serum albumin
  • TiterMax Gold adjuvant manufactured by TiterMax USA
  • Magic Mouse adjuvant manufactured by Creative Diagnostics
  • mice On day 34, the mice were sacrificed and peripheral lymph nodes were collected to prepare lymph node cells.
  • genomicmeONE-CF Ishihara Sangyo Co., Ltd.
  • the prepared lymph node cells and P3U1 myeloma cells were fused at a ratio of 5: 1.
  • the hybridoma was cultured in a 96-well plastic plate. After incubation for 7 days at 5% CO 2 and 37 ° C., the culture supernatant was collected.
  • the refolded rhAPOA4-His monomer was obtained by diluting rhAPOA4-His prepared in Example 1 twice with 50 mM Tris-buffered saline (pH 7.5) containing 8 M urea (manufactured by Wako Pure Chemical Industries, Ltd.) at room temperature. After leaving for 30 minutes, it was prepared by diluting 50 times with 50 mM Tris buffered saline (pH 7.5) (reaction solution) containing 4% Block Ace (DS Pharma Biomedical) and 0.01% Tween20. . The well was washed three times with a washing solution, and then the hybridoma culture supernatant was added to the well and incubated at room temperature for 1 hour.
  • hybridoma expressing mouse anti-human APOA4 antibody was cloned by limiting dilution from a well containing the culture supernatant of the hybridoma that showed reactivity to rhAPOA4-His.
  • a hybridoma clone expressing a mouse anti-human APOA4 antibody having activity to react with rhAPOA4-His monomer was obtained.
  • Example 4 Reactivity of anti-human APOA4 monoclonal antibody to rhAPOA4-His monomer and dimer 50 mM Tris buffer (pH 7) containing 2 ⁇ g / mL of anti-6 ⁇ His rabbit polyclonal antibody (anti-6 ⁇ His antibody, manufactured by Bethyl Laboratories) 5) was added to a well of a 96-well plate (manufactured by Nunc) at 50 ⁇ L / well and incubated at 4 ° C. overnight.
  • Tris-buffered saline (pH 7.5) (reaction solution) containing 0.01% Tween 20 was diluted to 450 ng / mL, and 50 ⁇ L / well was added. After incubating at room temperature for 1 hour, the wells were washed three times with a washing solution, and then the anti-human APOA4 monoclonal antibody obtained in Example 3 was diluted with the reaction solution to 1 ⁇ g / mL and added at 50 ⁇ L / well. After incubation for 1 hour at room temperature, the wells were washed 3 times with a washing solution.
  • Example 5 Analysis of APOA4 monomer and dimer in human serum
  • Two serum samples (N65S and N67S) were used as human specimens. After separation of the serum, 150 ⁇ L of the serum stored at 4 ° C. or ⁇ 80 ° C. for 2 weeks was subjected to size fractionation by size exclusion chromatography (SEC). APOA4 in the elution fraction was detected by Western blotting.
  • SEC column Superdex200 10/300 GL (manufactured by GE Healthcare) connected to an AKTA pure25 chromatography system was used, and the SEC column was carried out at 4 ° C.
  • the solvent used was D-PBS ( ⁇ ) (manufactured by Wako Pure Chemical Industries, Ltd.), and the flow rate was 0.5 ml / min.
  • the elution profile of APOA4 was detected by absorbance at 280 nm, and the elution fraction was fractionated into 0.3-well portions in 96-well plates.
  • SDS-PAGE contains fractions 1 to 16 (32 wells) collected from the well starting point (C1) by 2 wells. Provided.
  • the gel after electrophoresis was transferred onto a nitrocellulose membrane using an iBLOT2 drive blotting system (ThermoFisher Scientific) and 50 mM Tris-buffered saline containing 5% skim milk and 0.05% Tween 20 (hereinafter referred to as blocking solution).
  • the mixture was shaken at room temperature for 1 hour or more to perform blocking.
  • Rabbit polyclonal anti-APOA4 antibody (Fitzgerald, # 70R-5429) diluted with a blocking solution was used as a primary antibody and shaken at 4 ° C. overnight.
  • the amount of protein in the detected APOA4 band was quantified using ImageLab Software (manufactured by BIO-RAD) from the band intensity of the dilution series of the standard substance run on the same gel, and the dimer eluted in fractions 6-9, The ratio of monomers eluting in fractions 10-14 was calculated.
  • the result of SEC is shown in FIG.
  • the dimers in the serum stored at 4 ° C. were 17.9% and 17.0% for N65S and N67S, respectively, and 0% and 0.08% for the freeze-thawed serum, respectively.
  • Example 6 Preparation of peroxidase-labeled monoclonal antibody
  • Anti-human APOA4 monoclonal antibody no. 33, no. 36 and no. 37 was labeled with Peroxidase (HRP: Horseradish Peroxidase) using Peroxidase Labeling Kit-NH2 (manufactured by Dojindo Laboratories) (hereinafter referred to as “HRP labeled antibody No. 33”, “HRP labeled antibody No. 33”, respectively).
  • HRP labeled antibody No. 33 Horseradish Peroxidase
  • HRP-labeled antibody No. 37 HRP-labeled antibody No.
  • Example 7 Measurement of human serum APOA4 by ELISA
  • Anti-human APOA monoclonal antibody No. 30 was adjusted to a concentration of 2 ⁇ g / mL with 50 mM Tris buffer (pH 7.5).
  • the antibody No. 30 solution was added to a maxi soap cup (NUNC, manufactured by C8 Maxisorp) at 100 ⁇ L / well, and then the maxi soap cup was placed in a wet box and allowed to stand at 4 ° C. overnight. 30 was coated on the wells.
  • NUNC maxi soap cup
  • TMB 3,3 ′, 5,5′-tetramethylbenzidine
  • Example 8 Specimen Dilution Test Antibody No. as a capture antibody 30 and HRP-labeled antibody No. 30 as the labeled detection antibody.
  • APOA4 in the sample for specimen dilution test was measured. The results are shown in FIGS. 4 (A) and 4 (C).
  • the dilution line is a straight line parallel to the ideal curve. It was confirmed that APOA4 in the sample was accurately measured according to the dilution ratio of the sample.
  • Example 9 Addition recovery test 1 ng / mL or 2 ng / mL of rhAPOA-His as a standard and serum samples (N54 to N61) of 8 healthy persons diluted 32000 times or 64000 times with a reaction solution
  • APOA4 in the mixed specimen was measured.
  • the standard and diluted specimen were measured in the same manner, and the addition recovery rate (%) calculated by the following formula was calculated by each ELISA. The closer the addition recovery rate is to 100%, the more accurately it can be measured.
  • Additive recovery rate (%) (Measured value of mixed sample-measured value of diluted sample) ⁇ measured value of standard product x 100
  • the ELISA using No. 33 is shown in Table 1, and antibody No. 1 was used as a capture antibody.
  • the ELISA using No. 36 is shown in Table 2, and antibody No. 1 was used as the capture antibody.
  • the ELISA using 37 is shown in Table 3.
  • antibody No. 30 and HRP-labeled antibody No. 30 as the labeled detection antibody.
  • the recovery rate of addition was 79% or more, and it was confirmed that in any ELISA, APOA4 in the specimen can be measured accurately.
  • Example 10 Correlation of measured values of human serum APOA4 by ELISA and LC / MS ApOA4 in serum was measured in the same manner as in Example 6 using 5 samples of healthy human serum.
  • APOA4 of the same specimen was quantified by a selective reaction monitoring method using LC / MS (Liquid Chromatography / Mass Spectrometry), and the correlation between the measured value of human serum APOA4 by ELISA and the measured value of human serum APOA4 by LC / MS Examined. The result is shown in FIG. As shown in FIG. 5, the correlation coefficient was 0.8015, and a good correlation was observed between the measured value of APOA4 in the ELISA of the present invention and the measured value by LC / MS.
  • Example 11 Antibody No. 30, Antibody No. 33, Antibody No. 36 and antibody no. Amino acid sequence analysis of variable region 37 30, Antibody No. 33, Antibody No. 36 and antibody no.
  • the 37 heavy chain and light chain signal sequences and the DNA sequence encoding the variable region were amplified by the 5′-RACE (5′-rapid amplification of cDNA ends) method as follows.
  • Total RNA was prepared from the hybridoma obtained in Example 3 using RNeasy Mini kit (QIAGEN) and treated with DNase (RNase free DNase set, QIAGEN). Double-stranded cDNA was prepared from the obtained total RNA using SMARTER TM RACE 5 ′ / 3 ′ kit (manufactured by TAKARA).
  • the mouse Ig ⁇ light chain was amplified using a reverse primer comprising the nucleotide sequence represented by SEQ ID NO: 36), and the signal sequence of the antibody and the gene sequence encoding the variable region were amplified.
  • the amplified gene sequence was inserted into a pCR2.1 vector (Invitrogen / Life Technologies).
  • Antibody No. 30, Antibody No. 33, Antibody No. 36 and antibody, no. 37 gene sequences were analyzed using ABI3130XL. By this analysis, a gene sequence encoding a variable region including a signal sequence and a CDR sequence of each antibody was determined, and converted to an amino acid sequence using gene analysis software Genetyx (Genetix Corporation).
  • Example 12 Antibody No. 30, Antibody No. 33, Antibody No. 36 and antibody no. Analysis of amino acid sequence of 37 CDRs 30, Antibody No. 33, Antibody No. 36 and antibody no.
  • the 37 CDRs are numbered using the Abysis software (license from UCL) according to the Kabat numbering system (Kabat numbering system). In accordance with the Kabat definition for (Kabat definition).
  • the amino acid sequence of 37 CDRs was determined as follows.
  • Heavy chain CDR1 amino acid sequence represented by SEQ ID NO: 3
  • Heavy chain CDR2 amino acid sequence represented by SEQ ID NO: 4
  • Heavy chain CDR3 amino acid sequence represented by SEQ ID NO: 5
  • Light chain CDR2 Amino acid sequence represented by SEQ ID NO: 16
  • Light chain CDR3 Amino acid sequence represented by SEQ ID NO: 17
  • Heavy chain CDR1 amino acid sequence represented by SEQ ID NO: 6
  • Heavy chain CDR2 amino acid sequence represented by SEQ ID NO: 7
  • Heavy chain CDR3 amino acid sequence represented by SEQ ID NO: 8
  • Light chain CDR1 represented by SEQ ID NO: 18
  • Light chain CDR2 Amino acid sequence represented by SEQ ID NO: 19
  • Light chain CDR3 Amino acid sequence represented by SEQ ID NO: 20
  • Heavy chain CDR1 amino acid sequence represented by SEQ ID NO: 9
  • Heavy chain CDR2 amino acid sequence represented by SEQ ID NO: 10
  • heavy chain CDR3 amino acid sequence represented by SEQ ID NO: 11
  • light chain CDR1 represented by SEQ ID NO: 21
  • Light chain CDR2 Amino acid sequence represented by SEQ ID NO: 22
  • Light chain CDR3 Amino acid sequence represented by SEQ ID NO: 23
  • Heavy chain CDR1 amino acid sequence represented by SEQ ID NO: 12
  • Heavy chain CDR2 amino acid sequence represented by SEQ ID NO: 13
  • Heavy chain CDR3 amino acid sequence represented by SEQ ID NO: 14
  • light chain CDR1 represented by SEQ ID NO: 24
  • Light chain CDR2 Amino acid sequence represented by SEQ ID NO: 25
  • Light chain CDR3 Amino acid sequence represented by SEQ ID NO: 26
  • Example 13 Antibody No. 30, Antibody No. 33, Antibody No. 36 and antibody no. 37 isotypes
  • the hybridoma clone obtained in Example 2 (3) was cultured, and antibody No. 1 was prepared from the culture supernatant using Protein A (GE Healthcare). 30, Antibody No. 33, Antibody No. 36, Antibody No. 37 was purified. The isotype was determined using a monoclonal antibody isotyping kit (BD Biosciences). As a result, antibody No. The 30 isotypes were IgG2b, ⁇ . In addition, antibody No. 33, Antibody No. 36 and antibody no. The 37 isotypes were IgG1, ⁇ .
  • Example 14 Antibody No. 30, Antibody No. 33 Epitope map by HDX-MS 10 ⁇ M rhAPOA4-His, and about 10 ⁇ M antibody No. to rhAPOA4-His 30 or antibody no.
  • the solution obtained by adding 33 and mixed was diluted 20 times with a PBS deuterium solution to start the deuteration reaction.
  • a PBS solution was used as a control.
  • an equal amount of a cooled 500 mM TCEP solution (pH 3 or less) containing 2M guanidine hydrochloride was added to stop the reaction.
  • antibody No. Thirty epitopes were identified as the 89th to 110th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 2 and the 200th to 224th amino acid sequences.
  • antibody No. The 33 epitopes were identified as the amino acid sequence at positions 122 to 144, the amino acid sequence at positions 156 to 177, and the amino acid sequence at positions 242 to 252 in the amino acid sequence represented by SEQ ID NO: 2.
  • the identified peptides were mapped to the reported X-ray crystal structure of APOA4 dimer. 30 and antibody no.
  • Each of the 33 epitope peptides constitutes one epitope region in terms of the three-dimensional structure. 30 and antibody no. It became clear that it was different in 33.
  • an anti-APOA4 monoclonal antibody or antibody fragment thereof capable of accurately measuring APOA4 in a specimen, an immunoassay method for APOA4 using the monoclonal antibody or antibody fragment thereof, and the monoclonal antibody or antibody fragment thereof
  • a kit for measuring APOA4 is provided.

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See also references of EP3760641A4
STAN, S. ET AL.: "Apo A-IV: an update on regulation and physiologic functions", BIOCHIM. BIOPHYS. ACTA, vol. 1631, 2003, pages 177 - 187, XP004414267, DOI: 10.1016/S1388-1981(03)00004-0
TOKUHARA, D. ET AL.: "Specific expression of apolipoprotein A-IV in the follicle-associated epithelium of the small intestine", DIGESTIVE DISEASES AND SCIENCES, vol. 59, no. 11, 2014, pages 2682 - 2692, XP055635566 *
WU, A. -L. ET AL.: "Relative contributions by liver and intestine to individual plasma apolipoproteins in the rat.", J. BIOL. CHEM., vol. 254, 1979, pages 7316 - 7322

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113156134A (zh) * 2020-11-26 2021-07-23 江苏荃信生物医药有限公司 用于检测人白介素23的elisa试剂盒及检测方法
CN113156134B (zh) * 2020-11-26 2024-01-23 江苏荃信生物医药股份有限公司 用于检测人白介素23的elisa试剂盒及检测方法

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