WO2023008404A1 - ヒト化抗体を含む試薬 - Google Patents

ヒト化抗体を含む試薬 Download PDF

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WO2023008404A1
WO2023008404A1 PCT/JP2022/028698 JP2022028698W WO2023008404A1 WO 2023008404 A1 WO2023008404 A1 WO 2023008404A1 JP 2022028698 W JP2022028698 W JP 2022028698W WO 2023008404 A1 WO2023008404 A1 WO 2023008404A1
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antibody
antibodies
humanized antibody
humanized
reagent
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French (fr)
Japanese (ja)
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晴登 石川
公隆 山本
憲幸 泉谷
大輔 小笠原
ゆき 若林
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Denka Co Ltd
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Denka Co Ltd
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Priority to CN202280052194.2A priority patent/CN117716037A/zh
Priority to US18/290,904 priority patent/US20250102496A1/en
Priority to EP22849465.4A priority patent/EP4372009A4/en
Priority to KR1020237041539A priority patent/KR20240005839A/ko
Publication of WO2023008404A1 publication Critical patent/WO2023008404A1/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
    • G01N33/531—Production of immunochemical test materials
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • C12N15/8258—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon for the production of oral vaccines (antigens) or immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

  • the present invention provides humanized antibodies, fragments thereof (such as variable regions and scFv), conjugates (such as fusion proteins) comprising such humanized antibodies or fragments, and such humanization in diagnostic and therapeutic pretargeting methods and compositions. Concerning the use of antibodies or fragments.
  • the invention further relates to the use of such humanized antibodies in conventional immunotherapeutic and immunodiagnostic methods and compositions, eg, for treatment and imaging of disease.
  • heterophilic antibodies antibodies against non-human animal antibodies present in human blood are called heterophilic antibodies. Since mouse-derived monoclonal antibodies are often used in immunoassays, non-specific reactions due to human anti-mouse antibodies (HAMA) are often observed. As many as 10% of human specimens contain HAMA.
  • the present invention is to provide a reagent, an antigen measurement kit, and an antigen measurement method using an antibody capable of reducing non-specific reactions.
  • the present inventors have found that the use of humanized antibodies reduces non-specific reactions in antigen-antibody reactions, and have completed the present invention.
  • the present invention is as follows.
  • [5] A reagent according to any one of [1] to [4] for diagnosis.
  • [8] An immunoassay method comprising contacting a humanized antibody against a substance to be measured with the substance to be measured. [9] The immunoassay method of [8], wherein the humanized antibody suppresses non-specific reactions. [10] The immunoassay method of [8] or [9], wherein the humanized antibody is IgG. [11] The immunoassay method of [8] or [9], wherein the humanized antibody is produced using a plant expression system. [12] A method for suppressing non-specific antigen-antibody reaction in an immunoassay using a humanized antibody against a substance to be measured. [13] The method of suppressing a non-specific reaction of [12], wherein the non-specific reaction is suppressed by a humanized antibody.
  • FIG. 10 shows the results of measurement of physiological saline and CRP-negative serum with a reagent using an anti-CRP antibody derived from mice or a humanized anti-CRP antibody derived from mice.
  • a humanized antibody is used as an antibody that binds to a substance to be measured in an immunoassay method using an antigen-antibody reaction.
  • the invention provides reagents comprising humanized antibodies.
  • An antibody has a structure in which a total of four polypeptide chains, each consisting of two identical heavy chains and two identical light chains, are linked by disulfide bonds and arranged in a Y shape.
  • the "antibody” is not particularly limited as long as it can specifically bind to the target antigen, and may be any isotype of IgG, IgM, IgA, IgE, and IgD.
  • human immunoglobulins 9 types and 4 classes (isotypes) of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE and IgM are known.
  • the definition of IgG antibodies herein is broad and includes heavy chain antibodies (HCAbs), which consist only of heavy chains found in camelids such as llamas and alpacas.
  • IgG is composed of two heavy chains (H chains) and two light chains (L chains).
  • a heavy chain is composed of a variable region (VH), a first constant region (CH1), a second constant region (CH2) and a third constant region (CH3) in order from the N-terminus.
  • a light chain is composed of a variable region (VL) and a constant region (CL) in order from the N-terminus.
  • the portion composed of CH2 and CH3 is the Fc region.
  • One heavy chain and one light chain are linked by a disulfide bond between a cysteine residue present in CH1 and a cysteine residue present in CL. Also, the heavy chains are linked by disulfide bonds between cysteine residues present in the hinge region located between CH1 and CH2.
  • Antibodies may be fragments of IgG in which heavy chains are present, may be rIgG (reduced IgG) composed of one heavy chain and one light chain, It may be a fragment composed of a single heavy chain. In an antibody having two heavy chains, at least one heavy chain may have a sugar chain attached thereto, and both heavy chains may not have a sugar chain attached thereto. IgG fragments also include Fab, F(ab')2, scFv, Fab', single chain immunoglobulins, and the like.
  • a fragment of IgG is a fragment that binds to a substance to be detected, which is a target antigen, and is called a functional fragment of IgG.
  • Humanized antibody of the present invention It is an antibody derived from a non-human animal antibody (typically murine) or derived from a chimeric antibody that retains or substantially retains the antigen-binding properties of the parent antibody, but is immunogenic in humans. means weaker antibodies.
  • the chimeric antibody refers to an antibody consisting of a variable region derived from a non-human animal antibody and a constant region of a human antibody.
  • "humanized antibody” includes “chimeric antibody”.
  • the heavy chain constant region is composed of CH1, CH2 and CH3, and the light chain constant region is the ⁇ or ⁇ constant region.
  • humanized antibodies can be prepared by: (a) grafting only non-human CDRs onto human framework and constant regions (Jones et al., Nature 321: 522-25 (1986); Verhoeyen et al., Science 239: 1534-1536 ( 1988)); or (b) by grafting entire non-human variable domains (to retain their ligand binding properties) and yet "dressing" them with a human-like surface by replacing exposed residues to reduce immunogenicity. (also called “veneered antibodies”) (Padlan, Molec. Immun. 28: 489-498 (1991); Padlan, Molec. Immun. 31(3): 169-217 (1994)) , can potentially be manufactured. Antibodies produced by the method (a) are called CDR-grafted antibodies.
  • a further feature of the present invention is to humanize and manufacture the Fc portion of the antibody.
  • Exemplary sequences of heavy chain constant regions that make up the Fc portion are as follows.
  • Humanized antibodies of the present invention have multiple amino acid substitutions, deletions, additions and/or insertions in the amino acid sequences other than the CDRs, such as the heavy chain constant region amino acid sequences described above, as long as they bind to the substance to be measured. For example, 1, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 15 or less, Up to 20 amino acids may be substituted, deleted, added and/or inserted.
  • the amino acid sequence of such humanized antibodies is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% identical to the original amino acid sequence without substitutions, deletions, additions and/or insertions. , have 98% or greater than 99% amino acid sequence identity.
  • humanized antibody of the present invention may be any mammal, such as humans or non-human animals such as mice, rabbits, rats, guinea pigs, hamsters, goats, sheep and camels. Not only human antibodies, but also recombinant antibodies such as humanized antibodies and chimeric antibodies can be used. Antibodies may be polyclonal antibodies or monoclonal antibodies.
  • the humanized antibody of the present invention can be produced by, for example, expressing a DNA encoding a heavy chain variable region, a DNA encoding a heavy chain constant region, a DNA encoding a light chain variable region and/or a DNA encoding a light chain constant region. , transforming a host cell with the vector, and culturing the host cell, a recombinant antibody can be produced.
  • the above-described heavy chain-encoding DNA and light chain-encoding DNA may be introduced into the same expression vector and used to transform host cells, or the heavy chain-encoding DNA and light chain-encoding DNA may be transformed into the same expression vector.
  • the DNA may be inserted into separate vectors and the two vectors used to transform the host cell.
  • the vector may contain a DNA encoding a signal peptide that promotes antibody secretion from host cells, and the signal peptide can be cleaved and removed after antibody production to produce a mature protein.
  • Animal cells, bacteria, yeast, etc. can be used as host cells, and known expression vectors for each host cell can be used as expression vectors.
  • humanized antibodies can be produced by using plant expression systems, and can also be produced by using transgenic plants.
  • a known method can be used for the plant transient expression system. Examples of such methods include, but are not limited to, the agroinfiltration method, the plant virus vector method, the magnICON® system, and the particle gun method.
  • a plant expressing the protein can be obtained by transforming Agrobacterium with the vector and infecting the plant with the Agrobacterium.
  • RNA is obtained from the cDNA of the plant virus genome into which the DNA encoding the humanized antibody has been inserted.
  • RNA is used as a vector to inoculate and infect plants.
  • viral vectors include, for example, tobacco mosaic virus (TMV) vectors, plumpox virus (PPV) vectors, potato X virus (PVX) vectors, alfalfa mosaic virus (AIMV) vectors, cucumber mosaic virus (CMV) vectors, cowpea mosaic virus (CPMV) vectors, zucchini yellow mosaic virus (ZYMV) vectors, and the like.
  • plants expressing humanized antibodies can be obtained as follows. First, cDNA of TMV or PVX genome into which DNA encoding a humanized antibody is inserted is introduced into a T-DNA vector. Then, a plant body is infected with Agrobacterium transformed with the obtained T-DNA vector.
  • the produced antibodies can be purified by separation and purification methods used for ordinary proteins. Such methods include affinity chromatography, ion exchange chromatography and other chromatography, ultrafiltration, salting out, dialysis, and the like.
  • the present invention is a reagent used in an immunoassay method, and includes diagnostic agents and test agents such as in-vitro diagnostic agents.
  • the reagent may contain a buffer or the like for stably holding the antibody.
  • Other components can be appropriately determined by those skilled in the art according to the immunoassay method to be used.
  • immunological measurement methods include immunostaining (including fluorescent antibody method, enzyme antibody method, heavy metal-labeled antibody method, and radioisotope-labeled antibody method), electrophoretic separation and fluorescence, enzymes, radioisotopes, etc. method (including western blotting, fluorescence two-dimensional electrophoresis), enzyme-linked immunosorbent assay (ELISA), dot blotting, latex agglutination (LA: Latex Agglutination-Turbidimetric Immunoassay), immunochromatography law, etc.
  • immunostaining including fluorescent antibody method, enzyme antibody method, heavy metal-labeled antibody method, and radioisotope-labeled antibody method
  • electrophoretic separation and fluorescence enzymes, radioisotopes, etc.
  • ELISA enzyme-linked immunosorbent assay
  • LA Latex Agglutination-Turbidimetric Immunoassay
  • immunochromatography law etc.
  • Substances to be measured that can be detected and quantified by reagents include antigens such as proteins and polysaccharides, haptens including low-molecular physiologically active substances such as monosaccharides, oligosaccharides, amino acids, peptides, and substances having a steroid skeleton.
  • antigens such as proteins and polysaccharides, haptens including low-molecular physiologically active substances such as monosaccharides, oligosaccharides, amino acids, peptides, and substances having a steroid skeleton.
  • antigens such as proteins and polysaccharides, haptens including low-molecular physiologically active substances such as monosaccharides, oligosaccharides, amino acids, peptides, and substances having a steroid skeleton.
  • haptens including low-molecular physiologically active substances such as monosaccharides, oligosaccharides, amino acids, peptides, and substances
  • protein markers such as CRP (C-reactive protein), prostate specific antigen, ferritin, ⁇ -2 microglobulin, myoglobin, hemoglobin, albumin, creatinine, IgG, IgA, IgM, etc.
  • immunoglobulins various tumor markers, LDL, HDL, lipoproteins such as TG, influenza A virus, influenza B virus, respiratory syncytial virus (RSV), rhinovirus, rotavirus, norovirus, adenovirus, astrovirus, HAV, Viral antigens such as HBs, HCV, HIV, and EBV, bacterial antigens such as Chlamydia trachomatis, Streptococcus, Pertussis, Helicobacter pylori, Leptospira, Treponema pallidum, Toxoplasma gondii, Borrelia, Legionella, Bacillus anthracis, and MRSA , toxins produced by bacteria, mycoplasma lipid antigens, peptide hormones such as human chorionic gonadotropins, steroids such as steroid hormones, bioactive amines such as epinephrine and morphine, vitamins, prostaglandins, antibiotics such as tetracycl
  • the substances to be measured include the above protein markers, various tumor markers, lipoproteins, viral antigens, bacterial antigens, toxins produced by bacteria, peptide hormones, steroids, physiologically active amines, vitamins, antibiotics, pesticides, Examples include antibodies that specifically react with antigens such as endocrine disruptors. Moreover, IgE antibody etc. which exist in the living body can be mentioned as a to-be-measured substance. In this case, an antibody against the above antibody may be used as the humanized antibody.
  • the reagent is added to the specimen derived from the subject who needs to confirm the presence or absence of the substance to be measured.
  • the specimen is not particularly limited as long as it contains the substance to be measured. Serum, plasma, urine, stool, cerebrospinal fluid or dilutions thereof are preferred.
  • kits comprising the above reagents.
  • Kits can be used for diagnostics, research, and the like.
  • the kit may contain not only the antibody as a reagent but also a carrier for immobilizing the humanized antibody and a secondary antibody, depending on its intended use.
  • an insoluble carrier is preferable, and examples thereof include particles such as latex particles such as polyethylene and polystyrene, alumina particles, silica particles, colloidal gold, and magnetic particles. More specifically, microplates can be used as insoluble carriers for ELISA (direct method, indirect method, sandwich method, etc.), and latex and the like can be used for latex agglutination methods.
  • a blocking agent is preferably included in the kit from the viewpoint of preventing non-specific reaction between the antibody and the antigen.
  • the humanized antibody may be directly immobilized on the carrier by physical adsorption or the like, or may be indirectly immobilized via chemical cross-linking, such as a linker.
  • Kits for use in direct methods include, for example, reagents containing humanized antibodies for enzyme-labeled antibodies that bind to antigens, substrates for enzymatic reactions, standard antigens for use in preparing standard curves or control experiments, etc. is included.
  • Kits for use in indirect methods may include, for example, primary antibodies that bind to the antigen, secondary antibodies that bind to the primary antibodies, substrates for enzymatic reactions, as reagents containing humanized antibodies, standard curve generation or control experiments. Standard antigens and the like for use are included. Secondary antibodies may be labeled for detection.
  • Kits for use in sandwich methods include, for example, antibodies bound to solid phases as reagents containing humanized antibodies, antibodies for enzyme labeling, substrates for enzymatic reactions, and reagents for use in standard curve generation or control experiments. Standard antigens and the like are included.
  • the enzyme-labeled antibody may be a humanized antibody.
  • Kits for use in latex agglutination include, for example, binding detection antibodies as reagents containing humanized antibodies, standard antigens for use in standard curve generation or control experiments, and non-specific reactions between antibodies and antigens. Blocking agents and the like for prevention are included.
  • the present invention provides a method for measuring a substance to be measured using an antigen-antibody reaction using a humanized antibody.
  • the method can be performed using the reagent or kit described above, and is a measurement method by the immunological method described above.
  • the present invention provides a method for suppressing non-specific antigen-antibody reactions using humanized antibodies.
  • Non-specific reaction due to Fc receptor is a non-specific reaction that appears when the sample contains a receptor for the Fc region of the antibody, and non-specific reaction due to rheumatoid factor appears to the Fc region of the antibody.
  • a non-specific reaction caused by antibodies, and a non-specific reaction caused by heterophile antibodies is a non-specific reaction caused by antibodies against non-human animal antibodies present in human blood. Non-specific reaction may also occur when a substance unrelated to the desired antigen-antibody reaction forms an antigen-antibody reactant or exhibits cross-reaction. is preferably used in combination with
  • the non-specific reaction suppression method includes the step of contacting the humanized antibody with the specimen. Optional steps may be included before and after the contacting step.
  • ⁇ Preparation of anti-CRP (C-reactive protein) humanized antibody (expressed in plants) 1.
  • Vector preparation and protein expression A mouse-derived humanized anti-CRP monoclonal antibody (IgG) was selected as an antibody with a humanized Fc region.
  • IgG heavy chains were cloned into tobacco mosaic virus (TMV) vectors (Icon Genetics) and light chains were cloned into potato virus X (PVX) vectors (Icon Genetics). After introducing these vectors into separate Agrobacterium for transformation and culturing, the mixture of the culture solutions was infiltrated into leaves of Nicotiana benthamiana and harvested after about one week. Harvested leaves (infected leaves) were frozen.
  • TMV tobacco mosaic virus
  • PVX potato virus X
  • Vector Preparation and Protein Expression A mouse-derived humanized anti-CRP monoclonal antibody (IgG) was selected as an antibody with a humanized Fc region. The IgG heavy chain and light chain were cloned into pCHO1.0vector (manufactured by Thermo Fisher Scientific Co., Ltd.). After linearization of this vector with a restriction enzyme, Freedom TM CHO-S TM (manufactured by Thermo Fisher Scientific Co., Ltd.) was transfected.
  • mouse-derived humanized anti-CRP monoclonal antibody-producing CHO stable expression clones were obtained according to Freedom TM CHO-S TM Kit USER GUIDE (manufactured by Thermo Fisher Scientific Co., Ltd.). This clone was cultured in Fed-batch culture for 14 days, and the cell-removed liquid was collected from the culture medium.
  • the cell-free solution was centrifuged (4° C., 15,000 ⁇ g, 10 minutes) to collect the supernatant. This supernatant was filtered through a 0.22 ⁇ m filter and subjected to protein A column chromatography (HiTrap TM MabSelect SuRe TM manufactured by Global Life Science Technology) to separate and remove CHO cell-derived impurities.
  • a reagent for measurement by immunoagglutination was prepared as follows. i) Sensitized particles obtained by supporting 0.26 mg of a rabbit-derived anti-CRP polyclonal antibody with respect to 1 mL of a polystyrene latex suspension are added to a buffer solution (glycine, pH 7.3) so as to be 0.18 (w/v)%. to prepare a latex suspension. ii) A buffer (glycine, pH 7.0) was prepared.
  • Physiological saline and CRP-negative human serum (commercially available from Golden West Biologicals) were measured using the above-mentioned reagents.
  • 125 ⁇ L of buffer solution glycine, pH 7.0
  • sample solution glycine, pH 7.0
  • 125 ⁇ L of the latex suspension was added and mixed with stirring at 37°C.
  • the agglutination reaction for about 3 minutes was measured as a change in absorbance, and the CRP concentration of each sample was calculated from a calibration curve prepared using standard solutions (Table 1, Fig. 1).
  • a measurement reagent for immunoagglutination was prepared as follows. i) Sensitized particles obtained by supporting 0.26 mg of a rabbit-derived anti-CRP polyclonal antibody with respect to 1 mL of a polystyrene latex suspension are added to a buffer solution (glycine, pH 7.3) so as to be 0.18 (w/v)%. to prepare a latex suspension.
  • a latex test solution (anti-human CRP goat polyclonal antibody-sensitized latex particles) of "N-assay LA CRP-T Nittobo" (Nittobo Medical Co., Ltd.) using a goat-derived anti-CRP polyclonal antibody was prepared.
  • a buffer (phosphate, pH 7.4) was prepared.
  • Physiological saline and CRP-negative human serum (commercially available from Golden West Biologicals) were measured using the above-mentioned reagents. 240 ⁇ L of a buffer solution (phosphoric acid, pH 7.4) was added to 3.0 ⁇ L of the sample solution, and the mixture was stirred and mixed at 37°C. After standing for 5 minutes, 36 ⁇ L of the latex suspension was added and mixed with stirring at 37°C. Agglutination reaction for about 90 seconds was measured as absorbance change (Fig. 1).
  • a buffer solution phosphoric acid, pH 7.4
  • the amount of change in absorbance is considered to be close to that of physiological saline. be judged.
  • the mouse-derived humanized anti-CRP antibody gave an absorbance change similar to that of physiological saline, so the reagents using mouse-derived humanized anti-CRP antibody and goat-derived anti-CRP antibody suppressed non-specific reactions caused by heterophile antibodies. It is thought that non-specific agglutination is less likely to occur during sample measurement in a measurement method that does not add a compound for

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PCT/JP2022/028698 2021-07-26 2022-07-26 ヒト化抗体を含む試薬 Ceased WO2023008404A1 (ja)

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JP2023538536A JPWO2023008404A1 (enExample) 2021-07-26 2022-07-26
CN202280052194.2A CN117716037A (zh) 2021-07-26 2022-07-26 含有人源化抗体的试剂
US18/290,904 US20250102496A1 (en) 2021-07-26 2022-07-26 Reagent containing humanized antibody
EP22849465.4A EP4372009A4 (en) 2021-07-26 2022-07-26 REAGENT CONTAINING A HUMANIZED ANTIBODY
KR1020237041539A KR20240005839A (ko) 2021-07-26 2022-07-26 인간화 항체를 포함한 시약

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