WO2022186317A1 - Procédé de détection du cancer, procédé d'examen du cancer et kit l'utilisant - Google Patents

Procédé de détection du cancer, procédé d'examen du cancer et kit l'utilisant Download PDF

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WO2022186317A1
WO2022186317A1 PCT/JP2022/009047 JP2022009047W WO2022186317A1 WO 2022186317 A1 WO2022186317 A1 WO 2022186317A1 JP 2022009047 W JP2022009047 W JP 2022009047W WO 2022186317 A1 WO2022186317 A1 WO 2022186317A1
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cea
probe molecule
cancer
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galnac
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和訓 岡田
慎太郎 八木
克己 青柳
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株式会社先端生命科学研究所
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Priority claimed from JP2021033693A external-priority patent/JP2022134527A/ja
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07—ORGANIC CHEMISTRY
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • 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

Definitions

  • the present invention relates to a cancer detection method, a cancer examination method, and a kit used therefor, more specifically, a method for detecting at least one type of cancer selected from the group consisting of prostate cancer and colorectal cancer, and methods for examining cancer, and kits for use in these methods.
  • Biomarkers such as tumor markers are useful for detection of diseases such as malignant tumors, guidelines for deciding treatment policies, and monitoring markers for evaluating therapeutic effects, and have been extensively studied in recent years.
  • examples of such biomarkers include CA19-9 and CEA (Carcinoembryonic Antigen).
  • CA19-9 is a type I carbohydrate antigen recognized by a monoclonal antibody (NS19-9) obtained by Koprowski et al. The site is sialyl Lewis A in which sialic acid is added to Lewis A antigen (Le a ) of Lewis blood group antigens.
  • CA19-9 exists as a mucin-type glycoprotein in the blood (Non-Patent Document 1, etc.), and is detected in the blood even in healthy subjects (those not suffering from cancer, hereinafter the same).
  • the concentration is elevated in gastrointestinal cancer patients, particularly colon cancer, pancreatic cancer, bile duct cancer, gallbladder cancer, and the like (Non-Patent Document 1).
  • CEA established Gold P. and Freedman S. O. It is a glycoprotein with a molecular weight of about 200,000, and is also known as CD66e or CEACAM5.
  • the protein portion of CEA is a single-chain polypeptide consisting of about 700 amino acids, and one molecule of CEA has 28 N-type glycosylation sites, most of which are bound by complex-type sugar chains. has been reported to contain 50% or more sugar chains (Non-Patent Document 2).
  • CEA is detected in the blood of healthy individuals, but its concentration is known to increase in cancer patients with gastrointestinal cancer, particularly colon cancer, pancreatic cancer, and gastric cancer. , lung cancer, uterine cancer, ovarian cancer, fetal cancer, etc. have also been reported to show high concentrations in blood.
  • CA19-9 and CEA have been conventionally used as monitoring markers for detection of these cancers, guidelines for determining treatment policy, and determination of therapeutic effects.
  • or anti-CEA antibody (capture body) to capture CA19-9 and CEA present in the sample and a sandwich method in which labeled anti-CA19-9 antibody and anti-CEA antibody (labeled body) are bound to
  • kits for measuring CA19-9 and CEA "Lumipulse Presto (registered trademark) CA19-9” and “Lumipulse Presto (registered trademark) CEA” are manufactured and sold by Fujirebio.
  • Patent Document 1 discloses the amount of free PSA in a sample and a sugar chain ( ⁇ 2-3 A method for determining prostate cancer from the ratio of the amount of PSA containing sialic acid) is described.
  • CA19-9 detected in the blood of healthy subjects exists as a part of low- to medium-molecular-weight mucins of about 200,000 to 1,000,000 Da, whereas CA19 detected in the blood of cancer patients -9 has been reported to exist as part of a high molecular weight mucin of about 5-10 million Da. Potential as a marker is expected.
  • CA19-9 and CEA are detected in small amounts even in the blood of healthy subjects. Predict or discriminate between negative and positive cancers, etc. as described above.
  • conventional methods for measuring CA19-9 and CEA only one type of anti-CA19-9 antibody specific to sialyl Lewis A or only one type of anti-CEA antibody is used. sex is not enough.
  • CEA measurements are rarely elevated due to the presence of prostate cancer. Therefore, conventional methods for measuring CA19-9 and CEA are still insufficient to discriminate between prostate cancer patients and healthy subjects or prostatic hyperplasia patients, or between colon cancer patients and healthy subjects with higher accuracy. The inventors have found that this is insufficient.
  • the present invention has been made in view of the above problems, and a cancer detection method and cancer test capable of specifically detecting prostate cancer and/or colorectal cancer with high accuracy using a new biomarker as an index.
  • the object is to provide methods, as well as kits for use in these methods.
  • the present inventors for the purpose of searching for sugar chains as new tumor markers that could not be measured with existing antibodies, the present inventors, in addition to anti-CA19-9 antibodies and anti-CEA antibodies, used a water-soluble carrier consisting of a water-soluble polymer.
  • a complex (blocked labeled lectin) comprising a labeled substance immobilized on the water-soluble carrier and a lectin was also used for screening.
  • the amount of molecules detected when WFA was used as a lectin that is, the amount of lectin-binding GalNAc (N-acetylgalactosamine)-added CA19-9 (GalNAc-added CA19-9) was It was found to significantly increase in cancer patients and colorectal cancer patients.
  • the amount of molecules detected when MAM is used as a lectin that is, the amount of CEA with ⁇ 2-3 sialylated lectin-binding sugar chain ( ⁇ 2-3 sialylated CEA) was We also found a significant increase in prostate cancer patients compared to . Therefore, prostate cancer and colon cancer are specifically detected by measuring the amount of GalNAc-added CA19-9, or prostate cancer is specifically detected by measuring the amount of ⁇ 2-3 sialylated CEA.
  • prostate cancer patients and healthy subjects or prostatic hyperplasia patients, or colorectal cancer patients and healthy subjects compared to when only CA19-9 is measured or when CEA is measured only with anti-CEA antibody Further, the inventors have found that it is possible to perform discrimination with higher accuracy, and have completed the present invention.
  • a method for detecting at least one type of cancer selected from the group consisting of prostate cancer and colon cancer comprising a measuring step of measuring the amount of GalNAc-added CA19-9 in a sample.
  • a method of testing for at least one type of cancer selected from the group consisting of prostate cancer and colorectal cancer comprising a measuring step of measuring the amount of GalNAc-added CA19-9 in a sample derived from a subject. .
  • [3] A method for screening a subject predicted to have at least one type of cancer selected from the group consisting of prostate cancer and colorectal cancer, wherein GalNAc-added CA19-9 in a sample derived from the subject
  • the method of [2] comprising a measuring step of measuring the amount and a selecting step of selecting subjects using the measured amount of GalNAc-added CA19-9 as an indicator.
  • the measuring step is a step of contacting the sample with a first probe molecule capable of specifically binding to CA19-9 and a second probe molecule capable of specifically binding to GalNAc [1] The method according to any one of to [4].
  • the second probe molecule is a lectin capable of specifically binding to GalNAc.
  • the measuring step is a step of contacting the sample with a capturing body and a labeling body;
  • the capture body comprises a water-insoluble carrier and either one of a first probe molecule and a second probe molecule immobilized on the water-insoluble carrier, and wherein the label comprises a labeling substance and the other of the first probe molecule and the second probe molecule;
  • the capture body comprises a water-insoluble carrier and a first probe molecule immobilized on the water-insoluble carrier, and Blocking wherein the label comprises a water-soluble carrier, a labeling substance and a second probe molecule immobilized on the water-soluble carrier, and the second probe molecule is a lectin capable of specifically binding to GalNAc is a labeled lectin,
  • the method according to [8]. [10] A kit for use in the method according to any one of [5] to [9], comprising a first probe molecule capable of specifically binding to CA19-9 and capable of specifically binding to GalNAc and a second probe molecule. [11] The kit of [10], wherein the first probe molecule is an antibody capable of specifically binding to CA19-9.
  • the capture body comprises a water-insoluble carrier and a first probe molecule immobilized on the water-insoluble carrier, and Blocking wherein the label comprises a water-soluble carrier, a labeling substance and a second probe molecule immobilized on the water-soluble carrier, and the second probe molecule is a lectin capable of specifically binding to GalNAc is a labeled lectin,
  • the kit according to [13].
  • a method of detecting prostate cancer comprising the step of measuring the amount of ⁇ 2-3 sialylated CEA in a sample.
  • a method of testing for prostate cancer comprising the step of measuring the amount of ⁇ 2-3 sialylated CEA in a sample derived from a subject.
  • a method for screening a subject predicted to have prostate cancer comprising a measuring step of measuring the amount of ⁇ 2-3 sialylated CEA in a sample derived from the subject; The method of [15], comprising a screening step of screening subjects using the amount of acid-added CEA as an index.
  • the measuring step is a step of contacting the sample with a first probe molecule capable of specifically binding to CEA and a second probe molecule capable of specifically binding to ⁇ 2-3 sialic acid.
  • the measuring step is a step of contacting the sample with a capturing body and a labeling body;
  • the capture body comprises a water-insoluble carrier and either one of a first probe molecule and a second probe molecule immobilized on the water-insoluble carrier, and wherein the label comprises a labeling substance and the other of the first probe molecule and the second probe molecule;
  • the capture body comprises a water-insoluble carrier and a first probe molecule immobilized on the water-insoluble carrier
  • the label comprises a water-soluble carrier, a labeling substance immobilized on the water-soluble carrier, and a second probe molecule
  • the second probe molecule is a lectin capable of specifically binding to ⁇ 2-3 sialic acid. is a blocked labeled lectin that is The method according to [22].
  • the kit of [24] wherein the first probe molecule is an antibody capable of specifically binding to CEA.
  • a capture body comprising a water-insoluble carrier and either one of a first probe molecule and a second probe molecule immobilized on the water-insoluble carrier; and a label comprising a labeling substance and the other of the first probe molecule and the second probe molecule;
  • the capture body comprises a water-insoluble carrier and a first probe molecule immobilized on the water-insoluble carrier, and
  • the label comprises a water-soluble carrier, a labeling substance immobilized on the water-soluble carrier, and a second probe molecule, and the second probe molecule is a lectin capable of specifically binding to ⁇ 2-3 sialic acid. is a blocked labeled lectin that is The kit of [27].
  • a cancer detection method and a cancer detection method capable of specifically detecting prostate cancer and/or colorectal cancer with high accuracy using a new biomarker as an index, and to these methods It becomes possible to provide a kit for use.
  • FIG. 1 is a graph showing measurement results of CA19-9 in a healthy subject group and a prostate cancer group.
  • 2 is a graph showing measurement results of CA19-9/WFA in a healthy subject group and a prostate cancer group.
  • FIG. 10 is a graph showing measurement results of CA19-9 in a prostatic hyperplasia group and a prostatic cancer group.
  • FIG. 10 is a graph showing the measurement results of CA19-9/WFA in a prostatic hyperplasia group and a prostatic cancer group.
  • FIG. 1 is a graph showing the relationship between measured values of CA19-9 and measured values of CA19-9/WFA.
  • 1 is a graph showing the relationship between PSA measurements and CA19-9/WFA measurements.
  • 1 is a graph showing measurement results of CA19-9 in a healthy subject group and a colorectal cancer group.
  • 2 is a graph showing measurement results of CA19-9/WFA in a healthy subject group and a colorectal cancer group.
  • 1 is a graph showing the relationship between measured values of CA19-9 and measured values of CA19-9/WFA.
  • 1 is a graph showing measurement results of CA19-9/WFA in a healthy subject group and a breast cancer group.
  • 2 is a graph showing measurement results of CA19-9/MAM in a healthy subject group and a prostate cancer group.
  • 2 is a graph showing measurement results of CA19-9/MAM in a healthy subject group and a colorectal cancer group.
  • FIG. 2 is a graph showing CEA measurement results for a healthy subject group and a prostate cancer group.
  • 2 is a graph showing measurement results of CEA/MAM in a healthy subject group and a prostate cancer group.
  • FIG. 10 is a graph showing the measurement results of CEA in the prostatic hyperplasia group and the prostatic cancer group.
  • FIG. 10 is a graph showing the measurement results of CEA/MAM in the prostatic hyperplasia group and the prostatic cancer group;
  • FIG. 4 is a graph showing the relationship between measured values of CEA and measured values of CEA/MAM;
  • 4 is a graph showing the relationship between PSA measurements and CEA/MAM measurements.
  • 4 is a graph showing measurement results of CEA/WFA in a healthy subject group and a prostate cancer group.
  • a first cancer detection method of the present invention is a method for detecting at least one type of cancer selected from the group consisting of prostate cancer and colorectal cancer, and measures the amount of GalNAc-added CA19-9 in a sample.
  • the method includes the step of measuring
  • a first cancer testing method of the present invention is a method of testing for at least one cancer selected from the group consisting of prostate cancer and colorectal cancer, wherein GalNAc in a sample derived from a subject is tested. It is a method including a measuring step of measuring the amount of added CA19-9 (hereinafter, the first cancer detection method and the first cancer examination method are collectively referred to as “first method” as the case may be).
  • the second cancer detection method of the present invention is a method for detecting prostate cancer, and is a method including a measurement step of measuring the amount of ⁇ 2-3 sialylated CEA in a sample.
  • a second cancer testing method of the present invention is a method of testing for prostate cancer, and includes a measuring step of measuring the amount of ⁇ 2-3 sialylated CEA in a sample derived from a subject.
  • the second cancer detection method and the second cancer examination method are collectively referred to as the "second method” in some cases, and further, the first method and the second method are collectively referred to in some cases. "Method of the Invention").
  • cancer includes epithelial malignant tumors (cancer) and non-epithelial malignant tumors (sarcoma).
  • the cancer to be detected or tested in the first method of the present invention is at least one type of cancer selected from the group consisting of prostate cancer and colon cancer.
  • Cancer to be detected or tested in the second method of the present invention is prostate cancer.
  • Prostate cancer according to the present invention refers to cancer that develops in the prostate, such as prostate cancer, prostate small cell carcinoma, prostate ductal carcinoma, prostate sarcoma, prostate squamous cell carcinoma, prostate adenosquamous cell carcinoma, and prostate basal cell. cancer, prostatic mucinous carcinoma, prostatic signet ring cell carcinoma.
  • prostate cancer is preferable as the prostate cancer according to the present invention.
  • colorectal cancer according to the present invention refers to cancer occurring in the large intestine (particularly the colon or rectum). Tumors, non-epithelial malignancies, and lymphomas. Among these, colorectal adenocarcinoma is preferable as the colorectal cancer according to the present invention.
  • GalNAc-added CA19-9 refers to a molecule comprising CA19-9 and GalNAc (N-acetylgalactosamine) attached to CA19-9.
  • CA19-9 refers to a type I sugar chain antigen recognized by a monoclonal antibody (NS19-9) obtained using human colon cancer cultured cell line SW-1116 as an immunogen.
  • CA19-9 has sialyl Lewis A in which sialic acid is added to the Lewis A antigen (Le a ) of the Lewis blood group antigens as the antigen-determining site for the following anti-CA19-9 antibody (Koprowski et al., Somat. Cell Genet., 5, 957, 1979).
  • GalNAc N-acetylgalactosamine
  • GalNAc-added CA19-9 preferably contains GalNAc as a WFA-linked sugar chain that binds to WFA, preferably as a terminal GalNAc residue.
  • a plurality of CA19-9 and GalNAc may be present, and even if CA19-9 and GalNAc are directly bound, they are bound via the core protein or other sugar chains.
  • the core protein include mucin, apolipoprotein, kininogen, and ARCVF.
  • the average mass of GalNAc-added CA19-9 is not particularly limited, but is 25,000 to 10,000,000 Da. more preferably 1,000,000 to 10,000,000 Da.
  • CA19-9 and GalNAc may be present on particles even larger than the core protein, such as extracellular vesicles such as exosomes or viruses.
  • the particle size of the GalNAc-added CA19-9 (including the particles described above) in this case is not particularly limited, but is about 50 to 500 nm.
  • ⁇ 2-3 sialylated CEA means a CEA molecule containing a sugar chain ⁇ 2-3 sialic acid ( ⁇ 2,3-linked sialic acid) attached thereto. is also referred to as “ ⁇ 2-3 sialic acid-containing CEA”.
  • CEA refers to carcinoembryonic antigen, which is a glycoprotein.
  • CEA is one of the molecules constituting the CEACAM (carcinoembryonic antigen-associated adhesion molecule) family involved in various processes such as cell adhesion, proliferation, differentiation, and tumor suppression, and is labeled as "CD66e” or "CEACAM5 (carcinoembryonic antigen CD66e or CEACAM5 and CEA are used synonymously herein.
  • CEA typically consists of a protein portion, which is a single-chain polypeptide consisting of about 700 amino acids, and a sugar chain portion, which is bound thereto and consists of multiple sugar chains.
  • the ⁇ 2-3 sialylated CEA according to the present invention contains at least ⁇ 2-3 sialic acid as the sugar chain moiety.
  • ⁇ 2-3 sialic acid refers to a sugar chain ( ⁇ 2-3Gal sialic acid) in which the 2-position carbon of sialic acid is glycoside-bonded to the 3-position carbon of galactose (Gal), which is a monosaccharide.
  • Gal galactose
  • a plurality of ⁇ 2-3 sialic acids may be present.
  • the ⁇ 2-3 sialylated CEA according to the present invention preferably contains ⁇ 2-3 sialic acid as a MAM-linked sugar chain that binds to MAM, and the terminal sialic acid residue is 2 from the end of the sugar chain. It is more preferable to contain a sugar chain ( ⁇ 2-3Gal sialic acid) linked to the second galactose residue as an ⁇ 2,3-linked sugar chain ( ⁇ 2-3Gal sialic acid). It is more preferably contained as a terminal ⁇ 2-3 sialic acid ( ⁇ 2-3Gal ⁇ 1-4GlcNAc sialic acid) of the glycosylation site.
  • the average mass of such ⁇ 2-3 sialylated CEA is not particularly limited, but is 100,000 to 300,000 Da. preferably 180,000 to 200,000 Da.
  • the content of the sugar chain moiety in one molecule of ⁇ 2-3 sialylated CEA is not particularly limited, but is preferably 40 to 60% by mass.
  • the term "subject” refers to a subject, preferably a person, on whom the cancer examination method of the present invention is performed.
  • a subject according to the present invention may be a healthy subject for the purpose of screening or the like, or a subject suffering from prostate cancer or colon cancer but without subjective symptoms.
  • the term "healthy subject” refers to cancer to be tested (i.e., prostate cancer and/or colon cancer in the first method, prostate cancer in the second method). cancer). It should be noted that whether the subject truly suffers from prostate cancer and/or colorectal cancer is determined (definitive diagnosis) by biopsy of prostate tissue and/or colorectal tissue collected from the subject. .
  • sample used in the first method of the present invention is not particularly limited as long as it is a sample in which GalNAc-added CA19-9 can be present.
  • sample used in the second method of the present invention is not particularly limited as long as it is a sample in which ⁇ 2-3 sialylated CEA can be present.
  • sample generally include blood samples such as serum, plasma, and whole blood collected from cancer test subjects such as the subject; urine, sputum, saliva, sweat, cerebrospinal fluid, and digestive juice. , body fluid specimens other than blood, such as semen, lymph, and ascitic fluid; mucosa specimens such as oral mucosa, pharyngeal mucosa, and intestinal mucosa; and various biopsy specimens.
  • the sample may be a cultured cell or a cell culture solution.
  • the sample according to the present invention is preferably a blood sample, more preferably serum (also referred to as "serum sample").
  • samples may be diluted or suspended with a diluent as necessary, or may be pretreated as appropriate.
  • diluent include buffers such as phosphate buffer, Tris buffer, Good's buffer, borate buffer, acetate buffer, citrate buffer, glycine buffer, succinate buffer, and phthalate buffer. liquid.
  • pretreatment include pulverization, freezing, heating, concentration, fractionation, desalting, and the like; addition of pH adjusters, stabilizers, preservatives, preservatives, surfactants, and the like; purification, and the like. These may be used alone or in combination of two or more.
  • the purification treatment is not particularly limited, but includes, for example, column treatment; adsorption of contaminants with a substance such as an antibody that adsorbs contaminants in a sample immobilized on a water-insoluble carrier to obtain GalNAc-added CA19-9.
  • a sample containing (in the first method) or a sample containing ⁇ 2-3 sialylated CEA (in the second method) is removed from the sample; CA19-9 containing GalNAc-added CA19-9 is captured, contaminants are removed by washing or the like, and then released (in the first method) or ⁇ 2- by an anti-CEA antibody immobilized on a water-insoluble carrier.
  • a treatment of capturing CEA containing tri-sialylated CEA, removing contaminants by washing or the like, and then liberating the CEA may be mentioned.
  • a first method of the present invention includes a measuring step of measuring the amount of GalNAc-added CA19-9 in a sample. Also, the second method of the present invention includes a measuring step of measuring the amount of ⁇ 2-3 sialylated CEA in the sample.
  • the method for measuring the amount of GalNAc-added CA19-9 and the method for measuring the amount of ⁇ 2-3 sialylated CEA include a method capable of measuring the amount of GalNAc-added CA19-9 and measuring the amount of ⁇ 2-3 sialylated CEA, respectively. It is not particularly limited as long as the method can be used, and a conventionally known method, a method based thereon, or a combination thereof can be employed as appropriate.
  • a probe molecule capable of specifically binding to GalNAc-added CA19-9 (in the first method) or a probe molecule capable of specifically binding to ⁇ 2-3 sialylated CEA (in the second method) is used.
  • the measurement step according to the present invention includes a method using a probe molecule capable of specifically binding to GalNAc-added CA19-9 (in the case of the first method) and a method capable of specifically binding to ⁇ 2-3 sialylated CEA. It is preferable that the method (in the case of the second method) using a probe molecule with a Examples of such probe molecules include antibodies; binding proteins such as protein A, protein G and protein L; avidins such as avidin D and streptavidin; lectins; be done.
  • antibody includes not only complete antibodies, but also antibody fragments (eg, Fab, Fab', F(ab') 2 , Fv, single-chain antibodies, diabodies, etc.) and variable regions of antibodies. Also included are low-molecular-weight antibodies conjugated with
  • the probe molecule capable of specifically binding to GalNAc-added CA19-9 is a combination of a probe molecule capable of specifically binding to CA19-9 and a probe molecule capable of specifically binding to GalNAc.
  • the probe molecule capable of specifically binding to ⁇ 2-3 sialylated CEA includes a probe molecule capable of specifically binding to CEA and a probe molecule capable of specifically binding to ⁇ 2-3 sialic acid. A combination with a probe molecule is preferred.
  • the probe molecule capable of specifically binding to CA19-9 and the probe molecule capable of specifically binding to CEA are referred to as "first probe molecule", and the probe molecule capable of specifically binding to GalNAc and A probe molecule capable of specifically binding to ⁇ 2-3 sialic acid is referred to as a "second probe molecule”.
  • first probe molecule capable of specifically binding to CA19-9
  • examples of the first probe molecule capable of specifically binding to CA19-9 include an antibody capable of specifically binding to CA19-9 (herein sometimes referred to as "anti-CA19-9 antibody”), CA19 Antibodies capable of specifically binding to proteins to which -9 binds (such as the aforementioned core protein), among which anti-CA19-9 antibodies are preferred.
  • anti-CA19-9 antibody an antibody capable of specifically binding to CA19-9
  • CA19 Antibodies capable of specifically binding to proteins to which -9 binds such as the aforementioned core protein
  • anti-CA19-9 antibody refers to an antibody that can specifically recognize and bind to sialyl Lewis A of CA19-9.
  • Such an anti-CA19-9 antibody is not particularly limited as long as it has the ability to bind to CA19-9, and may be a polyclonal antibody or a monoclonal antibody. Monoclonal antibodies are preferred.
  • the anti-CA19-9 antibody can be produced by appropriately adopting and improving conventionally known production methods, and commonly available methods may be used as appropriate.
  • the term "capable of binding specifically to CEA” refers to sites other than ⁇ 2-3 sialic acid (more preferably, sialic acid ⁇ 2-3Gal ⁇ 1-4GlcNAc sugar chain) in CEA. It shows that it can specifically bind to
  • site other than ⁇ 2-3 sialic acid may be the whole or part of the protein moiety, or the whole or part of the sugar chain moiety other than ⁇ 2-3 sialic acid. It may be a combination of two or more.
  • Examples of the first probe molecule capable of specifically binding to CEA include antibodies capable of specifically binding to CEA (anti-protein antibody having a recognition site for the protein portion of CEA, an anti-protein antibody having a recognition site for the sugar chain portion of CEA, and antibodies whose recognition sites are the protein portion and sugar chain portion of CEA.
  • anti-CEA antibody anti-CEACAM family antibody
  • CEA and other CEACAM Antibodies whose recognition sites are family molecules anti-CEA antibodies are preferred
  • anti-CEA protein antibodies whose recognition sites are at least part of the CEA protein portion are more preferred
  • ⁇ 2- Antibodies that do not interfere with the binding of trisialic acid to the second probe molecule are preferred.
  • the anti-CEA antibody is not particularly limited as long as it has the ability to bind to CEA, and may be a polyclonal antibody or a monoclonal antibody, but from the viewpoint of homogeneity and stability, a monoclonal antibody is preferable. preferable.
  • Anti-CEA antibodies can be produced by appropriately adopting and improving conventionally known production methods, and commonly available methods may be used as appropriate.
  • the anti-CA19-9 antibody and anti-CEA antibody for example, when the following lectin is used as the second probe molecule, the lectin also recognizes the antibody sugar chain and the detection sensitivity is lowered.
  • Antibodies are preferably produced under conditions in which glycosylation does not occur, such as by using genetically modified E. coli or cells in which antibody genes are expressed, or by restricting nutritional conditions in culturing antibody-producing cells.
  • Examples of the second probe molecule capable of specifically binding to GalNAc include, for example, an antibody capable of specifically binding to GalNAc (herein sometimes referred to as "anti-GalNAc antibody”), lectins (e.g., Noda Fuji Lectin (WFA), Soybean Lectin (SBA), Nayokusa Fuji Lectin (VVA), Himalayan Fuji Bean Lectin (DBA), Purple Saxocinka Lectin (BPL), Apple Potato Lectin (HPA)), GalNAc-specific Among them, lectins that can specifically bind to GalNAc are preferred, and Nodafuji lectin (WFA) is particularly preferred.
  • WFA Nodafuji lectin
  • Wisteria floribunda agglutinin is a protein that recognizes the sugar chain structure of GalNAc and exhibits binding activity, and is a leguminous lectin derived from the plant Noda wisteria.
  • the lectin may be a modified lectin into which a mutation has been introduced or an artificially synthesized lectin for the purpose of increasing the specificity of sugar chain recognition activity. Moreover, you may use suitably what is generally distribute
  • Examples of the second probe molecule capable of specifically binding to ⁇ 2-3 sialic acid include, for example, an antibody capable of specifically binding to ⁇ 2-3 sialic acid ⁇ 2-3 sialic acid-binding artificial proteins such as Lectenz (registered trademark), lectins capable of specifically binding to ⁇ 2-3 sialic acid (e.g., canine endu lectin (MAM/MAL/MAA-II ), canine cochlea lectins such as canine cochlea lectin (MAH/MAA-I); willow pine mushroom lectin (ACG); jackfruit lectin (Jacalin)).
  • an antibody capable of specifically binding to ⁇ 2-3 sialic acid ⁇ 2-3 sialic acid-binding artificial proteins such as Lectenz (registered trademark), lectins capable of specifically binding to ⁇ 2-3 sialic acid (e.g., canine endu lectin (MAM/MAL/MAA-II ), canine cochle
  • MAM Mesackia amurensis lectin
  • MAA-II is a protein that recognizes the sugar chain structure of ⁇ 2-3 sialic acid (especially sialic acid ⁇ 2-3Gal ⁇ 1-4GlcNAc) and exhibits binding activity. It is a leguminous lectin derived from the seeds of a plant, the dog pagoda.
  • the lectin may be a modified lectin into which a mutation has been introduced or an artificially synthesized lectin for the purpose of increasing the specificity of sugar chain recognition activity. Moreover, you may use suitably what is generally distribute
  • the sample When using the first probe molecule and the second probe molecule, the sample is brought into contact with the first probe molecule and the second probe molecule in the measurement step.
  • both CA19-9 and GalNAc are recognized, and GalNAc-added CA19-9 containing both can be detected and measured (in the case of the first method).
  • both ⁇ 2-3 sialic acid and portions of CEA other than ⁇ 2-3 sialic acid are recognized, and ⁇ 2-3 sialylated CEA containing both can be detected and measured (the second method case).
  • the contact between the sample and the first probe molecule and the contact between the sample and the second probe molecule may be at the same time or at different times. may come first.
  • the amount of GalNAc-added CA19-9 is measured using a probe molecule (preferably, the first probe molecule and/or the second probe molecule) capable of specifically binding to GalNAc-added CA19-9. It is preferably carried out by detecting a signal generated by a labeling substance attached or attached to a molecule (eg, secondary antibody or protein A) that recognizes these molecules.
  • a probe molecule capable of specifically binding to ⁇ 2-3 sialylated CEA (preferably, the first probe molecule and/or the 2 probe molecules) or molecules that recognize these molecules (eg, secondary antibody or protein A).
  • the amount of GalNAc-added CA19-9 or ⁇ 2-3 sialylated CEA can be measured by measuring the amount of the detected signal and, if necessary, semi-quantifying or quantifying it.
  • the above-mentioned "signal” includes coloration (color development), quenching, reflected light, luminescence, fluorescence, radiation from radioactive isotopes, etc., and in addition to those that can be confirmed with the naked eye, measurement methods and devices according to the type of signal This includes items that can be verified.
  • the amounts of GalNAc-added CA19-9 and ⁇ 2-3 sialylated CEA to be measured may be semi-quantified or quantified using standard samples or the like. Alternatively, the signal amount may be directly used as the amount of GalNAc-added CA19-9 or ⁇ 2-3 sialylated CEA of the present invention.
  • labeling substance As the labeling substance according to the present invention, those used as labeling substances in known immunoassay methods and methods based thereon can be used without particular limitation.
  • enzymes luminescent substances such as acridinium derivatives; fluorescent substances such as europium; fluorescent proteins such as allophycocyanin (APC) and phycoerythrin (R-PE); radioactive substances such as 125 I; low molecular weight labeling substances such as rhodamine isothiocyanate (RITC); gold particles; avidin; biotin; latex; dinitrophenyl (DNP); A combination of the above may also be used.
  • luminescent substances such as acridinium derivatives
  • fluorescent substances such as europium
  • fluorescent proteins such as allophycocyanin (APC) and phycoerythrin (R-PE)
  • radioactive substances such as 125 I
  • low molecular weight labeling substances such as rhodamine isothiocyanate (RITC)
  • various measurements can be performed by adding a chromogenic substrate, a fluorescent substrate, a chemiluminescent substrate, or the like as the substrate.
  • the enzyme include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (ALP), ⁇ -galactosidase ( ⁇ -gal), glucose oxidase, and luciferase.
  • Measurement methods using the first probe molecule and the second probe molecule include immunological measurement methods such as the sandwich method, competitive method, and immunoturbidimetric method, and measurement methods based on these principles. There are no particular restrictions. Such measurement methods include, for example, generally ELISA, digital ELISA, CLEIA (chemiluminescent enzyme immunoassay), CLIA (chemiluminescence immunoassay), ECLIA (electrochemical immunoassay), RIA (radioimmunoassay). ) using microplates, particles, etc. as carriers; immunochromatography; surface plasmon resonance analysis; detection methods based on fluorescence resonance energy transfer;
  • the sandwich method is preferable from the viewpoint of the possibility of constructing a measurement system with higher sensitivity and specificity.
  • a more specific aspect of the measuring process according to the present invention will be described by taking the sandwich method as an example.
  • the measuring step is a step of contacting the sample with a capturing body and a labeling body;
  • the capture body comprises a water-insoluble carrier and either one of a first probe molecule and a second probe molecule immobilized on the water-insoluble carrier, and the label comprises a labeling substance and the other of the first probe molecule and the second probe molecule;
  • first aspect can be mentioned.
  • the first probe molecule and the second probe molecule may be provided in either the capturing body or the labeling body, respectively. One is included in the label.
  • GalNAc-added CA19-9 can be captured and detected with high accuracy and ease (in the case of the first method).
  • ⁇ 2-3 sialic acid and CEA in this way, ⁇ 2-3 sialylated CEA can be easily captured and detected with high accuracy (in the case of the second method).
  • the label comprises a first labeling substance and either one of a first probe molecule and a second probe molecule.
  • a second labeled body comprising a first labeled body, a second labeled substance, and the other of the first probe molecule and the second probe molecule, and the capture body is a non-
  • An aspect hereinafter sometimes referred to as a “second aspect” that is a capturing body comprising a water-soluble carrier and probe molecules immobilized on the water-insoluble carrier may be employed.
  • the first labeling substance and the second labeling substance produce signals different from each other.
  • the probe molecules provided in the capture body are probe molecules capable of specifically binding to GalNAc-added CA19-9 (the first probe molecule and the second probe molecule are a probe molecule capable of specifically binding to the first probe molecule and/or the second probe molecule, and in the case of the second method, capable of specifically binding to ⁇ 2-3 sialylated CEA Probe molecules (including first probe molecules and second probe molecules); probe molecules that can specifically bind to first probe molecules and/or second probe molecules.
  • Examples of such a sandwich method include the forward sandwich method, which is a two-step method (reaction between the capture body and GalNAc-added CA19-9 in the sample, reaction between the captured body and GalNAc-added CA19-9 bound to the capture body and the label (Section 1). method 1), or the reaction between the capturing body and ⁇ 2-3 sialylated CEA in the sample, or the reaction between the ⁇ 2-3 sialylated CEA bound to the capturing body and the labeled body (in the case of the second method ) sequentially), reverse sandwich method (preliminarily reacting the label with GalNAc-added CA19-9 or ⁇ 2-3 sialylated CEA in the sample, and reacting the resulting complex with the capturing agent).
  • the forward sandwich method which is a two-step method (reaction between the capture body and GalNAc-added CA19-9 in the sample, reaction between the captured body and GalNAc-added CA19-9 bound to the capture body and the label (Section
  • the sample is brought into contact with the capture body, and binding between the probe molecule of the capture body and GalNAc-added CA19-9 (for example, binding of the first probe molecule and CA19-9 ) to capture the GalNAc-attached CA19-9 with the capturing body (in the case of the first method).
  • the sample is brought into contact with the capture body, and ⁇ 2-3 binding occurs via binding between the probe molecule of the capture body and ⁇ 2-3 sialylated CEA (for example, binding between the first probe molecule and CEA).
  • the sialylated CEA is captured by the capturing body (in the case of the second method) (primary reaction: capturing step).
  • the labeled substance is brought into contact with the GalNAc-added CA19-9 captured by the capture substance, and binding between the probe molecule of the labeled substance and GalNAc-added CA19-9 (for example, binding of the second probe molecule and GalNAc to binding) (for the first method).
  • the label is brought into contact with ⁇ 2-3 sialylated CEA captured by the capturer, and binding of the probe molecule of the label to ⁇ 2-3 sialylated CEA (for example, binding of the second probe molecule and ⁇ 2-3 sialic acid) (in the case of the second method) (secondary reaction: labeling step).
  • the "capture body” comprises a water-insoluble carrier and a probe molecule capable of specifically binding to GalNAc-added CA19-9 immobilized on the water-insoluble carrier. or, in the case of the second method, a probe molecule capable of specifically binding to ⁇ 2-3 sialylated CEA immobilized on said water-insoluble carrier, respectively, wherein said It is a conjugate in which a water-insoluble carrier and the probe molecule are directly or indirectly bound.
  • the probe molecules provided in the capture body according to the first aspect are preferably either one of the first probe molecules and the second probe molecules.
  • the probes provided in the capture body The molecule may be either the first probe molecule or the second probe molecule, but when a lectin is used as the second probe molecule, the capture In the first method, the probe molecule provided in the body is preferably a first probe molecule capable of specifically binding to CA19-9, more preferably an anti-CA19-9 antibody, and In the case of method 2, the first probe molecule capable of specifically binding to CEA is preferred, and an anti-CEA antibody is more preferred.
  • the water-insoluble carrier contained in the capturing body is a water-insoluble substance that mainly supports the probe molecule and functions as a carrier for immobilization.
  • water-insoluble substance refers to a substance that is insoluble in water at normal temperature and pressure (solubility in water is 0.001 g/mL or less, preferably 0.0001 g/mL or less, the same shall apply hereinafter).
  • the material of such a water-insoluble carrier those commonly used in immunoassays and measurements based thereon can be used, and are not particularly limited. (Meth)acrylate, polymethyl methacrylate, polyimide, nylon, etc.), gelatin, glass, latex, silica, metals (gold, platinum, etc.), and metal compounds (iron oxide, cobalt oxide, nickel ferrite, etc.) At least one selected from, the material of the water-insoluble carrier may be a composite material of these substances or a composite material of these substances and other substances.
  • the water-insoluble carrier includes a carboxy group, an epoxy group, a tosyl group, an amino group, a hydroxyl group, an isothiocyanate group, an isocyanate group, an azide group, an aldehyde group, a carbonate group, an allyl group, an aminooxy group, and a maleimide group. , the surface of which is modified with an active group such as a thiol group.
  • the shape of the water-insoluble carrier is not particularly limited, and may be, for example, a plate, fiber, film, particle, etc. However, from the viewpoint of reaction efficiency, it is a particle. From the viewpoint of automation and shortening of the time, magnetic particles are more preferable.
  • a water-insoluble carrier conventionally known ones can be used as appropriate, and commercially available ones can also be used as appropriate.
  • the content of the probe molecule is not particularly limited, and may be appropriately adjusted according to the ease of binding between the probe molecule and GalNAc-added CA19-9 or ⁇ 2-3 sialylated CEA.
  • the mass of the probe molecule is preferably 0.1 to 10 parts by mass, more preferably 1 to 5 parts by mass.
  • the capture body can be produced by immobilizing the probe molecule on the water-insoluble carrier.
  • a conventionally known method or a method analogous thereto can be appropriately employed, and the probe molecule may be directly or indirectly immobilized on the water-insoluble carrier.
  • the water-insoluble carrier and/or the probe molecule include a carboxy group, an epoxy group, a tosyl group, an amino group, a hydroxy group, an isothiocyanate group, an isocyanate group, an azide group, an aldehyde group,
  • active groups such as carbonate groups, allyl groups, aminooxy groups, maleimide groups, thiol groups, etc., or by imparting the active groups as necessary, and binding them together, the probe molecules are converted into the It can be immobilized directly to a water-insoluble carrier.
  • a linker that binds to the probe molecule is immobilized to the water-insoluble carrier, and the probe molecule is bound to the linker to bind the probe molecule to the water-insoluble carrier.
  • the linker is not particularly limited. hydrazide) and the like.
  • the probe molecule may be modified in some way, a substance that captures the modified portion may be immobilized on the water-insoluble carrier, and the probe molecule may be immobilized on the water-insoluble carrier.
  • a representative example of the modifying moiety is biotin, and a representative example of a substance that captures the modifying moiety is streptavidin, but the present invention is not limited to these.
  • the ratio of the water-insoluble carrier and the probe molecule to be subjected to these reactions can be appropriately selected so as to achieve the preferred range of the above-mentioned ratio in the capturing body.
  • a suitable blocking agent eg, bovine serum albumin, gelatin, etc.
  • Blocking may be performed.
  • a commercially available one may be used as appropriate.
  • the "label” according to the present invention is a complex comprising a labeling substance and a probe molecule capable of specifically binding to GalNAc-added CA19-9 in the first method; or in the second method. a complex comprising a labeling substance and a probe molecule capable of specifically binding to ⁇ 2-3 sialylated CEA, wherein the labeling substance and the probe molecule are respectively bound directly or indirectly It is a conjugate.
  • the probe molecule provided in the label according to the first aspect is preferably the other molecule of the molecules provided in the capture body.
  • the probe molecule provided in the label may be either the first probe molecule or the second probe molecule.
  • a second probe molecule capable of specifically binding to GalNAc.
  • lectins are more preferred, and WFA is particularly preferred.
  • the labeled substance includes the labeled substance and the antibody (anti-CA19-9 antibody, anti-GalNAc antibody (for the first method); anti-CEA antibody, anti- ⁇ 2-3 sialic acid antibody (for the second method). method), etc.), a labeled lectin comprising the labeling substance and a lectin, and a blocked labeled lectin.
  • a blocked labeled lectin is preferable as the labeled substance in the present invention, particularly in the first aspect.
  • the term "blocked labeled lectin” refers to a complex comprising a water-soluble carrier made of a water-soluble polymer, and a labeling substance and a lectin immobilized on the water-soluble carrier, wherein the water-soluble carrier and the label It is a conjugate in which a substance and a lectin are bound directly or indirectly.
  • the blocked labeled lectin comprises a lectin capable of specifically binding to GalNAc as the second probe molecule in the first method, and ⁇ 2 as the second probe molecule in the second method.
  • a lectin capable of specifically binding to -3 sialic acid is provided.
  • the probe molecule provided in the capturing body is the first probe molecule.
  • the lectin is as described above, preferably WFA (for the first method) or MAM (for the second method).
  • the labeling substance is as described above, including its preferred embodiments.
  • the labeling substance and the lectin are supported on the water-soluble carrier. Even if both the substance and the lectin are bound to the other two, even if the lectin is bound to the water-soluble carrier via the labeling substance, the labeling substance is bound to the water-soluble carrier via the lectin.
  • the affinity at each binding point between a lectin and a lectin-binding sugar chain structure is weak. to form Therefore, by using this, multiple binding points are generated in one conjugate, which improves the overall affinity and enables highly sensitive detection of the target GalNAc or ⁇ 2-3 sialic acid.
  • the water-soluble carrier contained in the blocked labeled lectin mainly functions as a carrier for carrying the labeling substance and the lectin, and is composed of a water-soluble polymer.
  • the water-soluble polymer constituting the water-soluble carrier according to the present invention (hereinafter referred to as "first water-soluble polymer”) is particularly a water-soluble polymer capable of immobilizing and supporting the labeling substance and lectin.
  • first water-soluble polymer is particularly a water-soluble polymer capable of immobilizing and supporting the labeling substance and lectin.
  • water-soluble polymer means that the solubility in water at normal temperature and normal pressure exceeds 0.01 g/mL, preferably 0.05 g/mL or more, more preferably 0.1 g/mL or more. A polymer compound is shown.
  • the weight-average molecular weight is from the viewpoint of sensitivity of measurement and water solubility. From this point of view, it is preferably 6,000 to 4,000,000, more preferably 20,000 to 1,000,000.
  • the first water-soluble polymer according to the present invention has an average mass of 70,000 to 1,000,000 Da from the viewpoint of obtaining a blocked labeled antibody with a more preferable average particle size. and more preferably 150,000 to 700,000 Da.
  • one blocked labeled lectin may contain a plurality of types of water-soluble polymers having different weight average molecular weights as the first water-soluble polymer.
  • a high molecular weight blocked labeled lectin in which the weight average molecular weight of the first water-soluble polymer is 200,000 or more and a high molecular weight blocked labeled lectin in which the weight average molecular weight of the first water-soluble polymer is 100, 000 (more preferably 100,000 or less) with a low molecular weight blocked labeled lectin, and the weight average molecular weight of the first water-soluble polymer is 200,000 to 700,000 (further A high molecular weight blocked labeled lectin, preferably 250,000 to 500,000), and a first water-soluble polymer having a weight average molecular weight of 20,000 to 100,000 (more preferably 50,000 to 70,000) ) with a low-molecular-
  • the mass ratio (mass of high-molecular-weight blocked labeled lectin:mass of low-molecular-weight blocked labeled lectin) is preferably 10:1 to 1:10, more preferably 5:1 to 1:5, even more preferably 3:1 to 1:3.
  • Examples of the first water-soluble polymer according to the present invention include dextran, aminodextran, Ficoll (trade name), dextrin, agarose, pullulan, various celluloses (eg, hemicellulose, ligrin, etc.), chitin, chitosan, and the like. polysaccharide; ⁇ -galactosidase; thyroglobulin; hemocyanin; polylysine; or a combination of two or more.
  • the first water-soluble polymer according to the present invention is inexpensive and available in large quantities, and is relatively easy to chemically process such as addition of functional groups and coupling reactions. is preferably at least one selected from the group consisting of polysaccharides and modifications thereof, and at least one selected from the group consisting of dextran and aminodextran, and modifications thereof More preferred, dextran is even more preferred.
  • the content of the labeling substance is not particularly limited, and can be appropriately adjusted according to the measurement mechanism and the like. It is preferable to set the number of molecules of the labeling substance to be bound to one molecule as large as possible.
  • the labeling substance is an enzyme
  • the mass of the first water-soluble polymer is a combination of two or more, the same shall apply hereinafter
  • the mass of the labeling substance per 100 parts by mass (the sum of them when the labeling substance is a combination of two or more) is 100 to 1, It is preferably 000 parts by mass, more preferably 300 to 800 parts by mass.
  • the lectin content is not particularly limited, but is set so that the number of lectin molecules that bind to one molecule of the first water-soluble polymer is as large as possible in order to further improve the measurement sensitivity.
  • the mass of the lectin relative to 100 parts by mass of the first water-soluble polymer is 100 to 2,000 parts by mass. preferably 300 to 1,500 parts by mass.
  • the blocked labeled lectin preferably has a weight average molecular weight of 1,000,000 to 10,000,000, preferably 1,500,000 to 5,000, per molecule of the blocked labeled lectin. 000 is more preferred. When the weight-average molecular weight is 1,000,000 or more, the measurement sensitivity tends to be higher. be.
  • the blocked labeled lectin can be produced by immobilizing the labeled substance and lectin on the water-soluble carrier.
  • a conventionally known method or a method analogous thereto can be appropriately adopted, and the labeling substance and lectin (hereinafter collectively referred to as “supported substance” in some cases) are directly immobilized on the water-soluble carrier. may be fixed or indirectly fixed.
  • the first water-soluble polymer constituting the to-be-supported substance and/or the water-soluble carrier has a carboxy group, an epoxy group, or a tosyl group.
  • a method of using a water-soluble polymer having these active groups as the substance to be supported and/or the water-soluble carrier and binding them together for immobilization can be exemplified.
  • the material to be supported and the first water-soluble polymer to which the active groups are attached commercially available products may be used as they are, or the active groups may be added to the surface of the material to be supported and the water-soluble polymer under appropriate reaction conditions. may be introduced and prepared.
  • thiol groups can be introduced using commercially available reagents such as S-acetylmercaptosuccinic anhydride and 2-iminothiolane hydrochloride.
  • introduction of a maleimide group to an amino group on the first water-soluble polymer constituting the material to be supported and/or the water-soluble carrier can be performed by, for example, N-(6-maleimidocaproyloxy) succinimide or N It can be carried out using commercially available reagents such as -(4-maleimidobutyryloxy)succinimide.
  • a pyridyl disulfide group can be performed by, for example, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N- ⁇ 6-[3-(2-pyridyldithio)propionamido]hexanoyloxy ⁇ sulfosuccinimide, sodium-ac-sulfonate (SPDP), SPDP) and other commercially available reagents can be used.
  • SPDP N-succinimidyl 3-(2-pyridyldithio)propionate
  • SPDP sodium-ac-sulfonate
  • SPDP sodium-ac-sulfonate
  • a pyridyl disulfide group can also be introduced by reducing it to a thiol group after introduction.
  • Methods for indirectly immobilizing the substance to be supported on the water-soluble carrier include, for example, oligopeptides containing polyhistidine, polyethylene glycol, cysteine and/or lysine, linker molecules having the active group (for example, the above capture method of fixing via a linker such as those mentioned in the method for producing the body).
  • the selection and size of the linker can be appropriately set in consideration of the strength of binding to the substance to be supported, steric hindrance due to immobilization of the substance to be supported on the water-soluble carrier, and the like.
  • the labeling substance and the lectin may be immobilized on the water-soluble carrier at once, or they may be immobilized separately and sequentially. From the viewpoint of easiness in controlling the amounts of substances and lectins, it is preferable to immobilize one of them on the water-soluble carrier before immobilizing the other.
  • the blocked labeling lectin is obtained by immobilizing the labeling substance and the lectin on separate water-soluble carriers, and the labeling substance (blocked labeling substance) immobilized on one water-soluble carrier and the other water-soluble carrier. It can also be produced by binding to a lectin (blocked lectin) immobilized on a protein directly or via the linker or the like.
  • the method for producing the blocked labeled lectin is not particularly limited.
  • the labeling substance is an enzyme
  • the first water-soluble polymer is a polysaccharide or glycoprotein.
  • the first water-soluble polymer is oxidized with an oxidizing agent such as sodium periodate to give an aldehyde group, reacted with hydrazine hydrochloride, and then treated with dimethylamine borane (DMAB).
  • oxidizing agent such as sodium periodate
  • a reducing agent such as sodium periodate
  • the enzyme is also oxidized with an oxidizing agent such as sodium periodate to impart an aldehyde group to its sugar chain.
  • the hydrazine residue and the aldehyde group provided above are reacted to form a hydrazone bond to obtain the first water-soluble polymer-enzyme conjugate.
  • the resulting first water-soluble polymer-enzyme conjugate is treated with a crosslinker having N-hydroxysuccinimide and a maleimide group at each end (e.g., SM(PEG) 4 , SMCC, etc.) to introduce a maleimide group. do.
  • a crosslinker having N-hydroxysuccinimide and a maleimide group at each end e.g., SM(PEG) 4 , SMCC, etc.
  • a lectin is thiolated with a thiolating reagent to give a thiol group, or in the case of a lectin having a disulfide bond in its molecule, a thiol group is obtained by reduction.
  • the first water-soluble polymer (water-soluble carrier)-enzyme-lectin is formed by binding the maleimide group introduced into the first water-soluble polymer-enzyme conjugate with the thiol group provided to the lectin. can be covalently bonded.
  • a lectin is obtained by binding two or more molecules of the first water-soluble polymer via an enzyme. The ratios of the first water-soluble polymer, the labeling substance, and the lectin subjected to these reactions can be appropriately selected so as to achieve the preferred range of each content in the blocked labeling lectin.
  • the method for contacting the sample with the capturing body is not particularly limited, and a conventionally known method or a method based thereon can be employed as appropriate.
  • the method of injecting the sample into this (probe molecule-immobilized plate), or when the water-insoluble carrier is a particle, the capturing body (probe molecule-immobilized particle) is added to the sample. method.
  • the content (final concentration) of the capturing body in the reaction solution containing the capturing body and the sample is not particularly limited, and is not particularly limited because it is appropriately adjusted according to the type of sample, concentration, etc., but from the viewpoint of efficient capture in a short time, for example, It is preferably 0.01 to 1.5% by mass, more preferably 0.05 to 1% by mass, even more preferably 0.1 to 0.5% by mass.
  • the conditions for the capture step are not particularly limited, and can be adjusted as appropriate. It can be carried out for about 10 minutes, preferably about 30 seconds to 5 minutes, but is not limited to these conditions.
  • the content (final concentration) of the label in the reaction solution containing the label and GalNAc-added CA19-9 is not particularly limited, and is adjusted appropriately according to the type of sample, concentration, etc., so is not particularly limited. , from the viewpoint that excessive use may cause a high background signal, for example, it is preferably 0.001 to 10 ⁇ g / mL, more preferably 0.01 to 5 ⁇ g / mL, 0.1 More preferably ⁇ 1 ⁇ g/mL.
  • the labeled product in the reaction solution containing the labeled product and ⁇ 2-3 sialylated CEA is not particularly limited, and may be appropriately adjusted according to the type of sample, concentration, etc.
  • 0.001 to 10 ⁇ g/mL is preferable, and 0.01 to 5 ⁇ g/mL is preferable. More preferably, it is 0.1 to 1 ⁇ g/mL.
  • the heating can be performed for about 3 minutes to 120 minutes, preferably about 5 minutes to 10 minutes, but the conditions are not limited to these.
  • Examples include a method of removing the liquid phase (supernatant) from above, and a method of recovering the particles by centrifugation or magnet collection and removing the liquid phase (supernatant) in the case of the probe molecule-immobilized particles.
  • the injection and removal of the cleaning liquid may be repeated as necessary.
  • the washing solution include neutral (preferably pH 6 to 9) known buffers (sodium phosphate buffer, MES, Tris, CFB, MOPS, PIPES, HEPES, tricine buffer, bicine buffer, glycine buffer, etc.). and may contain a stabilizing protein such as BSA, a surfactant, or the like.
  • the sample may be diluted with a diluent as described above. may be used by suspending it in the particle suspension medium (particle liquid), and further, another reaction buffer may be appropriately added to the reaction system between the sample and the capturing body and/or the labeling body.
  • particle suspension media and reaction buffers are not particularly limited. buffer, glycine buffer, etc.), and may be independently added with a stabilizing protein such as BSA, serum, or the like.
  • a water-soluble polymer (hereinafter referred to as "second different from the first water-soluble polymer constituting the water-soluble carrier in that it is a free water-soluble polymer that does not carry the labeling substance and lectin), free lectin ( It differs from the lectin contained in the blocked labeled lectin in that it is neither immobilized on the water-soluble carrier nor the labeling substance.
  • the second water-soluble polymer examples include those mentioned as the first water-soluble polymer. good. Moreover, it may be the same type of polymer as the first water-soluble polymer. Among these, the second water-soluble polymer is preferably at least one selected from the group consisting of polysaccharides and modifications thereof, from the viewpoint that the measurement sensitivity tends to be further improved, and dextran and aminodextran, and at least one selected from the group consisting of modifications thereof, more preferably dextran.
  • the weight average molecular weight of the second water-soluble polymer is preferably 500,000 to 5,000,000, more preferably 1,000,000 to It is more preferably 3,000,000, and even more preferably 1,500,000 to 2,500,000.
  • the amount of the second water-soluble polymer is not particularly limited, but the content of the second water-soluble polymer in the reaction solution containing the sample, the blocked labeled lectin, and the second water-soluble polymer (When the second water-soluble polymer is a combination of two or more types, the total thereof) is preferably 0.01 to 10 w/v%, and preferably 0.5 to 3 w/v%. More preferred (w/v %: weight/volume (g/mL) percentage, same below).
  • the free lectin examples include those similar to those listed above as the lectin, and one of these may be used alone or two or more may be used in combination. Moreover, it may be of the same kind as the lectin contained in the blocked labeled lectin. Among these, the free lectin is particularly preferably of the same type as the lectin contained in the blocked labeled lectin, from the viewpoint of improving reactivity and suppressing background.
  • the amount of the free lectin is not particularly limited, but the content of the free lectin (free When the lectin is a combination of two or more, the total thereof) is preferably 1 to 10,000 parts by mass, more preferably 10 to 5,000 parts by mass.
  • a signal is measured according to the labeling substance.
  • the labeling substance is an enzyme
  • a signal for example, color development or luminescence
  • the amount of GalNAc-added CA19-9 in the sample can be detected as a signal amount, and if necessary, the amount of GalNAc-added CA19-9 in the sample can be determined by comparing with the measured value of a standard sample. can be quantified (for the first method).
  • the amount of ⁇ 2-3 sialylated CEA in the sample can be detected as a signal amount by this, and if necessary, the ⁇ 2-3 sialylated CEA in the sample can be detected by comparing with the measured value of a standard sample.
  • the amount of acid-added CEA can be quantified (for the second method).
  • the amount of GalNAc-added CA19-9 or the amount of ⁇ 2-3 sialylated CEA measured in the above-described measurement step is used as an indicator, and the sample or the subject from which the sample is derived is used as an index.
  • the presence or absence of prostate cancer and/or colon cancer in a person can be detected.
  • Such a cancer detection method can be used for research purposes such as drug discovery, as well as for the cancer examination method of the present invention.
  • the amount of GalNAc-added CA19-9 in a sample derived from a subject is measured, and the presence or absence of prostate cancer and/or colon cancer is detected using it as an index.
  • prediction of whether the subject is afflicted with prostate cancer and / or colorectal cancer, determination of the presence or absence of the affliction of the cancer or the possibility thereof, or progression or severity of the cancer degree can be evaluated.
  • aspects of the first cancer examination method of the present invention include, for example, the following aspects: A method for screening a subject predicted to have at least one type of cancer selected from the group consisting of prostate cancer and colorectal cancer, wherein GalNAc-added CA19-9 in a sample derived from the subject A first screening method comprising a measuring step of measuring the amount and a selecting step of selecting subjects using the measured amount of GalNAc-added CA19-9 as an indicator; A method for determining the presence or absence of at least one cancer selected from the group consisting of prostate cancer and colorectal cancer in a subject, wherein the amount of GalNAc-added CA19-9 in a sample derived from the subject is A first discrimination method comprising a measuring step of measuring and a discriminating step of discriminating a subject using the measured amount of GalNAc-added CA19-9 as an index; A method for evaluating the progression or severity of at least one type of cancer selected from the group consisting of prostate cancer and colorectal cancer in a subject, wherein
  • the amount of ⁇ 2-3 sialylated CEA in a sample derived from a subject is measured, and the presence or absence of prostate cancer is detected using this as an index, thereby detecting the presence of prostate cancer.
  • the subject it is possible to predict whether or not the subject is afflicted with prostate cancer, determine the presence or absence of the affliction of the cancer or the possibility thereof, or evaluate the progress or severity of the cancer. .
  • aspects of the second cancer examination method of the present invention include, for example, the following aspects: A method for screening a subject predicted to have prostate cancer, comprising a measuring step of measuring the amount of ⁇ 2-3 sialylated CEA in a sample derived from the subject; A second screening method comprising a selection step of selecting subjects using the amount of acid-added CEA as an index; A method for discriminating the presence or absence of prostate cancer in a subject, comprising a measuring step of measuring the amount of ⁇ 2-3 sialylated CEA in a sample derived from the subject, and the measured ⁇ 2-3 sialylated CEA.
  • a second discrimination method comprising a discrimination step of discriminating a subject using the CEA amount as an index;
  • a method for evaluating the degree of progression or severity of prostate cancer in a subject comprising a measuring step of measuring the amount of ⁇ 2-3 sialylated CEA in a sample derived from the subject, and the measured ⁇ 2-3 sialyl a second evaluation method comprising an evaluation step of evaluating the subject using the amount of acid-added CEA as an index; is mentioned.
  • screening means selection of subjects who may have the cancer (prostate cancer and/or colorectal cancer). Specifically, in the present invention, the screening method includes predicting that the subject is likely to be afflicted with prostate cancer and/or colorectal cancer (including recurrence), Methods of sorting from groups of no or low sex are included. In the screening method, depending on the purpose, for example, subjects may be screened at a level at which a medium possibility can be expected.
  • a negative concordance rate (percentage of subjects predicted to be in the normal group by the screening that are truly in the normal group) is preferably 95% or more.
  • the discrimination method includes determining whether or not the subject is suffering from prostate cancer or colorectal cancer, regardless of the presence or absence of symptoms, or whether or not there is a high possibility of having the same.
  • a method of determining whether the cancer that the subject is suffering from is prostate cancer or colon cancer, or a method of determining that the cancer is likely to be; It also includes a method for determining whether prostate cancer or colorectal cancer, which the examiner had suffered in the past, has recurred, or a method for determining that there is a high possibility that the cancer has recurred.
  • two groups a group consisting of persons truly affected by the cancer (positive group) and a group consisting of persons not affected by the cancer (normal group: negative group). Then, it is determined whether the subject is included in the positive group or the negative group. Such differentiation can be applied to assist doctors in diagnosing prostate cancer and/or colorectal cancer.
  • the negative concordance rate percentage of subjects who are discriminated as the normal group by the discrimination actually belong to the normal group is 95% or more.
  • the evaluation method includes, for example, a method of evaluating the degree of progression or severity of prostate cancer and/or colorectal cancer that a subject has; A method for providing an index for determining a therapeutic policy for cancer; and a method for determining therapeutic efficacy for cancer.
  • the amount of GalNAc-added CA19-9 at the time of discovery of the cancer in the subject or before treatment is compared with the current amount of GalNAc-added CA19-9, and if the amount is increased, the therapeutic effect is evaluated as low. However, if it decreases, it can be evaluated that the therapeutic effect is high.
  • the amount of added CEA and the amount of ⁇ 2-3 sialylated CEA in the subject are compared. can be evaluated as low.
  • the amount of ⁇ 2-3 sialylated CEA at the time of the cancer discovery or before treatment in the subject is compared with the current amount of ⁇ 2-3 sialylated CEA, and if the amount is increased, the therapeutic effect can be evaluated as low, and if it decreases, the therapeutic effect can be evaluated as high.
  • the amount of GalNAc-added CA19-9 measured in the measurement step is compared with a predetermined cutoff value.
  • the subject having a higher GalNAc-added CA19-9 amount than the cutoff value is predicted or discriminated as having (or having a high possibility of having) prostate cancer and/or colon cancer do.
  • the amount of ⁇ 2-3 sialylated CEA measured in the measuring step is compared with a predetermined cutoff value, and the amount of ⁇ 2-3 sialylated CEA is A subject having a higher value than the cutoff value is predicted or discriminated as having (or having a high possibility of having) prostate cancer.
  • the "cutoff value" is a predetermined value that serves as a reference for determination based on the amount of GalNAc-added CA19-9 or the amount of ⁇ 2-3 sialylated CEA, and the positive group and the negative group. Indicates a boundary value for judging a group.
  • Such a cut-off value depends on the purpose of the cancer testing method of the present invention, the method for measuring the amount of GalNAc-added CA19-9 or the amount of ⁇ 2-3 sialylated CEA, the properties of the subject and sample, and the dilution conditions. It is not particularly limited because it is appropriately set by, for example.
  • the detection sensitivity is high, that is, patients in the early stages of cancer can be picked up to some extent, and early detection becomes possible.
  • GalNAc-added CA19-9 and ⁇ 2-3 sialylated CEA are detected in small amounts even in samples derived from healthy subjects. It becomes possible to perform with higher precision.
  • the sample is a serum specimen, diluted to 1/10 by volume, anti-CA19-9 antibody immobilized particles are used as the capturing body, and ,
  • the amount of GalNAc-added CA19-9 measured by sandwich immunoassay using blocked labeled WFA as the labeled substance is expressed by the emission intensity (count) of light having maximum absorption at a wavelength of 463 nm with ALP as the labeled substance and AMPPD as the substrate.
  • 50,000 to 80,000 counts are mentioned, preferably 60,000 to 70,000 counts, but not limited thereto.
  • the cut-off value determined in the above range depending on the purpose of the cancer test method of the present invention, the method for measuring the amount of GalNAc-added CA19-9, the properties of the subject and the sample, any one from within the range A point is selected and applied.
  • the sample is a serum sample, diluted to 1/10 in volume ratio, anti-CEA antibody-immobilized particles are used as the capturing agent, and ,
  • the amount of ⁇ 2-3 sialylated CEA measured by a sandwich immunoassay using blocked labeled MAM as the labeled substance is calculated as the luminescence intensity (count ) (in the case of Example 2), 210,000 to 320,000 counts, preferably 270,000 to 300,000 counts, but not limited thereto.
  • the cut-off value determined in the above range can be any value within the range depending on the purpose of the cancer testing method of the present invention, the method for measuring the amount of ⁇ 2-3 sialylated CEA, and the properties of the subject and sample. or one point is selected and applied.
  • information for determining a treatment policy specific to prostate cancer and/or colorectal cancer can be provided by distinguishing it from other cancers (Section 1).
  • method 1) it is possible to provide information for determining a treatment policy specific to prostate cancer (in the case of the second method).
  • the second method by identifying subjects who have or are likely to have (including recurrence) prostate cancer and/or colorectal cancer, and conducting further tests and definitive diagnoses, prostate cancer and/or Alternatively, early detection and early therapeutic intervention of colorectal cancer become possible.
  • the method of the present invention is a method of assisting a doctor or the like in diagnosing prostate cancer and/or colorectal cancer, or a method of providing information for the diagnosis of prostate cancer and/or colorectal cancer by a doctor or the like. But also.
  • the method of the present invention is also suitable as a method for combining with a conventional CA19-9 measurement method or a conventional CEA measurement method, or a method for measuring other monitoring markers, thereby preventing prostate cancer and / or It is possible to further improve the detection sensitivity and diagnostic accuracy of colorectal cancer.
  • the first kit of the present invention is a kit for use in the cancer detection method or cancer examination method of the present invention, comprising a first probe molecule capable of specifically binding to CA19-9 and a GalNAc-specific and a second probe molecule that is physically bindable.
  • the second kit of the present invention is a kit for use in the cancer detection method or cancer examination method of the present invention, comprising a first probe molecule capable of specifically binding to CEA, ⁇ 2-3 and a second probe molecule capable of specifically binding to sialic acid (hereinafter, the first kit and the second kit are sometimes collectively referred to as "the kit of the present invention").
  • the first probe molecule and the second probe molecule are as described above, including their preferred embodiments.
  • the kit of the present invention includes a capture body comprising a water-insoluble carrier and either one of a first probe molecule and a second probe molecule immobilized on the water-insoluble carrier; and It is also preferred to provide a label comprising a labeling substance and the other of the first probe molecule and the second probe molecule.
  • the capturing body and the labeling body are as described above, including their preferred embodiments.
  • the first probe molecule, the second probe molecule, the capturing body, and the labeling body are each independently solid (powder) or liquid dissolved in a buffer solution.
  • a buffer solution may be
  • the concentrations of the first probe molecule, the second probe molecule, the capturing body, and the labeling body in each solution are not particularly limited, but are each independently, for example, 0.01 to 10 ⁇ g/ It is preferably mL, more preferably 0.1 to 5.0 ⁇ g/mL, even more preferably 0.5 to 3.0 ⁇ g/mL.
  • the kit of the present invention may further comprise components that should be included in conventional immunological measurement methods such as ELISA, CLEIA, immunochromatography, and methods based thereon.
  • immunological measurement methods such as ELISA, CLEIA, immunochromatography, and methods based thereon.
  • magnetic beads or a plate for immobilizing the probe molecule, a sensor chip, the standard sample (each concentration), a control reagent It may further include at least one selected from the group consisting of the particle suspension medium, reaction buffer, washing solution, second water-soluble polymer, and free lectin.
  • the labeling substance is an enzyme, it may further contain a substrate, a reaction stopping solution, and the like necessary for detection and quantification of the labeling substance.
  • the kit of the present invention may optionally include the diluent, a pretreatment liquid for pretreatment of the sample; a cartridge for dilution; and a pretreatment reaction stopping solution or neutralizing solution.
  • a device including a zone carrying the capturing body and/or the labeling body may be further included.
  • the device can include other components suitable for immunochromatography, such as a developing pad and an absorbent pad.
  • the kit of the present invention may further include instructions for use of the kit.
  • % means weight/volume (w/v: g/mL) percentage unless otherwise specified.
  • Example 1 Measurement of WFA-linked glycosylated CA19-9 (CA19-9/WFA) contained in a serum sample using blocked labeled lectin (WFA) (1) Preparation of hydrazinated dextran 4.8 mL 240.0 mg of dextran (manufactured by CarboMer) having a molecular weight of 250 k was added to 0.1 M phosphate buffer (pH 7.0) and dissolved by stirring in a dark place at 25° C. for 30 minutes. Then, 2.664 mL of 150 mM NaIO 4 and 0.536 mL of deionized water were added and stirred in the dark at 25° C. for 30 minutes.
  • WFA blocked labeled lectin
  • RC50K regenerated cellulose with a molecular weight of 50,000 dialysis membrane
  • dialysis with 4 L of ion-exchanged water was performed in a dark place for 3 hours, and then allowed to stand overnight at 4°C.
  • Buffer exchange was performed by gel filtration (Sephadex G-25) using 0.1 M sodium phosphate buffer (pH 6.0) to obtain 85.0 mL of solution.
  • the concentration of dextran in the resulting solution was adjusted to 1.0 mg/mL to obtain a solution of hydrazinated dextran.
  • hydrazide dextran prepared in (1) of Example 1 was added so that the hydrazide group (amino group) concentration was 25 ⁇ M, and the mixture was stirred at 25° C. in the dark for 16 hours.
  • 85 mg of DMAB was added and stirred in the dark at 25° C. for 2 hours.
  • 10.1 mL of 1.5 M Tris buffer (pH 9.0) was added and stirred for 2 hours in the dark at 25°C.
  • An ultrafiltration module (Pellicon XL50, manufactured by Merck Millipore) was attached to Labscale TFF System (manufactured by Merck Millipore), concentrated to 15 mL, and subjected to gel filtration (Superdex 200 pg ) to obtain 14 mL of a 3.0 mg/mL dextran-enzyme conjugate solution.
  • the buffer was exchanged with 0.1 M sodium phosphate buffer (pH 6.3) containing 20 mM EDTA/2Na and 0.5% CHAPS using a PD-10 column (Sephadex G-25).
  • a PD-10 column Sephadex G-25
  • the maleimide-PEGylated dextran-enzyme conjugate was concentrated using a centrifugal filter (Merck, Amicon Ultra 50K) to adjust the final concentration to 2 mg/mL.
  • the buffer was exchanged with 0.1 M sodium phosphate buffer (pH 6.3) containing 20 mM EDTA/2Na and 0.5% CHAPS using a PD-10 column (Sephadex G-25). WFA after buffer exchange was adjusted to 650 ⁇ g/mL.
  • the solution was concentrated using a centrifugal filter (Merck, Amicon Ultra 50K), passed through a ⁇ 0.22 ⁇ m filter, and subjected to gel filtration chromatography (column: Superose 6 Increase 10/300 GL, buffer: 0.000).
  • WFA blocked labeled lectin
  • the washed antibody-bound particles were replaced with 0.1 M sodium phosphate buffer (pH 6.0) containing 10 mM glycine, and mixed by inversion for 1 hour at 25°C in the dark to remove aldehydes produced by oxidation of antibody sugar chains.
  • the group was blocked with glycine.
  • 100 ⁇ L of 10 mg/mL DMAB was added to the antibody-bound particle solution after the reaction and mixed by inversion for 30 minutes in the dark at 25° C. to stabilize the unstable bond between the aldehyde group derived from the sugar chain of the antibody and glycine.
  • the antibody-bound particles after stabilization were washed three times with 0.6 mL of a buffer containing 2% BSA (50 mM MES, 1 mM EDTA, 150 mM NaCl, 2% BSA, 0.1% ProClin 300, pH 6.0), BSA was physically adsorbed by inverting and mixing the buffer at 37° C. for 16 hours.
  • the antibody-bound particles to which BSA was physically adsorbed were washed three times with a storage buffer (50 mM Tris, 2% BSA, 150 mM NaCl, 1 mM EDTA, 0.1% ProClin 300, pH 7.2), and stored in the same buffer at 4°C. saved.
  • the obtained oxidized anti-CA19-9 antibody-bound magnetic particles were diluted in a 50 mM Tris-based solution so that the concentration of the antibody-bound particles was 0.005%, and the oxidized anti-CA19-9 antibody was added.
  • a combined particle solution was prepared.
  • the blocked labeled lectin (WFA) obtained in (5) of Example 1 was diluted to a concentration of 0.5 ⁇ g/mL in a 50 mM MES-based solution to prepare a labeled body fluid.
  • CA19-9 and WFA-linked glycosylated CA19-9 contained in serum samples 10 serum samples collected from healthy subjects (group of healthy subjects: 1 to 10 healthy subjects) 10 serum specimens collected from patients with prostatic hyperplasia (prostatic hypertrophy group: prostatic hyperplasia 1-10), 15 serum specimens collected from prostate cancer patients (prostate cancer group: prostate cancer 1-15), and colorectal cancer patients 15 serum specimens (colonic cancer group: colon cancer 1 to 15) collected from each were diluted to a concentration of 1/10 by volume using a specimen diluent (manufactured by Fujirebio).
  • WFA-linked glycosylated CA19-9 contained in the same sample was measured using Lumipulse (registered trademark) L-2400 (manufactured by Fujirebio). . That is, 50 ⁇ L of each diluted sample solution was mixed with 50 ⁇ L of the oxidized anti-CA19-9 antibody-bound particle solution prepared in (6) of Example 1, and reacted at 37° C. for 8 minutes. Next, the magnetic particles were collected and washed five times with Lumipulse (registered trademark) washing solution (manufactured by Fujirebio). Next, 50 ⁇ L of the labeled body fluid prepared in (6) of Example 1 was added to each well, and reacted at 37° C.
  • Lumipulse registered trademark
  • AMPPD (3-(2′-spiroadamantane)-4-methoxy-4-(3′-phosphoryloxy)phenyl-1,2-dioxetane disodium 50 ⁇ L of LUMIPULSE (registered trademark) substrate solution (manufactured by Fujirebio) containing salt) was added and reacted at 37° C. for 4 minutes.
  • CA19-9 contained in each sample diluted above is Lumipulse Presto (registered trademark) CA19-9 (Fujirebio Co., Ltd.) comprising anti-CA19-9 antibody-binding particles and alkaline phosphatase (ALP)-labeled anti-CA19-9 antibody. (manufactured by Fujirebio Co., Ltd.) and measured by Lumipulse (registered trademark) L-2400 (manufactured by Fujirebio Co., Ltd.). The measurement results were output as the luminescence intensity (count) of the substrate (AMPPD) in the same manner as described above. The measurement results for each specimen are shown in Table 1 below. The results shown are the average values of duplicate measurements.
  • FIG. 1 shows the measurement results of CA19-9 in the healthy subject group and the prostate cancer group
  • FIG. 2 shows the measurement results of CA19-9/WFA in the healthy subject group and the prostate cancer group
  • FIG. -9 shows the measurement results of CA19-9/WFA in the prostatic hyperplasia group and prostatic cancer group, respectively.
  • FIG. 5 shows the relationship between the measured values of CA19-9 and the measured values of CA19-9/WFA.
  • CA19-9/WFA serves as a novel index for discriminating between healthy subjects and prostate cancer patients, or between prostatic hyperplasia patients and prostate cancer patients.
  • the cut-off value was calculated from the measured values of healthy subjects, and the ability to detect prostate cancer patients was tested when samples exceeding the cut-off value were found to be positive. did.
  • the cut-off value was calculated as 67,659 counts or less by adding the value obtained by multiplying the standard deviation of the measured values of the healthy subject group by 2 to the average of the measured values of the healthy subject group. As a result, 10 out of 15 specimens in the prostate cancer group, or 66.7%, were determined to be positive at this cut-off value.
  • 1 specimen out of 20 specimens in both groups was determined to be false positive, giving a specificity of 95.0%.
  • FIG. 6 shows the relationship between the measured values of PSA and the measured values of CA19-9/WFA.
  • FIG. 7 shows the measurement results of CA19-9 in the healthy subject group and the colon cancer group
  • FIG. 8 shows the measurement results of CA19-9/WFA in the healthy subject group and the colon cancer group
  • FIG. Relations with CA19-9/WFA measurements are shown, respectively.
  • CA19-9/WFA is a novel index for discriminating between healthy subjects and colorectal cancer patients.
  • Example 1 Verification of reactivity of CA19-9/WFA against cancers other than prostate cancer and colorectal cancer
  • CA19-9/WFA was measured in the same manner as in Example 1 (7) for 15 serum specimens (breast cancer group: breast cancer 1 to 15) collected from breast cancer patients. The measurement results for each sample are shown in Table 3 below.
  • FIG. 10 shows the measurement results of CA19-9/WFA in the healthy subject group and the breast cancer group.
  • Example 2 Measurement of sugar chain-attached CA19-9 by blocking labeled lectin using lectin other than WFA ) was conjugated to a blocked labeled lectin (MAM). That is, in the same manner as (1) to (5) of Example 1, except that MAM was used instead of WFA and the dextran with a molecular weight of 500 k (manufactured by Fluka) was used, the final 382 A 2.0 mL solution of 1 ⁇ g/mL blocked labeled lectin (MAM) (dextran-enzyme-MAM conjugate) was obtained. The size of dextran does not affect the specificity of lectin sugar chains.
  • Example 2 In the same manner as in (6) to (7) of Example 1, except that the obtained blocked enzyme lectin (MAM) was used instead of the blocked labeled lectin (WFA), ( 10 serum specimens collected from healthy subjects similar to those used in 7) (healthy subject group: healthy subjects 1 to 10), 15 serum specimens collected from prostate cancer patients (prostate cancer group: prostate cancer 1 to 15) , and, for 15 serum specimens collected from colorectal cancer patients (colonic cancer group: colon cancer 1 to 15), MAM-linked glycosylated CA19-9 (CA19-9/MAM) contained in each specimen was measured. The measurement results for each specimen are shown in Table 4 below.
  • FIG. 11 shows the CA19-9/MAM measurement results for the healthy subject group and prostate cancer group
  • FIG. 12 shows the CA19-9/MAM measurement results for the healthy subject group and colon cancer group.
  • FIG. 12, p 0.3340.
  • the measurement of CA19-9/WFA which measures the combination of CA19-9 and WFA-linked sugar chains, is a new specific index for discriminating between healthy subjects and patients with prostate cancer or colon cancer. was shown.
  • Example 2 Measurement of MAM-linked glycosylated CEA (CEA/MAM) contained in a serum sample using blocked labeled lectin (MAM)
  • CEA/MAM blocked labeled lectin
  • the buffer was exchanged with 0.1 M sodium phosphate buffer (pH 6.3) containing 20 mM EDTA/2Na and 0.5% CHAPS using a PD-10 column (Sephadex G-25). MAM after buffer exchange was adjusted to 650 ⁇ g/mL.
  • the solution was concentrated using a centrifugal filter (Merck, Amicon Ultra 50K), passed through a ⁇ 0.22 ⁇ m filter, and subjected to gel filtration chromatography (column: Superose 6 Increase 10/300 GL, buffer: 0.000).
  • MAM blocked labeled lectin
  • the washed particles were replaced with 0.1 M sodium phosphate buffer (pH 6.0) containing 10 mM glycine, and mixed by inversion for 1 hour at 25°C in the dark to remove aldehyde groups generated by oxidation of sugar chains of antibodies. blocked with glycine. Furthermore, 100 ⁇ L of 10 mg/mL DMAB was added to the antibody-bound particle solution after the reaction, and mixed by inversion for 30 minutes in the dark at 25° C. to stabilize the unstable bond between the aldehyde group derived from the antibody sugar chain and glycine.
  • the particles were washed three times with 0.6 mL of a buffer containing 2% BSA (50 mM MES, 1 mM EDTA, 150 mM NaCl, 2% BSA, 0.1% ProClin 300, pH 6.0). BSA was physically adsorbed by mixing by inversion for 16 hours at °C. The antibody-bound particles to which BSA was physically adsorbed were washed three times with a storage buffer (50 mM Tris, 2% BSA, 150 mM NaCl, 1 mM EDTA, 0.1% ProClin 300, pH 7.2), and stored in the same buffer at 4°C. saved. The obtained oxidized anti-CEA antibody-bound magnetic particles were diluted with a 50 mM Tris-based solution so that the concentration of the antibody-bound particles was 0.005% to prepare an oxidized anti-CEA antibody-bound particle solution. did.
  • a storage buffer 50 mM Tris, 2% BSA, 150 mM NaCl, 1 m
  • the blocked labeled lectin (MAM) obtained in (5) of Example 2 was diluted in a 50 mM MES-based solution to a concentration of 0.5 ⁇ g/mL to prepare a labeled body fluid.
  • CEA/MAM MAM-linked glycosylated CEA
  • Lumipulse (registered trademark) L-2400 manufactured by Fujirebio was used to measure MAM-linked glycosylated CEA (CEA/MAM) contained in the serum sample. That is, 50 ⁇ L of each diluted sample solution was mixed with 50 ⁇ L of the oxidized anti-CEA antibody-bound particle liquid prepared in Example 2 (6), and reacted at 37° C. for 8 minutes. Next, the magnetic particles were collected and washed five times with Lumipulse (registered trademark) washing solution (manufactured by Fujirebio). Next, 50 ⁇ L of each of the labeled body fluids prepared in (6) of Example 2 was added and allowed to react at 37° C. for 8 minutes.
  • AMPPD (3-(2′-spiroadamantane)-4-methoxy-4-(3′-phosphoryloxy)phenyl-1,2-dioxetane disodium 50 ⁇ L of LUMIPULSE (registered trademark) substrate solution (manufactured by Fujirebio) containing salt) was added and reacted at 37° C. for 4 minutes.
  • LUMIPULSE registered trademark
  • CEA contained in each sample diluted above was obtained using Lumipulse Presto (registered trademark) CEA (Fujirebio) equipped with anti-CEA antibody-binding particles and alkaline phosphatase (ALP)-labeled anti-CEA antibody.
  • Lumipulse Presto registered trademark
  • CEA Flujirebio
  • ALP alkaline phosphatase
  • L-2400 manufactured by Fujirebio Co., Ltd.
  • the measurement results were output as the luminescence intensity (count) of the substrate (AMPPD) in the same manner as described above.
  • the measurement results for each sample are shown in Table 5 below. The results shown are the average values of duplicate measurements.
  • FIG. 13 shows the CEA measurement results for the healthy subject group and the prostate cancer group
  • FIG. 14 shows the CEA/MAM measurement results for the healthy subject group and the prostate cancer group
  • FIG. 15 shows the CEA measurement results for the prostatic hyperplasia group and the prostate cancer group
  • FIG. 16 shows the measurement results of CEA/MAM in the prostatic hyperplasia group and the prostatic cancer group, respectively.
  • FIG. 17 shows the relationship between the measured values of CEA and the measured values of CEA/MAM.
  • the measured value of CEA/MAM does not simply represent the abundance of CEA, but CEA (recognized by an anti-CEA antibody capable of specifically binding to sites other than MAM-linked sugar chains). were shown to be different biomarkers.
  • CEA/MAM is a novel index for discriminating between healthy subjects and prostate cancer patients, or between prostatic hyperplasia patients and prostate cancer patients.
  • the cutoff value was calculated from the measured values of the healthy subject group, and the ability to detect prostate cancer patients was tested when samples exceeding the cutoff value were considered positive.
  • the cut-off value was calculated as 299,216 counts or less by adding the value obtained by multiplying the standard deviation of the measured values of the healthy subject group by 2 to the average of the measured values of the healthy subject group.
  • 8 out of 15 specimens in the prostate cancer group were determined to be positive at this cut-off value, accounting for 53.3%.
  • all 20 samples from the healthy subject group and the prostatic hyperplasia group were determined to be negative.
  • Example 2 In the same manner as in (6) to (7) of Example 2, except that the obtained blocked enzyme lectin (WFA) was used instead of the blocked enzyme lectin (MAM), ( 10 serum specimens collected from healthy subjects similar to those used in 7) (healthy subject group: healthy subjects 1 to 10), and 15 serum specimens collected from prostate cancer patients (prostate cancer group: prostate cancer 1 to 15 ), WFA-linked glycosylated CEA (CEA/WFA) contained in each sample was measured. The measurement results for each specimen are shown in Table 6 below.
  • FIG. 19 shows the CEA/WFA measurement results for the healthy subject group and the prostate cancer group.
  • a cancer detection method and a cancer detection method capable of specifically detecting prostate cancer and/or colorectal cancer with high sensitivity using a new biomarker as an index, and to these methods It becomes possible to provide a kit for use.

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Abstract

La présente invention concerne : un procédé de détection d'au moins un type de cancer choisi dans le groupe constitué par le cancer de la prostate et le cancer du côlon, le procédé comprenant une étape de mesure pour mesurer la quantité de CA19-9 attachée à GalNAc dans un échantillon ; et un procédé d'examen du cancer de la prostate, le procédé comprenant une étape de mesure pour mesurer la quantité de CEA sialylé α2-3 dans un échantillon.
PCT/JP2022/009047 2021-03-03 2022-03-03 Procédé de détection du cancer, procédé d'examen du cancer et kit l'utilisant WO2022186317A1 (fr)

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