WO2022186317A1 - Cancer detection method, cancer examination method, and kit using same - Google Patents

Cancer detection method, cancer examination method, and kit using same 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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Japanese (ja)
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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 JP2021033694A external-priority patent/JP2022134528A/en
Priority claimed from JP2021033693A external-priority patent/JP2022134527A/en
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    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
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    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C—CHEMISTRY; METALLURGY
    • 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
    • C07K14/42—Lectins, e.g. concanavalin, phytohaemagglutinin
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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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
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—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

The present invention provides: a method for detecting at least one type of cancer selected from the group consisting of prostate cancer and colon cancer, wherein the method includes a measurement step for measuring the quantity of GalNAc-attached CA19-9 in a sample; and a method for examining prostate cancer, wherein the method includes a measurement step for measuring the quantity of α2-3 sialylated CEA in a sample.

Description

がん検出方法、がん検査方法、及びこれらに用いるキットCancer detection method, cancer test method, and kit used therefor
　本発明は、がん検出方法、がん検査方法、及びこれらに用いるキットに関し、より詳しくは、前立腺がん及び大腸がんからなる群から選択される少なくとも１種のがんを検出する方法、及び前記がんを検査する方法、並びに、これらの方法に用いるキットに関する。 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.
　腫瘍マーカー等のバイオマーカーは、悪性腫瘍等の疾患の検出や、治療方針決定の指針、治療効果判定のためのモニタリングマーカーとして有用であり、近年盛んに研究されている。このようなバイオマーカーとしては、例えば、ＣＡ１９－９、ＣＥＡ（癌胎児性抗原：Ｃａｒｃｉｎｏｅｍｂｒｙｏｎｉｃ　Ａｎｔｉｇｅｎ）が挙げられる。ＣＡ１９－９は、１９７９年にＫｏｐｒｏｗｓｋｉらによってヒト大腸がん培養細胞株ＳＷ－１１１６を免疫原として得られたモノクローナル抗体（ＮＳ１９－９）に認識されるＩ型糖鎖抗原であり、その抗原決定部位は、ルイス式血液型抗原のうちのルイスＡ抗原（Ｌｅａ）にシアル酸が付加したシアリルルイスＡである。ＣＡ１９－９は、血中ではムチン型糖タンパク質として存在しており（非特許文献１等）、健常者（がんに罹患していない者の意、以下同じ）においても血中に検出されるが、消化器がん、特に大腸がん、膵がん、胆管がん、胆嚢がん等のがん患者においてはその濃度が上昇することが知られている（非特許文献１）。 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). However, it is known that 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).
　また、ＣＥＡは、１９６５年にＧｏｌｄ　Ｐ．とＦｒｅｅｄｍａｎ　Ｓ．Ｏ．とによってヒト大腸癌組織抽出液から見出された、分子量約２０万の糖タンパク質であり、ＣＤ６６ｅ又はＣＥＡＣＡＭ５としても知られている。ＣＥＡのタンパク質部分は約７００個のアミノ酸よりなる１本鎖のポリペプチドであり、１分子のＣＥＡには２８箇所のＮ型糖鎖修飾部位があり、その大部分に複合型糖鎖が結合して５０％以上の糖鎖を含むことが報告されている（非特許文献２）。ＣＥＡは、健常者においても血中に検出されるが、消化器がん、特に大腸がん、膵がん、胃がん等のがん患者においてはその濃度が上昇することが知られており、また、肺がん、子宮がん、卵巣がん、胎児性癌がん等でも血中の濃度が高値を示す例が報告されている。 Also, in 1965, 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.
　そのため、ＣＡ１９－９やＣＥＡは、これらのがんの検出や、治療方針決定の指針、治療効果判定のためのモニタリングマーカーとして従来から用いられており、例えば、固相化した抗ＣＡ１９－９抗体や抗ＣＥＡ抗体（捕捉体）で試料中に存在するＣＡ１９－９やＣＥＡを捕捉し、そこに標識化した抗ＣＡ１９－９抗体や抗ＣＥＡ抗体（標識体）を結合させるサンドイッチ法で試料中のＣＡ１９－９やＣＥＡを測定するためのキットとして、「ルミパルスプレスト（登録商標）ＣＡ１９－９」や「ルミパルスプレスト（登録商標）ＣＥＡ」が富士レビオ株式会社によって製造販売されている。 Therefore, 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 As 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.
　また、前立腺がんの検出や、治療方針決定の指針、治療効果判定には、例えば、従来から血中のＰＳＡ（前立腺特異抗原：Ｐｒｏｓｔａｔｅ　Ｓｐｅｃｉｆｉｃ　Ａｎｔｉｇｅｎ）量が指標として用いられている。さらに、前立腺がん患者のＰＳＡにおいては、健常者に比べてＧａｌＮＡｃ（Ｎ－アセチルガラクトサミン）が有意に増加することが報告されている（非特許文献３等）。また、例えば、特許文献１には、試料中の遊離型ＰＳＡ量と糖鎖の末端シアル酸残基が糖鎖の末端から２番目のガラクトース残基にα２、３結合した糖鎖（α２－３シアル酸）を有するＰＳＡ量との比率から前立腺がんを判定する方法が記載されている。しかしながら、ＰＳＡ量は前立腺がん患者のみならず前立腺肥大患者においても上昇することから、低濃度帯にカットオフ値を設定した場合には、前立腺がんの的中率が低くなる傾向にあることが課題となっている。また、ホルモン治療等によってＰＳＡ量が上昇しなくなった前立腺がん患者においては、これをモニタリングマーカーとして採用することができないという課題もあった。 In addition, for example, the amount of PSA (Prostate Specific Antigen) in blood has conventionally been used as an indicator for the detection of prostate cancer, the guideline for determining treatment policy, and the determination of therapeutic effects. Furthermore, it has been reported that GalNAc (N-acetylgalactosamine) is significantly increased in PSA of prostate cancer patients compared to healthy subjects (Non-Patent Document 3, etc.). In addition, for example, Patent Document 1 discloses the amount of free PSA in a sample and a sugar chain (α2-3 A method for determining prostate cancer from the ratio of the amount of PSA containing sialic acid) is described. However, since the amount of PSA increases not only in patients with prostate cancer but also in patients with prostatic hyperplasia, when the cut-off value is set in the low concentration range, the prostatic cancer hit rate tends to be low. is an issue. In addition, there is also the problem that it cannot be used as a monitoring marker in prostate cancer patients whose PSA level has stopped increasing due to hormone therapy or the like.
国際公開第２０１９／２２１２７９号WO2019/221279
　健常者の血中に検出されるＣＡ１９－９は、約２０万～１００万Ｄａの低～中分子のムチンの一部として存在するのに対して、がん患者の血中に検出されるＣＡ１９－９は、約５００万～１０００万Ｄａの高分子のムチンの一部として存在することが報告されており、ＣＡ１９－９以外の型の糖鎖についても、がん検出のための新たなバイオマーカーとしての可能性が期待されている。 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.
　また、上記のように、ＣＥＡにおける糖鎖の含有率は５０％にも達するものの、その糖鎖構造は未だ十分に明らかになっておらず、これらの糖鎖には、がん検出のための新たなバイオマーカーとしての可能性が期待される。 In addition, as described above, although the sugar chain content in CEA reaches 50%, its sugar chain structure has not yet been fully elucidated. Potential as a new biomarker is expected.
　さらに、上記のようにＣＡ１９－９やＣＥＡは健常者の血中においても少量は検出されるため、従来のＣＡ１９－９やＣＥＡを測定する方法では、通常カットオフ値を設定し、それを指標に上記のがん等の陰性及び陽性の予測又は判別を行う。しかしながら、かかる従来のＣＡ１９－９やＣＥＡを測定する方法では、シアリルルイスＡに特異的な抗ＣＡ１９－９抗体の１種のみや、抗ＣＥＡ抗体の１種のみを用いるため、がん種ごとの特異性が未だ十分ではない。さらに、前立腺がん患者において、ＣＥＡの測定値が前立腺がんの罹患を原因として上昇することは稀である。したがって、従来のＣＡ１９－９やＣＥＡを測定する方法では、前立腺がん患者と健常者又は前立腺肥大患者と、或いは、大腸がん患者と健常者と、をより高精度で判別するためには未だ不十分であることを本発明者らは見出した。 Furthermore, as described above, 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. However, in such 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. Furthermore, in prostate cancer patients, 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.
　本発明は上記課題に鑑みてなされたものであり、新たなバイオマーカーを指標とした、高精度で前立腺がん及び／又は大腸がんを特異的に検出可能ながん検出方法及びがん検査方法、並びに、これらの方法に用いるキットを提供することを目的とする。 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.
　本発明者らは、既存抗体では測定できなかった新たな腫瘍マーカーとしての糖鎖の探索を目的として、抗ＣＡ１９－９抗体や抗ＣＥＡ抗体に加えて、水溶性高分子からなる水溶性担体と前記水溶性担体に固定された標識物質及びレクチンとを備える複合体（ブロック化標識レクチン）も用いてスクリーニングを行った。その結果、レクチンとしてＷＦＡを用いた場合に検出される分子の量、すなわち、レクチン結合性のＧａｌＮＡｃ（Ｎ－アセチルガラクトサミン）が付加したＣＡ１９－９（ＧａｌＮＡｃ付加ＣＡ１９－９）の量が、前立腺がん患者及び大腸がん患者で有意に増加することを見出した。また、レクチンとしてＭＡＭを用いた場合に検出される分子の量、すなわち、レクチン結合性糖鎖であるα２－３シアル酸が付加したＣＥＡ（α２－３シアル酸付加ＣＥＡ）の量が、健常者に比べて、前立腺がん患者で有意に増加することも見出した。そのため、ＧａｌＮＡｃ付加ＣＡ１９－９量を測定することにより前立腺がん及び大腸がんを特異的に検出し、又は、α２－３シアル酸付加ＣＥＡ量を測定することにより前立腺がんを特異的に検出し、かつ、前立腺がん患者と健常者又は前立腺肥大患者と、或いは、大腸がん患者と健常者とを、ＣＡ１９－９のみを測定した場合や抗ＣＥＡ抗体のみでＣＥＡを測定した場合よりもさらに高精度で判別することが可能となることを見出し、本発明を完成するに至った。 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. As a result, 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. In addition, the amount of molecules detected when MAM is used as a lectin, that is, the amount of CEA with α2-3 sialylated lectin-binding sugar chain (α2-3 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 α2-3 sialylated CEA. And, 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.
　かかる知見により得られた本発明の態様は次のとおりである。
［１］
　前立腺がん及び大腸がんからなる群から選択される少なくとも１種のがんを検出する方法であり、試料中のＧａｌＮＡｃ付加ＣＡ１９－９量を測定する測定工程を含む、方法。
［２］
　前立腺がん及び大腸がんからなる群から選択される少なくとも１種のがんを検査する方法であり、被検者由来の試料中のＧａｌＮＡｃ付加ＣＡ１９－９量を測定する測定工程を含む、方法。
［３］
　前立腺がん及び大腸がんからなる群から選択される少なくとも１種のがんであると予測される被検者をスクリ－ニングする方法であり、被検者由来の試料中のＧａｌＮＡｃ付加ＣＡ１９－９量を測定する測定工程と、測定されたＧａｌＮＡｃ付加ＣＡ１９－９量を指標として被検者を選別する選別工程と、を含む、［２］に記載の方法。
［４］
　ＧａｌＮＡｃ付加ＣＡ１９－９において、ＧａｌＮＡｃがＷＦＡに結合するＷＦＡ結合型糖鎖である、［１］～［３］のうちのいずれか一項に記載の方法。
［５］
　前記測定工程が、前記試料と、ＣＡ１９－９に特異的に結合可能な第１のプローブ分子及びＧａｌＮＡｃに特異的に結合可能な第２のプローブ分子と、を接触させる工程である、［１］～［４］のうちのいずれか一項に記載の方法。
［６］
　第１のプローブ分子が、ＣＡ１９－９に特異的に結合可能な抗体である、［５］に記載の方法。
［７］
　第２のプローブ分子が、ＧａｌＮＡｃに特異的に結合可能なレクチンである、［５］又は［６］に記載の方法。
［８］
　前記測定工程が、前記試料と、捕捉体及び標識体と、を接触させる工程であり、
　前記捕捉体が、非水溶性担体と、前記非水溶性担体に固定された第１のプローブ分子及び第２のプローブ分子のうちのいずれか一方と、を備えるものであり、かつ、
　前記標識体が、標識物質と、第１のプローブ分子及び第２のプローブ分子のうちの他方と、を備えるものである、
［５］～［７］のうちのいずれか一項に記載の方法。
［９］
　前記捕捉体が、非水溶性担体と、前記非水溶性担体に固定された第１のプローブ分子と、を備えるものであり、かつ、
　前記標識体が、水溶性担体と、前記水溶性担体に固定された標識物質及び第２のプローブ分子と、を備え、第２のプローブ分子がＧａｌＮＡｃに特異的に結合可能なレクチンであるブロック化標識レクチンである、
［８］に記載の方法。
［１０］
　［５］～［９］のうちのいずれか一項に記載の方法に用いるためのキットであり、ＣＡ１９－９に特異的に結合可能な第１のプローブ分子と、ＧａｌＮＡｃに特異的に結合可能な第２のプローブ分子と、を備える、キット。
［１１］
　第１のプローブ分子が、ＣＡ１９－９に特異的に結合可能な抗体である、［１０］に記載のキット。
［１２］
　第２のプローブ分子が、ＧａｌＮＡｃに特異的に結合可能なレクチンである、［１０］又は［１１］に記載のキット。
［１３］
　非水溶性担体と、前記非水溶性担体に固定された第１のプローブ分子及び第２のプローブ分子のうちのいずれか一方と、を備える捕捉体、並びに、
　標識物質と、第１のプローブ分子及び第２のプローブ分子のうちの他方と、を備える標識体、
を備える、［１０］～［１２］のうちのいずれか一項に記載のキット。
［１４］
　前記捕捉体が、非水溶性担体と、前記非水溶性担体に固定された第１のプローブ分子と、を備えるものであり、かつ、
　前記標識体が、水溶性担体と、前記水溶性担体に固定された標識物質及び第２のプローブ分子と、を備え、第２のプローブ分子がＧａｌＮＡｃに特異的に結合可能なレクチンであるブロック化標識レクチンである、
［１３］に記載のキット。
［１５］
　前立腺がんを検出する方法であり、試料中のα２－３シアル酸付加ＣＥＡ量を測定する測定工程を含む、方法。
［１６］
　前立腺がんを検査する方法であり、被検者由来の試料中のα２－３シアル酸付加ＣＥＡ量を測定する測定工程を含む、方法。
［１７］
　前立腺がんであると予測される被検者をスクリ－ニングする方法であり、被検者由来の試料中のα２－３シアル酸付加ＣＥＡ量を測定する測定工程と、測定されたα２－３シアル酸付加ＣＥＡ量を指標として被検者を選別する選別工程と、を含む、［１５］に記載の方法。
［１８］
　α２－３シアル酸付加ＣＥＡにおいて、α２－３シアル酸がＭＡＭに結合するＭＡＭ結合型糖鎖である、［１５］～［１７］のうちのいずれか一項に記載の方法。
［１９］
　前記測定工程が、前記試料と、ＣＥＡに特異的に結合可能な第１のプローブ分子及びα２－３シアル酸に特異的に結合可能な第２のプローブ分子と、を接触させる工程である、［１５］～［１８］のうちのいずれか一項に記載の方法。
［２０］
　第１のプローブ分子が、ＣＥＡに特異的に結合可能な抗体である、［１９］に記載の方法。
［２１］
　第２のプローブ分子が、α２－３シアル酸に特異的に結合可能なレクチンである、［１９］又は［２０］に記載の方法。
［２２］
　前記測定工程が、前記試料と、捕捉体及び標識体と、を接触させる工程であり、
　前記捕捉体が、非水溶性担体と、前記非水溶性担体に固定された第１のプローブ分子及び第２のプローブ分子のうちのいずれか一方と、を備えるものであり、かつ、
　前記標識体が、標識物質と、第１のプローブ分子及び第２のプローブ分子のうちの他方と、を備えるものである、
［１９］～［２１］のうちのいずれか一項に記載の方法。
［２３］
　前記捕捉体が、非水溶性担体と、前記非水溶性担体に固定された第１のプローブ分子と、を備えるものであり、かつ、
　前記標識体が、水溶性担体と、前記水溶性担体に固定された標識物質及び第２のプローブ分子と、を備え、第２のプローブ分子がα２－３シアル酸に特異的に結合可能なレクチンであるブロック化標識レクチンである、
［２２］に記載の方法。
［２４］
　［１９］～［２３］のうちのいずれか一項に記載の方法に用いるためのキットであり、ＣＥＡに特異的に結合可能な第１のプローブ分子と、α２－３シアル酸に特異的に結合可能な第２のプローブ分子と、を備える、キット。
［２５］
　第１のプローブ分子が、ＣＥＡに特異的に結合可能な抗体である、［２４］に記載のキット。
［２６］
　第２のプローブ分子が、α２－３シアル酸に特異的に結合可能なレクチンである、［２４］又は［２５］に記載のキット。
［２７］
　非水溶性担体と、前記非水溶性担体に固定された第１のプローブ分子及び第２のプローブ分子のうちのいずれか一方と、を備える捕捉体、並びに、
　標識物質と、第１のプローブ分子及び第２のプローブ分子のうちの他方と、を備える標識体、
を備える、［２４］～［２６］のうちのいずれか一項に記載のキット。
［２８］
　前記捕捉体が、非水溶性担体と、前記非水溶性担体に固定された第１のプローブ分子と、を備えるものであり、かつ、
　前記標識体が、水溶性担体と、前記水溶性担体に固定された標識物質及び第２のプローブ分子と、を備え、第２のプローブ分子がα２－３シアル酸に特異的に結合可能なレクチンであるブロック化標識レクチンである、
［２７］に記載のキット。 The aspects of the present invention obtained from such findings are as follows.
[1]
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.
[2]
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.
[4]
The method according to any one of [1] to [3], wherein GalNAc in GalNAc-added CA19-9 is a WFA-linked sugar chain that binds to WFA.
[5]
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].
[6]
The method of [5], wherein the first probe molecule is an antibody capable of specifically binding to CA19-9.
[7]
The method of [5] or [6], wherein the second probe molecule is a lectin capable of specifically binding to GalNAc.
[8]
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;
[5] The method according to any one of [7].
[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 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.
[12]
The kit of [10] or [11], wherein the second probe molecule is a lectin capable of specifically binding to GalNAc.
[13]
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 kit according to any one of [10] to [12].
[14]
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].
[15]
A method of detecting prostate cancer, comprising the step of measuring the amount of α2-3 sialylated CEA in a sample.
[16]
A method of testing for prostate cancer, comprising the step of measuring the amount of α2-3 sialylated CEA in a sample derived from a subject.
[17]
A method for screening a subject predicted to have prostate cancer, comprising a measuring step of measuring the amount of α2-3 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.
[18]
The method according to any one of [15] to [17], wherein in α2-3 sialylated CEA, α2-3 sialic acid is a MAM-linked sugar chain that binds to MAM.
[19]
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 α2-3 sialic acid. 15] The method according to any one of [18].
[20]
The method of [19], wherein the first probe molecule is an antibody capable of specifically binding to CEA.
[21]
The method of [19] or [20], wherein the second probe molecule is a lectin capable of specifically binding to α2-3 sialic acid.
[22]
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;
[19] The method according to any one of [21].
[23]
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 α2-3 sialic acid. is a blocked labeled lectin that is
The method according to [22].
[24]
A kit for use in the method according to any one of [19] to [23], comprising: a first probe molecule capable of specifically binding to CEA; and a bindable second probe molecule.
[25]
The kit of [24], wherein the first probe molecule is an antibody capable of specifically binding to CEA.
[26]
The kit of [24] or [25], wherein the second probe molecule is a lectin capable of specifically binding to α2-3 sialic acid.
[27]
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 kit of any one of [24]-[26], comprising:
[28]
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 α2-3 sialic acid. is a blocked labeled lectin that is
The kit of [27].
　本発明によれば、新たなバイオマーカーを指標とした、高精度で前立腺がん及び／又は大腸がんを特異的に検出可能ながん検出方法及びがん検査方法、並びに、これらの方法に用いるキットを提供することが可能となる。 According to the present invention, 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.
健常人群及び前立腺癌群のＣＡ１９－９の測定結果を示すグラフである。1 is a graph showing measurement results of CA19-9 in a healthy subject group and a prostate cancer group. 健常人群及び前立腺癌群のＣＡ１９－９／ＷＦＡの測定結果を示すグラフである。2 is a graph showing measurement results of CA19-9/WFA in a healthy subject group and a prostate cancer group. 前立腺肥大群及び前立腺癌群のＣＡ１９－９の測定結果を示すグラフである。FIG. 10 is a graph showing measurement results of CA19-9 in a prostatic hyperplasia group and a prostatic cancer group. FIG. 前立腺肥大群及び前立腺癌群のＣＡ１９－９／ＷＦＡの測定結果を示すグラフである。FIG. 10 is a graph showing the measurement results of CA19-9/WFA in a prostatic hyperplasia group and a prostatic cancer group. FIG. ＣＡ１９－９の測定値とＣＡ１９－９／ＷＦＡの測定値との関係を示すグラフである。1 is a graph showing the relationship between measured values of CA19-9 and measured values of CA19-9/WFA. ＰＳＡの測定値とＣＡ１９－９／ＷＦＡの測定値との関係を示すグラフである。1 is a graph showing the relationship between PSA measurements and CA19-9/WFA measurements. 健常人群及び大腸がん群のＣＡ１９－９の測定結果を示すグラフである。1 is a graph showing measurement results of CA19-9 in a healthy subject group and a colorectal cancer group. 健常人群及び大腸がん群のＣＡ１９－９／ＷＦＡの測定結果を示すグラフである。2 is a graph showing measurement results of CA19-9/WFA in a healthy subject group and a colorectal cancer group. ＣＡ１９－９の測定値とＣＡ１９－９／ＷＦＡの測定値との関係を示すグラフである。1 is a graph showing the relationship between measured values of CA19-9 and measured values of CA19-9/WFA. 健常人群及び乳癌群のＣＡ１９－９／ＷＦＡの測定結果を示すグラフである。1 is a graph showing measurement results of CA19-9/WFA in a healthy subject group and a breast cancer group. 健常人群及び前立腺癌群のＣＡ１９－９／ＭＡＭの測定結果を示すグラフである。2 is a graph showing measurement results of CA19-9/MAM in a healthy subject group and a prostate cancer group. 健常人群及び大腸がん群のＣＡ１９－９／ＭＡＭの測定結果を示すグラフである。2 is a graph showing measurement results of CA19-9/MAM in a healthy subject group and a colorectal cancer group. 健常人群及び前立腺癌群のＣＥＡの測定結果を示すグラフである。2 is a graph showing CEA measurement results for a healthy subject group and a prostate cancer group. 健常人群及び前立腺癌群のＣＥＡ／ＭＡＭの測定結果を示すグラフである。2 is a graph showing measurement results of CEA/MAM in a healthy subject group and a prostate cancer group. 前立腺肥大群及び前立腺癌群のＣＥＡの測定結果を示すグラフである。FIG. 10 is a graph showing the measurement results of CEA in the prostatic hyperplasia group and the prostatic cancer group. FIG. 前立腺肥大群及び前立腺癌群のＣＥＡ／ＭＡＭの測定結果を示すグラフである。FIG. 10 is a graph showing the measurement results of CEA/MAM in the prostatic hyperplasia group and the prostatic cancer group; FIG. ＣＥＡの測定値とＣＥＡ／ＭＡＭの測定値との関係を示すグラフである。4 is a graph showing the relationship between measured values of CEA and measured values of CEA/MAM; ＰＳＡの測定値とＣＥＡ／ＭＡＭの測定値との関係を示すグラフである。4 is a graph showing the relationship between PSA measurements and CEA/MAM measurements. 健常人群及び前立腺癌群のＣＥＡ／ＷＦＡの測定結果を示すグラフである。4 is a graph showing measurement results of CEA/WFA in a healthy subject group and a prostate cancer group.
　以下、本発明をその好適な実施形態に即して詳細に説明する。 The present invention will be described in detail below in accordance with its preferred embodiments.
　＜がん検出方法、がん検査方法＞
　本発明の第１のがん検出方法は、前立腺がん及び大腸がんからなる群から選択される少なくとも１種のがんを検出する方法であり、試料中のＧａｌＮＡｃ付加ＣＡ１９－９量を測定する測定工程を含む方法である。また、本発明の第１のがん検査方法は、前立腺がん及び大腸がんからなる群から選択される少なくとも１種のがんを検査する方法であり、被検者由来の試料中のＧａｌＮＡｃ付加ＣＡ１９－９量を測定する測定工程を含む方法である（以下、場合により第１のがん検出方法及び第１のがん検査方法を総称して「第１の方法」という）。 <Cancer detection method, cancer examination method>
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 In addition, 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).
　本発明の第２のがん検出方法は、前立腺がんを検出する方法であり、試料中のα２－３シアル酸付加ＣＥＡ量を測定する測定工程を含む方法である。また、本発明の第２のがん検査方法は、前立腺がんを検査する方法であり、被検者由来の試料中のα２－３シアル酸付加ＣＥＡ量を測定する測定工程を含む方法である（以下、場合により第２のがん検出方法及び第２のがん検査方法を総称して「第２の方法」という、さらに、場合により、第１の方法及び第２の方法を総称して「本発明の方法」という）。 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 α2-3 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 α2-3 sialylated CEA in a sample derived from a subject. (Hereinafter, 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").
　［がん］
　本発明において、「がん」には、上皮性の悪性腫瘍（癌）及び非上皮性の悪性腫瘍（肉腫）が含まれる。本発明の第１の方法において検出又は検査の対象となるがんは、前立腺がん及び大腸がんからなる群から選択される少なくとも１種のがんである。また、本発明の第２の方法において検出又は検査の対象となるがんは、前立腺がんである。本発明に係る前立腺がんとは、前立腺に発生するがんを示し、例えば、前立腺癌、前立腺小細胞癌、前立腺導管癌、前立腺肉腫、前立腺扁平上皮癌、前立腺腺扁平上皮癌、前立腺基底細胞癌、前立腺粘液癌、前立腺印環細胞癌が挙げられる。これらの中でも、本発明に係る前立腺がんとしては、前立腺癌が好ましい。また、本発明に係る大腸がんとは、大腸（特に結腸又は直腸）に発生するがんを示し、例えば、当該領域における、腺癌、内分泌細胞癌、腺扁平上皮癌、扁平上皮癌、カルチノイド腫瘍、非上皮性悪性腫瘍、リンパ腫が挙げられる。これらの中でも、本発明に係る大腸がんとしては、大腸腺癌が好ましい。 [cancer]
In the present 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. Among these, prostate cancer is preferable as the prostate cancer according to the present invention. In addition, 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付加ＣＡ１９－９］
　本発明において、「ＧａｌＮＡｃ付加ＣＡ１９－９」とは、ＣＡ１９－９と、ＣＡ１９－９に結合して付加されたＧａｌＮＡｃ（Ｎ－アセチルガラクトサミン）と、を含む分子のことをいう。 [GalNAc-added CA19-9]
In the present invention, "GalNAc-attached CA19-9" refers to a molecule comprising CA19-9 and GalNAc (N-acetylgalactosamine) attached to CA19-9.
　本発明において、「ＣＡ１９－９」とは、ヒト大腸癌培養細胞株ＳＷ－１１１６を免疫原として得られたモノクローナル抗体（ＮＳ１９－９）により認識されるＩ型糖鎖抗原のことを示す。ＣＡ１９－９は、下記の抗ＣＡ１９－９抗体に対する抗原決定部位として、ルイス式血液型抗原のうちのルイスＡ抗原（Ｌｅａ）にシアル酸が付加したシアリルルイスＡを有する（Ｋｏｐｒｏｗｓｋｉら、Ｓｏｍａｔ．Ｃｅｌｌ　Ｇｅｎｅｔ．，５，９５７，１９７９）。 In the present invention, “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－アセチルガラクトサミン）」は、ＩＵＰＡＣ名「２－（アセチルアミノ）－２－デオキシ－Ｄ－ガラクトース」で示される単糖である。本発明に係るＧａｌＮＡｃ付加ＣＡ１９－９としては、ＧａｌＮＡｃを、ＷＦＡに結合するＷＦＡ結合型糖鎖として含んでいることが好ましく、末端ＧａｌＮＡｃ残基として含んでいることが好ましい。 "GalNAc (N-acetylgalactosamine)" is a monosaccharide indicated by the IUPAC name "2-(acetylamino)-2-deoxy-D-galactose". GalNAc-added CA19-9 according to the present invention preferably contains GalNAc as a WFA-linked sugar chain that binds to WFA, preferably as a terminal GalNAc residue.
　１分子のＧａｌＮＡｃ付加ＣＡ１９－９において、ＣＡ１９－９及びＧａｌＮＡｃはそれぞれ複数存在していてよく、ＣＡ１９－９とＧａｌＮＡｃとは直接結合していても、コアタンパク質や他の糖鎖を介して結合していてもよい。前記コアタンパク質としては、例えば、ムチン、アポリポプロテイン、キニノーゲン、ＡＲＣＶＦが挙げられる。この場合のＧａｌＮＡｃ付加ＣＡ１９－９の平均質量（ゲル濾過クロマトグラフィー（ＧＦＣ）によるマーカーでの検量による平均質量、以下同じ）としては、特に限定されないが、２５，０００～１０，０００，０００Ｄａであることが好ましく、１，０００，０００～１０，０００，０００Ｄａであることがより好ましい。 In one molecule of GalNAc-added CA19-9, 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. may be Examples of the core protein include mucin, apolipoprotein, kininogen, and ARCVF. In this case, the average mass of GalNAc-added CA19-9 (average mass by calibration with a gel filtration chromatography (GFC) marker, hereinafter the same) is not particularly limited, but is 25,000 to 10,000,000 Da. more preferably 1,000,000 to 10,000,000 Da.
　また、ＣＡ１９－９とＧａｌＮＡｃとは、エクソソームのような細胞外小胞やウイルスのように、前記コアタンパク質よりもさらに巨大な粒子上に存在する形態であってもよい。この場合のＧａｌＮＡｃ付加ＣＡ１９－９（前記粒子を含む）の粒子径としては、特に限定されないが、５０～５００ｎｍ程度である。 In addition, 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.
　［α２－３シアル酸付加ＣＥＡ］
　本発明において、「α２－３シアル酸付加ＣＥＡ」とは、糖鎖であるα２－３シアル酸（α２，３結合型シアル酸）が結合して付加されており、これを含むＣＥＡ分子のことをいい、「α２－３シアル酸含有ＣＥＡ」ともいう。本発明において、「ＣＥＡ」とは、糖タンパク質である癌胎児性抗原（Ｃａｒｃｉｎｏｅｍｂｒｙｏｎｉｃ　Ａｎｔｉｇｅｎ）のことを示す。ＣＥＡは、細胞接着、増殖、分化、腫瘍抑制等の様々なプロセスに関与するＣＥＡＣＡＭ（癌胎児性抗原関連接着分子）ファミリーを構成する分子の一つであり、「ＣＤ６６ｅ」又は「ＣＥＡＣＡＭ５（ｃａｒｃｉｎｏｅｍｂｒｙｏｎｉｃ　ａｎｔｉｇｅｎ　ｒｅｌａｔｅｄ　ｃｅｌｌ　ａｄｈｅｓｉｏｎ　ｍｏｌｅｃｕｌｅ　５）」としても知られるが、本明細書中では、ＣＤ６６ｅ又はＣＥＡＣＡＭ５とＣＥＡとを同義で用いる。ＣＥＡは、典型的には、約７００個のアミノ酸よりなる１本鎖のポリペプチドであるタンパク質部分と、それに結合した複数の糖鎖からなる糖鎖部分とからなる。 [α2-3 sialylated CEA]
In the present invention, “α2-3 sialylated CEA” means a CEA molecule containing a sugar chain α2-3 sialic acid (α2,3-linked sialic acid) attached thereto. is also referred to as “α2-3 sialic acid-containing CEA”. In the present invention, "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.
　本発明に係るα２－３シアル酸付加ＣＥＡは、前記糖鎖部分として少なくともα２－３シアル酸を含む。本発明において、「α２－３シアル酸」とは、単糖であるガラクトース（Ｇａｌ）の３位炭素に、シアル酸の２位炭素がグリコシド結合した糖鎖（シアル酸α２－３Ｇａｌ）を示す。１分子のα２－３シアル酸付加ＣＥＡにおいて、α２－３シアル酸は複数存在していてよく、ＣＥＡのタンパク質部分とα２－３シアル酸とは直接結合していても、他の糖鎖を介して結合していてもよい。本発明に係るα２－３シアル酸付加ＣＥＡとしては、α２－３シアル酸を、ＭＡＭに結合するＭＡＭ結合型糖鎖として含んでいることが好ましく、末端シアル酸残基が糖鎖の末端から２番目のガラクトース残基にα２、３結合した糖鎖（シアル酸α２－３Ｇａｌ）として含んでいることがより好ましく、ＣＥＡのタンパク質部分のアミド部分にＮ－アセチルグルコサミン（ＧｌｃＮＡｃ）がβ結合したＮ型糖鎖修飾部位の末端α２－３シアル酸（シアル酸α２－３Ｇａｌβ１－４ＧｌｃＮＡｃ）として含んでいることがより好ましい。 The α2-3 sialylated CEA according to the present invention contains at least α2-3 sialic acid as the sugar chain moiety. In the present invention, “α2-3 sialic acid” refers to a sugar chain (α2-3Gal 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. In one molecule of α2-3 sialylated CEA, a plurality of α2-3 sialic acids may be present. may be combined with The α2-3 sialylated CEA according to the present invention preferably contains α2-3 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 (α2-3Gal sialic acid) linked to the second galactose residue as an α2,3-linked sugar chain (α2-3Gal sialic acid). It is more preferably contained as a terminal α2-3 sialic acid (α2-3Galβ1-4GlcNAc sialic acid) of the glycosylation site.
　このようなα２－３シアル酸付加ＣＥＡの平均質量（ゲル濾過クロマトグラフィー（ＧＦＣ）によるマーカーでの検量による平均質量、以下同じ）としては、特に限定されないが、１００，０００～３００，０００Ｄａであることが好ましく、１８０，０００～２００，０００Ｄａであることがより好ましい。また、１分子のα２－３シアル酸付加ＣＥＡにおける前記糖鎖部分の含有量としては、特に限定されないが、４０～６０質量％であることが好ましい。 The average mass of such α2-3 sialylated CEA (average mass calibrated with a gel filtration chromatography (GFC) marker, hereinafter the same) 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 α2-3 sialylated CEA is not particularly limited, but is preferably 40 to 60% by mass.
　［被検者］
　本発明において、「被検者」とは、本発明のがん検査方法を行う対象を示し、好ましくは人である。本発明に係る被検者としては、スクリーニング等を目的とした、健常者であっても、前立腺がん又は大腸がんに罹患しているが自覚症状のない者であってもよい。また、予後の決定、治療方針の決定、治療効果の確認、再発の確認等を目的とした、既に前記がんに罹患していることが既知の患者や過去に前記がんに罹患した患者であってもよい。また、本発明において、「健常者」とは、検査の対象となるがん（すなわち、第１の方法の場合には前立腺がん及び／又は大腸がん、第２の方法の場合には前立腺がん）に罹患していない者を示す。なお、被検者が真に前立腺がん及び／又は大腸がんに罹患していることは、被検者から採取された前立腺組織及び／又は大腸組織の生検によって決定（確定診断）される。 [Subject]
In the present invention, 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. In addition, for the purpose of determining prognosis, determining treatment policy, confirming therapeutic effect, confirming recurrence, etc. There may be. In the present invention, 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. .
　［試料］
　本発明の第１の方法に用いられる「試料」としては、ＧａｌＮＡｃ付加ＣＡ１９－９が存在し得る試料である限り特に制限はない。また、本発明の第２の方法に用いられる「試料」としては、α２－３シアル酸付加ＣＥＡが存在し得る試料である限り特に制限はない。 [sample]
The "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. Moreover, the "sample" used in the second method of the present invention is not particularly limited as long as it is a sample in which α2-3 sialylated CEA can be present.
　前記試料としては、一般的には、前記被検者等、がん検査の対象から採取された血清、血漿、及び全血等の血液検体；尿、痰、唾液、汗、髄液、消化液、精液、リンパ液、腹水等の血液以外の体液検体；口腔粘膜、咽頭粘膜、腸管粘膜等の粘膜検体；並びに各種生検検体等が挙げられる。また、前記試料としては、培養細胞や細胞培養液であってもよい。本発明に係る試料としては血液検体が好ましく、血清（「血清検体」ともいう）がより好ましい。 Examples of the 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. Moreover, 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").
　これらの試料は、必要に応じて希釈液で希釈又は懸濁したものであっても、適宜前処理が施されたものであってもよい。前記希釈液としては、例えば、リン酸緩衝液、Ｔｒｉｓ緩衝液、グッド緩衝液、ホウ酸緩衝液、酢酸緩衝液、クエン酸緩衝液、グリシン緩衝液コハク酸緩衝液、フタル酸緩衝液等の緩衝液が挙げられる。前記前処理としては、粉砕、凍結、加熱、濃縮、分画、脱塩等の処理；ｐＨ調整剤、安定化剤、保存剤、防腐剤、界面活性剤等の添加処理；精製処理等が挙げられ、これらのうちの１種を単独であっても２種以上の組み合わせであってもよい。前記精製処理としては、特に制限されないが、例えば、カラムによる処理；非水溶性担体に固定された試料中の夾雑物に吸着する抗体等の物質により夾雑物を吸着してＧａｌＮＡｃ付加ＣＡ１９－９を含む試料（第１の方法の場合）中やα２－３シアル酸付加ＣＥＡを含む試料（第２の方法の場合）中から除去する処理；非水溶性担体に固定された抗ＣＡ１９－９抗体によってＧａｌＮＡｃ付加ＣＡ１９－９を含むＣＡ１９－９を捕捉して夾雑物を洗浄等により除去してから遊離させる処理（第１の方法の場合）や非水溶性担体に固定された抗ＣＥＡ抗体によってα２－３シアル酸付加ＣＥＡを含むＣＥＡを捕捉して夾雑物を洗浄等により除去してから遊離させる処理（第２の方法の場合）等が挙げられる。 These samples may be diluted or suspended with a diluent as necessary, or may be pretreated as appropriate. Examples of the 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. Examples of the 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 α2-3 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 α2- 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 (in the case of the second method) may be mentioned.
　［測定工程］
　本発明の第１の方法は、試料中のＧａｌＮＡｃ付加ＣＡ１９－９量を測定する測定工程を含む。また、本発明の第２の方法は、試料中のα２－３シアル酸付加ＣＥＡ量を測定する測定工程を含む。ＧａｌＮＡｃ付加ＣＡ１９－９量の測定方法及びα２－３シアル酸付加ＣＥＡ量の測定方法としては、それぞれ、ＧａｌＮＡｃ付加ＣＡ１９－９量を測定することができる方法及びα２－３シアル酸付加ＣＥＡ量を測定することができる方法である限り特に制限されず、従来公知の方法やそれに準じた方法、又はそれらの組み合わせを適宜採用することができる。例えば、ＧａｌＮＡｃ付加ＣＡ１９－９に特異的に結合可能なプローブ分子（第１の方法の場合）やα２－３シアル酸付加ＣＥＡに特異的に結合可能なプローブ分子（第２の方法の場合）を用いる方法；高速液体クロマトグラフィー同位体希釈質量分析法（ＬＣ－ＩＤＭＳ）；誘導結合プラズマ発光分析法（ＩＣＰ－ＯＥＳ）；誘導結合プラズマ質量分析法（ＩＣＰ－ＭＳ）；原子吸光分光法（Ａｔｏｍｉｃ　Ａｂｓｏｒｐｔｉｏｎ　Ｓｐｅｃｔｒｏｍｅｔｒｙ、ＡＡＳ）；ＨＰＬＣ；ＦＰＬＣ；ＮＭＲ；ＩＲ；ＦＴＩＲ；ＵＶ－ＶＩＳ吸光光度計測法；フローサイトメトリ；質量分析法；及びこれらを組み合わせた方法が挙げられるが、これらに限定されるものではない。 [Measurement process]
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 α2-3 sialylated CEA in the sample. The method for measuring the amount of GalNAc-added CA19-9 and the method for measuring the amount of α2-3 sialylated CEA include a method capable of measuring the amount of GalNAc-added CA19-9 and measuring the amount of α2-3 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. For example, 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 α2-3 sialylated CEA (in the second method) is used. Methods used; high performance liquid chromatography isotope dilution mass spectrometry (LC-IDMS); inductively coupled plasma optical emission spectrometry (ICP-OES); inductively coupled plasma mass spectrometry (ICP-MS); atomic absorption spectroscopy (Atomic Absorption) HPLC; FPLC; NMR; IR; FTIR; UV-VIS spectrophotometry; flow cytometry; .
　中でも、本発明に係る測定工程としては、ＧａｌＮＡｃ付加ＣＡ１９－９に特異的に結合可能なプローブ分子を用いる方法（第１の方法の場合）やα２－３シアル酸付加ＣＥＡに特異的に結合可能なプローブ分子を用いる方法（第２の方法の場合）であることが好ましい。このようなプローブ分子としては、例えば、抗体；プロテインＡ、プロテインＧ、プロテインＬ等の結合タンパク質；アビジンＤ、ストレプトアビジン等のアビジン類；レクチン；ガレクチン、糖鎖受容体、免疫受容体等が挙げられる。なお、本発明において、「抗体」には、完全な抗体の他、抗体断片（例えば、Ｆａｂ、Ｆａｂ’、Ｆ（ａｂ’）２、Ｆｖ、単鎖抗体、ダイアボディー等）や抗体の可変領域を結合させた低分子化抗体も含まれる。 Among them, 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 α2-3 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. In the present invention, the term "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
　第１の方法において、ＧａｌＮＡｃ付加ＣＡ１９－９に特異的に結合可能なプローブ分子としては、ＣＡ１９－９に特異的に結合可能なプローブ分子とＧａｌＮＡｃに特異的に結合可能なプローブ分子との組み合わせであることが好ましい。また、第２の方法において、α２－３シアル酸付加ＣＥＡに特異的に結合可能なプローブ分子としては、ＣＥＡに特異的に結合可能なプローブ分子とα２－３シアル酸に特異的に結合可能なプローブ分子との組み合わせであることが好ましい。以下、場合により、ＣＡ１９－９に特異的に結合可能なプローブ分子及びＣＥＡに特異的に結合可能なプローブ分子を「第１のプローブ分子」といい、ＧａｌＮＡｃに特異的に結合可能なプローブ分子及びα２－３シアル酸に特異的に結合可能なプローブ分子を「第２のプローブ分子」という。 In the first method, 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. Preferably. In the second method, the probe molecule capable of specifically binding to α2-3 sialylated CEA includes a probe molecule capable of specifically binding to CEA and a probe molecule capable of specifically binding to α2-3 sialic acid. A combination with a probe molecule is preferred. Hereinafter, in some cases, 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 α2-3 sialic acid is referred to as a "second probe molecule".
　〔第１のプローブ分子〕
　ＣＡ１９－９に特異的に結合可能な第１のプローブ分子としては、例えば、ＣＡ１９－９に特異的に結合可能な抗体（本明細書中、場合により「抗ＣＡ１９－９抗体」という）、ＣＡ１９－９が結合するタンパク質（前記コアタンパク質等）に特異的に結合可能な抗体が挙げられ、中でも、抗ＣＡ１９－９抗体が好ましい。 [First probe molecule]
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.
　本発明において、「抗ＣＡ１９－９抗体」とは、ＣＡ１９－９のシアリルルイスＡを特異的に認識し、結合することができる抗体を示す。このような抗ＣＡ１９－９抗体としては、ＣＡ１９－９への結合能を有する限り特に制限はなく、ポリクローナル抗体であってもモノクローナル抗体であってもよいが、均質性や安定性の観点からはモノクローナル抗体であることが好ましい。抗ＣＡ１９－９抗体は、従来公知の産生方法を適宜採用、改良することによって産生することができ、また、一般に流通されているものを適宜用いてもよい。 In the present invention, "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.
　本発明において、「ＣＥＡに特異的に結合可能」とは、特に断りの無い場合、ＣＥＡのうち、α２－３シアル酸（より好ましくは、シアル酸α２－３Ｇａｌβ１－４ＧｌｃＮＡｃ糖鎖）以外の部位に対して特異的に結合可能なことを示す。「α２－３シアル酸以外の部位」としては、タンパク質部分の全体又はその一部であっても、α２－３シアル酸以外の糖鎖部分の全体又は一部であっても、これらのうちの２種以上の組み合わせであってもよい。ＣＥＡに特異的に結合可能な第１のプローブ分子としては、例えば、ＣＥＡに特異的に結合可能な抗体（ＣＥＡのタンパク質部分を認識部位とする抗タンパク質抗体、ＣＥＡの糖鎖部分を認識部位とする抗糖鎖抗体、及びＣＥＡのタンパク質部分と糖鎖部分とを認識部位とする抗体を含む。本明細書中、場合により「抗ＣＥＡ抗体」という）、抗ＣＥＡＣＡＭファミリー抗体（ＣＥＡと他のＣＥＡＣＡＭファミリー分子とを認識部位とする抗体）が挙げられ、中でも、抗ＣＥＡ抗体が好ましく、ＣＥＡのタンパク質部分の少なくとも一部を認識部位とする抗ＣＥＡタンパク質抗体であることがより好ましく、また、α２－３シアル酸と第２のプローブ分子との結合に干渉しない抗体であることが好ましい。 In the present invention, unless otherwise specified, the term "capable of binding specifically to CEA" refers to sites other than α2-3 sialic acid (more preferably, sialic acid α2-3Galβ1-4GlcNAc sugar chain) in CEA. It shows that it can specifically bind to The “site other than α2-3 sialic acid” may be the whole or part of the protein moiety, or the whole or part of the sugar chain moiety other than α2-3 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.Herein, sometimes referred to as "anti-CEA antibody"), anti-CEACAM family antibody (CEA and other CEACAM Antibodies whose recognition sites are family molecules), among which anti-CEA antibodies are preferred, and anti-CEA protein antibodies whose recognition sites are at least part of the CEA protein portion are more preferred, and α2- Antibodies that do not interfere with the binding of trisialic acid to the second probe molecule are preferred.
　前記抗ＣＥＡ抗体としては、ＣＥＡへの結合能を有する限り特に制限はなく、ポリクローナル抗体であってもモノクローナル抗体であってもよいが、均質性や安定性の観点からはモノクローナル抗体であることが好ましい。抗ＣＥＡ抗体は、従来公知の産生方法を適宜採用、改良することによって産生することができ、また、一般に流通されているものを適宜用いてもよい。 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.
　本発明に係る抗ＣＡ１９－９抗体及び抗ＣＥＡ抗体としては、それぞれ、例えば、第２のプローブ分子として下記のレクチンを用いる場合に、当該レクチンが抗体糖鎖も認識して検出感度が低下することを抑制するために、酸化処理、グリコシダーゼ処理、プロテアーゼ処理等によって、レクチン結合性の糖鎖が除去若しくは破壊されたもの；抗体分子中の糖鎖付加アミノ酸を含む領域が除去されたもの；又は、抗体遺伝子を発現させた遺伝子組み換え大腸菌や細胞を用いたり抗体産生細胞の培養における栄養条件を制限したりすることよって糖鎖付加が起こらない条件で産生された抗体であることが好ましい。 For the anti-CA19-9 antibody and anti-CEA antibody according to the present invention, 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. In order to suppress 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.
　〔第２のプローブ分子〕
　ＧａｌＮＡｃに特異的に結合可能な第２のプローブ分子としては、例えば、ＧａｌＮＡｃに特異的に結合可能な抗体（本明細書中、場合により「抗ＧａｌＮＡｃ抗体」という）、ＧａｌＮＡｃに特異的に結合可能なレクチン（例えば、ノダフジレクチン（ＷＦＡ）、ダイズレクチン（ＳＢＡ）、ナヨクサフジレクチン（ＶＶＡ）、ヒマラヤフジマメレクチン（ＤＢＡ）、ムラサキソシンカレクチン（ＢＰＬ）、リンゴマイマイレクチン（ＨＰＡ））、ＧａｌＮＡｃに特異的に結合可能なガレクチンが挙げられ、中でも、ＧａｌＮＡｃに特異的に結合可能なレクチンが好ましく、ノダフジレクチン（ＷＦＡ）が特に好ましい。なお、ノダフジレクチン（ＷＦＡ：Ｗｉｓｔｅｒｉａ　ｆｌｏｒｉｂｕｎｄａ　ａｇｇｌｕｔｉｎｉｎ）は、ＧａｌＮＡｃの糖鎖構造を認識して結合活性を示すタンパク質であり、植物であるノダフジに由来するマメ科レクチンである。前記レクチンとしては、糖鎖認識活性の特異性の増大等を目的として、変異が導入された改変レクチンとしたものや人工的に合成されたものであってもよい。また、一般に流通されているものを適宜用いてもよい。 [Second probe molecule]
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. Wisteria floribunda agglutinin (WFA) 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|circulating.
　α２－３シアル酸に特異的に結合可能な第２のプローブ分子としては、例えば、α２－３シアル酸に特異的に結合可能な抗体（本明細書中、場合により「抗α２－３シアル酸抗体」という）、Ｌｅｃｔｅｎｚ（登録標章）のようなα２－３シアル酸結合型人工タンパク質、α２－３シアル酸に特異的に結合可能なレクチン（例えば、イヌエンジュレクチン（ＭＡＭ／ＭＡＬ／ＭＡＡ－ＩＩ）、イヌエンジュレクチン（ＭＡＨ／ＭＡＡ－Ｉ）等のイヌエンジュレクチン；ヤナギマツタケレクチン（ＡＣＧ）；ジャックフルーツレクチン（Ｊａｃａｌｉｎ））が挙げられ、中でも、α２－３シアル酸に特異的に結合可能なレクチンが好ましく、イヌエンジュレクチンがより好ましく、ＭＡＭが特に好ましい。なお、ＭＡＭ（Ｍａａｃｋｉａ　ａｍｕｒｅｎｓｉｓ　Ｌｅｃｔｉｎ；ＭＡＬ又はＭＡＡ－ＩＩともいう）は、α２－３シアル酸の糖鎖構造（特に、シアル酸α２－３Ｇａｌβ１－４ＧｌｃＮＡｃ）を認識して結合活性を示すタンパク質であり、植物であるイヌエンジュの種子に由来するマメ科レクチンである。前記レクチンとしては、糖鎖認識活性の特異性の増大等を目的として、変異が導入された改変レクチンとしたものや人工的に合成されたものであってもよい。また、一般に流通されているものを適宜用いてもよい。 Examples of the second probe molecule capable of specifically binding to α2-3 sialic acid include, for example, an antibody capable of specifically binding to α2-3 sialic acid α2-3 sialic acid-binding artificial proteins such as Lectenz (registered trademark), lectins capable of specifically binding to α2-3 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)). Preferred, more preferred is canine endulectin, and particularly preferred is MAM. MAM (Maackia amurensis lectin; also referred to as MAL or MAA-II) is a protein that recognizes the sugar chain structure of α2-3 sialic acid (especially sialic acid α2-3Galβ1-4GlcNAc) 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|circulating.
　第１のプローブ分子及び第２のプローブ分子を用いる場合、前記測定工程では、前記試料と、第１のプローブ分子及び第２のプローブ分子と、を接触させる。これにより、ＣＡ１９－９とＧａｌＮＡｃとがいずれも認識され、両者を含むＧａｌＮＡｃ付加ＣＡ１９－９を検出して測定することができる（第１の方法の場合）。又は、α２－３シアル酸とＣＥＡのα２－３シアル酸以外の部分とがいずれも認識され、両者を含むα２－３シアル酸付加ＣＥＡを検出して測定することができる（第２の方法の場合）。前記試料と第１のプローブ分子との接触及び前記試料と第２のプローブ分子との接触は、互いに同時であってもよいし、別時であってもよいし、別時の場合にはいずれの接触が先であってもよい。 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. As a result, 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). Alternatively, both α2-3 sialic acid and portions of CEA other than α2-3 sialic acid are recognized, and α2-3 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.
　〔標識物質〕
　第１の方法の場合、ＧａｌＮＡｃ付加ＣＡ１９－９量の測定は、ＧａｌＮＡｃ付加ＣＡ１９－９に特異的に結合可能なプローブ分子（好ましくは、第１のプローブ分子及び／又は第２のプローブ分子）に付加した、又はこれらの分子を認識する分子（例えば、二次抗体やプロテインＡ）に付加した標識物質によって生じるシグナルを検出することによって行うことが好ましい。又は、第２の方法の場合、α２－３シアル酸付加ＣＥＡ量の測定は、α２－３シアル酸付加ＣＥＡに特異的に結合可能なプローブ分子（好ましくは、第１のプローブ分子及び／又は第２のプローブ分子）に付加した、又はこれらの分子を認識する分子（例えば、二次抗体やプロテインＡ）に付加した標識物質によって生じるシグナルを検出することによって行うことが好ましい。検出したシグナルの量を測定し、必要に応じてこれを半定量又は定量することによって、ＧａｌＮＡｃ付加ＣＡ１９－９量又はα２－３シアル酸付加ＣＥＡ量を測定することができる。前記「シグナル」には、呈色（発色）、消光、反射光、発光、蛍光、放射性同位体による放射線等が含まれ、肉眼で確認できるものの他、シグナルの種類に応じた測定方法・装置によって確認できるものも含まれる。本発明において、測定されるＧａｌＮＡｃ付加ＣＡ１９－９量及びα２－３シアル酸付加ＣＥＡ量としては、それぞれ、標準試料等を用いて半定量又は定量してもよいが、より簡便かつ迅速な観点からは、前記シグナル量をそのまま本発明のＧａｌＮＡｃ付加ＣＡ１９－９量又はα２－３シアル酸付加ＣＥＡ量としてよい。 [Labeling substance]
In the case of the first method, 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. Alternatively, in the case of the second method, the amount of α2-3 sialylated CEA is measured using a probe molecule capable of specifically binding to α2-3 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 α2-3 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. In the present invention, the amounts of GalNAc-added CA19-9 and α2-3 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 α2-3 sialylated CEA of the present invention.
　本発明に係る標識物質としては、公知の免疫学的測定方法やそれに準じた方法において標識物質として用いられているものを特に制限なく用いることができる。例えば、酵素；アクリジニウム誘導体等の発光物質；ユーロピウム等の蛍光物質；アロフィコシアニン（ＡＰＣ）及びフィコエリスリン（Ｒ－ＰＥ）等の蛍光蛋白質；１２５Ｉ等の放射性物質；フルオレセインイソチオシアネート（ＦＩＴＣ）及びローダミンイソチオシアネート（ＲＩＴＣ）等の低分子量標識物質；金粒子；アビジン；ビオチン；ラテックス；ジニトロフェニル（ＤＮＰ）；ジゴキシゲニン（ＤＩＧ）が挙げられ、これらのうちの１種を単独であっても２種以上の組み合わせであってもよい。例えば、前記標識物質として酵素を用いた場合には、発色基質、蛍光基質、化学発光基質等を基質として添加することにより、当該基質に応じて種々の測定を行うことができる。前記酵素としては、例えば、西洋ワサビペルオキシダーゼ（ＨＲＰ）、アルカリホスファターゼ（ＡＬＰ）、β－ガラクトシダーゼ（β－ｇａｌ）、グルコースオキシダーゼ、ルシフェラーゼを挙げることができるが、これらに限定されるものではない。 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. For example, 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. For example, when an enzyme is used as the labeling substance, various measurements can be performed by adding a chromogenic substrate, a fluorescent substrate, a chemiluminescent substrate, or the like as the substrate. Examples of the enzyme include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (ALP), β-galactosidase (β-gal), glucose oxidase, and luciferase.
　〔サンドイッチ法〕
　第１のプローブ分子及び第２のプローブ分子を用いる測定方法としては、サンドイッチ法、競合法、及び免疫比濁法等の免疫学的測定方法や、これらの原理に準じた測定方法が挙げられ、特に制限されない。かかる測定方法では、例えば、一般的に、ＥＬＩＳＡ、デジタルＥＬＩＳＡ、ＣＬＥＩＡ（化学発光酵素免疫測定法）、ＣＬＩＡ（化学発光免疫測定法）、ＥＣＬＩＡ（電気化学免疫測定法）、ＲＩＡ（放射免疫測定法）等の、マイクロプレートや粒子等を担体とする方法；イムノクロマト；表面プラズモン共鳴分析法；蛍光共鳴エネルギー移動による検出方法等を採用することができる。 [Sandwich method]
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;
　本発明に係る測定方法としては、より感度及び特異度の高い測定システムを構築可能な傾向にある観点からは、サンドイッチ法が好ましい。以下、本発明に係る測定工程について、サンドイッチ法を例に挙げてより具体的な態様を説明する。 As the measurement method according to the present invention, the sandwich method is preferable from the viewpoint of the possibility of constructing a measurement system with higher sensitivity and specificity. Hereinafter, a more specific aspect of the measuring process according to the present invention will be described by taking the sandwich method as an example.
　前記サンドイッチ法を用いる場合の態様としては、
　前記測定工程が、前記試料と、捕捉体及び標識体と、を接触させる工程であり、
　前記捕捉体が、非水溶性担体と、前記非水溶性担体に固定された第１のプローブ分子及び第２のプローブ分子のうちのいずれか一方と、を備えるものであり、かつ、
　前記標識体が、標識物質と、第１のプローブ分子及び第２のプローブ分子のうちの他方と、を備える、
態様（以下、場合により「第１の態様」という）が挙げられる。第１の態様において、第１のプローブ分子及び第２のプローブ分子は、それぞれ、前記捕捉体及び前記標識体のうちのいずれに備えられていてもよいが、一方が捕捉体に含まれ、もう一方が標識体に含まれる。これによりＧａｌＮＡｃ及びＣＡ１９－９をいずれも捕捉することで、ＧａｌＮＡｃ付加ＣＡ１９－９を高精度かつ簡便に捕捉して検出することができる（第１の方法の場合）。又は、これによりα２－３シアル酸及びＣＥＡをいずれも捕捉することで、α２－３シアル酸付加ＣＥＡを高精度かつ簡便に捕捉して検出することができる（第２の方法の場合）。 As an aspect when using the sandwich method,
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;
Aspect (hereinafter sometimes referred to as "first aspect") can be mentioned. In the first aspect, 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. By capturing both GalNAc and CA19-9 in this manner, GalNAc-added CA19-9 can be captured and detected with high accuracy and ease (in the case of the first method). Alternatively, by capturing both α2-3 sialic acid and CEA in this way, α2-3 sialylated CEA can be easily captured and detected with high accuracy (in the case of the second method).
　前記サンドイッチ法の他の態様としては、上記に限られず、例えば、前記標識体が、第１の標識物質と、第１のプローブ分子及び第２のプローブ分子のうちのいずれか一方と、を備える第１の標識体、並びに、第２の標識物質と、第１のプローブ分子及び第２のプローブ分子のうちの他方と、を備える第２の標識体であり、かつ、前記捕捉体が、非水溶性担体と、前記非水溶性担体に固定されたプローブ分子と、を備える捕捉体である態様（以下、場合により「第２の態様」という）としてもよい。第２の態様において、第１の標識物質と第２の標識物質とは互いに異なるシグナルを生じるものである。また、この場合の前記捕捉体に備えられるプローブ分子としては、第１の方法の場合、ＧａｌＮＡｃ付加ＣＡ１９－９に特異的に結合可能なプローブ分子（第１のプローブ分子及び第２のプローブ分子を含む）；第１のプローブ分子及び／又は第２のプローブ分子に特異的に結合可能なプローブ分子が挙げられ、第２の方法の場合、α２－３シアル酸付加ＣＥＡに特異的に結合可能なプローブ分子（第１のプローブ分子及び第２のプローブ分子を含む）；第１のプローブ分子及び／又は第２のプローブ分子に特異的に結合可能なプローブ分子が挙げられる。 Other aspects of the sandwich method are not limited to those described above. For example, 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. In the second aspect, the first labeling substance and the second labeling substance produce signals different from each other. In the first method, the probe molecules provided in the capture body in this case 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 α2-3 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.
　このようなサンドイッチ法としては、２ステップ法であるフォワードサンドイッチ法（捕捉体と試料中のＧａｌＮＡｃ付加ＣＡ１９－９との反応、捕捉体に結合したＧａｌＮＡｃ付加ＣＡ１９－９と標識体との反応（第１の方法の場合）、又は捕捉体と試料中のα２－３シアル酸付加ＣＥＡとの反応、捕捉体に結合したα２－３シアル酸付加ＣＥＡと標識体との反応（第２の方法の場合）を逐次的に行う方法）、リバースサンドイッチ法（予め標識体と試料中のＧａｌＮＡｃ付加ＣＡ１９－９又はα２－３シアル酸付加ＣＥＡとを反応させ、生成した複合体を捕捉体と反応させる方法）、及び１ステップ法（試料中のＧａｌＮＡｃ付加ＣＡ１９－９又はα２－３シアル酸付加ＣＥＡ、捕捉体、標識体の反応を同時に１ステップで行う方法）を挙げることができるが、これらのいずれも採用可能である。 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 α2-3 sialylated CEA in the sample, or the reaction between the α2-3 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 α2-3 sialylated CEA in the sample, and reacting the resulting complex with the capturing agent). , and a one-step method (a method in which the reaction of GalNAc-added CA19-9 or α2-3 sialylated CEA in the sample, the capturing agent, and the labeling agent are simultaneously performed in one step), but any of these methods can be used. It is possible.
　例えば、フォワードサンドイッチ法では、先ず、前記試料と前記捕捉体とを接触させ、前記捕捉体のプローブ分子とＧａｌＮＡｃ付加ＣＡ１９－９との結合（例えば、第１のプローブ分子とＣＡ１９－９との結合）を介してＧａｌＮＡｃ付加ＣＡ１９－９を当該捕捉体に捕捉させる（第１の方法の場合）。又は、前記試料と前記捕捉体とを接触させ、前記捕捉体のプローブ分子とα２－３シアル酸付加ＣＥＡとの結合（例えば、第１のプローブ分子とＣＥＡとの結合）を介してα２－３シアル酸付加ＣＥＡを当該捕捉体に捕捉させる（第２の方法の場合）（１次反応：捕捉ステップ）。次いで、前記捕捉体に捕捉されたＧａｌＮＡｃ付加ＣＡ１９－９に前記標識体を接触させて、前記標識体のプローブ分子とＧａｌＮＡｃ付加ＣＡ１９－９との結合（例えば、第２のプローブ分子とＧａｌＮＡｃとの結合）を介して標識する（第１の方法の場合）。又は、前記捕捉体に捕捉されたα２－３シアル酸付加ＣＥＡに前記標識体を接触させて、前記標識体のプローブ分子とα２－３シアル酸付加ＣＥＡとの結合（例えば、第２のプローブ分子とα２－３シアル酸との結合）を介して標識する（第２の方法の場合）（２次反応：標識ステップ）。これらの反応により、捕捉体－ＧａｌＮＡｃ付加ＣＡ１９－９－標識体を含む複合体が形成される（第１の方法の場合）。又は、捕捉体－α２－３シアル酸付加ＣＥＡ－標識体を含む複合体が形成される（第２の方法の場合）。結合しなかった試料及び標識体を必要に応じて洗浄して除去（洗浄ステップ）した後、前記標識物質に応じた所定の方法で、当該標識物質に由来するシグナルを測定する（測定ステップ）。 For example, in the forward sandwich method, first, 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). Alternatively, the sample is brought into contact with the capture body, and α2-3 binding occurs via binding between the probe molecule of the capture body and α2-3 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). Next, 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). Alternatively, the label is brought into contact with α2-3 sialylated CEA captured by the capturer, and binding of the probe molecule of the label to α2-3 sialylated CEA (for example, binding of the second probe molecule and α2-3 sialic acid) (in the case of the second method) (secondary reaction: labeling step). These reactions form a complex containing the captor-GalNAc-attached CA19-9-label (for the first method). Alternatively, a complex containing capture entity-α2-3 sialylated CEA-label is formed (in the case of the second method). After removing unbound sample and label by washing as necessary (washing step), a signal derived from the labeling substance is measured by a predetermined method according to the labeling substance (measurement step).
　（捕捉体）
　本発明に係る「捕捉体」は、第１の方法の場合、非水溶性担体と、前記非水溶性担体に固定されたＧａｌＮＡｃ付加ＣＡ１９－９に特異的に結合可能なプローブ分子と、を備える複合体であり；又は第２の方法の場合、前記非水溶性担体に固定されたα２－３シアル酸付加ＣＥＡに特異的に結合可能なプローブ分子と、を備える複合体であり、それぞれ、前記非水溶性担体と前記プローブ分子とが直接的又は間接的に結合した結合体である。第１の態様に係る捕捉体に備えられる前記プローブ分子としては、第１のプローブ分子及び第２のプローブ分子のうちのいずれか一方であることが好ましく、この場合、当該捕捉体に備えられるプローブ分子としては、第１のプローブ分子及び第２のプローブ分子のうちのいずれであってもよいが、第２のプローブ分子としてレクチンを用いる場合には、より捕捉性が高い観点からは、前記捕捉体に備えられるプローブ分子としては、第１の方法の場合、ＣＡ１９－９に特異的に結合可能な第１のプローブ分子であることが好ましく、抗ＣＡ１９－９抗体であることがより好ましく、第２の方法の場合、ＣＥＡに特異的に結合可能な第１のプローブ分子であることが好ましく、抗ＣＥＡ抗体であることがより好ましい。 (trapping body)
In the first method, the "capture body" according to the present invention 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 α2-3 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. In this case, 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.
　〈非水溶性担体〉
　前記捕捉体に含まれる非水溶性担体は、主に前記プローブ分子を担持し、固相化する担体として機能するものであり、非水溶性の物質である。本発明において、「非水溶性の物質」とは、常温常圧下において水に不溶（水に対する溶解度が０．００１ｇ／ｍＬ以下、好ましくは０．０００１ｇ／ｍＬ以下、以下同様）である物質を示す。 <Water-insoluble carrier>
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. In the present invention, the term "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). .
　このような非水溶性担体の材質としては、一般的に免疫学的測定及びそれに準じた測定に用いられているものを用いることができ、特に制限はされず、例えば、高分子ポリマー（ポリスチレン、（メタ）アクリル酸エステル、ポリメチルメタクリレート、ポリイミド、ナイロン等）、ゼラチン、ガラス、ラテックス、シリカ、金属（金、白金等）、及び金属化合物（酸化鉄、酸化コバルト、ニッケルフェライト等）からなる群から選択される少なくとも１種が挙げられる。また、前記非水溶性担体の材質としては、これらの複合材や、これら物質と他の物質との複合材であってもよく、例えば、前記高分子ポリマー、ゼラチン、及びラテックスからなる群から少なくとも１種の有機高分子と、酸化鉄（スピネルフェライト等）、酸化コバルト、及びニッケルフェライトからなる群から少なくとも１種の金属化合物と、からなる有機無機複合材であってもよい。さらに、前記非水溶性担体としては、カルボキシ基、エポキシ基、トシル基、アミノ基、ヒドロキシ基、イソチオシアネート基、イソシアネート基、アジド基、アルデヒド基、カーボネート基、アリル基、アミノオキシ基、マレイミド基、チオール基等の活性基で表面修飾されたものであってもよい。 As 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 In addition, 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. It may be an organic-inorganic composite material comprising one kind of organic polymer and at least one kind of metal compound selected from the group consisting of iron oxide (such as spinel ferrite), cobalt oxide, and nickel ferrite. Further, 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.
　また、本発明において、前記非水溶性担体の形状としても特に制限はされず、例えば、プレート、繊維、膜、粒子等のいずれであってもよいが、反応効率の観点からは、粒子であることが好ましく、自動化・短時間化の観点からは、磁性粒子であることがより好ましい。このような非水溶性担体としては、従来公知のものを適宜用いることができ、市販のものを適宜用いることもできる。 In the present invention, 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. As such a water-insoluble carrier, conventionally known ones can be used as appropriate, and commercially available ones can also be used as appropriate.
　〈捕捉体の構成及び製造方法〉
　前記捕捉体において、前記プローブ分子の含有量としては特に制限されず、該プローブ分子とＧａｌＮＡｃ付加ＣＡ１９－９又はα２－３シアル酸付加ＣＥＡとの結合のしやすさ等に応じて適宜調整することができるが、例えば、前記非水溶性担体（好ましくは粒子）の質量（非水溶性担体が２種以上の組み合わせである場合にはそれらの合計）１００質量部に対するプローブ分子の質量（プローブ分子が２種以上の組み合わせである場合にはそれらの合計）が、０．１～１０質量部であることが好ましく、１～５質量部であることがより好ましい。 <Structure and manufacturing method of capturing body>
In the capturing body, 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 α2-3 sialylated CEA. However, for example, the mass of the probe molecule (the probe molecule is In the case of a combination of two or more, the total thereof) is preferably 0.1 to 10 parts by mass, more preferably 1 to 5 parts by mass.
　前記捕捉体は、前記非水溶性担体に前記プローブ分子を固定することによって製造することができる。かかる製造方法としては、適宜従来公知の方法又はそれに準じた方法を採用することができ、前記非水溶性担体に前記プローブ分子を直接固定してもよく、間接的に固定してもよい。 The capture body can be produced by immobilizing the probe molecule on the water-insoluble carrier. As such a production method, 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.
　直接固定する場合には、例えば、前記非水溶性担体及び／又は前記プローブ分子として、カルボキシ基、エポキシ基、トシル基、アミノ基、ヒドロキシ基、イソチオシアネート基、イソシアネート基、アジド基、アルデヒド基、カーボネート基、アリル基、アミノオキシ基、マレイミド基、チオール基等の活性基を有するものを用い、又は必要に応じて前記活性基を付与して、これらを結合させることにより、前記プローブ分子を前記非水溶性担体に直接固定することができる。 In the case of direct immobilization, for example, 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, By using those having 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.
　間接的に固定する場合には、例えば、前記プローブ分子に結合するリンカーを前記非水溶性担体に固定し、該リンカーに前記プローブ分子を結合させることにより、前記プローブ分子を前記非水溶性担体に間接的に固定することができる。前記リンカーとしては、特に制限されず、例えば、プローブ分子に結合可能な二次抗体、プロテインＧ、プロテインＡ、光分解可能な光切断型リンカー、前記活性基を有するリンカー分子（例えば、ヒドラジン塩、ヒドラジド）等が挙げられる。また、前記プローブ分子に何らかの修飾を行い、その修飾部分を捕捉する物質を前記非水溶性担体に固定化して、前記非水溶性担体上にプローブ分子を固定してもよい。例えば、前記修飾部分の代表例としてはビオチンが、その修飾部分を捕捉する物質の代表例としてはストレプトアビジンが、それぞれ挙げられるが、これらに限定されるものではない。 In the case of indirect immobilization, for example, 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. Can be fixed indirectly. The linker is not particularly limited. hydrazide) and the like. Alternatively, 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. For example, 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.
　これらの反応に供する非水溶性担体及びプローブ分子の比率は、上記の捕捉体における比率の好ましい範囲を達成するように適宜選択することができる。また、必要に応じて、前記プローブ分子や非水溶性担体への非特異的な吸着を防ぐことを目的として、適当なブロッキング剤（例えば、牛血清アルブミンやゼラチン等）で前記非水溶性担体のブロッキングを行ってもよい。さらに、このような捕捉体としては、市販のものを適宜用いてもよい。 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. In addition, if necessary, for the purpose of preventing non-specific adsorption to the probe molecule or water-insoluble carrier, a suitable blocking agent (eg, bovine serum albumin, gelatin, etc.) may be added to the water-insoluble carrier. Blocking may be performed. Furthermore, as such a trapping body, a commercially available one may be used as appropriate.
　（標識体）
　本発明に係る「標識体」は、第１の方法の場合、標識物質と、ＧａｌＮＡｃ付加ＣＡ１９－９に特異的に結合可能なプローブ分子と、を備える複合体であり；又は第２の方法の場合、標識物質と、α２－３シアル酸付加ＣＥＡに特異的に結合可能なプローブ分子と、を備える複合体であり、それぞれ、前記標識物質と前記プローブ分子とが直接的又は間接的に結合した結合体である。第１の態様に係る標識体に備えられる前記プローブ分子としては、第１のプローブ分子及び第２のプローブ分子のうち、捕捉体に備えられる分子の他方の分子であることが好ましく、この場合、当該標識体に備えられるプローブ分子としては、第１のプローブ分子及び第２のプローブ分子のうちのいずれであってもよいが、第１の方法の場合、抗原に対する親和性の高い抗ＣＡ１９－９抗体のような分子を第１のプローブ分子として捕捉体に備えて使用するという観点からは、ＧａｌＮＡｃに特異的に結合可能な第２のプローブ分子であることが好ましく、ＧａｌＮＡｃに特異的に結合可能なレクチンであることがより好ましく、ＷＦＡであることが特に好ましい。また、第２の方法の場合、抗原に対する親和性の高い抗ＣＥＡ抗体のような分子を第１のプローブ分子として捕捉体に備えて使用するという観点からは、α２－３シアル酸に特異的に結合可能な第２のプローブ分子であることが好ましく、α２－３シアル酸に特異的に結合可能なレクチンであることがより好ましく、ＭＡＭであることが特に好ましい。 (Label)
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 α2-3 sialylated CEA, wherein the labeling substance and the probe molecule are respectively bound directly or indirectly It is a conjugate. Among the first probe molecule and the second probe molecule, 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. In this case, The probe molecule provided in the label may be either the first probe molecule or the second probe molecule. From the viewpoint of using a molecule such as an antibody as the first probe molecule in the capture body, it is preferably a second probe molecule capable of specifically binding to GalNAc. lectins are more preferred, and WFA is particularly preferred. In the case of the second method, from the viewpoint of using a molecule such as an anti-CEA antibody that has a high affinity for the antigen as the first probe molecule, it is necessary to prepare the capturing body for the specific α2-3 sialic acid. It is preferably a second probe molecule capable of binding, more preferably a lectin capable of specifically binding to α2-3 sialic acid, and particularly preferably MAM.
　（ブロック化標識レクチン）
　本発明において、前記標識体としては、前記標識物質と前記抗体（抗ＣＡ１９－９抗体、抗ＧａｌＮＡｃ抗体（第１の方法の場合）；抗ＣＥＡ抗体、抗α２－３シアル酸抗体（第２の方法の場合）等）とを備える標識化抗体、前記標識物質とレクチンとを備える標識化レクチン、ブロック化標識レクチンが挙げられ、これらのうちの１種のみであっても２種以上の組み合わせであってもよい。これらの中でも、本発明、特に第１の態様における標識体としては、ブロック化標識レクチンであることが好ましい。本発明において、「ブロック化標識レクチン」とは、水溶性高分子からなる水溶性担体と、前記水溶性担体に固定された標識物質及びレクチンとを備える複合体であって、水溶性担体と標識物質とレクチンとが直接的又は間接的に結合した結合体である。本発明において、前記ブロック化標識レクチンでは、第１の方法の場合、第２のプローブ分子としてＧａｌＮＡｃに特異的に結合可能なレクチンを備え、第２の方法の場合、第２のプローブ分子としてα２－３シアル酸に特異的に結合可能なレクチンを備える。この場合、前記捕捉体に備えられるプローブ分子は第１のプローブ分子であることが好ましい。前記レクチンとしては上述したとおりであり、好ましくはＷＦＡ（第１の方法の場合）又はＭＡＭ（第２の方法の場合）である。また、前記標識物質としては、その好ましい態様も含めて、上述したとおりである。 (blocked labeled lectin)
In the present invention, 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-α2-3 sialic acid antibody (for the second method). method), etc.), a labeled lectin comprising the labeling substance and a lectin, and a blocked labeled lectin. There may be. Among these, a blocked labeled lectin is preferable as the labeled substance in the present invention, particularly in the first aspect. In the present invention, 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. In the present invention, the blocked labeled lectin comprises a lectin capable of specifically binding to GalNAc as the second probe molecule in the first method, and α2 as the second probe molecule in the second method. A lectin capable of specifically binding to -3 sialic acid is provided. In this case, it is preferable that 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). In addition, the labeling substance is as described above, including its preferred embodiments.
　前記ブロック化標識レクチンにおいては、前記水溶性担体に前記標識物質及びレクチンが担持されていればよく、水溶性担体に標識物質及びレクチンが互いに独立して結合していても、水溶性担体、標識物質、及びレクチンのいずれもが他の２者と結合していても、水溶性担体に標識物質を介してレクチンが結合していても、水溶性担体にレクチンを介して標識物質が結合していてもよい。一般に、レクチンとレクチン結合性糖鎖構造との各々の結合点における親和性は弱いが、このようなブロック化標識レクチンでは、複数のレクチンが高分子である水溶性担体によって一連となった複合体を形成する。そのため、これを用いることで一つの複合体に複数の結合点が生じるため、全体としての親和性が向上し、対象であるＧａｌＮＡｃ又はα２－３シアル酸を高感度で検出可能になる。 In the blocked labeled lectin, it is sufficient that 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. may In general, 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 α2-3 sialic acid.
　〈水溶性担体〉
　前記ブロック化標識レクチンに含まれる水溶性担体は、主に前記標識物質及びレクチンを担持する担体として機能するものであり、水溶性高分子からなる。本発明に係る水溶性担体を構成する水溶性高分子（以下、「第１の水溶性高分子」という）としては、前記標識物質及びレクチンを固定させて担持できる水溶性高分子である限り特に制限はない。本発明において、「水溶性高分子」とは、常温常圧下で水に対する溶解度が０．０１ｇ／ｍＬを超える、好ましくは０．０５ｇ／ｍＬ以上である、より好ましくは０．１ｇ／ｍＬ以上である高分子化合物を示す。 <Water-soluble carrier>
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. There are no restrictions. In the present invention, the term "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.
　本発明に係る第１の水溶性高分子としては、重量平均分子量（ゲルパーミエーションクロマトグラフィー（ＧＰＣ）によるポリスチレン換算での重量平均分子量、以下同じ）が、測定の感度の観点及び水溶性であるという観点から、６，０００～４，０００，０００であることが好ましく、２０，０００～１，０００，０００であることがより好ましい。 As the first water-soluble polymer according to the present invention, the weight-average molecular weight (weight-average molecular weight in terms of polystyrene by gel permeation chromatography (GPC), hereinafter the same) 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.
　また、本発明に係る第１の水溶性高分子としては、平均質量が、より好ましい平均粒子径のブロック化標識抗体を得られる傾向にある観点から、７０，０００～１，０００，０００Ｄａであることも好ましく、１５０，０００～７００，０００Ｄａであることもより好ましい。 Further, 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.
　また、前記ブロック化標識レクチンとしては、一つのブロック化標識レクチンに、第１の水溶性高分子として、重量平均分子量が異なる複数種の水溶性高分子が含まれていてもよい。さらに、前記ブロック化標識レクチンとしては、第１の水溶性高分子の重量平均分子量が２００，０００以上である高分子量ブロック化標識レクチンと、第１の水溶性高分子の重量平均分子量が１００，０００未満（より好ましくは１００，０００以下）である低分子量ブロック化標識レクチンと、の組み合わせであることも好ましく、第１の水溶性高分子の重量平均分子量が２００，０００～７００，０００（さらに好ましくは２５０，０００～５００，０００）である高分子量ブロック化標識レクチンと、第１の水溶性高分子の重量平均分子量が２０，０００～１００，０００（さらに好ましくは５０，０００～７０，０００）である低分子量ブロック化標識レクチンと、の組み合わせであることがより好ましい。前記ブロック化標識レクチンとして、かかる高分子量ブロック化標識レクチンと前記低分子量ブロック化標識レクチンとを組み合わせる場合、これらの質量比（高分子量ブロック化標識レクチンの質量：低分子量ブロック化標識レクチンの質量）としては、１０：１～１：１０であることが好ましく、５：１～１：５であることがより好ましく、３：１～１：３であることがさらに好ましい。 In addition, as the blocked labeled lectin, 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. Furthermore, as the blocked labeled lectin, 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-weight blocked labeled lectin. When the high-molecular-weight blocked labeled lectin and the low-molecular-weight blocked labeled lectin are combined as the blocked-labeled lectin, their 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.
　本発明に係る第１の水溶性高分子としては、例えば、デキストラン、アミノデキストラン、フィコール（商品名）、デキストリン、アガロース、プルラン、各種セルロース（例えば、ヘミセルロースやリグリン等）、キチン、及びキトサン等の多糖類；β―ガラクトシダーゼ；サイログロブリン；ヘモシアニン；ポリリジン；ポリペプチド；ＤＮＡ；並びに、これらの修飾体（例えば、ジエチルアミノエチルデキストランやデキストラン硫酸ナトリウム等）が挙げられ、これらのうちの１種を単独であっても２種以上の組み合わせであってもよい。これらの中でも、本発明に係る第１の水溶性高分子としては、安価で大量に入手可能であり、また、官能基の付加、カップリング反応などの化学的な加工が比較的容易である観点からは、多糖類及びその修飾体からなる群から選択される少なくとも１種であることが好ましく、デキストラン及びアミノデキストラン、並びに、これらの修飾体からなる群から選択される少なくとも１種であることがより好ましく、デキストランであることがさらに好ましい。 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; β-galactosidase; thyroglobulin; hemocyanin; polylysine; or a combination of two or more. Among these, 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.
　〈ブロック化標識レクチンの構成及び製造方法〉
　前記ブロック化標識レクチンにおいて、前記標識物質の含有量としては特に制限されず、測定機構等に応じて適宜調整することができるが、測定感度をより向上させるために、第１の水溶性高分子１分子に結合する標識物質の分子数ができるだけ多くなるように設定することが好ましく、例えば、前記標識物質が酵素である場合、第１の水溶性高分子の質量（第１の水溶性高分子が２種以上の組み合わせである場合にはそれらの合計、以下同じ）１００質量部に対する標識物質の質量（標識物質が２種以上の組み合わせである場合にはそれらの合計）が、１００～１，０００質量部であることが好ましく、３００～８００質量部であることがより好ましい。 <Construction and production method of blocked labeled lectin>
In the blocked labeled lectin, 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. For example, when 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.
　前記ブロック化標識レクチンにおいて、レクチンの含有量としては特に制限されないが、測定感度をより向上させるために、第１の水溶性高分子１分子に結合するレクチンの分子数ができるだけ多くなるように設定することが好ましく、例えば、第１の水溶性高分子の質量１００質量部に対するレクチンの質量（レクチンが２種以上の組み合わせである場合にはそれらの合計）が、１００～２，０００質量部であることが好ましく、３００～１，５００質量部であることがより好ましい。 In the blocked labeled lectin, 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. For example, the mass of the lectin relative to 100 parts by mass of the first water-soluble polymer (when the lectins are a combination of two or more, the total thereof) is 100 to 2,000 parts by mass. preferably 300 to 1,500 parts by mass.
　また、前記ブロック化標識レクチンとしては、ブロック化標識レクチン１分子あたりの重量平均分子量が、１，０００，０００～１０，０００，０００であることが好ましく、１，５００，０００～５，０００，０００であることがより好ましい。前記重量平均分子量が１，０００，０００以上であると、測定感度がより高くなる傾向にあり、他方、１０，０００，０００以下であると、水溶液中における凝集等をより十分に抑制できる傾向にある。 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.
　前記ブロック化標識レクチンは、前記水溶性担体に前記標識物質及びレクチンを固定することによって製造することができる。かかる製造方法としては、適宜従来公知の方法又はそれに準じた方法を採用することができ、前記水溶性担体に前記標識物質及びレクチン（以下、場合により「被担持物質」と総称する）を直接固定してもよく、間接的に固定してもよい。 The blocked labeled lectin can be produced by immobilizing the labeled substance and lectin on the water-soluble carrier. As such a production method, 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.
　前記被担持物質を前記水溶性担体に直接固定する方法としては、例えば、前記被担持物質及び／又は前記水溶性担体を構成する第１の水溶性高分子に、カルボキシ基、エポキシ基、トシル基、アミノ基、ヒドロキシ基、イソチオシアネート基、イソシアネート基、アジド基、アルデヒド基、カーボネート基、アリル基、アミノオキシ基、マレイミド基、チオール基、ピリジルジスルフィド基等の活性基を付与し、或いは、前記被担持物質及び／又は水溶性担体としてこれらの活性基を有する水溶性高分子を用い、これらを結合させることによって固定する方法が挙げられる。前記活性基を付与した被担持物質及び第１の水溶性高分子としては、市販のものをそのまま用いてもよいし、適切な反応条件で被担持物質及び水溶性高分子表面に前記活性基を導入して調製してもよい。一例として、チオール基の導入は、例えば、Ｓ－アセチルメルカプト無水コハク酸や２－イミノチオラン塩酸塩等の市販の試薬を用いて行うことができる。また、前記被担持物質及び／又は前記水溶性担体を構成する第１の水溶性高分子上のアミノ基へのマレイミド基の導入は、例えば、Ｎ－（６－マレイミドカプロイルオキシ）スクシンイミドやＮ－（４－マレイミドブチリロキシ）スクシンイミド等の市販の試薬を用いて行うことができる。ピリジルジスルフィド基の導入は、例えば、Ｎ－Ｓｕｃｃｉｎｉｍｉｄｙｌ　３－（２－ｐｙｒｉｄｙｌｄｉｔｈｉｏ）ｐｒｏｐｉｏｎａｔｅ（ＳＰＤＰ）やＮ－｛６－［３－（２－Ｐｙｒｉｄｙｌｄｉｔｈｉｏ）ｐｒｏｐｉｏｎａｍｉｄｏ］ｈｅｘａｎｏｙｌｏｘｙ｝ｓｕｌｆｏｓｕｃｃｉｎｉｍｉｄｅ、ｓｏｄｉｕｍ　ｓａｌｔ（Ｓｕｌｆｏ－ＡＣ５－ＳＰＤＰ）等の市販の試薬を用いて行うことができる。また、ピリジルジスルフィド基の導入後に還元してチオール基とすることで、これを導入することもできる。 As a method for directly fixing the to-be-supported substance to the water-soluble carrier, for example, 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. , an amino group, a hydroxy group, an isothiocyanate group, an isocyanate group, an azide group, an aldehyde group, a carbonate group, an allyl group, an aminooxy group, a maleimide group, a thiol group, an active group such as a pyridyl disulfide group, or the above 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. As 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. As an example, thiol groups can be introduced using commercially available reagents such as S-acetylmercaptosuccinic anhydride and 2-iminothiolane hydrochloride. Further, 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. Introduction of 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. A pyridyl disulfide group can also be introduced by reducing it to a thiol group after introduction.
　前記被担持物質を前記水溶性担体に間接的に固定する方法としては、例えば、ポリヒスチジン、ポリエチレングリコール、システイン及び／又はリジンを含むオリゴペプチド、前記活性基を有するリンカー分子（例えば、上記の捕捉体の製造方法で挙げたもの）等のリンカーを介して固定する方法が挙げられる。前記リンカーの選択及びその大きさは、前記被担持物質との結合の強さや前記被担持物質を前記水溶性担体に固定化したことによる立体障害等を考慮して適宜設定することができる。 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.
　前記ブロック化標識レクチンの製造方法においては、前記水溶性担体に前記標識物質及びレクチンを一度に固定しても、これらを別々に順次固定してもよいが、製造のしやすさ、並びに、標識物質及びレクチン量の制御しやすさの観点からは、前記水溶性担体にいずれか一方を固定してから他方を固定することが好ましい。また、前記ブロック化標識レクチンは、前記標識物質とレクチンとをそれぞれ別の水溶性担体に固定し、一の水溶性担体に固定された標識物質（ブロック化標識物質）と、他の水溶性担体に固定されたレクチン（ブロック化レクチン）とを直接、又は前記リンカー等を介して、結合させることで製造することもできる。 In the method for producing the blocked labeled lectin, 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. In addition, 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.
　前記ブロック化標識レクチンの製造方法としては、特に制限されず、例えば、下記の実施例に記載のように、前記標識物質が酵素であり、前記第１の水溶性高分子が多糖類や糖タンパク質である場合、先ず、第１の水溶性高分子を過ヨウ素酸ナトリウムのような酸化剤で酸化してアルデヒド基を付与し、ヒドラジン塩酸塩と反応させた後、これをジメチルアミンボラン（ＤＭＡＢ）等の還元剤と反応させてヒドラジン化する。一方、前記酵素も過ヨウ素酸ナトリウムのような酸化剤で酸化してその糖鎖にアルデヒド基を付与させる。次いで、上記で付与したヒドラジン残基とアルデヒド基とを反応させてヒドラゾン結合させ、第１の水溶性高分子－酵素結合体を得る。得られた第１の水溶性高分子－酵素結合体を、Ｎ－ヒドロキシスクシンイミド及びマレイミド基を各末端に有するクロスリンカー（例えば、ＳＭ（ＰＥＧ）４、ＳＭＣＣ等）で処理し、マレイミド基を導入する。一方、レクチンをチオール化試薬でチオール化してチオール基を付与し、又は分子内にジスルフィド結合を持つレクチンの場合は還元によってチオール基を得る。最後に、第１の水溶性高分子－酵素結合体に導入したマレイミド基と、レクチンに付与したチオール基とを結合させることにより、第１の水溶性高分子（水溶性担体）－酵素－レクチンの三者を共有結合させることができる。この方法によれば、２分子以上の第１の水溶性高分子が酵素を介して結合したものに、レクチンが結合したものが得られる。これらの反応に供する第１の水溶性高分子、標識物質、レクチンの比率は、上記のブロック化標識レクチンにおける各含有量の好ましい範囲を達成するように適宜選択することができる。 The method for producing the blocked labeled lectin is not particularly limited. For example, as described in Examples below, the labeling substance is an enzyme, and the first water-soluble polymer is a polysaccharide or glycoprotein. , first, 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). hydrazinization by reacting with a reducing agent such as On the other hand, the enzyme is also oxidized with an oxidizing agent such as sodium periodate to impart an aldehyde group to its sugar chain. Next, 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. On the other hand, 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. Finally, 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. According to this method, 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.
　（捕捉ステップ）
　前記捕捉ステップにおいて、前記試料と前記捕捉体とを接触させる方法としては、特に制限されず、適宜従来公知の方法又はそれに準じた方法を採用することができ、例えば、前記非水溶性担体がプレートである場合にはこれ（プローブ分子固定化プレート）に前記試料を注入する方法や、前記非水溶性担体が粒子である場合には前記試料中に前記捕捉体（プローブ分子固定化粒子）を添加する方法が挙げられる。 (capturing step)
In the capturing step, 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. In the case of , 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.
　前記捕捉ステップにおける前記試料と前記捕捉体との反応において、前記捕捉体及び前記試料を含む反応液中の、捕捉体の含有量（終濃度）（捕捉体が２種以上の組み合わせである場合にはそれらの合計、以下同じ）としては、特に制限されず、試料の種類、濃度等に応じて適宜調整されるものであるため特に制限されないが、短時間に効率よく捕捉する観点から、例えば、０．０１～１．５質量％であることが好ましく、０．０５～１質量％であることがより好ましく、０．１～０．５質量％であることがさらに好ましい。 In the reaction between the sample and the capturing body in the capturing step, the content (final concentration) of the capturing body in the reaction solution containing the capturing body and the sample (when the capturing body is a combination of two or more is the total of them, the same applies hereinafter) 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.
　また、前記捕捉ステップの条件としても特に制限されず、適宜調整することができ、例えば、室温～４５℃、好ましくは２０～３７℃、ｐＨ６～９程度、好ましくはｐＨ７～８で、５秒～１０分程度、好ましくは３０秒～５分程度行うことができるが、これらの条件に限定されるものではない。 In addition, 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.
　（標識ステップ）
　第１の方法の場合、前記標識ステップにおける前記標識体とＧａｌＮＡｃ付加ＣＡ１９－９との反応において、前記標識体及びＧａｌＮＡｃ付加ＣＡ１９－９を含む反応液中の、標識体の含有量（終濃度）（標識体が２種以上の組み合わせである場合にはそれらの合計、以下同じ）としては、特に制限されず、試料の種類、濃度等に応じて適宜調整されるものであるため特に制限されないが、過剰に用いると高いバックグラウンドシグナルを生じる可能性がある観点から、例えば、０．００１～１０μｇ／ｍＬであることが好ましく、０．０１～５μｇ／ｍＬであることがより好ましく、０．１～１μｇ／ｍＬであることがさらに好ましい。 (labeled step)
In the first method, in the reaction between the label and GalNAc-added CA19-9 in the labeling step, the content (final concentration) of the label in the reaction solution containing the label and GalNAc-added CA19-9 (If the label is a combination of two or more, the total thereof, the same shall apply hereinafter) 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 μg / mL, more preferably 0.01 to 5 μg / mL, 0.1 More preferably ~1 μg/mL.
　また、第２の方法の場合、前記標識ステップにおける前記標識体とα２－３シアル酸付加ＣＥＡとの反応において、前記標識体及びα２－３シアル酸付加ＣＥＡを含む反応液中の、標識体の含有量（終濃度）（標識体が２種以上の組み合わせである場合にはそれらの合計、以下同じ）としては、特に制限されず、試料の種類、濃度等に応じて適宜調整されるものであるため特に制限されないが、過剰に用いると高いバックグラウンドシグナルを生じる可能性がある観点から、例えば、０．００１～１０μｇ／ｍＬであることが好ましく、０．０１～５μｇ／ｍＬであることがより好ましく、０．１～１μｇ／ｍＬであることがさらに好ましい。 In the case of the second method, in the reaction between the labeled product and α2-3 sialylated CEA in the labeling step, the labeled product in the reaction solution containing the labeled product and α2-3 sialylated CEA The content (final concentration) (if the label is a combination of two or more, the total thereof; the same shall apply hereinafter) is not particularly limited, and may be appropriately adjusted according to the type of sample, concentration, etc. However, from the viewpoint that excessive use may cause a high background signal, for example, 0.001 to 10 μg/mL is preferable, and 0.01 to 5 μg/mL is preferable. More preferably, it is 0.1 to 1 μg/mL.
　さらに、前記標識ステップの他の条件としても特に制限されず、適宜調整することができ、例えば、室温～３７℃、好ましくは２０～３７℃、ｐＨ５．０～７．０、好ましくは５．５～６．５で、３分～１２０分程度、好ましくは、５分～１０分程度行うことができるが、これらの条件に限定されるものではない。 Furthermore, other conditions for the labeling step are not particularly limited and can be adjusted as appropriate. to 6.5, 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.
　（洗浄ステップ）
　前記サンドイッチ法には、前記捕捉ステップ及び／又は前記標識ステップの後に、前記捕捉体に結合したＧａｌＮＡｃ付加ＣＡ１９－９と、それ以外の前記捕捉体に結合していない（捕捉されていない）夾雑物とを分離（第１の方法の場合）、前記捕捉体に結合したα２－３シアル酸付加ＣＥＡと、それ以外の前記捕捉体に結合していない（捕捉されていない）夾雑物とを分離（第２の方法の場合）し、前記夾雑物を除去する洗浄ステップをさらに含むことが好ましい。前記夾雑物を除去する方法としては、特に制限されず、適宜従来公知の方法又はそれに準じた方法を採用することができ、例えば、前記捕捉体が前記プローブ分子固定化プレートである場合にはプレート上から液相（上清）を除去する方法や、前記プローブ分子固定化粒子である場合には前記粒子を遠心や集磁によって回収して液相（上清）を除去する方法が挙げられる。また、前記洗浄ステップにおいては、必要に応じて、洗浄液の注入及び除去を繰り返してもよい。前記洗浄液としては、例えば、中性（好ましくは、ｐＨ６～９）の公知の緩衝液（ナトリウムリン酸バッファー、ＭＥＳ、Ｔｒｉｓ、ＣＦＢ、ＭＯＰＳ、ＰＩＰＥＳ、ＨＥＰＥＳ、トリシンバッファー、ビシンバッファー、グリシンバッファー等）が挙げられ、また、ＢＳＡ等の安定化蛋白や界面活性剤等が添加されたものであってもよい。 (Washing step)
In the sandwich method, after the capturing step and/or the labeling step, GalNAc-attached CA19-9 bound to the capture body and other contaminants not bound to the capture body (uncaptured) are separated (in the case of the first method), and α2-3 sialylated CEA bound to the capture body and other contaminants not bound to the capture body (not captured) are separated ( In the case of the second method), it is preferable to further include a washing step for removing said contaminants. The method for removing the contaminants is not particularly limited, and conventionally known methods or methods based thereon can be employed as appropriate. 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. In addition, in the cleaning step, the injection and removal of the cleaning liquid may be repeated as necessary. Examples of 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.
　本発明に係る測定工程として前記サンドイッチ法を用いる場合、前記試料としては、上記のように希釈液で希釈して用いてもよいが、前記捕捉体が前記プローブ分子固定化粒子等である場合には、その粒子懸濁媒（粒子液）に懸濁して用いてもよく、さらに、前記試料と前記捕捉体及び／又は前記標識体との反応系には、他の反応用バッファーを適宜添加してもよい。これらの粒子懸濁媒、反応用バッファーとしては、特に制限されないが、例えば、それぞれ独立に、公知の緩衝液（ナトリウムリン酸バッファー、ＭＥＳ、Ｔｒｉｓ、ＣＦＢ、ＭＯＰＳ、ＰＩＰＥＳ、ＨＥＰＥＳ、トリシンバッファー、ビシンバッファー、グリシンバッファー等）が挙げられ、また、それぞれ独立に、ＢＳＡ等の安定化蛋白や血清等が添加されたものであってもよい。 When the sandwich method is used as the measurement step according to the present invention, 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. may These 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.
　また、前記標識体として前記ブロック化標識レクチンを用いる場合、前記試料と前記標識体との反応系には、測定感度をさらに向上させる観点から、他に、水溶性高分子（以下、「第２の水溶性高分子」という；前記標識物質及びレクチンを担持していない遊離の水溶性高分子である点で前記水溶性担体を構成する第１の水溶性高分子とは異なる）、遊離レクチン（前記水溶性担体にも前記標識物質にも固定されていない点で前記ブロック化標識レクチン等に含まれるレクチンとは異なる）等を共存させてもよい。 Further, when the blocked labeled lectin is used as the label, 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.
　第２の水溶性高分子としては、第１の水溶性高分子として挙げたものと同様のものが挙げられ、これらのうちの１種を単独であっても２種以上の組み合わせであってもよい。また、第１の水溶性高分子と同種の高分子であってよい。これらの中でも、第２の水溶性高分子としては、測定感度がより向上する傾向にある観点からは、多糖類及びその修飾体からなる群から選択される少なくとも１種であることが好ましく、デキストラン及びアミノデキストラン、並びに、これらの修飾体からなる群から選択される少なくとも１種であることがより好ましく、デキストランであることがさらに好ましい。 Examples of the second water-soluble polymer 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.
　また、第２の水溶性高分子の重量平均分子量としては、測定感度がより向上する傾向にある観点からは、５００，０００～５，０００，０００であることが好ましく、１，０００，０００～３，０００，０００であることがより好ましく、１，５００，０００～２，５００，０００であることがさらに好ましい。 Further, 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.
　第２の水溶性高分子の量としては、特に限定されないが、前記試料、ブロック化標識レクチン、及び第２の水溶性高分子を含む反応液中における第２の水溶性高分子の含有量（第２の水溶性高分子が２種以上の組み合わせである場合にはそれらの合計）が、０．０１～１０ｗ／ｖ％であることが好ましく、０．５～３ｗ／ｖ％であることがより好ましい（ｗ／ｖ％：重量／容量（ｇ／ｍＬ）パーセント、以下同じ）。 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).
　前記遊離レクチンとしては、上記でレクチンとして挙げたものと同様のものが挙げられ、これらのうちの１種を単独であっても２種以上の組み合わせであってもよい。また、ブロック化標識レクチンに含まれるレクチンと同種のものであってよい。これらの中でも、前記遊離レクチンとしては、反応性の向上とバックグラウンドの抑制の観点からは、ブロック化標識レクチンに含まれるレクチンと同種のものであることが特に好ましい。 Examples of the free lectin 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.
　前記遊離レクチンの量としては、特に限定されないが、前記試料、ブロック化標識レクチン、及び前記遊離レクチンを含む反応液中において、ブロック化標識レクチンの含有量１００質量部に対する遊離レクチンの含有量（遊離レクチンが２種以上の組み合わせである場合にはそれらの合計）が、１～１０，０００質量部であることが好ましく、１０～５，０００質量部であることがより好ましい。 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.
　（測定ステップ）
　前記測定ステップにおいては、前記標識物質に応じてシグナルを測定する。例えば、前記標識物質が酵素である場合には、当該酵素に対応する発色基質や発光基質を添加して反応させることによって生じるシグナル（例えば、発色や発光）を測定する。これにより、試料中のＧａｌＮＡｃ付加ＣＡ１９－９量をシグナル量として検出することができ、また、必要に応じて標準試料における測定値との比較をすることによって試料中のＧａｌＮＡｃ付加ＣＡ１９－９量を定量することができる（第１の方法の場合）。又は、これによって試料中のα２－３シアル酸付加ＣＥＡ量をシグナル量として検出することができ、また、必要に応じて標準試料における測定値との比較をすることによって試料中のα２－３シアル酸付加ＣＥＡ量を定量することができる（第２の方法の場合）。 (measurement step)
In the measuring step, a signal is measured according to the labeling substance. For example, when the labeling substance is an enzyme, a signal (for example, color development or luminescence) generated by adding a chromogenic or luminescent substrate corresponding to the enzyme and reacting is measured. As a result, 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). Alternatively, the amount of α2-3 sialylated CEA in the sample can be detected as a signal amount by this, and if necessary, the α2-3 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).
　［がん検出方法］
　本発明のがん検出方法によれば、上記の測定工程によって測定されたＧａｌＮＡｃ付加ＣＡ１９－９量又はα２－３シアル酸付加ＣＥＡ量を指標とすることにより、前記試料又は試料が由来する被検者における前立腺がん及び／又は大腸がんの存在の有無を検出することができる。かかるがん検出方法は、創薬等の研究目的の他、本発明のがん検査方法に用いることができる。 [Cancer detection method]
According to the cancer detection method of the present invention, the amount of GalNAc-added CA19-9 or the amount of α2-3 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.
　［がん検査方法］
　本発明の第１のがん検査方法では、被検者由来の試料中におけるＧａｌＮＡｃ付加ＣＡ１９－９量を測定し、それを指標として前立腺がん及び／又は大腸がんの有無を検出することで、当該被検者において、前立腺がん及び／又は大腸がんに罹患しているか否かの予測、前記がんの罹患の有無若しくはその可能性の判別、又は、前記がんの進行度若しくは重症度の評価をすることができる。 [Cancer test method]
In the first cancer testing 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.
　すなわち、本発明の第１のがん検査方法の態様としては具体的に、例えば、次の態様：
　前立腺がん及び大腸がんからなる群から選択される少なくとも１種のがんであると予測される被検者をスクリ－ニングする方法であり、被検者由来の試料中のＧａｌＮＡｃ付加ＣＡ１９－９量を測定する測定工程と、測定されたＧａｌＮＡｃ付加ＣＡ１９－９量を指標として被検者を選別する選別工程と、を含む、第１のスクリーニング方法；
　被検者における前立腺がん及び大腸がんからなる群から選択される少なくとも１種のがんの罹患の有無を鑑別する方法であり、被検者由来の試料中のＧａｌＮＡｃ付加ＣＡ１９－９量を測定する測定工程と、測定されたＧａｌＮＡｃ付加ＣＡ１９－９量を指標として被検者を判別する判別工程と、を含む、第１の鑑別方法；
　被検者における前立腺がん及び大腸がんからなる群から選択される少なくとも１種のがんの進行度又は重症度を評価する方法であり、被検者由来の試料中のＧａｌＮＡｃ付加ＣＡ１９－９量を測定する測定工程と、測定されたＧａｌＮＡｃ付加ＣＡ１９－９量を指標として被検者を評価する評価工程と、を含む、第１の評価方法；
が挙げられる。 That is, specific 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 GalNAc-added CA19-9 in a sample derived from the subject A first evaluation method comprising a measurement step of measuring the amount and an evaluation step of evaluating the subject using the measured amount of GalNAc-added CA19-9 as an index;
is mentioned.
　また、本発明の第２のがん検査方法では、被検者由来の試料中におけるα２－３シアル酸付加ＣＥＡ量を測定し、それを指標として前立腺がんの有無を検出することで、当該被検者において、前立腺がんに罹患しているか否かの予測、前記がんの罹患の有無若しくはその可能性の判別、又は、前記がんの進行度若しくは重症度の評価をすることができる。 In addition, in the second cancer testing method of the present invention, the amount of α2-3 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. In a 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. .
　すなわち、本発明の第２のがん検査方法の態様としては具体的に、例えば、次の態様：
　前立腺がんであると予測される被検者をスクリ－ニングする方法であり、被検者由来の試料中のα２－３シアル酸付加ＣＥＡ量を測定する測定工程と、測定されたα２－３シアル酸付加ＣＥＡ量を指標として被検者を選別する選別工程と、を含む、第２のスクリーニング方法；
　被検者における前立腺がんの罹患の有無を鑑別する方法であり、被検者由来の試料中のα２－３シアル酸付加ＣＥＡ量を測定する測定工程と、測定されたα２－３シアル酸付加ＣＥＡ量を指標として被検者を判別する判別工程と、を含む、第２の鑑別方法；
　被検者における前立腺がんの進行度又は重症度を評価する方法であり、被検者由来の試料中のα２－３シアル酸付加ＣＥＡ量を測定する測定工程と、測定されたα２－３シアル酸付加ＣＥＡ量を指標として被検者を評価する評価工程と、を含む、第２の評価方法；
が挙げられる。 That is, specific 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 α2-3 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 α2-3 sialylated CEA in a sample derived from the subject, and the measured α2-3 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 α2-3 sialylated CEA in a sample derived from the subject, and the measured α2-3 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.
　〔スクリ－ニング方法〕
　本発明において、「スクリ－ニング」とは、前記がん（前立腺がん及び／又は大腸がん）に罹患している可能性がある被検者を選別することを示す。前記スクリ－ニング方法には、本発明では具体的に、被検者を、前立腺がん及び／又は大腸がんに罹患（再発を含む）している可能性が高いと予測して、前記可能性が無い又は低い群から選別する方法が含まれる。前記スクリーニング方法においては、その目的に応じて、例えば、中程度の可能性が期待できるレベルで被検者を選別してもよい。 [Screening method]
In the present invention, "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.
　より具体的には、例えば、前記がんに真に罹患している者からなる群（陽性群）と前記がんに罹患していない者からなる群（正常群：陰性群）との２群に切り分け、被検者を、陽性群に含まれると予測して、正常群（陰性群）から選別する。この場合の予測基準としては、例えば、陰性一致率（当該スクリ－ニングによって正常群と予測された被検者が真に正常群にある割合）が９５％以上であることが好ましい。 More specifically, for example, 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). , and the subjects are sorted out from the normal group (negative group) by predicting that they will be included in the positive group. As a criterion for prediction in this case, for example, 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.
　〔鑑別方法〕
　本発明において、「鑑別」とは、前立腺がん及び／又は大腸がんを他の疾患又は状態と区別して、被検者が当該がんに罹患しているか否かを判別することを示す。また、前記罹患の有無の判別のみならず、罹患の可能性がある場合におけるその程度の評価（高い／中程度／低い等の評価）を含む。前記鑑別方法には、本発明では具体的に、症状の有無に関わらず、被検者において、前立腺がん若しくは大腸がんに罹患しているか否か又はその可能性が高いか否かを判別する方法を含み、例えば、被検者が罹患しているがんが、前立腺がん若しくは大腸がんであるか否かを判別する方法、又は前記がんである可能性が高いと判別する方法；被検者が過去に罹患していた前立腺がん若しくは大腸がんが再発したか否かを判別する方法、又は前記がんが再発した可能性が高いと判別する方法も含む。 [Distinction method]
In the present invention, "differential" means distinguishing prostate cancer and/or colorectal cancer from other diseases or conditions to determine whether or not a subject is affected by the cancer. In addition, it includes not only the determination of the presence or absence of the disease, but also the evaluation of the degree of the possibility of the disease (evaluation such as high/moderate/low). Specifically, in the present invention, 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. For example, 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.
　より具体的には、例えば、前記がんに真に罹患している者からなる群（陽性群）と前記がんに罹患していない者からなる群（正常群：陰性群）との２群に切り分け、被検者が、前記陽性群に含まれるのか陰性群に含まれるのかを判別する。かかる鑑別は、医師等による前立腺がん及び／又は大腸がんの診断の補助に適用することができる。この場合の判別基準としては、例えば、陰性一致率（当該鑑別によって正常群と判別された被検者が真に正常群にある割合）が９５％以上であることが好ましい。 More specifically, for example, 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. As a discrimination criterion in this case, for example, it is preferable that 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.
　〔評価方法〕
　本発明において、前記評価方法には、例えば、被検者が罹患している前立腺がん及び／又は大腸がんの進行度又は重症度を評価する方法；被検者が罹患している前記がんの治療方針決定のための指標を提供する方法；前記がんの治療効果の判定方法を含む。 〔Evaluation method〕
In the present invention, 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.
　より具体的に、第１の評価方法の場合には、例えば、前記がんに罹患していない者又は前記がんの進行度若しくは重症度の低い者からなる群におけるＧａｌＮＡｃ付加ＣＡ１９－９量と、被検者のＧａｌＮＡｃ付加ＣＡ１９－９量とを比較し、被検者における量の方が多ければ進行度若しくは重症度が高いと評価し、少なければ進行度若しくは重症度が低いと評価することができる。また、被検者の前記がんの発見当初若しくは治療前におけるＧａｌＮＡｃ付加ＣＡ１９－９量と現在のＧａｌＮＡｃ付加ＣＡ１９－９量とを比較し、量が増加していればその治療効果は低いと評価し、減少していればその治療効果は高いと評価することができる。 More specifically, in the case of the first evaluation method, for example, the amount of GalNAc-added CA19-9 and , Compared with the amount of GalNAc-added CA19-9 in the subject, if the amount in the subject is higher, the degree of progression or severity is evaluated as high, and if the amount is lower, the degree of progression or severity is evaluated as low can be done. In addition, 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.
　また、第２の評価方法の場合には、より具体的に、例えば、前記がんに罹患していない者又は前記がんの進行度若しくは重症度の低い者からなる群におけるα２－３シアル酸付加ＣＥＡ量と、被検者のα２－３シアル酸付加ＣＥＡ量とを比較し、被検者における量の方が多ければ進行度若しくは重症度が高いと評価し、少なければ進行度若しくは重症度が低いと評価することができる。また、被検者の前記がんの発見当初若しくは治療前におけるα２－３シアル酸付加ＣＥＡ量と現在のα２－３シアル酸付加ＣＥＡ量とを比較し、量が増加していればその治療効果は低いと評価し、減少していればその治療効果は高いと評価することができる。 In addition, in the case of the second evaluation method, more specifically, for example, α2-3 sialic acid The amount of added CEA and the amount of α2-3 sialylated CEA in the subject are compared. can be evaluated as low. In addition, the amount of α2-3 sialylated CEA at the time of the cancer discovery or before treatment in the subject is compared with the current amount of α2-3 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.
　（カットオフ値）
　前記スクリーニング方法の選別工程及び前記鑑別方法の判別工程では、第１の方法の場合、例えば、前記測定工程で測定されたＧａｌＮＡｃ付加ＣＡ１９－９量を、予め定められたカットオフ値と比較して、前記ＧａｌＮＡｃ付加ＣＡ１９－９量が前記カットオフ値よりも高い被検者を、前立腺がん及び／又は大腸がんに罹患している（又は罹患している可能性が高い）と予測又は判別する。第２の方法の場合には、例えば、前記測定工程で測定されたα２－３シアル酸付加ＣＥＡ量を、予め定められたカットオフ値と比較して、前記α２－３シアル酸付加ＣＥＡ量が前記カットオフ値よりも高い被検者を、前立腺がんに罹患している（又は罹患している可能性が高い）と予測又は判別する。 (cutoff value)
In the selection step of the screening method and the determination step of the identification method, in the case of the first method, for example, 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. In the case of the second method, for example, the amount of α2-3 sialylated CEA measured in the measuring step is compared with a predetermined cutoff value, and the amount of α2-3 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.
　本発明において、「カットオフ値」とは、前記ＧａｌＮＡｃ付加ＣＡ１９－９量又は前記α２－３シアル酸付加ＣＥＡ量によって判定するための基準となる予め定められた値であり、上記陽性群と陰性群とを判定するための境界値のことを示す。このようなカットオフ値は、本発明のがん検査方法の目的、ＧａｌＮＡｃ付加ＣＡ１９－９量の測定方法又はα２－３シアル酸付加ＣＥＡ量の測定方法、被検者や試料の性質、希釈条件等によって適宜設定されるものであるため、特に限定されるものではない。例えば、カットオフ値を比較的低値に設定することで、検出感度を高く、すなわち、前記がんの初期段階にある患者をある程度広く拾集でき、早期発見が可能となる。他方、ＧａｌＮＡｃ付加ＣＡ１９－９及びα２－３シアル酸付加ＣＥＡは健常者由来の試料においても少量は検出されるため、前記カットオフ値を比較的高値に設定することで、前記スクリーニング及び前記鑑別をより高精度で行うことが可能となる。 In the present invention, 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 α2-3 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 α2-3 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. For example, by setting the cut-off value to a relatively low value, 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. On the other hand, GalNAc-added CA19-9 and α2-3 sialylated CEA are detected in small amounts even in samples derived from healthy subjects. It becomes possible to perform with higher precision.
　カットオフ値の一例としては、第１の方法の場合、例えば、前記試料を血清検体とし、体積比で１／１０に希釈し、前記捕捉体として抗ＣＡ１９－９抗体固定化粒子を用い、かつ、前記標識体としてブロック化標識ＷＦＡを用いたサンドイッチイムノアッセイで測定したＧａｌＮＡｃ付加ＣＡ１９－９量を、前記標識物質をＡＬＰ及び基質をＡＭＰＰＤとして波長４６３ｎｍに極大吸収を有する光の発光強度（カウント）で示す場合（実施例１の場合）には、５０，０００～８０，０００カウントが挙げられ、好ましくは６０，０００～７０，０００カウントが挙げられるが、これに限定されるものではない。なお、前記範囲で定められるカットオフ値は、本発明のがん検査方法の目的、ＧａｌＮＡｃ付加ＣＡ１９－９量の測定方法、被検者や試料の性質に応じて、その範囲内からいずれか１点が選択されて適用される。 As an example of the cut-off value, in the case of the first method, for example, 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. In the case indicated (in the case of Example 1), 50,000 to 80,000 counts are mentioned, preferably 60,000 to 70,000 counts, but not limited thereto. It should be noted that 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.
　また、第２の方法の場合、カットオフ値の一例としては、例えば、前記試料を血清検体とし、体積比で１／１０に希釈し、前記捕捉体として抗ＣＥＡ抗体固定化粒子を用い、かつ、前記標識体としてブロック化標識ＭＡＭを用いたサンドイッチイムノアッセイで測定したα２－３シアル酸付加ＣＥＡ量を、前記標識物質をＡＬＰ及び基質をＡＭＰＰＤとして波長４６３ｎｍに極大吸収を有する光の発光強度（カウント）で示す場合（実施例２の場合）には、２１０，０００～３２０，０００カウントが挙げられ、好ましくは２７０，０００～３００，０００カウントが挙げられるが、これに限定されるものではない。なお、前記範囲で定められるカットオフ値は、本発明のがん検査方法の目的、α２－３シアル酸付加ＣＥＡ量の測定方法、被検者や試料の性質に応じて、その範囲内からいずれか１点が選択されて適用される。 In the case of the second method, as an example of the cutoff value, for example, 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 α2-3 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. It should be noted that 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 α2-3 sialylated CEA, and the properties of the subject and sample. or one point is selected and applied.
　このような本発明のがん検査方法によれば、他のがんと区別して、前立腺がん及び／又は大腸がんに特異的な治療方針の決定等のための情報を提供すること（第１の方法の場合）又は前立腺がんに特異的な治療方針の決定等のための情報を提供すること（第２の方法の場合）が可能となる。また、前立腺がん及び／又は大腸がんに罹患（再発を含む）している、又はその可能性が高い被検者を特定し、さらなる検査や確定診断を行うことにより、前立腺がん及び／又は大腸がんの早期発見や早期治療介入等が可能となる。さらに、がんに由来する糖鎖構造の情報はがんの浸潤性の指標となることが報告されていることから、本発明のがん検査方法によってがんの悪性度や予後についての情報を得ることも可能となる。なお、本発明の方法は、医師等による前立腺がん及び／又は大腸がんの診断を補助する方法、又は医師等による前立腺がん及び／又は大腸がんの診断のための情報を提供する方法でもある。 According to such a cancer examination method of the present invention, 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). In the case of method 1), it is possible to provide information for determining a treatment policy specific to prostate cancer (in the case of the second method). In addition, 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. Furthermore, since it has been reported that information on the sugar chain structure derived from cancer serves as an index of cancer invasiveness, information on cancer malignancy and prognosis can be obtained by the cancer testing method of the present invention. It is also possible to obtain 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.
　さらに、本発明の方法は、従来のＣＡ１９－９測定方法又は従来のＣＥＡ測定方法や、他のモニタリングマーカーの測定方法と組み合わせるための方法としても好適であり、これにより、前立腺がん及び／又は大腸がんの検出感度や診断の精度をさらに向上させることが可能となる。 Furthermore, 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.
　＜キット＞
　本発明の第１のキットは、上記本発明のがん検出方法又はがん検査方法に用いるためのキットであり、ＣＡ１９－９に特異的に結合可能な第１のプローブ分子と、ＧａｌＮＡｃに特異的に結合可能な第２のプローブ分子と、を備えるキットである。また、本発明の第２のキットは、上記本発明のがん検出方法又はがん検査方法に用いるためのキットであり、ＣＥＡに特異的に結合可能な第１のプローブ分子と、α２－３シアル酸に特異的に結合可能な第２のプローブ分子と、を備えるキットである（以下、場合により第１のキット及び第２のキットを総称して「本発明のキット」という）。第１のプローブ分子及び第２のプローブ分子としては、それぞれ、これらの好ましい態様も含めて、上述のとおりである。 <Kit>
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, α2-3 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.
　また、本発明のキットとしては、非水溶性担体と、前記非水溶性担体に固定された第１のプローブ分子及び第２のプローブ分子のうちのいずれか一方と、を備える捕捉体、並びに、標識物質と、第１のプローブ分子及び第２のプローブ分子のうちの他方と、を備える標識体、を備えることも好ましい。前記捕捉体及び前記標識体としては、それぞれ、これらの好ましい態様も含めて、上述のとおりである。 In addition, 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.
　本発明のキットにおいて、第１のプローブ分子、第２のプローブ分子、前記捕捉体、及び前記標識体としては、それぞれ独立に、固体（粉末）状であっても緩衝液に溶解された液体状であってもよい。液体状である場合、各溶液における第１のプローブ分子、第２のプローブ分子、前記捕捉体、及び前記標識体の濃度は、特に限定されないが、それぞれ独立に、例えば、０．０１～１０μｇ／ｍＬであることが好ましく、０．１～５．０μｇ／ｍＬであることがより好ましく、０．５～３．０μｇ／ｍＬであることがさらに好ましい。 In the kit of the present invention, 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. may be When liquid, 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 μg/ It is preferably mL, more preferably 0.1 to 5.0 μg/mL, even more preferably 0.5 to 3.0 μg/mL.
　本発明のキットとしては、他に、ＥＬＩＳＡ、ＣＬＥＩＡ、イムノクロマト等の通常の免疫学的測定方法及びそれに準じた方法で備えるべき構成をさらに備えていてもよい。例えば、上記のサンドイッチ法を本発明に係る測定方法の原理とする場合には、前記プローブ分子を固相化するための磁性ビーズやプレート、センサーチップ、前記標準試料（各濃度）、対照試薬、前記粒子懸濁媒、前記反応用バッファー、前記洗浄液、第２の水溶性高分子、遊離レクチンからなる群から選択される少なくとも１種をさらに備えていてもよい。また、前記標識物質が酵素である場合には、当該標識物質の検出・定量に必要な基質や反応停止液等をさらに含んでいてもよい。 In addition, 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. For example, when the above sandwich method is used as the principle of the measurement method according to the present invention, 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. Moreover, when 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.
　さらに、本発明のキットは、必要に応じて、前記希釈液や、試料の前処理を行うための前処理液；希釈用カートリッジ；前処理の反応停止液又は中和液を備えていてもよい。また、前記サンドイッチ法としてイムノクロマトを採用する場合には、前記捕捉体及び／又は前記標識体を担持したゾーンを含むデバイスをさらに含んでいてもよい。前記デバイスとしては、展開液パッドや吸収パッド等、イムノクロマトに適したその他の構成要素を備えることができる。さらに、本発明のキットには、当該キットの使用説明書をさらに含んでいてもよい。 Furthermore, 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. . Moreover, when immunochromatography is employed as the sandwich method, 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. Furthermore, the kit of the present invention may further include instructions for use of the kit.
　以下、実施例及び比較例に基づいて本発明をより具体的に説明するが、本発明は以下の実施例に限定されるものではない。なお、各実施例及び比較例において、「％」の表示は、特に記載のない場合、重量／容量（ｗ／ｖ：ｇ／ｍＬ）パーセントを示す。 The present invention will be described in more detail below based on examples and comparative examples, but the present invention is not limited to the following examples. In each example and comparative example, "%" means weight/volume (w/v: g/mL) percentage unless otherwise specified.
　（実施例１）　ブロック化標識レクチン（ＷＦＡ）を用いた血清検体に含まれるＷＦＡ結合型糖鎖付加ＣＡ１９－９（ＣＡ１９－９／ＷＦＡ）の測定
　（１）ヒドラジン化デキストランの調製
　４．８ｍＬの０．１Ｍリン酸バッファー（ｐＨ７．０）に分子量２５０ｋのデキストラン（ＣａｒｂｏＭｅｒ社製）を２４０．０ｍｇ添加し、２５℃の暗所で３０分間攪拌して溶解させた。次いで、１５０ｍＭ　ＮａＩＯ４を２．６６４ｍＬ、及びイオン交換水を０．５３６ｍＬ添加して、２５℃の暗所で３０分間攪拌した。次いで、ＰＤ－１０カラム（ＧＥ　Ｈｅａｌｔｈｃａｒｅ社製、Ｓｅｐｈａｄｅｘ　Ｇ－２５充填カラム：以下単に「Ｓｅｐｈａｄｅｘ　Ｇ－２５」）を用いて、０．１Ｍナトリウムリン酸バッファー（ｐＨ６．０）でバッファー交換を行い、２０．０ｍＬの溶液を得た。得られた溶液に、５．０４ｇのＮＨ２ＮＨ２・ＨＣｌを添加し、２５℃の暗所で２時間攪拌した。８００ｍｇのＤＭＡＢ（ジメチルアミンボラン）を加え、さらに２５℃の暗所で２時間攪拌した。ＲＣ５０Ｋ（分子量５万の再生セルロース）透析膜を用いてイオン交換水４Ｌによる透析を暗所で３時間行った後、４℃で一晩静置した。０．１Ｍナトリウムリン酸バッファー（ｐＨ６．０）を用いたゲル濾過（Ｓｅｐｈａｄｅｘ　Ｇ－２５）でバッファー交換を行い、８５．０ｍＬの溶液を得た。得られた溶液においてデキストランの濃度が１．０ｍｇ／ｍＬとなるように調整し、ヒドラジン化デキストランの溶液を得た。 (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. Then, using a PD-10 column (manufactured by GE Healthcare, Sephadex G-25 packed column: hereinafter simply "Sephadex G-25"), the buffer was exchanged with 0.1 M sodium phosphate buffer (pH 6.0), 20.0 mL of solution was obtained. To the resulting solution was added 5.04 g of NH 2 NH 2 .HCl and stirred at 25° C. in the dark for 2 hours. 800 mg of DMAB (dimethylamine borane) was added and further stirred at 25° C. in the dark for 2 hours. Using an 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.
　（２）デキストラン－酵素結合体の調製
　１０ｍｇ／ｍＬのアルカリホスファターゼ（オリエンタル酵母社製、ＡＬＰ－５０）３０．０ｍＬについて、０．１Ｍナトリウムリン酸バッファー（ｐＨ６．０）を用いたゲル濾過（Ｓｅｐｈａｄｅｘ　Ｇ－２５）でバッファー交換を行い、３．０ｍｇ／ｍＬの溶液を９０．６ｍＬ調製した。次いで、２７ｍＭ　ＮａＩＯ４を４５．３ｍＬ添加して、２５℃の暗所で３分間攪拌した。次いで、０．１Ｍナトリウムリン酸バッファー（ｐＨ６．０）を用いたゲル濾過（Ｓｅｐｈａｄｅｘ　Ｇ－２５）でバッファー交換を行い、０．５ｍｇ／ｍＬの溶液を調製した。実施例１の（１）で調製した１．０ｍｇ／ｍＬヒドラジン化デキストランをヒドラジド基（アミノ基）の濃度が２５μＭになるように添加し、２５℃の暗所で１６時間攪拌した。ＤＭＡＢを８５ｍｇ添加し、２５℃の暗所で２時間攪拌した。次いで、１．５Ｍ　Ｔｒｉｓバッファー（ｐＨ９．０）を１０．１ｍＬ添加し、２５℃の暗所で２時間攪拌した。Ｌａｂｓｃａｌｅ　ＴＦＦ　Ｓｙｓｔｅｍ（Ｍｅｒｃｋ　Ｍｉｌｌｉｐｏｒｅ社製）に限外濾過モジュール（ペリコンＸＬ５０、Ｍｅｒｃｋ　Ｍｉｌｌｉｐｏｒｅ社製）を取り付け１５ｍＬまで濃縮し、０．１Ｍナトリウムリン酸バッファー（ｐＨ７．０）を用いたゲル濾過（Ｓｕｐｅｒｄｅｘ　２００ｐｇ）を行い、３．０ｍｇ／ｍＬのデキストラン－酵素結合体の溶液１４ｍＬを得た。 (2) Preparation of dextran-enzyme conjugate 30.0 mL of 10 mg/mL alkaline phosphatase (ALP-50, manufactured by Oriental Yeast Co., Ltd.) was subjected to gel filtration (Sephadex) using 0.1 M sodium phosphate buffer (pH 6.0). G-25), buffer exchange was performed to prepare 90.6 mL of a 3.0 mg/mL solution. Then 45.3 mL of 27 mM NaIO 4 was added and stirred for 3 min at 25° C. in the dark. Then, buffer exchange was performed by gel filtration (Sephadex G-25) using 0.1 M sodium phosphate buffer (pH 6.0) to prepare a 0.5 mg/mL solution. 1.0 mg/mL hydrazide dextran prepared in (1) of Example 1 was added so that the hydrazide group (amino group) concentration was 25 μM, 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. Then, 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.
　（３）デキストラン－酵素結合体のマレイミド－ＰＥＧ化
　実施例１の（２）で調製したデキストラン－酵素結合体に０．１Ｍナトリウムリン酸バッファー（ｐＨ７．０）を加えて２ｍｇ／ｍＬデキストラン－酵素結合体を７５０μＬ調製した。これにＤＭＳＯ中に溶解した２５０ｍＭのＳＭ（ＰＥＧ）４（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製、ＳＭ（ＰＥＧ）４）を８．３５μＬ添加、混合し、２５℃の暗所で１時間転倒混和した。反応後、ＰＤ－１０カラム（Ｓｅｐｈａｄｅｘ　Ｇ－２５）を用いて２０ｍＭ　ＥＤＴＡ・２Ｎａ、０．５％　ＣＨＡＰＳを含む０．１Ｍナトリウムリン酸バッファー（ｐＨ６．３）にバッファー交換を行った。バッファー交換後に遠心式フィルター（Ｍｅｒｃｋ社製、Ａｍｉｃｏｎ　Ｕｌｔｒａ　５０Ｋ）を用いてマレイミド－ＰＥＧ化デキストラン－酵素結合体の濃縮を行い、最終濃度を２ｍｇ／ｍＬに調整した。 (3) Maleimide-PEGylation of dextran-enzyme conjugate 0.1 M sodium phosphate buffer (pH 7.0) was added to the dextran-enzyme conjugate prepared in (2) of Example 1 to give 2 mg/mL dextran-enzyme. 750 μL of conjugate was prepared. To this, 8.35 μL of 250 mM SM(PEG) 4 (SM(PEG) 4 manufactured by Thermo Fisher Scientific) dissolved in DMSO was added, mixed, and mixed by inversion in a dark place at 25° C. for 1 hour. After the reaction, 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). After buffer exchange, 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.
　（４）レクチンのチオール化
　５ｍｇのノダフジレクチン（ＷＦＡ；ＶＥＣＴＯＲ社製）を２．５ｍＬの０．１Ｍナトリウムリン酸バッファー（ｐＨ７．０）中に溶解し、２ｍｇ／ｍＬ　ＷＦＡ溶液を得た。２．５ｍＬのＷＦＡ溶液に１００μＬの０．５Ｍ　ＥＤＴＡ・２Ｎａ（ｐＨ８．０）を添加して混和し、次いで、７５μＬの１０ｍｇ／ｍＬ　２－イミノチオラン塩酸塩溶液を添加し、２５℃の暗所で１時間転倒混和した。反応後、ＰＤ－１０カラム（Ｓｅｐｈａｄｅｘ　Ｇ－２５）を用いて２０ｍＭ　ＥＤＴＡ・２Ｎａ、０．５％　ＣＨＡＰＳを含む０．１Ｍナトリウムリン酸バッファー（ｐＨ６．３）にバッファー交換を行った。バッファー交換後のＷＦＡは６５０μｇ／ｍＬに調整した。 (4) Thiolation of Lectin 5 mg of Nodafuji lectin (WFA; manufactured by VECTOR) was dissolved in 2.5 mL of 0.1 M sodium phosphate buffer (pH 7.0) to obtain a 2 mg/mL WFA solution. Add 100 μL of 0.5 M EDTA.2Na (pH 8.0) to 2.5 mL of WFA solution and mix, then add 75 μL of 10 mg/mL 2-iminothiolane hydrochloride solution and incubate at 25° C. in the dark. Mixed by inversion for 1 hour. After the reaction, 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 μg/mL.
　（５）カップリング
　実施例１の（４）で得たチオール化して６５０μｇ／ｍＬに調整したＷＦＡ　２ｍＬに対して、実施例１の（３）で得たマレイミド－ＰＥＧ化デキストラン－酵素結合体の溶液（２ｍｇ／ｍＬ）を５００μＬ添加し、２５℃の暗所で１時間転倒混和し、ＷＦＡとデキストラン－酵素結合体とをカップリングさせた。反応後、２５μＬの２００ｍＭ　３－Ｍｅｒｃａｐｔｏ－１，２－ｐｒｏｐａｎｅｄｉｏｌを添加し、２５℃の暗所で３０分間転倒混和した。さらにその後、５０μＬの２００ｍＭ　２－Ｉｏｄｏａｃｅｔａｍｉｄｅを添加し、２５℃の暗所で３０分間転倒混和した。反応後の溶液は遠心式フィルター（Ｍｅｒｃｋ社製、Ａｍｉｃｏｎ　Ｕｌｔｒａ　５０Ｋ）を用いて濃縮した後にφ０．２２μｍフィルターを通過させ、ゲル濾過クロマトグラフィー（カラム：Ｓｕｐｅｒｏｓｅ　６　Ｉｎｃｒｅａｓｅ　１０／３００　ＧＬ、バッファー：０．１Ｍ　ＭＥＳ、０．５Ｍ　ＮａＣｌ、１ｍＭ　ＭｇＣｌ２、０．１ｍＭ　ＺｎＣｌ２、５ｍＭ　Ｇｌｕｃｏｓｅ、０．０５％　ＣＨＡＰＳ、ｐＨ６．８）によって精製し、最終的に３７３．９μｇ／ｍＬのブロック化標識レクチン（ＷＦＡ）（デキストラン－酵素－ＷＦＡ結合体）の溶液を２．０ｍＬ得た。 (5) Coupling The maleimide-PEGylated dextran-enzyme conjugate obtained in (3) of Example 1 was added to 2 mL of the WFA thiolated to 650 µg/mL obtained in (4) of Example 1. 500 μL of the solution (2 mg/mL) was added and mixed by inversion in the dark at 25° C. for 1 hour to couple WFA with the dextran-enzyme conjugate. After the reaction, 25 μL of 200 mM 3-Mercapto-1,2-propanediol was added and mixed by inversion for 30 minutes at 25° C. in the dark. After that, 50 μL of 200 mM 2-Iodoacetamide was added and mixed by inversion for 30 minutes in the dark at 25°C. After the reaction, the solution was concentrated using a centrifugal filter (Merck, Amicon Ultra 50K), passed through a φ0.22 μm filter, and subjected to gel filtration chromatography (column: Superose 6 Increase 10/300 GL, buffer: 0.000). 1 M MES, 0.5 M NaCl, 1 mM MgCl 2 , 0.1 mM ZnCl 2 , 5 mM Glucose, 0.05% CHAPS, pH 6.8) to a final concentration of 373.9 μg/mL blocked labeled lectin (WFA ) (dextran-enzyme-WFA conjugate) solution was obtained in 2.0 mL.
　（６）測定試薬の調製
　２４ｍｇの抗ＣＡ１９－９抗体結合磁性粒子（富士レビオ社製、以下単に「抗体結合粒子」）に対して６ｍＬの２０ｍＭ　過ヨウ素酸ナトリウム溶液（２０ｍＭ　ＮａＩＯ４、１００ｍＭ　ＮａＯＡｃ、１５０ｍＭ　ＮａＣｌ、ｐＨ５．５）を添加して混合し、４℃遮光下で３０分間転倒混和を行うことで粒子に結合した抗体の糖鎖を酸化した。酸化処理後の抗体結合粒子は６ｍＬの０．１Ｍ　リン酸ナトリウムバッファー（ｐＨ６．０）で３回洗浄した。洗浄した抗体結合粒子は１０ｍＭ　グリシンを含む０．１Ｍ　リン酸ナトリウムバッファー（ｐＨ６．０）に置換し、２５℃遮光下で１時間転倒混和を行うことで、抗体の糖鎖の酸化によって生じたアルデヒド基をグリシンでブロックした。さらに、反応後の抗体結合粒子液に１００μＬの１０ｍｇ／ｍＬ　ＤＭＡＢを添加して２５℃遮光下で３０分間転倒混和を行い、抗体の糖鎖由来のアルデヒド基とグリシンとの不安定な結合を安定化させた。安定化後の抗体結合粒子は０．６ｍＬの２％　ＢＳＡを含むバッファー（５０ｍＭ　ＭＥＳ、１ｍＭ　ＥＤＴＡ、１５０ｍＭ　ＮａＣｌ、２％　ＢＳＡ、０．１％　ＰｒｏＣｌｉｎ　３００、ｐＨ６．０）で３回洗浄し、同バッファー、３７℃の条件で１６時間転倒混和することにより、ＢＳＡを物理吸着させた。ＢＳＡを物理吸着させた抗体結合粒子は保存バッファー（５０ｍＭ　Ｔｒｉｓ、２％　ＢＳＡ、１５０ｍＭ　ＮａＣｌ、１ｍＭ　ＥＤＴＡ、０．１％　ＰｒｏＣｌｉｎ　３００、ｐＨ７．２）で３回洗浄し、同バッファー中に４℃で保存した。得られた酸化処理済みの抗ＣＡ１９－９抗体結合磁性粒子を抗体結合粒子の濃度が０．００５％となるように５０ｍＭ　Ｔｒｉｓをベースとする溶液に希釈して、酸化処理済み抗ＣＡ１９－９抗体結合粒子液を調製した。 (6) Preparation of measurement reagent 6 mL of 20 mM sodium periodate solution (20 mM NaIO 4 , 100 mM NaOAc, 20 mM NaIO 4 , 100 mM NaOAc, 150 mM NaCl, pH 5.5) was added and mixed, and the sugar chains of the antibody bound to the particles were oxidized by inversion mixing for 30 minutes at 4° C. in the dark. After the oxidation treatment, the antibody-bound particles were washed three times with 6 mL of 0.1 M sodium phosphate buffer (pH 6.0). 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. Furthermore, 100 μL 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. made it 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.
　また、実施例１の（５）で得られたブロック化標識レクチン（ＷＦＡ）を、濃度が０．５μｇ／ｍＬとなるように５０ｍＭ　ＭＥＳをベースとする溶液に希釈して標識体液を調製した。 In addition, the blocked labeled lectin (WFA) obtained in (5) of Example 1 was diluted to a concentration of 0.5 μg/mL in a 50 mM MES-based solution to prepare a labeled body fluid.
　（７）血清検体に含まれるＣＡ１９－９及びＷＦＡ結合型糖鎖付加ＣＡ１９－９（ＣＡ１９－９／ＷＦＡ）の測定
　健常人から採取した血清検体１０例（健常人群：健常人１～１０）、前立腺肥大患者から採取した血清検体１０例（前立腺肥大群：前立腺肥大１～１０）、前立腺癌患者から採取した血清検体１５例（前立腺癌群：前立腺癌１～１５）、及び、大腸がん患者から採取した血清検体１５例（大腸がん群：大腸がん１～１５）について、それぞれ、検体希釈液（富士レビオ社製）を用いて体積比で１／１０濃度に希釈した。 (7) Measurement of CA19-9 and WFA-linked glycosylated CA19-9 (CA19-9/WFA) 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).
　希釈した各検体について、ルミパルス（登録商標）Ｌ－２４００（富士レビオ社製）を用いて、同検体に含まれるＷＦＡ結合型糖鎖付加ＣＡ１９－９（ＣＡ１９－９／ＷＦＡ）の測定を行った。すなわち、５０μＬの各希釈検体溶液を、実施例１の（６）で調製した酸化処理済み抗ＣＡ１９－９抗体結合粒子液５０μＬと混和し、３７℃で８分間反応させた。次いで、磁性粒子を集磁し、ルミパルス（登録商標）洗浄液（富士レビオ社製）で５回洗浄した。次いで、実施例１の（６）で調製した標識体液をそれぞれ５０μＬ添加し、３７℃で８分間反応させた。次いで、磁性粒子を集磁し、５回洗浄した後、ＡＭＰＰＤ（３－（２’－スピロアダマンタン）－４－メトキシ－４－（３’－ホスホリルオキシ）フェニル－１，２－ジオキセタン・２ナトリウム塩）を含むルミパルス（登録商標）基質液（富士レビオ社製）を５０μＬ添加し、３７℃で４分間反応させた。磁性粒子に結合したブロック化標識レクチン（ＷＦＡ）のアルカリホスファターゼの触媒作用によりＡＭＰＰＤが分解されることで放出される、波長４６３ｎｍに極大吸収を有する光の発光強度（カウント）を、ルミパルス（登録商標）Ｌ－２４００（富士レビオ社製）を用いて計測し、測定結果とした。 For each diluted sample, WFA-linked glycosylated CA19-9 (CA19-9/WFA) contained in the same sample was measured using Lumipulse (registered trademark) L-2400 (manufactured by Fujirebio). . That is, 50 μL of each diluted sample solution was mixed with 50 μL 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 μL of the labeled body fluid prepared in (6) of Example 1 was added to each well, and reacted at 37° C. for 8 minutes. Next, the magnetic particles were collected, washed five times, and then AMPPD (3-(2′-spiroadamantane)-4-methoxy-4-(3′-phosphoryloxy)phenyl-1,2-dioxetane disodium 50 μL of LUMIPULSE (registered trademark) substrate solution (manufactured by Fujirebio) containing salt) was added and reacted at 37° C. for 4 minutes. The luminous intensity (count) of light having a maximum absorption at a wavelength of 463 nm, which is emitted by the decomposition of AMPPD by the catalytic action of alkaline phosphatase of blocked labeled lectin (WFA) bound to magnetic particles, was measured using Lumipulse (registered trademark). ) L-2400 (manufactured by Fujirebio Co., Ltd.) was used for measurement, and the measurement results were obtained.
　また、上記で希釈した各検体に含まれるＣＡ１９－９は、抗ＣＡ１９－９抗体結合粒子及びアルカリホスファターゼ（ＡＬＰ）標識抗ＣＡ１９－９抗体を備えるルミパルスプレスト（登録商標）ＣＡ１９－９（富士レビオ社製）を用いて、ルミパルス（登録商標）Ｌ－２４００（富士レビオ社製）によって測定した。測定結果は、上記と同様に基質（ＡＭＰＰＤ）の発光強度（カウント）で出力した。各検体における測定結果を下記の表１に示す。なお、表示の結果は、二重測定の平均値を示す。 In addition, 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.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　（８）健常人、前立腺肥大、及び前立腺癌の各群間での測定値の比較
　表１の結果について、健常人群と前立腺癌群との間で統計学的に有意な差が認められるか検討した。図１に健常人群及び前立腺癌群のＣＡ１９－９の測定結果を、図２に健常人群及び前立腺癌群のＣＡ１９－９／ＷＦＡの測定結果を、図３に前立腺肥大群及び前立腺癌群のＣＡ１９－９の測定結果を、図４に前立腺肥大群及び前立腺癌群のＣＡ１９－９／ＷＦＡの測定結果を、それぞれ示す。 (8) Comparison of measured values among healthy subjects, prostatic hyperplasia, and prostate cancer groups Regarding the results of Table 1, examine whether there is a statistically significant difference between the healthy subjects group and the prostate cancer group. did. 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, and FIG. -9, and FIG. 4 shows the measurement results of CA19-9/WFA in the prostatic hyperplasia group and prostatic cancer group, respectively.
　Ｗｉｌｃｏｘｏｎ検定によって検定を行ったところ、健常人群と前立腺癌群との間にはＣＡ１９－９の測定値については有意な差が認められなかった（図１、ｐ＝０．７６０３）のに対して、ＣＡ１９－９／ＷＦＡの測定値については有意な差が認められた（図２、ｐ＝０．００１０）。同様に、前立腺肥大群と前立腺癌群との間でも、ＣＡ１９－９の測定値については有意な差が認められなかった（図３、ｐ＝０．６７７４）のに対して、ＣＡ１９－９／ＷＦＡの測定値については有意な差が認められた（図４、ｐ＝０．００１７）。 When tested by the Wilcoxon test, there was no significant difference in CA19-9 measurement values between the healthy subject group and the prostate cancer group (Fig. 1, p = 0.7603). , CA19-9/WFA measurements were significantly different (FIG. 2, p=0.0010). Similarly, between the prostatic hyperplasia group and the prostatic cancer group, there was no significant difference in the measured value of CA19-9 (Fig. 3, p = 0.6774), whereas CA19-9/ A significant difference was observed for WFA measurements (Figure 4, p=0.0017).
　また、図５に、ＣＡ１９－９の測定値とＣＡ１９－９／ＷＦＡの測定値との関係を示す。図５に示されるように、今回の結果からは、ＣＡ１９－９の測定値とＣＡ１９－９／ＷＦＡの値との間に明確な相関関係は示されなかった（回帰直線：ＣＡ１９－９／ＷＦＡ（カウント）＝６６２３８．７８－１．６９９７２３６×ＣＡ１９－９（カウント）、Ｒ２＝０．００９３６９、分散分析のｐ値＝０．５８０２）。このことから、ＣＡ１９－９／ＷＦＡの測定値は単にＣＡ１９－９の存在量を示すものではなく、ＣＡ１９－９とは異なるバイオマーカーであることが示された。 Further, FIG. 5 shows the relationship between the measured values of CA19-9 and the measured values of CA19-9/WFA. As shown in FIG. 5, the results of this time did not show a clear correlation between the measured values of CA19-9 and the values of CA19-9/WFA (regression line: CA19-9/WFA (counts) = 66238.78-1.6997236 x CA19-9 (counts), R 2 = 0.009369, ANOVA p-value = 0.5802). This indicates that the measured value of CA19-9/WFA is not simply indicative of the abundance of CA19-9, but is a biomarker different from CA19-9.
　以上より、ＣＡ１９－９／ＷＦＡの測定は、健常人と前立腺癌患者との判別、又は、前立腺肥大患者と前立腺癌患者との判別の新規の指標となることが示された。 From the above, it was shown that the measurement of 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.
　（９）ＣＡ１９－９／ＷＦＡの測定値とＰＳＡ測定値との比較
　先ず、実施例１の（７）で用いたものと同様の前立腺肥大患者から採取した血清検体１０例、前立腺癌患者から採取した血清検体１５例について、検体中に含まれるＰＳＡ量を測定した。測定は、各検体について、希釈は行わず、抗ＰＳＡ抗体結合粒子及びアルカリホスファターゼ（ＡＬＰ）標識抗ＰＳＡ抗体を備えるルミパルスプレスト（登録商標）ＰＳＡ（富士レビオ社製）を用いて、ルミパルス（登録商標）Ｌ－２４００（富士レビオ社製）によって測定した。測定結果は、ＰＳＡ濃度（ｎｇ／ｍＬ）で出力した。各検体における測定結果を下記の表２に示す。 (9) Comparison of measured values of CA19-9/WFA and measured values of PSA First, 10 serum specimens collected from patients with benign prostatic hyperplasia similar to those used in (7) of Example 1, collected from patients with prostate cancer. The amount of PSA contained in the 15 serum specimens obtained was measured. Measurement was carried out for each sample without dilution, using Lumipulse Presto (registered trademark) PSA (manufactured by Fujirebio) equipped with anti-PSA antibody-binding particles and alkaline phosphatase (ALP)-labeled anti-PSA antibody, Lumipulse (registered trademark) ) Measured by L-2400 (manufactured by Fujirebio). The measurement results were output as PSA concentrations (ng/mL). The measurement results for each sample are shown in Table 2 below.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　次いで、表２の結果に対して、ＰＳＡ測定値の一般的なカットオフ値である４ｎｇ／ｍＬを用い、カットオフ値を上回る検体を陽性とした場合の前立腺癌患者の検出能力を試験した。その結果、前記カットオフ値で陽性と判定されたのは前立腺癌群では１５検体中４検体であり、２６．７％であった。また、前立腺肥大群では１０検体中１検体が前記カットオフ値で陽性と判定され、擬陽性となった。 Next, for the results in Table 2, a general cutoff value of 4 ng/mL for PSA measurement was used to test the ability to detect prostate cancer patients when samples exceeding the cutoff value were considered positive. As a result, 4 out of 15 specimens in the prostate cancer group, or 26.7%, were determined to be positive at the cut-off value. In addition, in the prostatic hyperplasia group, 1 out of 10 specimens was determined to be positive at the cutoff value, which was a false positive.
　他方、ＣＡ１９－９／ＷＦＡの測定値（表１）についても、健常人群の測定値からカットオフ値を算出し、カットオフ値を上回る検体を陽性とした場合の前立腺癌患者の検出能力を試験した。カットオフ値は、健常人群の測定値の平均に健常人群の測定値の標準偏差の値に２を乗じた値を加算して、６７，６５９カウント以下と算出された。その結果、このカットオフ値で陽性と判定されたのは前立腺癌群では１５検体中１０検体であり、６６．７％であった。また、健常人群及び前立腺肥大群において擬陽性と判定されたのは、両群を合わせた２０検体中１検体であり、特異度９５．０％となった。 On the other hand, for the measured values of CA19-9/WFA (Table 1), 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. In the healthy subject group and the prostatic hyperplasia group, 1 specimen out of 20 specimens in both groups was determined to be false positive, giving a specificity of 95.0%.
　さらに、図６に、ＰＳＡの測定値とＣＡ１９－９／ＷＦＡの測定値との関係を示す。図６に示されるように、今回の結果からは、ＰＳＡの測定値とＣＡ１９－９／ＷＦＡの測定値との間に明確な相関関係は示されなかった（回帰直線：ＣＡ１９－９／ＷＦＡ（カウント）＝６７１６３．７９９－１４３３．０７７４×ＰＳＡ（ｎｇ／ｍＬ）、Ｒ２＝０．０１６６７、分散分析のｐ値＝０．５３８４）。 Furthermore, FIG. 6 shows the relationship between the measured values of PSA and the measured values of CA19-9/WFA. As shown in FIG. 6, the results of this time did not show a clear correlation between the measured values of PSA and the measured values of CA19-9/WFA (regression line: CA19-9/WFA ( counts) = 67163.799-1433.0774 x PSA (ng/mL), R 2 = 0.01667, ANOVA p-value = 0.5384).
　以上より、ＣＡ１９－９／ＷＦＡの測定による前立腺癌の検出能力（感度、特異度）はＰＳＡと比較しても十分に高く、さらに、ＰＳＡとは独立した診断指標として利用できることが示された。 From the above, it was shown that the detection ability (sensitivity, specificity) of prostate cancer by measuring CA19-9/WFA is sufficiently high compared to PSA, and that it can be used as a diagnostic index independent of PSA.
　（１０）健常人群と大腸がん群との間での測定値の比較
　表１の結果について、健常人群と大腸がん群との間で統計学的に有意な差が認められるか検討した。図７に健常人群及び大腸がん群のＣＡ１９－９の測定結果を、図８に健常人群及び大腸がん群のＣＡ１９－９／ＷＦＡの測定結果を、図９にＣＡ１９－９の測定値とＣＡ１９－９／ＷＦＡの測定値との関係を、それぞれ示す。 (10) Comparison of measured values between healthy subject group and colorectal cancer group Regarding the results in Table 1, it was examined whether a statistically significant difference was observed between the healthy subject group and the colorectal cancer group. 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, and FIG. Relations with CA19-9/WFA measurements are shown, respectively.
　Ｗｉｌｃｏｘｏｎ検定によって検定を行ったところ、健常人群と大腸がん群との間にはＣＡ１９－９の測定値については有意な差が認められなかった（図７、ｐ＝０．１７１４）のに対して、ＣＡ１９－９／ＷＦＡの測定値については有意な差が認められた（図８、ｐ＝０．０１８４）。また、ＣＡ１９－９／ＷＦＡの測定値について、実施例１の（９）で算出されたカットオフ値を上回る検体を陽性とした場合の前立腺癌患者の検出能力を試験したところ、前記カットオフ値で陽性と判定されたのは大腸がん群で１５検体中１１検体であり、７３．３％であった。他方、健常人群の中で偽陽性と判定されたものは無かった。このことから、ＣＡ１９－９／ＷＦＡの測定による大腸がんの検出能力（感度、特異度）は十分に高いことが示された。 When tested by the Wilcoxon test, there was no significant difference in CA19-9 measurement values between the healthy subject group and the colorectal cancer group (Fig. 7, p = 0.1714). , there was a significant difference in the CA19-9/WFA measurements (Fig. 8, p = 0.0184). In addition, regarding the measured value of CA19-9/WFA, the ability to detect prostate cancer patients was tested when samples exceeding the cutoff value calculated in (9) of Example 1 were positive, and the cutoff value In the colorectal cancer group, 11 out of 15 specimens (73.3%) were positive. On the other hand, none of the healthy subjects were determined to be false positives. This indicates that the CA19-9/WFA measurement has sufficiently high detectability (sensitivity, specificity) for colorectal cancer.
　さらに、図９に示されるように、今回の結果からは、ＣＡ１９－９の測定値とＣＡ１９－９／ＷＦＡの測定値との間に明確な相関関係は示されなかった（回帰直線：ＣＡ１９－９／ＷＦＡ（カウント）＝７００６８．６８９＋１．８７３０９５４×ＣＡ１９－９（カウント）、Ｒ２＝０．０４１３９２、分散分析のｐ値＝０．３２９３）。このことから、ＣＡ１９－９／ＷＦＡの測定値は単にＣＡ１９－９の存在量を示すものではなく、ＣＡ１９－９とは異なるバイオマーカーであることが示された。 Furthermore, as shown in FIG. 9, the results of this time did not show a clear correlation between the measured values of CA19-9 and the measured values of CA19-9/WFA (regression line: CA19- 9/WFA(counts)=70068.689+1.8730954×CA19-9(counts), R 2 =0.041392, ANOVA p-value=0.3293). This indicates that the measured value of CA19-9/WFA is not simply indicative of the abundance of CA19-9, but is a biomarker different from CA19-9.
　以上より、ＣＡ１９－９／ＷＦＡの測定は、健常人と大腸がん患者との判別の新規の指標となることが示された。 From the above, it was shown that the measurement of CA19-9/WFA is a novel index for discriminating between healthy subjects and colorectal cancer patients.
　（比較例１）　前立腺癌及び大腸がん以外の癌に対するＣＡ１９－９／ＷＦＡの反応性の検証
　ＣＡ１９－９／ＷＦＡの測定値が高値であった前立腺癌及び大腸がん以外のがんの一例として、乳癌患者から採取された血清検体１５例（乳癌群：乳癌１～１５）について、実施例１の（７）と同様にしてＣＡ１９－９／ＷＦＡを測定した。各検体における測定結果を下記の表３に示す。 (Comparative Example 1) Verification of reactivity of CA19-9/WFA against cancers other than prostate cancer and colorectal cancer An example of cancers other than prostate cancer and colorectal cancer in which the measured value of CA19-9/WFA was high As such, 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.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　表３に示した乳癌群の測定結果及び表１に示した健常人群の測定結果について、健常人群と乳癌群との間で統計学的に有意な差が認められるか検討した。図１０に健常人群及び乳癌群のＣＡ１９－９／ＷＦＡの測定結果を示す。Ｗｉｌｃｏｘｏｎ検定によって検定を行ったところ、健常人群と乳癌群との間にはＣＡ１９－９／ＷＦＡの測定値について有意な差が認められなかった（図１０、ｐ＝０．０８０６）。この結果から、ＣＡ１９－９／ＷＦＡの測定値は全ての癌に対して無差別に高値を示すものではなく、癌種に特異的に高値となることが示された。 Regarding the measurement results of the breast cancer group shown in Table 3 and the measurement results of the healthy subject group shown in Table 1, we examined whether there was a statistically significant difference between the healthy subject group and the breast cancer group. FIG. 10 shows the measurement results of CA19-9/WFA in the healthy subject group and the breast cancer group. Wilcoxon's test showed no significant difference in CA19-9/WFA measurements between the healthy subject group and the breast cancer group (FIG. 10, p=0.0806). These results indicated that the CA19-9/WFA measured value did not indiscriminately show high values in all cancers, but showed high values specifically in cancer types.
　（比較例２）　ＷＦＡ以外のレクチンを用いたブロック化標識レクチンによる糖鎖付加ＣＡ１９－９の測定
　ＷＦＡに代えて、α２，３結合型シアル酸を認識するイヌエンジュレクチン（ＭＡＭ、Ｊ－ケミカル社製）を結合させたブロック化標識レクチン（ＭＡＭ）を調製した。すなわち、ＷＦＡに代えてＭＡＭを用い、かつ、デキストランを分子量５００ｋのもの（Ｆｌｕｋａ社製）に変更した以外は、実施例１の（１）～（５）と同様の方法で、最終的に３８２．１μｇ／ｍＬのブロック化標識レクチン（ＭＡＭ）（デキストラン－酵素－ＭＡＭ結合体）の溶液を２．０ｍＬ得た。なお、デキストランのサイズは、レクチンの糖鎖型特異性に影響するものではない。 (Comparative 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 μg/mL blocked labeled lectin (MAM) (dextran-enzyme-MAM conjugate) was obtained. The size of dextran does not affect the specificity of lectin sugar chains.
　ブロック化標識レクチン（ＷＦＡ）に代えて、得られたブロック化酵素レクチン（ＭＡＭ）を用いたこと以外は、実施例１の（６）～（７）と同様の方法で、実施例１の（７）で用いたものと同様の健常人から採取した血清検体１０例（健常人群：健常人１～１０）、前立腺癌患者から採取した血清検体１５例（前立腺癌群：前立腺癌１～１５）、及び、大腸がん患者から採取した血清検体１５例（大腸がん群：大腸がん１～１５）について、各検体に含まれるＭＡＭ結合型糖鎖付加ＣＡ１９－９（ＣＡ１９－９／ＭＡＭ）の測定を行った。各検体における測定結果を下記の表４に示す。 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.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　表４の結果について、健常人群と前立腺癌群又は大腸がん群との間で統計学的に有意な差が認められるか検討した。図１１に健常人群及び前立腺癌群のＣＡ１９－９／ＭＡＭの測定結果を、図１２に健常人群及び大腸がん群のＣＡ１９－９／ＭＡＭの測定結果を、それぞれ示す。Ｗｉｌｃｏｘｏｎ検定によって検定を行ったところ、健常人群と前立腺癌群との間にはＣＡ１９－９／ＭＡＭの測定値について有意な差が認められなかった（図１１、ｐ＝０．１９２４）。また、同様に、健常人群と大腸がん群との間でもＣＡ１９－９／ＭＡＭの測定値について有意な差が認められなかった（図１２、ｐ＝０．３３４０）。以上より、ＣＡ１９－９とＷＦＡ結合型糖鎖との組み合わせを測定するＣＡ１９－９／ＷＦＡの測定は、健常人と前立腺癌患者又は大腸がん患者との判別に特異的な新規の指標となることが示された。 Regarding the results in Table 4, we examined whether there was a statistically significant difference between the healthy subject group and the prostate cancer group or colon cancer group. FIG. 11 shows the CA19-9/MAM measurement results for the healthy subject group and prostate cancer group, and FIG. 12 shows the CA19-9/MAM measurement results for the healthy subject group and colon cancer group. Wilcoxon's test showed no significant difference in CA19-9/MAM measurements between the healthy subject group and the prostate cancer group (FIG. 11, p=0.1924). Similarly, no significant difference was observed in the measured values of CA19-9/MAM between the healthy subject group and the colorectal cancer group (Fig. 12, p = 0.3340). Based on the above, 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.
　（実施例２）　ブロック化標識レクチン（ＭＡＭ）を用いた血清検体に含まれるＭＡＭ結合型糖鎖付加ＣＥＡ（ＣＥＡ／ＭＡＭ）の測定
　（１）ヒドラジン化デキストランの調製
　デキストランを分子量を２５０ｋのデキストラン（ＣａｒｂｏＭｅｒ社製）に代えたこと以外は実施例１の（１）と同様にして、ヒドラジン化デキストランの溶液（デキストラン濃度：１．０ｍｇ／ｍＬを得た）。 (Example 2) Measurement of MAM-linked glycosylated CEA (CEA/MAM) contained in a serum sample using blocked labeled lectin (MAM) (1) Preparation of hydrazinated dextran A solution of hydrazinated dextran (dextran concentration: 1.0 mg/mL) was obtained in the same manner as in (1) of Example 1, except that it was replaced with CarboMer).
　（２）デキストラン－酵素結合体の調製
　実施例１の（２）と同様にして、３．０ｍｇ／ｍＬのデキストラン－酵素結合体の溶液１４ｍＬを得た。 (2) Preparation of dextran-enzyme conjugate In the same manner as in Example 1 (2), 14 mL of a 3.0 mg/mL dextran-enzyme conjugate solution was obtained.
　（３）デキストラン－酵素結合体のマレイミド－ＰＥＧ化
　実施例１の（３）と同様にして、マレイミド－ＰＥＧ化デキストラン－酵素結合体の溶液（最終濃度：２ｍｇ／ｍＬ）を得た。 (3) Maleimide-PEGylation of dextran-enzyme conjugate A solution of maleimide-PEGylated dextran-enzyme conjugate (final concentration: 2 mg/mL) was obtained in the same manner as in Example 1 (3).
　（４）レクチンのチオール化
　６ｍｇのイヌエンジュレクチン（ＭＡＭ；Ｊ－ケミカル社製）を３ｍＬの０．１Ｍナトリウムリン酸バッファー（ｐＨ７．０）中に溶解し、２ｍｇ／ｍＬ　ＭＡＭ溶液を得た。２．５ｍＬのＭＡＭ溶液に１００μＬの０．５Ｍ　ＥＤＴＡ・２Ｎａ（ｐＨ８．０）を添加して混和し、次いで、７５μＬの１０ｍｇ／ｍＬ　２－イミノチオラン塩酸塩溶液を添加し、２５℃の暗所で１時間転倒混和した。反応後、ＰＤ－１０カラム（Ｓｅｐｈａｄｅｘ　Ｇ－２５）を用いて２０ｍＭ　ＥＤＴＡ・２Ｎａ、０．５％　ＣＨＡＰＳを含む０．１Ｍナトリウムリン酸バッファー（ｐＨ６．３）にバッファー交換を行った。バッファー交換後のＭＡＭは６５０μｇ／ｍＬに調整した。 (4) Thiolation of Lectin 6 mg of canine endurance lectin (MAM; manufactured by J-Chemical) was dissolved in 3 mL of 0.1 M sodium phosphate buffer (pH 7.0) to obtain a 2 mg/mL MAM solution. Add 100 μL of 0.5 M EDTA.2Na (pH 8.0) to 2.5 mL of MAM solution and mix, then add 75 μL of 10 mg/mL 2-iminothiolane hydrochloride solution and incubate at 25° C. in the dark. Mixed by inversion for 1 hour. After the reaction, 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 μg/mL.
　（５）カップリング
　実施例２の（４）で得たチオール化して６５０μｇ／ｍＬに調整したＭＡＭ　２ｍＬに対して、実施例２の（３）で得たマレイミド－ＰＥＧ化デキストラン－酵素結合体の溶液（２ｍｇ／ｍＬ）を５００μＬ添加し、２５℃の暗所で１時間転倒混和し、ＭＡＭとデキストラン－酵素結合体とをカップリングさせた。反応後、２５μＬの２００ｍＭ　３－Ｍｅｒｃａｐｔｏ－１，２－ｐｒｏｐａｎｅｄｉｏｌを添加し、２５℃の暗所で３０分間転倒混和した。さらにその後、５０μＬの２００ｍＭ　２－Ｉｏｄｏａｃｅｔａｍｉｄｅを添加し、２５℃の暗所で３０分間転倒混和した。反応後の溶液は遠心式フィルター（Ｍｅｒｃｋ社製、Ａｍｉｃｏｎ　Ｕｌｔｒａ　５０Ｋ）を用いて濃縮した後にφ０．２２μｍフィルターを通過させ、ゲル濾過クロマトグラフィー（カラム：Ｓｕｐｅｒｏｓｅ　６　Ｉｎｃｒｅａｓｅ　１０／３００　ＧＬ、バッファー：０．１Ｍ　ＭＥＳ、０．５Ｍ　ＮａＣｌ、１ｍＭ　ＭｇＣｌ２、０．１ｍＭ　ＺｎＣｌ２、５ｍＭ　Ｇｌｕｃｏｓｅ、０．０５％　ＣＨＡＰＳ、ｐＨ６．８）によって精製し、最終的に３８２．１μｇ／ｍＬのブロック化標識レクチン（ＭＡＭ）（デキストラン－酵素－ＭＡＭ結合体）の溶液を２．０ｍＬ得た。 (5) Coupling Maleimide-PEGylated dextran-enzyme conjugate obtained in (3) of Example 2 was added to 2 mL of thiolated MAM adjusted to 650 μg/mL obtained in (4) of Example 2. 500 μL of solution (2 mg/mL) was added and mixed by inversion in the dark at 25° C. for 1 hour to couple MAM and dextran-enzyme conjugate. After the reaction, 25 μL of 200 mM 3-Mercapto-1,2-propanediol was added and mixed by inversion for 30 minutes at 25° C. in the dark. After that, 50 μL of 200 mM 2-Iodoacetamide was added and mixed by inversion for 30 minutes in the dark at 25°C. After the reaction, the solution was concentrated using a centrifugal filter (Merck, Amicon Ultra 50K), passed through a φ0.22 μm filter, and subjected to gel filtration chromatography (column: Superose 6 Increase 10/300 GL, buffer: 0.000). 1 M MES, 0.5 M NaCl, 1 mM MgCl 2 , 0.1 mM ZnCl 2 , 5 mM Glucose, 0.05% CHAPS, pH 6.8) to give a final concentration of 382.1 μg/mL blocked labeled lectin (MAM ) (dextran-enzyme-MAM conjugate) solution was obtained in 2.0 mL.
　（６）測定試薬の調製
　２４ｍｇの抗ＣＥＡ抗体結合磁性粒子（富士レビオ社製）に対して６ｍＬの２０ｍＭ　過ヨウ素酸ナトリウム溶液（２０ｍＭ　ＮａＩＯ４、１００ｍＭ　ＮａＯＡｃ、１５０ｍＭ　ＮａＣｌ、ｐＨ５．５）を添加して混合し、４℃遮光下で３０分間転倒混和を行うことで粒子に結合した抗体の糖鎖を酸化した。酸化処理後の粒子は６ｍＬの０．１Ｍ　リン酸ナトリウムバッファー（ｐＨ６．０）で３回洗浄した。洗浄した粒子は１０ｍＭ　グリシンを含む０．１Ｍ　リン酸ナトリウムバッファー（ｐＨ６．０）に置換し、２５℃遮光下で１時間転倒混和を行うことで、抗体の糖鎖の酸化によって生じたアルデヒド基をグリシンでブロックした。さらに、反応後の抗体結合粒子液に１００μＬの１０ｍｇ／ｍＬ　ＤＭＡＢを添加して２５℃遮光下で３０分間転倒混和を行い、抗体糖鎖由来のアルデヒド基とグリシンとの不安定な結合を安定化させた。反応後の粒子は０．６ｍＬの２％　ＢＳＡを含むバッファー（５０ｍＭ　ＭＥＳ、１ｍＭ　ＥＤＴＡ、１５０ｍＭ　ＮａＣｌ、２％　ＢＳＡ、０．１％　ＰｒｏＣｌｉｎ　３００、ｐＨ６．０）で３回洗浄し、同バッファー、３７℃の条件で１６時間転倒混和することにより、ＢＳＡを物理吸着させた。ＢＳＡを物理吸着させた抗体結合粒子は保存バッファー（５０ｍＭ　Ｔｒｉｓ、２％　ＢＳＡ、１５０ｍＭ　ＮａＣｌ、１ｍＭ　ＥＤＴＡ、０．１％　ＰｒｏＣｌｉｎ　３００、ｐＨ７．２）で３回洗浄し、同バッファー中に４℃で保存した。得られた酸化処理済み抗ＣＥＡ抗体結合磁性粒子を抗体結合粒子の濃度が０．００５％となるように５０ｍＭ　Ｔｒｉｓをベースとする溶液に希釈して、酸化処理済み抗ＣＥＡ抗体結合粒子液を調製した。 (6) Preparation of Measurement Reagent 6 mL of 20 mM sodium periodate solution (20 mM NaIO 4 , 100 mM NaOAc, 150 mM NaCl, pH 5.5) was added to 24 mg of anti-CEA antibody-bound magnetic particles (manufactured by Fujirebio). The particles were mixed by mixing under a light-shielding condition at 4° C. for 30 minutes by inversion mixing to oxidize the sugar chains of the antibody bound to the particles. The oxidized particles were washed three times with 6 mL of 0.1 M sodium phosphate buffer (pH 6.0). 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 μL 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. let me After the reaction, 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 ℃. 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.
　また、実施例２の（５）で得られたブロック化標識レクチン（ＭＡＭ）を、濃度が０．５μｇ／ｍＬとなるように５０ｍＭ　ＭＥＳをベースとする溶液に希釈して標識体液を調製した。 In addition, 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 μg/mL to prepare a labeled body fluid.
　（７）血清検体に含まれるＣＥＡ及びＭＡＭ結合型糖鎖付加ＣＥＡ（ＣＥＡ／ＭＡＭ）の測定
　健常人から採取した血清検体１０例（健常人群：健常人１～１０）、前立腺肥大患者から採取した血清検体１０例（前立腺肥大群：前立腺肥大１～１０）、及び、前立腺癌患者から採取した血清検体１５例（前立腺癌群：前立腺癌１～１５）について、それぞれ、検体希釈液（富士レビオ社製）を用いて体積比で１／１０濃度に希釈した。 (7) Measurement of CEA and MAM-linked glycosylated CEA (CEA/MAM) contained in serum specimens 10 serum specimens collected from healthy subjects (group of healthy subjects: 1 to 10 healthy subjects) and from patients with benign prostatic hyperplasia For 10 serum specimens (prostate hypertrophy group: prostatic hyperplasia 1 to 10) and 15 serum specimens collected from prostate cancer patients (prostate cancer group: prostate cancer 1 to 15), respectively, a sample diluent (Fujirebio Co., Ltd.) (manufactured) to a concentration of 1/10 by volume.
　希釈した各検体について、ルミパルス（登録商標）Ｌ－２４００（富士レビオ社製）を用いて、血清検体に含まれるＭＡＭ結合型糖鎖付加ＣＥＡ（ＣＥＡ／ＭＡＭ）の測定を行った。すなわち、５０μＬの各希釈検体溶液を、実施例２の（６）で調製した酸化処理済み抗ＣＥＡ抗体結合粒子液５０μＬと混和し、３７℃で８分間反応させた。次いで、磁性粒子を集磁し、ルミパルス（登録商標）洗浄液（富士レビオ社製）で５回洗浄した。次いで、実施例２の（６）で調製した標識体液をそれぞれ５０μＬ添加し、３７℃で８分間反応させた。次いで、磁性粒子を集磁し、５回洗浄した後、ＡＭＰＰＤ（３－（２’－スピロアダマンタン）－４－メトキシ－４－（３’－ホスホリルオキシ）フェニル－１，２－ジオキセタン・２ナトリウム塩）を含むルミパルス（登録商標）基質液（富士レビオ社製）を５０μＬ添加し、３７℃で４分間反応させた。磁性粒子に結合したブロック化標識レクチン（ＭＡＭ）のアルカリホスファターゼの触媒作用によりＡＭＰＰＤが分解されることで放出される、波長４６３ｎｍに極大吸収を有する光の発光強度（カウント）を、ルミパルス（登録商標）Ｌ－２４００（富士レビオ社製）を用いて計測し、測定結果とした。 For each diluted sample, 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 μL of each diluted sample solution was mixed with 50 μL 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 μL 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. Next, the magnetic particles were collected, washed five times, and then AMPPD (3-(2′-spiroadamantane)-4-methoxy-4-(3′-phosphoryloxy)phenyl-1,2-dioxetane disodium 50 μL of LUMIPULSE (registered trademark) substrate solution (manufactured by Fujirebio) containing salt) was added and reacted at 37° C. for 4 minutes. The luminescence intensity (count) of light having a maximum absorption at a wavelength of 463 nm, which is emitted by the decomposition of AMPPD by the catalytic action of alkaline phosphatase of blocked labeled lectin (MAM) bound to magnetic particles, was measured using Lumipulse (registered trademark). ) L-2400 (manufactured by Fujirebio Co., Ltd.) was used for measurement, and the measurement results were obtained.
　また、上記で希釈した各検体に含まれるＣＥＡは、抗ＣＥＡ抗体結合粒子及びアルカリホスファターゼ（ＡＬＰ）標識抗ＣＥＡ抗体を備えるルミパルスプレスト（登録商標）ＣＥＡ（富士レビオ社製）を用いて、上記と同様にルミパルス（登録商標）Ｌ－２４００（富士レビオ社製）によって測定した。測定結果は、上記と同様に基質（ＡＭＰＰＤ）の発光強度（カウント）で出力した。各検体における測定結果を下記の表５に示す。なお、表示の結果は、二重測定の平均値を示す。 In addition, the 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. Similarly, it was 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 sample are shown in Table 5 below. The results shown are the average values of duplicate measurements.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　（８）健常人、前立腺肥大、及び前立腺癌の各群間での測定値の比較
　表５の結果について、健常人群と前立腺癌群との間で統計学的に有意な差が認められるか検討した。図１３に健常人群及び前立腺癌群のＣＥＡの測定結果を、図１４に健常人群及び前立腺癌群のＣＥＡ／ＭＡＭの測定結果を、図１５に前立腺肥大群及び前立腺癌群のＣＥＡの測定結果を、図１６に前立腺肥大群及び前立腺癌群のＣＥＡ／ＭＡＭの測定結果を、それぞれ示す。 (8) Comparison of measured values among healthy subjects, prostatic hyperplasia, and prostate cancer groups Regarding the results of Table 5, examine whether there is a statistically significant difference between the healthy subjects group and the prostate cancer group. did. 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, and FIG. 15 shows the CEA measurement results for the prostatic hyperplasia group and the prostate cancer group. , and FIG. 16 shows the measurement results of CEA/MAM in the prostatic hyperplasia group and the prostatic cancer group, respectively.
　Ｗｉｌｃｏｘｏｎ検定によって検定を行ったところ、健常人群と前立腺癌群との間にはＣＥＡの測定値については有意な差が認められなかった（図１３、ｐ＝０．１０１８）のに対して、ＣＥＡ／ＭＡＭの測定値については有意な差が認められた（図１４、ｐ＝０．０２４７）。同様に、前立腺肥大群と前立腺癌群との間でも、ＣＥＡの測定値については有意な差が認められなかった（図１５、ｐ＝０．９３３７）のに対して、ＣＥＡ／ＭＡＭの測定値については有意な差が認められた（図１６、ｐ＝０．０００６）。 When tested by the Wilcoxon test, no significant difference was observed between the healthy subject group and the prostate cancer group in the measured CEA values (Fig. 13, p = 0.1018). A significant difference was observed for the measured value of /MAM (Fig. 14, p = 0.0247). Similarly, between the prostatic hyperplasia group and the prostatic cancer group, no significant difference was observed in the CEA measurement values (Fig. 15, p = 0.9337), whereas the CEA/MAM measurement values A significant difference was observed for (Figure 16, p = 0.0006).
　また、図１７に、ＣＥＡの測定値とＣＥＡ／ＭＡＭの測定値との関係を示す。図１７に示されるように、ＣＥＡの測定値とＣＥＡ／ＭＡＭの測定値との間に明確な相関関係は示されなかった（回帰直線：ＣＥＡ／ＭＡＭ（カウント）＝２５７１７３．５８－１０．６４３３８８×ＣＥＡ（カウント）、Ｒ２＝０．０１５１１２、分散分析のｐ値＝０．４８１７）。このことから、ＣＥＡ／ＭＡＭの測定値は単にＣＥＡの存在量を表すものではなく、ＣＥＡ（ＭＡＭ結合型糖鎖以外の部位に対して特異的に結合可能な抗ＣＥＡ抗体に認識される）とは異なるバイオマーカーであることが示された。 Also, FIG. 17 shows the relationship between the measured values of CEA and the measured values of CEA/MAM. As shown in FIG. 17, no clear correlation was shown between measured CEA and CEA/MAM (regression line: CEA/MAM (count)=257173.58-10.643388 x CEA (counts), R2 = 0.015112, ANOVA p-value = 0.4817). For this reason, 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.
　以上より、ＣＥＡ／ＭＡＭの測定は、健常人と前立腺癌患者との判別、又は、前立腺肥大患者と前立腺癌患者との判別の新規の指標となることが示された。 From the above, it was shown that the measurement of CEA/MAM is a novel index for discriminating between healthy subjects and prostate cancer patients, or between prostatic hyperplasia patients and prostate cancer patients.
　（９）ＣＥＡ／ＭＡＭの測定値とＰＳＡ測定値との比較
　実施例２の（７）で用いたものと同様の前立腺肥大患者から採取した血清検体１０例、前立腺癌患者から採取した血清検体１５例について、検体中に含まれるＰＳＡ量を測定した結果は、上記の表２に示すとおりである。表２の結果に対して、ＰＳＡ測定値の一般的なカットオフ値である４ｎｇ／ｍＬを用い、カットオフ値を上回る検体を陽性とした場合の前立腺癌患者の検出能力を試験した。その結果、前記カットオフ値で陽性と判定されたのは前立腺癌群では１５検体中４検体であり、２６．７％であった。また、前立腺肥大群では１０検体中１検体が前記カットオフ値で陽性と判定され、擬陽性となった。 (9) Comparison between measured values of CEA/MAM and measured values of PSA 10 serum specimens collected from patients with benign prostatic hyperplasia similar to those used in (7) of Example 2, 15 serum specimens collected from patients with prostate cancer For examples, the results of measuring the amount of PSA contained in the sample are shown in Table 2 above. For the results in Table 2, a general PSA cutoff value of 4 ng/mL was used to test the ability to detect prostate cancer patients when samples exceeding the cutoff value were considered positive. As a result, 4 out of 15 specimens in the prostate cancer group, or 26.7%, were determined to be positive at the cut-off value. In addition, in the prostatic hyperplasia group, 1 out of 10 specimens was determined to be positive at the cutoff value, which was a false positive.
　他方、ＣＥＡ／ＭＡＭの測定値（表５）についても、健常人群の測定値からカットオフ値を算出し、カットオフ値を上回る検体を陽性とした場合の前立腺癌患者の検出能力を試験した。カットオフ値は、健常人群の測定値の平均に健常人群の測定値の標準偏差の値に２を乗じた値を加算して、２９９，２１６カウント以下と算出された。その結果、このカットオフ値で陽性と判定されたのは前立腺癌群では１５検体中８検体であり、５３．３％であった。また、健常人群と前立腺肥大群とを合わせた２０検体全てが陰性と判定された。 On the other hand, for the measured values of CEA/MAM (Table 5), 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. As a result, 8 out of 15 specimens in the prostate cancer group were determined to be positive at this cut-off value, accounting for 53.3%. In addition, all 20 samples from the healthy subject group and the prostatic hyperplasia group were determined to be negative.
　さらに、図１８に、ＰＳＡの測定値とＣＥＡ／ＭＡＭの測定値との関係を示す。図１８に示されるように、今回の結果からは、ＰＳＡの測定値とＣＥＡ／ＭＡＭの測定値との間に明確な相関関係は示されなかった（回帰直線：ＣＥＡ／ＭＡＭ（カウント）＝２３３８５３．６８－４０５６．１９５２×ＰＳＡ（ｎｇ／ｍＬ）、Ｒ２＝０．００８８５１、分散分析のｐ値＝０．６５４６）。 Furthermore, FIG. 18 shows the relationship between the measured values of PSA and the measured values of CEA/MAM. As shown in FIG. 18, the results of this time did not show a clear correlation between the measured values of PSA and the measured values of CEA/MAM (regression line: CEA/MAM (count) = 233853 .68-4056.1952×PSA (ng/mL), R 2 =0.008851, ANOVA p-value=0.6546).
　以上より、ＣＥＡ／ＭＡＭの測定による前立腺癌の検出能力（感度、特異度）はＰＳＡと比較しても十分に高く、さらに、ＰＳＡとは独立した診断指標として利用できることが示された。 From the above, it was shown that the detection ability (sensitivity, specificity) of prostate cancer by measuring CEA/MAM is sufficiently high compared to PSA, and that it can be used as a diagnostic index independent of PSA.
　（比較例３）　ＭＡＭ以外のレクチンを用いたブロック化標識レクチンによる糖鎖付加ＣＥＡの測定
　ＭＡＭに代えて、Ｎ－アセチルガラクトサミンを認識するノダフジレクチン（ＷＦＡ、ＶＥＣＴＯＲ社製）を結合させたブロック化標識レクチン（ＷＦＡ）を調製した。すなわち、ＭＡＭに替えてＷＦＡを用い、かつ、デキストランを分子量２５０ｋのもの（ＣａｒｂｏＭｅｒ社製）に変更した以外は、実施例２の（１）～（５）と同様の方法で、最終的に３７３．９μｇ／ｍＬのブロック化標識レクチン（ＷＦＡ）（デキストラン－酵素－ＷＦＡ結合体）の溶液を２．０ｍＬ得た。なお、デキストランのサイズは、レクチンの糖鎖型特異性に影響するものではない。 (Comparative Example 3) Measurement of sugar chain-added CEA by blocking labeled lectin using lectin other than MAM Blocking by binding Nodafuji lectin (WFA, manufactured by VECTOR) that recognizes N-acetylgalactosamine instead of MAM A labeled lectin (WFA) was prepared. That is, except that WFA was used instead of MAM, and the dextran was changed to one with a molecular weight of 250 k (CarboMer), the same method as in (1) to (5) of Example 2 was performed, and finally 373 A 2.0 mL solution of 0.9 μg/mL blocked labeled lectin (WFA) (dextran-enzyme-WFA conjugate) was obtained. The size of dextran does not affect the specificity of lectin sugar chains.
　ブロック化酵素レクチン（ＭＡＭ）に代えて、得られたブロック化酵素レクチン（ＷＦＡ）を用いたこと以外は、実施例２の（６）～（７）と同様の方法で、実施例２の（７）で用いたものと同様の健常人から採取した血清検体１０例（健常人群：健常人１～１０）、及び前立腺癌患者から採取した血清検体１５例（前立腺癌群：前立腺癌１～１５）について、各検体に含まれるＷＦＡ結合型糖鎖付加ＣＥＡ（ＣＥＡ／ＷＦＡ）の測定を行った。各検体における測定結果を下記の表６に示す。 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.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　表６の結果について、健常人群と前立腺癌群との間で統計学的に有意な差が認められるか検討した。図１９に健常人群及び前立腺癌群のＣＥＡ／ＷＦＡの測定結果を示す。Ｗｉｌｃｏｘｏｎ検定によって検定を行ったところ、健常人群と前立腺癌群との間にはＣＥＡ／ＷＦＡの測定値について有意な差が認められなかった（図１９、ｐ＝０．４５４０）。以上より、ＣＥＡとＭＡＭ結合型糖鎖との組み合わせを測定するＣＥＡ／ＭＡＭの測定は、健常人と前立腺癌患者との判別に特異的な新規の指標となることが示された。 Regarding the results in Table 6, we examined whether there was a statistically significant difference between the healthy subject group and the prostate cancer group. FIG. 19 shows the CEA/WFA measurement results for the healthy subject group and the prostate cancer group. Wilcoxon's test showed no significant difference in CEA/WFA measurements between the healthy and prostate cancer groups (Fig. 19, p = 0.4540). From the above, it was shown that the measurement of CEA/MAM, which measures the combination of CEA and MAM-linked sugar chains, is a new specific indicator for discriminating between healthy subjects and prostate cancer patients.
　本発明によれば、新たなバイオマーカーを指標とした、高感度で前立腺がん及び／又は大腸がんを特異的に検出可能ながん検出方法及びがん検査方法、並びに、これらの方法に用いるキットを提供することが可能となる。 According to the present invention, 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.

Claims (14)

  1. 　前立腺がん及び大腸がんからなる群から選択される少なくとも１種のがんを検出する方法であり、試料中のＧａｌＮＡｃ付加ＣＡ１９－９量を測定する測定工程を含む、方法。 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.
  2. 　前立腺がん及び大腸がんからなる群から選択される少なくとも１種のがんを検査する方法であり、被検者由来の試料中のＧａｌＮＡｃ付加ＣＡ１９－９量を測定する測定工程を含む、方法。 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. 　前立腺がん及び大腸がんからなる群から選択される少なくとも１種のがんであると予測される被検者をスクリ－ニングする方法であり、被検者由来の試料中のＧａｌＮＡｃ付加ＣＡ１９－９量を測定する測定工程と、測定されたＧａｌＮＡｃ付加ＣＡ１９－９量を指標として被検者を選別する選別工程と、を含む、請求項２に記載の方法。 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 3. The method according to claim 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 index.
  4. 　ＧａｌＮＡｃ付加ＣＡ１９－９において、ＧａｌＮＡｃがＷＦＡに結合するＷＦＡ結合型糖鎖である、請求項１～３のうちのいずれか一項に記載の方法。 The method according to any one of claims 1 to 3, wherein in GalNAc-added CA19-9, GalNAc is a WFA-linked sugar chain that binds to WFA.
  5. 　前記測定工程が、前記試料と、ＣＡ１９－９に特異的に結合可能な第１のプローブ分子及びＧａｌＮＡｃに特異的に結合可能な第２のプローブ分子と、を接触させる工程である、請求項１～４のうちのいずれか一項に記載の方法。 Claim 1, wherein said measuring step is a step of contacting said sample with a first probe molecule capable of specifically binding to CA19-9 and a second probe molecule capable of specifically binding to GalNAc. 5. The method of any one of 4.
  6. 　前記測定工程が、前記試料と、捕捉体及び標識体と、を接触させる工程であり、
    　前記捕捉体が、非水溶性担体と、前記非水溶性担体に固定された第１のプローブ分子と、を備えるものであり、かつ、
    　前記標識体が、水溶性担体と、前記水溶性担体に固定された標識物質及び第２のプローブ分子と、を備え、第２のプローブ分子がＧａｌＮＡｃに特異的に結合可能なレクチンであるブロック化標識レクチンである、
    請求項５に記載の方法。     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 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,
    6. The method of claim 5.
  7. 　請求項５～６のうちのいずれか一項に記載の方法に用いるためのキットであり、ＣＡ１９－９に特異的に結合可能な第１のプローブ分子と、ＧａｌＮＡｃに特異的に結合可能な第２のプローブ分子と、を備える、キット。 A kit for use in the method according to any one of claims 5 to 6, comprising a first probe molecule capable of specifically binding to CA19-9 and a first probe molecule capable of specifically binding to GalNAc. 2 probe molecules.
  8. 　前立腺がんを検出する方法であり、試料中のα２－３シアル酸付加ＣＥＡ量を測定する測定工程を含む、方法。 A method for detecting prostate cancer, comprising a measuring step of measuring the amount of α2-3 sialylated CEA in a sample.
  9. 　前立腺がんを検査する方法であり、被検者由来の試料中のα２－３シアル酸付加ＣＥＡ量を測定する測定工程を含む、方法。 A method for examining prostate cancer, comprising a measuring step of measuring the amount of α2-3 sialylated CEA in a sample derived from a subject.
  10. 　前立腺がんであると予測される被検者をスクリ－ニングする方法であり、被検者由来の試料中のα２－３シアル酸付加ＣＥＡ量を測定する測定工程と、測定されたα２－３シアル酸付加ＣＥＡ量を指標として被検者を選別する選別工程と、を含む、請求項９に記載の方法。 A method for screening a subject predicted to have prostate cancer, comprising a measuring step of measuring the amount of α2-3 sialylated CEA in a sample derived from the subject; 10. The method according to claim 9, comprising a screening step of screening subjects using the amount of acid-added CEA as an index.
  11. 　α２－３シアル酸付加ＣＥＡにおいて、α２－３シアル酸がＭＡＭに結合するＭＡＭ結合型糖鎖である、請求項８～１０のうちのいずれか一項に記載の方法。 The method according to any one of claims 8 to 10, wherein in the α2-3 sialylated CEA, the α2-3 sialic acid is a MAM-linked sugar chain that binds to MAM.
  12. 　前記測定工程が、前記試料と、ＣＥＡに特異的に結合可能な第１のプローブ分子及びα２－３シアル酸に特異的に結合可能な第２のプローブ分子と、を接触させる工程である、請求項８～１１のうちのいずれか一項に記載の方法。 wherein said measuring step is a step of contacting said sample with a first probe molecule capable of specifically binding to CEA and a second probe molecule capable of specifically binding to α2-3 sialic acid. 12. The method of any one of paragraphs 8-11.
  13. 　前記測定工程が、前記試料と、捕捉体及び標識体と、を接触させる工程であり、
    　前記捕捉体が、非水溶性担体と、前記非水溶性担体に固定された第１のプローブ分子と、を備えるものであり、かつ、
    　前記標識体が、水溶性担体と、前記水溶性担体に固定された標識物質及び第２のプローブ分子と、を備え、第２のプローブ分子がα２－３シアル酸に特異的に結合可能なレクチンであるブロック化標識レクチンである、
    請求項１２に記載の方法。     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 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 α2-3 sialic acid. is a blocked labeled lectin that is
    13. The method of claim 12.
  14. 　請求項１２～１３のうちのいずれか一項に記載の方法に用いるためのキットであり、ＣＥＡに特異的に結合可能な第１のプローブ分子と、α２－３シアル酸に特異的に結合可能な第２のプローブ分子と、を備える、キット。 A kit for use in the method according to any one of claims 12 to 13, comprising a first probe molecule capable of specifically binding to CEA and capable of specifically binding to α2-3 sialic acid and a second probe molecule.
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