Cancer detection method, cancer examination method, and kit used therefor

Abstract

To provide a cancer detection method which allows for specifically and accurately detecting pancreatic cancer and bile duct cancer using a novel biomarker as an indicator.SOLUTION: A method of detecting at least one cancer selected from a group consisting of pancreatic cancer and bile duct cancer is provided, the method comprising a measurement step of measuring the level of fucose-added hemopexin in a sample.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a cancer detection method, a cancer examination method, and a kit used therefor, and more particularly, to a method for detecting at least one cancer selected from the group consisting of pancreatic cancer and bile duct cancer, a method for examining said cancer, and a kit used for these methods.

Background Art

[0002] Biomarkers such as tumor markers are useful as markers for detecting diseases such as malignant tumors, guiding treatment decisions, and monitoring treatment effects, and have been actively studied in recent years. As such biomarkers, CA19-9, CEA, etc. are known. For example, CA19-9 is known to have an increased blood concentration in cancer patients such as digestive tract cancers, particularly colorectal cancer, pancreatic cancer, bile duct cancer, gallbladder cancer, etc., and has been conventionally used as a marker for detecting these cancers, guiding treatment decisions, and monitoring treatment effects.

[0003] In recent years, changes in the sugar chain structure of glycoproteins in cancer have attracted attention. For example, Non-Patent Document 1 reports that fucosylated haptoglobin (Fuc-Hpt) in pancreatic cancer patients is significantly increased compared to healthy subjects. Furthermore, Non-Patent Document 2 suggests that fucosylated hemopexin may be a useful biomarker for hepatocellular carcinoma.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

[0005] However, in the above-described conventional monitoring markers, the specificity for each type of cancer is not yet sufficient. Among the above cancers, pancreatic cancer and bile duct cancer in particular are cancers for which early diagnosis is difficult. However, it has still been insufficient for more accurately discriminating between pancreatic cancer patients and healthy subjects, or between bile duct cancer patients and healthy subjects. In addition, as described above, structural changes in the sugar chains of glycoproteins in cancer have attracted attention, but have not yet been fully elucidated, and these sugar chains are expected to have the potential as new biomarkers for cancer detection.

[0006] The present invention has been made in view of the above problems, and an object thereof is to provide a cancer detection method and a cancer test method that can specifically detect pancreatic cancer and bile duct cancer with high precision using a new biomarker as an index, and a kit used for these methods. [Means for Solving the Problems]

[0007] The present inventors, for the purpose of searching for sugar chains as new tumor markers that could not be measured with existing antibodies, in addition to anti-hempexin antibodies, also used a complex (blocked labeled lectin) comprising a water-soluble carrier made of a water-soluble polymer, a labeling substance fixed to the water-soluble carrier, and a lectin for screening. As a result, it was found that the amount of molecules detected when using AOL as the lectin, that is, hemopexin to which fucose having lectin-binding property was added (fucose-added hemopexin), was significantly increased in pancreatic cancer patients and bile duct cancer patients compared to healthy subjects. Therefore, it was found that by measuring fucose-added hemopexin, it is possible to specifically detect pancreatic cancer and bile duct cancer, and to accurately discriminate between pancreatic cancer patients and healthy subjects, or between bile duct cancer patients and healthy subjects, and the present invention has been completed.

[0008] Aspects of the present invention obtained based on such findings are as follows. [1] A method for detecting at least one cancer selected from the group consisting of pancreatic cancer and bile duct cancer, the method comprising a measurement step of measuring fucose - added hemopexin in a sample. [2] A method for examining at least one cancer selected from the group consisting of pancreatic cancer and bile duct cancer, the method comprising a measurement step of measuring fucose - added hemopexin in a sample derived from a subject. [3] A method for screening a subject predicted to have at least one cancer selected from the group consisting of pancreatic cancer and bile duct cancer, the method comprising: a measurement step of measuring fucose - added hemopexin in a sample derived from the subject; and a selection step of selecting the subject using the measured fucose - added hemopexin as an index, the method according to [2]. [4] A method for predicting the risk that a subject has at least one cancer selected from the group consisting of pancreatic cancer and bile duct cancer, the method comprising: a measurement step of measuring fucose - added hemopexin in a sample derived from the subject; and a prediction step of predicting the risk that the subject has the cancer using the measured fucose - added hemopexin as an index, the method according to [2]. [5] The method according to any one of [1] to [4], wherein in the fucose - added hemopexin, the fucose is at least one selected from the group consisting of α1 - 6 fucose and α1 - 2 fucose. [6] The method according to any one of [1] to [5], wherein in the fucose - added hemopexin, the fucose is at least one selected from the group consisting of an AOL - binding sugar chain that binds to AOL and an AAL - binding sugar chain that binds to AAL. [7] The method according to any one of [1] to [6], wherein the measurement step is a step of bringing the sample into contact with a first probe molecule that can specifically bind to hemopexin and a second probe molecule that can specifically bind to fucose. [8] The method according to [7], wherein the first probe molecule is an antibody capable of specifically binding to haptoglobin. [9] The method according to [7] or [8], wherein the second probe molecule is a lectin capable of specifically binding to fucose.

[10] The measurement step is a step of bringing the sample into contact with a capture agent and a labeling agent, The capture agent includes a water-insoluble carrier and either one of the first probe molecule and the second probe molecule immobilized on the water-insoluble carrier, and The labeling agent includes a labeling substance and the other of the first probe molecule and the second probe molecule, The method according to any one of [7] to [9], characterized by the above.

[11] The capture agent includes a water-insoluble carrier and the first probe molecule immobilized on the water-insoluble carrier, and The labeling agent includes a water-soluble carrier, a labeling substance immobilized on the water-soluble carrier, and the second probe molecule, and is a blocked labeling lectin in which the second probe molecule is a lectin capable of specifically binding to fucose. The method according to

[10] , characterized by the above.

[12] A kit for use in the method according to any one of [7] to

[11] , comprising a first probe molecule capable of specifically binding to haptoglobin and a second probe molecule capable of specifically binding to fucose.

[13] The kit according to

[12] , wherein the first probe molecule is an antibody capable of specifically binding to haptoglobin.

[14] The kit according to

[12] or

[13] , wherein the second probe molecule is a lectin capable of specifically binding to fucose.

[15] A capture agent comprising a water-insoluble carrier and either the first probe molecule or the second probe molecule immobilized on the water-insoluble carrier, and A labeling agent comprising a labeling substance and the other of the first probe molecule and the second probe molecule, The kit according to any one of

[12] to

[14] , characterized by comprising the above.

[16] The capture agent comprises a water-insoluble carrier and the first probe molecule immobilized on the water-insoluble carrier, and The labeling agent 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 blocked labeling lectin which is a lectin capable of specifically binding to fucose. The kit according to

[15] , characterized by the above.

Advantages of the Invention

[0009] According to the present invention, it is possible to provide a cancer detection method and a cancer test method capable of specifically detecting pancreatic cancer and cholangiocarcinoma with high accuracy using a new biomarker as an index, and a kit for use in these methods.

Brief Description of the Drawings

[0010]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Embodiments for Carrying Out the Invention

[0011] Hereinafter, the present invention will be described in detail according to its preferred embodiments.

[0012] <Cancer detection method, cancer examination method> The cancer detection method of the present invention is a method for detecting at least one cancer selected from the group consisting of pancreatic cancer and bile duct cancer, and includes a measurement step of measuring fucose - added hemopexin in a sample. Further, the cancer examination method of the present invention is a method for examining at least one cancer selected from the group consisting of pancreatic cancer and bile duct cancer, and includes a measurement step of measuring fucose - added hemopexin in a sample derived from a subject.

[0013] [Cancer] In the present invention, "cancer" includes epithelial malignant tumors (cancer) and non - epithelial malignant tumors (sarcoma). The cancer to be detected or examined in the method of the present invention is at least one cancer selected from the group consisting of pancreatic cancer and bile duct cancer. The pancreatic cancer according to the present invention refers to cancer occurring in the pancreas, and examples include invasive ductal carcinoma of the pancreas (ordinary - type pancreatic cancer), pancreatic endocrine tumor, intraductal papillary mucinous tumor, mucinous cystic tumor, acinar cell carcinoma, undifferentiated carcinoma, serous cystadenocarcinoma, metastatic pancreatic cancer. Among these, representative pancreatic cancer is invasive ductal carcinoma of the pancreas, which is preferred in the present invention. The bile duct cancer according to the present invention refers to cancer occurring in the bile duct (intrahepatic bile duct or extrahepatic bile duct in the liver), and examples include intrahepatic cholangiocarcinoma, extrahepatic bile duct cancer (hilar bile duct cancer, upper bile duct cancer, middle bile duct cancer, lower bile duct cancer), papillary cancer, cystic duct cancer in this region. Among these, as the bile duct cancer according to the present invention, extrahepatic bile duct cancer occurring in the extrahepatic bile duct of the liver is preferred.

[0014] [Fucose - added hemopexin] In the present invention, "fucose - added hemopexin (hereinafter, also referred to as 'fucosylated hemopexin' in some cases)" refers to a molecule containing hemopexin and fucose bound and added to hemopexin. A plurality of fucoses may be added to one molecule of hemopexin.

[0015] "Hemopexin" is a glycoprotein that has a specific affinity for heme or iron and is involved in the transport of hemoglobin. It has conventionally been used as a monitoring marker for the diagnosis of iron deficiency anemia and liver disorders. Hemopexin typically consists of a protein portion composed of about 440 amino acids and a sugar chain portion composed of multiple sugar chains bound thereto, has a molecular weight of about 70,000, and about 20% thereof is the sugar chain portion. In the present invention, when simply referred to as "hemopexin", the following "fucose" is not included in the sugar chain portion of such hemopexin.

[0016] "Fucose" is a monosaccharide represented by the IUPAC name "(3S,4R,5R,6S)-6-methyloxane-2,3,4,5-tetrol" and is preferably of the L type. Fucose may be directly bound to the protein portion of hemopexin, but it is preferable that fucose is bound and added to the sugar chain constituting the sugar chain portion of hemopexin.

[0017] In addition, as the fucose-added hemopexin according to the present invention, it is preferable to contain fucose as at least one selected from the group consisting of α1-6 fucose and α1-2 fucose. In the present invention, "α1-6 fucose" refers to a sugar chain in which fucose is added to N-acetylglucosamine (GlcNAc) present at the reducing end (root) of an N-type sugar chain by an α1,6 bond, and is also referred to as "core fucose". Further, in the present invention, "α1-2 fucose" refers to a sugar chain in which fucose is added to galactose (Gal), which is a monosaccharide, by an α1,2 bond, and it is preferable that the terminal fucose residue is an α1,2 bond to the second galactose residue from the end of the sugar chain (Fucα1-2Gal). The structure of the other part of the sugar chain to which fucose binds at this time is not particularly limited, but as the fucose-added hemopexin according to the present invention, it is more preferable to contain fucose as fucose constituting at least one sugar chain selected from the group consisting of an AOL-binding type sugar chain that binds to AOL and an AAL-binding type sugar chain that binds to AAL, and it is even more preferable to contain fucose as fucose constituting an AOL-binding type sugar chain that binds to AOL.

[0018] The average mass of such fucose - added hemopexin (average mass determined by calibration with markers by gel filtration chromatography (GFC), the same hereinafter) is not particularly limited, but is preferably 50,000 to 80,000 Da, and more preferably 60,000 to 70,000 Da.

[0019] [Subject] In the present invention, the "subject" refers to the object for which the cancer detection method of the present invention is performed, and is preferably a human. As the subject according to the present invention, a healthy person for the purpose of screening or risk assessment, or a person suffering from pancreatic cancer or bile duct cancer without any subjective symptoms may be used. Further, a patient who is already known to have suffered from the above - mentioned cancer or a patient who has suffered from the above - mentioned cancer in the past for the purpose of determining prognosis, determining treatment policy, confirming treatment effect, confirming recurrence, etc. may also be used. In the present invention, the "healthy person" refers to a person who does not suffer from the cancer to be examined (that is, pancreatic cancer and / or bile duct cancer). Whether the subject is truly suffering from pancreatic cancer and / or bile duct cancer is determined (definitely diagnosed) by biopsy of pancreatic tissue and / or bile duct tissue collected from the subject.

[0020] [Sample] The "sample" used in the cancer detection method and cancer examination method of the present invention (in this specification, sometimes collectively simply referred to as "the method of the present invention") is not particularly limited as long as it is a sample in which fucose - added hemopexin may be present. Generally, blood specimens such as serum, plasma, and whole blood collected from the subject of cancer examination, etc.; body fluid specimens other than blood such as urine, sputum, saliva, sweat, cerebrospinal fluid, digestive fluid, semen, lymph fluid, ascites, etc.; mucosal specimens such as oral mucosa, pharyngeal mucosa, intestinal mucosa, etc.; and various biopsy specimens, etc. may be mentioned. Further, the sample may be a cultured cell or a cell culture solution. As the sample according to the present invention, a blood specimen is preferable, and serum (also referred to as "serum specimen") is more preferable.

[0021] These samples may be diluted or suspended with a diluent as necessary. Examples of the diluent include buffers such as phosphate buffer, Tris buffer, Good buffer, borate buffer, acetate buffer, citrate buffer, glycine buffer, succinate buffer, and phthalate buffer. Since hemopexin is usually contained in blood at a high concentration (for example, 63 to 109 mg / dL), the sample according to the present invention may be highly diluted (for example, 1 / 10 to 1 / 1,000,000 by volume ratio in a blood specimen). Thereby, even if the sample is trace, measurement is possible, and it is possible to further improve the detection accuracy by reducing the influence of contaminants.

[0022] In addition, the sample according to the present invention may be appropriately pretreated as necessary. Examples of the pretreatment include treatments such as grinding, freezing, heating, concentration, fractionation, and desalting; addition treatments such as a pH adjuster, a stabilizer, a preservative, an antiseptic, and a surfactant; and purification treatments. One of these may be used alone or in combination of two or more. The purification treatment is not particularly limited. For example, treatment with a column; treatment for adsorbing contaminants with a substance such as an antibody that adsorbs contaminants in a sample fixed to a water-insoluble carrier and removing them from the sample containing fucose-added hemopexin; treatment for capturing hemopexin containing fucose-added hemopexin with an anti-hemopexin antibody fixed to a water-insoluble carrier, removing contaminants by washing, etc., and then releasing them.

[0023] [Measurement step] The method of the present invention includes a measurement step of measuring fucose-added hemopexin in a sample. In the present invention, "measurement" includes detection of the presence and amount of fucose-added hemopexin in a sample as a signal, and quantification or semi-quantification of the amount of fucose-added hemopexin according to the signal amount.

[0024] As a method for measuring fucose-added hemopexin, there is no particular limitation as long as it is a method capable of measuring fucose-added hemopexin, and conventionally known methods, methods analogous thereto, or combinations thereof can be appropriately employed. For example, a method using a probe molecule capable of specifically binding to fucose-added hemopexin; 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 spectrometry (AAS); HPLC; FPLC; NMR; IR; FTIR; UV-VIS absorption photometry; flow cytometry; mass spectrometry; and methods combining these can be mentioned, but it is not limited thereto.

[0025] Among these, as the measurement step according to the present invention, it is preferably a method using a probe molecule capable of specifically binding to fucose-added hemopexin. 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; galectins, sugar chain receptors, immune receptors, and the like. In the present invention, the "antibody" includes not only complete antibodies but also antibody fragments (for example, Fab, Fab’, F(ab’)2, Fv, single-chain antibodies, diabodies, etc.) and small molecule antibodies obtained by binding the variable regions of antibodies.

[0026] The probe molecule capable of specifically binding to fucose-added hemopexin is preferably a combination of a probe molecule capable of specifically binding to hemopexin (referred to as the "first probe molecule" in the present invention) and a probe molecule capable of specifically binding to fucose (referred to as the "second probe molecule" in the present invention).

[0027] 〔First probe molecule〕 Examples of the first probe molecule capable of specifically binding to hemopexin include antibodies capable of specifically binding to hemopexin (including anti-protein antibodies that recognize the protein part of hemopexin as the recognition site, anti-glycan antibodies that recognize the sugar chain part of hemopexin as the recognition site, and antibodies that recognize both the protein part and the sugar chain part of hemopexin. In this specification, sometimes referred to as "anti-hemopexin antibody"). Among them, an anti-hemopexin protein antibody that recognizes at least a part of the protein part of hemopexin as the recognition site is preferred, and an antibody that does not interfere with the binding between fucose and the second probe molecule is also preferred.

[0028] The anti-hemopexin antibody is not particularly limited as long as it has the ability to bind to hemopexin, and it may be a polyclonal antibody or a monoclonal antibody. However, from the viewpoints of homogeneity and stability, a monoclonal antibody is preferred. The anti-hemopexin antibody can be produced by appropriately adopting and improving conventionally known production methods, or commercially available ones can be appropriately used.

[0029] Examples of the anti-hemopexin antibody according to the present invention include those in which the lectin-binding sugar chain is removed or destroyed by oxidation treatment, glycosidase treatment, protease treatment, etc. in order to suppress the decrease in detection sensitivity caused by the lectin also recognizing the antibody sugar chain when the following lectin is used as the second probe molecule; those in which the region containing the sugar chain-added amino acid in the antibody molecule is removed; or those produced under conditions where sugar chain addition does not occur by using genetically engineered Escherichia coli or cells expressing the antibody gene or restricting the nutritional conditions in the culture of antibody-producing cells.

[0030] 〔Second probe molecule〕 Examples of the second probe molecule that can specifically bind to fucose include an antibody that can specifically bind to fucose (hereinafter sometimes referred to as "anti-fucose antibody" in this specification), a lectin that can specifically bind to fucose (for example, Aspergillus oryzae Lectin (AOL), Aleuria aurantia Lectin (AAL), Lens culinaris Lectin (LCA), Limax flavus Lectin (LTL), Ulex europaeus Lectin I (UEA-I), Pholiota nameko Lectin (PhoSL), Anguilla anguilla Lectin (AAA)). Among them, a lectin that can specifically bind to fucose is preferable, a lectin that can specifically bind to at least one selected from the group consisting of α1-6 fucose and α1-2 fucose is more preferable, and a lectin that can specifically bind to α1-6 fucose is even more preferable. More specifically, Aspergillus oryzae Lectin (AOL) and / or Aleuria aurantia Lectin (AAL) are preferable, and Aspergillus oryzae Lectin (AOL) is particularly preferable.

[0031] Aspergillus oryzae Lectin (AOL) is a protein that mainly recognizes the sugar chain structure of α1-6 fucose (core fucose) and exhibits binding activity. Aleuria aurantia Lectin (AAL) is a protein that mainly recognizes the sugar chain structures of α1-6 fucose (core fucose) and α1-2 fucose and exhibits binding activity. The lectin may be a modified lectin into which mutations are introduced or an artificially synthesized one for the purpose of increasing the specificity of sugar chain recognition activity or the like. Also, those that are generally distributed may be appropriately used.

[0032] When using the first probe molecule and the second probe molecule, in the measurement step, the sample is brought into contact with the first probe molecule and the second probe molecule. As a result, both hemopexin and fucose are recognized, and fucose - added hemopexin containing both can be detected and measured. The contact between the sample and the first probe molecule and the contact between the sample and the second probe molecule may be simultaneous with each other, may be at different times, and in the case of different times, either contact may be prior.

[0033] 〔Labeling substance〕 In the present invention, the measurement of fucose - added hemopexin is preferably carried out by detecting a signal generated by a labeling substance added to a probe molecule (preferably, the first probe molecule and / or the second probe molecule) that can specifically bind to fucose - added hemopexin or added to a molecule (such as a secondary antibody or protein A) that recognizes these molecules. By measuring the amount of the detected signal and semi - quantifying or quantifying it as necessary, the amount of fucose - added hemopexin can be obtained. The "signal" includes color development (chromogenic reaction), quenching, reflected light, luminescence, fluorescence, radiation by radioisotopes, etc. In addition to those that can be confirmed with the naked eye, it also includes those that can be confirmed by a measurement method and apparatus according to the type of signal. In the present invention, as the amount of fucose - added hemopexin, the amount may be calibrated by a calibration curve using a standard sample, etc., but from a more convenient and rapid viewpoint, the amount of the signal may be directly used as the amount of fucose - added hemopexin of the present invention.

[0034] As the labeling substance according to the present invention, those used as labeling substances in known immunological measurement methods and methods analogous thereto 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); 125Radioactive substances such as I; low molecular weight labeling substances such as fluorescein isothiocyanate (FITC) and rhodamine isothiocyanate (RITC); gold particles; avidin; biotin; latex; dinitrophenyl (DNP); digoxigenin (DIG) may be mentioned, and one of these may be used alone or in combination of two or more. For example, when an enzyme is used as the labeling substance, various measurements can be performed according to the substrate by adding a chromogenic substrate, a fluorescent substrate, a chemiluminescent substrate, etc. as a substrate. Examples of the enzyme include horseradish peroxidase (HRP), alkaline phosphatase (ALP), β-galactosidase (β-gal), glucose oxidase, and luciferase, but are not limited thereto.

[0035] 〔Sandwich method〕 Examples of the measurement method using the first probe molecule and the second probe molecule include immunological measurement methods such as the sandwich method, the competitive method, and immunoturbidimetry, and measurement methods according to these principles, and are not particularly limited. In such a measurement method, for example, generally, methods using microplates, particles, etc. as carriers such as ELISA, digital ELISA, CLEIA (chemiluminescent enzyme immunoassay), CLIA (chemiluminescent immunoassay), ECLIA (electrochemiluminescence immunoassay), RIA (radioimmunoassay), etc.; immunochromatography; surface plasmon resonance analysis method; detection method by fluorescence resonance energy transfer, etc. can be adopted.

[0036] As the measurement method according to the present invention, from the viewpoint that it tends to be possible to construct a measurement system with higher sensitivity and specificity, the sandwich method is preferable. Hereinafter, a more specific embodiment will be described by taking the sandwich method as an example for the measurement process according to the present invention.

[0037] As an embodiment when using the sandwich method, the measurement step is a step of bringing the sample into contact with a capture body and a labeled body, The capture body includes 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 labeling body includes a labeling substance and the other of the first probe molecule and the second probe molecule. There is an aspect (hereinafter, sometimes referred to as "the first aspect"). In the first aspect, the first probe molecule and the second probe molecule may be provided in either the capture body or the labeling body, but one is included in the capture body and the other is included in the labeling body. By capturing both fucose and hemopexin in this way, fucose - added hemopexin can be captured and detected with high precision and simplicity.

[0038] As another aspect of the sandwich method, it is not limited to the above. For example, the labeling body is a first labeling body including a first labeling substance and either one of the first probe molecule and the second probe molecule, and a second labeling body including a second labeling substance and the other of the first probe molecule and the second probe molecule, and the capture body is a capture body including a water-insoluble carrier and a probe molecule immobilized on the water-insoluble carrier (hereinafter, sometimes referred to as "the second aspect") may be used. In the second aspect, the first labeling substance and the second labeling substance produce different signals from each other. Also, as the probe molecule provided in the capture body in this case, a probe molecule capable of specifically binding to fucose - added hemopexin (including the first probe molecule and the second probe molecule); a probe molecule capable of specifically binding to the first probe molecule and / or the second probe molecule can be mentioned.

[0039] Examples of such sandwich methods include the two-step forward sandwich method (a method in which the reaction between the capture body and the fucose-added hemopexin in the sample and the reaction between the fucose-added hemopexin bound to the capture body and the labeling body are sequentially performed), the reverse sandwich method (a method in which the labeling body and the fucose-added hemopexin in the sample are reacted in advance, and the resulting complex is reacted with the capture body), and the one-step method (a method in which the reaction of the fucose-added hemopexin, the capture body, and the labeling body in the sample is simultaneously performed in one step). Any of these can be adopted.

[0040] For example, in the forward sandwich method, first, the sample and the capture body are brought into contact, and fucose-added hemopexin is captured by the capture body through the binding between the probe molecule of the capture body and fucose-added hemopexin (for example, the binding between the first probe molecule and hemopexin) (primary reaction: capture step). Next, the labeling body is brought into contact with the fucose-added hemopexin captured by the capture body, and labeling is performed through the binding between the probe molecule of the labeling body and fucose-added hemopexin (for example, the binding between the second probe molecule and fucose) (secondary reaction: labeling step). By this reaction, a complex containing the capture body - fucose-added hemopexin - labeling body is formed. After washing and removing the unbound sample and labeling body as needed (washing step), a signal derived from the labeling substance is measured by a predetermined method according to the labeling substance (measurement step).

[0041] (Capture body) The "capture body" according to the present invention is a complex comprising a water-insoluble carrier and a probe molecule capable of specifically binding to fucose-added hemopexin immobilized on the water-insoluble carrier, and is a conjugate in which the water-insoluble carrier and the probe molecule are directly or indirectly bound. As the probe molecule provided in the capture body according to the first aspect, it is preferably either one of the first probe molecule and the second probe molecule. In this case, as the probe molecule provided in the capture body, it may be either of the first probe molecule and the second probe molecule. However, when lectin is used as the second probe molecule, from the viewpoint of higher capture performance, as the probe molecule provided in the capture body, it is preferably a first probe molecule capable of specifically binding to hemopexin, and more preferably an anti-hemopexin antibody.

[0042] 〈Water-insoluble carrier〉 The water-insoluble carrier contained in the capture body mainly supports the probe molecule and functions as a carrier for solid-phase immobilization, and is a water-insoluble substance. In the present invention, the "water-insoluble substance" refers to a substance that is insoluble in water (the solubility in water is 0.001 g / mL or less, preferably 0.0001 g / mL or less, the same hereinafter) under normal temperature and pressure.

[0043] As the material of such a water-insoluble carrier, those generally used in immunological measurements and measurements similar thereto can be used without particular limitation. For example, at least one selected from the group consisting of polymer polymers (polystyrene, (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.) can be mentioned. Further, as the material of the water-insoluble carrier, composite materials thereof or composite materials of these substances and other substances may be used. For example, at least one organic polymer selected from the group consisting of the above polymer polymers, gelatin, and latex, and at least one metal compound selected from the group consisting of iron oxide (spinel ferrite, etc.), cobalt oxide, and nickel ferrite may be an organic-inorganic composite material. Furthermore, as the water-insoluble carrier, those surface-modified with active groups such as carboxy group, epoxy group, tosyl group, amino group, hydroxy group, isothiocyanate group, isocyanate group, azide group, aldehyde group, carbonate group, allyl group, aminooxy group, maleimide group, and thiol group may be used.

[0044] In the present invention, the shape of the water-insoluble carrier is not particularly limited either. For example, it may be any of plates, fibers, membranes, particles, etc. However, from the viewpoint of reaction efficiency, it is preferably particles, and from the viewpoints of automation and shortening of time, it is more preferably magnetic particles. As such a water-insoluble carrier, conventionally known ones can be appropriately used, and commercially available ones can also be appropriately used.

[0045] <Configuration and Manufacturing Method of Capture Agent> In the capturer, the content of the probe molecule is not particularly limited and can be appropriately adjusted according to, for example, the ease of binding between the probe molecule and fucose - added hemopexin. For example, the mass of the probe molecule (when the probe molecule is a combination of two or more types, the total thereof) relative to 100 parts by mass of the mass of the water - insoluble carrier (preferably particles) (when the water - insoluble carrier is a combination of two or more types, the total thereof) is preferably 0.1 to 10 parts by mass, and more preferably 1 to 5 parts by mass.

[0046] The capturer can be produced by immobilizing the probe molecule on the water - insoluble carrier. As such a production method, a conventionally well - known method or a method analogous thereto can be appropriately adopted. The probe molecule may be directly immobilized on the water - insoluble carrier or indirectly immobilized.

[0047] In the case of direct immobilization, for example, as the water - insoluble carrier and / or the probe molecule, those having active groups such as carboxy group, epoxy group, tosyl group, amino group, hydroxy group, isothiocyanate group, isocyanate group, azide group, aldehyde group, carbonate group, allyl group, aminooxy group, maleimide group, thiol group, etc. are used, or the active groups are imparted as necessary, and by binding these, the probe molecule can be directly immobilized on the water - insoluble carrier.

[0048] When immobilizing indirectly, for example, a linker that binds to the probe molecule can be immobilized on the water-insoluble carrier, and the probe molecule can be indirectly immobilized on the water-insoluble carrier by binding the probe molecule to the linker. The linker is not particularly limited, and examples thereof include a secondary antibody capable of binding to a probe molecule, Protein G, Protein A, a photocleavable photocleavage linker, a linker molecule having the active group (e.g., hydrazine salt, hydrazide), and the like. Further, the probe molecule may be modified in some way, and a substance that captures the modified portion may be immobilized on the water-insoluble carrier to immobilize the probe molecule on the water-insoluble carrier. For example, biotin is a representative example of the modified portion, and streptavidin is a representative example of the substance that captures the modified portion, but the present invention is not limited thereto.

[0049] 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 preferable range of the ratio in the above-mentioned capturer. Further, if necessary, for the purpose of preventing non-specific adsorption to the probe molecule or the water-insoluble carrier, the water-insoluble carrier may be blocked with an appropriate blocking agent (e.g., bovine serum albumin, gelatin, etc.). Furthermore, as such a capturer, commercially available ones may be appropriately used.

[0050] (Label) The "labeling entity" according to the present invention is a complex comprising a labeling substance and a probe molecule capable of specifically binding to fucose - added hemopexin, and is a conjugate in which the labeling substance and the probe molecule are directly or indirectly bound. As the probe molecule provided in the labeling entity according to the first aspect, it is preferably the other molecule of the molecules provided in the capturer among the first probe molecule and the second probe molecule. In this case, as the probe molecule provided in the labeling entity, it may be either of the first probe molecule and the second probe molecule. However, from the viewpoint of using a molecule such as an anti - hemopexin antibody with high affinity for an antigen as the first probe molecule in the capturer, it is preferably a second probe molecule capable of specifically binding to fucose, more preferably a lectin capable of specifically binding to fucose, still more preferably at least one selected from the group consisting of AOL and AAL, and particularly preferably AOL.

[0051] (Blocked labeled lectin) In the present invention, examples of the labeling entity include a labeled antibody comprising the labeling substance and an antibody (such as an anti - hemopexin antibody, an anti - fucose antibody, etc.), a labeled lectin comprising the labeling substance and a lectin, and a blocked labeled lectin, and it may be only one of these or a combination of two or more. Among these, as the labeling entity in the present invention, particularly in the first aspect, a blocked labeled lectin is preferably used. In the present invention, the "blocked labeled lectin" is a complex comprising a water - soluble carrier made of a water - soluble polymer, a labeling substance and a lectin fixed to the water - soluble carrier, and is a conjugate in which the water - soluble carrier, the labeling substance and the lectin are directly or indirectly bound. In the present invention, the blocked labeled lectin comprises a lectin capable of specifically binding to fucose as the second probe molecule. In this case, the probe molecule provided in the capturer is preferably the first probe molecule. The lectin is as described above, preferably AOL and / or AAL, and more preferably AOL. Also, the labeling substance is as described above, including its preferred embodiments.

[0052] In the blocked labeled lectin, it is sufficient that the labeling substance and the lectin are carried on the water-soluble carrier. Whether the labeling substance and the lectin are independently bound to the water-soluble carrier, whether any of the water-soluble carrier, the labeling substance, and the lectin are bound to the other two, whether the lectin is bound to the water-soluble carrier via the labeling substance, or whether the labeling substance is bound to the water-soluble carrier via the lectin may be acceptable. Generally, the affinity at each binding point between the lectin and the lectin-binding sugar chain structure is weak. However, in such a blocked labeled lectin, a plurality of lectins form a complex series by a water-soluble carrier that is a polymer. Therefore, by using this, a plurality of binding points are generated in one complex, so that the overall affinity is improved and the target fucose can be detected with high sensitivity.

[0053] 〈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 (hereinafter referred to as "the first water-soluble polymer") constituting the water-soluble carrier according to the present invention is not particularly limited as long as it is a water-soluble polymer capable of fixing and carrying the labeling substance and the lectin. In the present invention, the "water-soluble polymer" refers to a polymer compound having a solubility in water of more than 0.01 g / mL, preferably 0.05 g / mL or more, and more preferably 0.1 g / mL or more at normal temperature and pressure.

[0054] The first water-soluble polymer according to the present invention preferably has a weight average molecular weight (weight average molecular weight in terms of polystyrene conversion by gel permeation chromatography (GPC), the same hereinafter) of 6,000 to 4,000,000, and more preferably 20,000 to 1,000,000 from the viewpoints of measurement sensitivity and water solubility.

[0055] In addition, as the first water-soluble polymer according to the present invention, from the viewpoint of tending to obtain a blocked labeled antibody having a more preferable average particle size, the average mass is preferably 70,000 to 1,000,000 Da, and more preferably 150,000 to 700,000 Da.

[0056] In addition, as the blocked labeled lectin, a single blocked labeled lectin may contain a plurality of water-soluble polymers having different weight average molecular weights as the first water-soluble polymer. Further, as the blocked labeled lectin, a combination of a high molecular weight blocked labeled lectin having a weight average molecular weight of 200,000 or more of the first water-soluble polymer and a low molecular weight blocked labeled lectin having a weight average molecular weight of less than 100,000 (more preferably 100,000 or less) of the first water-soluble polymer is also preferable, and a combination of a high molecular weight blocked labeled lectin having a weight average molecular weight of 200,000 to 700,000 (more preferably 250,000 to 500,000) of the first water-soluble polymer and a low molecular weight blocked labeled lectin having a weight average molecular weight of 20,000 to 100,000 (more preferably 50,000 to 70,000) of the first water-soluble polymer is more preferable. When combining such a high molecular weight blocked labeled lectin and the low molecular weight blocked labeled lectin as the blocked labeled lectin, the mass ratio thereof (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, and even more preferably 3:1 to 1:3.

[0057] Examples of the first water-soluble polymer according to the present invention include polysaccharides such as dextran, aminodextran, ficoll (trade name), dextrin, agarose, pullulan, various celluloses (e.g., hemicellulose, lignin, etc.), chitin, and chitosan; β-galactosidase; thyroglobulin; hemocyanin; polylysine; polypeptide; DNA; and modified products thereof (e.g., diethylaminoethyl dextran, sodium dextran sulfate, etc.). One of these may be used alone or in combination of two or more. Among these, from the viewpoint that the first water-soluble polymer according to the present invention is inexpensive and can be obtained in large quantities, and chemical processing such as addition of functional groups and coupling reactions is relatively easy, it is preferably at least one selected from the group consisting of polysaccharides and their modified products, more preferably at least one selected from the group consisting of dextran, aminodextran, and their modified products, and even more preferably dextran.

[0058] <Constitution 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 measuring mechanism and the like. However, in order to further improve the measurement sensitivity, it is preferably set so that the number of molecules of the labeling substance bound to one molecule of the first water-soluble polymer is as large as possible. For example, when the labeling substance is an enzyme, the mass of the labeling substance (the total when the labeling substance is a combination of two or more, the same hereinafter) with respect to 100 parts by mass of the mass of the first water-soluble polymer (the total when the first water-soluble polymer is a combination of two or more, the same hereinafter) is preferably 100 to 1,000 parts by mass, and more preferably 300 to 800 parts by mass.

[0059] In the blocked labeled lectin, the content of lectin is not particularly limited, but in order to further improve the measurement sensitivity, it is preferably set so that the number of lectin molecules bound to one molecule of the first water-soluble polymer is as large as possible. For example, the mass of lectin (the total when there are two or more types of lectins in combination) relative to 100 parts by mass of the first water-soluble polymer is preferably 100 to 2,000 parts by mass, and more preferably 300 to 1,500 parts by mass.

[0060] Further, as the blocked labeled lectin, the weight average molecular weight per molecule of the blocked labeled lectin is preferably 1,000,000 to 10,000,000, and more preferably 1,500,000 to 5,000,000. When the weight average molecular weight is 1,000,000 or more, the measurement sensitivity tends to be higher. On the other hand, when it is 10,000,000 or less, aggregation in an aqueous solution and the like can be more sufficiently suppressed.

[0061] The blocked labeled lectin can be produced by immobilizing the labeling substance and lectin on the water-soluble carrier. As such a production method, a conventionally well-known method or a method analogous thereto can be appropriately employed, and the labeling substance and lectin (hereinafter, sometimes collectively referred to as "substances to be supported") may be directly immobilized on the water-soluble carrier or indirectly immobilized.

[0062] As a method for directly immobilizing the substance to be supported on the water-soluble carrier, for example, a carboxy group, epoxy group, tosyl group, amino group, hydroxy group, isothiocyanate group, isocyanate group, azide group, aldehyde group, carbonate group, allyl group, aminooxy group, maleimide group, thiol group, pyridyldisulfide group or the like is imparted to the substance to be supported and / or the first water-soluble polymer constituting the water-soluble carrier, or a water-soluble polymer having these active groups is used as the substance to be supported and / or the water-soluble carrier, and they are fixed by bonding them. As the substance to be supported and the first water-soluble polymer imparted with the active group, commercially available ones may be used as they are, or they may be prepared by introducing the active group onto the surface of the substance to be supported and the water-soluble polymer under appropriate reaction conditions. As an example, the introduction of a thiol group can be carried out using commercially available reagents such as S-acetylmercaptosuccinic anhydride and 2-iminothiolane hydrochloride. Also, the introduction of a maleimide group to the amino group on the first water-soluble polymer constituting the substance to be supported and / or the water-soluble carrier can be carried out using commercially available reagents such as N-(6-maleimidocaproyloxy)succinimide and N-(4-maleimidobutyryloxy)succinimide. The introduction of a pyridyldisulfide group can be carried out using commercially available reagents such as N-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-{6-[3-(2-Pyridyldithio)propionamido]hexanoyloxy}sulfosuccinimide, sodium salt (Sulfo-AC5-SPDP), etc. Also, it can be introduced by reducing the pyridyldisulfide group after introduction to a thiol group.

[0063] As a method for indirectly immobilizing the supported substance on the water-soluble carrier, for example, a method of immobilizing through a linker such as polyhistidine, polyethylene glycol, an oligopeptide containing cysteine and / or lysine, a linker molecule having the active group (for example, those mentioned in the method for producing the above capturer), etc. can be mentioned. The selection and size of the linker can be appropriately set in consideration of the strength of the bond with the supported substance and the steric hindrance caused by immobilizing the supported substance on the water-soluble carrier.

[0064] In the method for producing the blocked labeled lectin, the labeling substance and lectin may be immobilized on the water-soluble carrier at once or separately and sequentially. However, from the viewpoints of ease of production and ease of controlling the amounts of the labeling substance and lectin, it is preferable to immobilize one of them on the water-soluble carrier and then immobilize the other. Further, the blocked labeled lectin can also be produced by immobilizing the labeling substance and lectin on different water-soluble carriers respectively, and directly binding the labeling substance (blocked labeling substance) immobilized on one water-soluble carrier and the lectin (blocked lectin) immobilized on the other water-soluble carrier, or through the linker or the like.

[0065] The method for producing the blocked labeled lectin is not particularly limited. For example, when the labeling substance is an enzyme and the first water-soluble polymer is a polysaccharide or glycoprotein as described in the following examples, first, the first water-soluble polymer is oxidized with an oxidizing agent such as sodium periodate to impart an aldehyde group, reacted with hydrazine hydrochloride, and then reacted with a reducing agent such as dimethylamine borane (DMAB) to hydrazinate. 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 added above are reacted to form a hydrazone bond, and a first water-soluble polymer-enzyme conjugate is obtained. The obtained first water-soluble polymer-enzyme conjugate is treated with a crosslinker having N-hydroxysuccinimide and maleimide groups at each end (for example, SM(PEG)4, SMCC, etc.) to introduce maleimide groups. On the other hand, the lectin is thiolated with a thiolating reagent to impart a thiol group, or in the case of a lectin having an intramolecular disulfide bond, a thiol group is obtained by reduction. Finally, by binding the maleimide group introduced into the first water-soluble polymer-enzyme conjugate and the thiol group imparted to the lectin, the three components of the first water-soluble polymer (water-soluble carrier)-enzyme-lectin can be covalently bonded. According to this method, a product in which two or more molecules of the first water-soluble polymer are bound via an enzyme and a lectin is bound can be obtained. The ratios of the first water-soluble polymer, the labeling substance, and the lectin used in these reactions can be appropriately selected so as to achieve the preferred ranges of the respective contents in the above-described blocked labeled lectin.

[0066] (Capture step) In the capture step, the method of bringing the sample into contact with the capture body is not particularly limited, and a conventionally known method or a method analogous thereto can be appropriately employed. For example, when the water-insoluble carrier is a plate, a method of injecting the sample into this (probe molecule-immobilized plate), or when the water-insoluble carrier is particles, a method of adding the capture body (probe molecule-immobilized particles) into the sample can be mentioned.

[0067] In the reaction between the sample and the capturer in the capturing step, the content (final concentration) of the capturer in the reaction solution containing the capturer and the sample (when there are two or more types of capturers in combination, the total thereof, the same applies hereinafter) is not particularly limited and can be appropriately adjusted according to the type, concentration, etc. of the sample, so it is not particularly limited. However, from the viewpoint of efficiently capturing in a short time, for example, it is preferably 0.01 to 1.5% by mass, more preferably 0.05 to 1% by mass, and even more preferably 0.1 to 0.5% by mass.

[0068] Also, the conditions of the capturing step are not particularly limited and can be appropriately adjusted. For example, it can be carried out at room temperature to 45°C, preferably 20 to 37°C, at a pH of about 6 to 9, preferably pH 7 to 8, for about 5 seconds to 10 minutes, preferably about 30 seconds to 5 minutes, but it is not limited to these conditions.

[0069] (Labeling step) In the reaction between the label and the fucose-added hemopexin in the labeling step, the content (final concentration) of the label in the reaction solution containing the label and the fucose-added hemopexin (when there are two or more types of labels in combination, the total thereof, the same applies hereinafter) is not particularly limited and can be appropriately adjusted according to the type, concentration, etc. of the sample, so it is not particularly limited. However, 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, and even more preferably 0.1 to 1 μg / mL.

[0070] Also, the other conditions of the labeling step are not particularly limited and can be appropriately adjusted. For example, it can be carried out at room temperature to 37°C, preferably 20 to 37°C, at a pH of 5.0 to 7.0, preferably 5.5 to 6.5, for about 3 minutes to 120 minutes, preferably about 5 minutes to 10 minutes, but it is not limited to these conditions.

[0071] (Washing step) In the sandwich method, it is preferable that after the capture step and / or the labeling step, a washing step is further included to separate the fucose-added hemopexin bound to the capture body from other contaminants not bound to (not captured by) the capture body and remove the contaminants. The method for removing the contaminants is not particularly limited, and a conventionally known method or a method analogous thereto can be appropriately adopted. For example, when the capture body is the probe molecule-immobilized plate, a method of removing the liquid phase (supernatant) from the plate, or when the capture body is the probe molecule-immobilized particle, a method of recovering the particles by centrifugation or magnetic collection and removing the liquid phase (supernatant) can be mentioned. Also, in the washing step, the injection and removal of the washing liquid may be repeated as necessary. Examples of the washing liquid include known neutral (preferably pH 6 to 9) buffer solutions (sodium phosphate buffer, MES, Tris, CFB, MOPS, PIPES, HEPES, tricine buffer, bicine buffer, glycine buffer, etc.), and those added with a stabilizing protein such as BSA or a surfactant may also be used.

[0072] When using the sandwich method as the measurement step according to the present invention, the sample may be diluted with a diluent as described above and used, but when the capture body is the probe molecule-immobilized particle or the like, it may be suspended in the particle suspension medium (particle solution) and used. Further, other reaction buffers may be appropriately added to the reaction system of the sample, the capture body, and / or the labeling body. These particle suspension media and reaction buffers are not particularly limited, and examples include, for example, independently known buffer solutions (sodium phosphate buffer, MES, Tris, CFB, MOPS, PIPES, HEPES, tricine buffer, bicine buffer, glycine buffer, etc.), and those independently added with a stabilizing protein such as BSA or serum may also be used.

[0073] Also, when using the blocked labeled lectin as the labeling substance, in the reaction system of the sample and the labeling substance, from the viewpoint of further improving the measurement sensitivity, in addition, a water-soluble polymer (hereinafter referred to as "second water-soluble polymer"; different from the first water-soluble polymer constituting the water-soluble carrier in that it is a free water-soluble polymer not carrying the labeling substance and lectin), free lectin (different from the lectin contained in the blocked labeled lectin, etc. in that it is not fixed to the water-soluble carrier or the labeling substance), etc. may coexist.

[0074] Examples of the second water-soluble polymer include the same ones as those listed for the first water-soluble polymer, and one of these may be used alone or in combination of two or more. Also, it may be a polymer of the same type as the first water-soluble polymer. Among these, from the viewpoint that the measurement sensitivity tends to be further improved, the second water-soluble polymer is preferably at least one selected from the group consisting of polysaccharides and their modified products, more preferably at least one selected from the group consisting of dextran and aminodextran, and their modified products, and even more preferably dextran.

[0075] Also, from the viewpoint that the measurement sensitivity tends to be further improved, 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 3,000,000, and even more preferably 1,500,000 to 2,500,000.

[0076] The amount of the second water-soluble polymer is not particularly limited, but the content of the second water-soluble polymer (the total when the second water-soluble polymer is a combination of two or more) in the reaction solution containing the sample, blocked labeled lectin, and second water-soluble polymer is preferably 0.01 to 10 w / v%, more preferably 0.5 to 3 w / v% (w / v%: weight / volume (g / mL) percent, the same hereinafter).

[0077] Examples of the free lectin include the same ones as those listed as lectins above, and one of these may be used alone or in combination of two or more. Also, it may be of the same type as the lectin contained in the blocked labeled lectin. Among these, from the viewpoint of improving reactivity and suppressing background, it is particularly preferable that the free lectin is of the same type as the lectin contained in the blocked labeled lectin.

[0078] The amount of the free lectin is not particularly limited, but in the reaction solution containing the sample, the blocked labeled lectin, and the free lectin, the content of the free lectin (the total when the free lectin is a combination of two or more) relative to 100 parts by mass of the content of the blocked labeled lectin is preferably 1 to 10,000 parts by mass, and more preferably 10 to 5,000 parts by mass.

[0079] (Measurement step) In the measurement 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 substrate or a luminescent substrate corresponding to the enzyme and reacting is measured. Thereby, the presence or absence of fucose - added hemopexin in the sample is detected by the presence or absence of the signal, and further, when fucose - added hemopexin is present, the amount of fucose - added hemopexin in the sample can be obtained as the signal amount, and if necessary, the amount of fucose - added hemopexin in the sample can be calibrated by comparing with the measured value in the standard sample.

[0080] [Cancer detection method] According to the cancer detection method of the present invention, by using the presence or absence of fucosylated hemopexin measured in the above measurement step as an index, the presence or absence of pancreatic cancer and / or bile duct cancer in the subject from whom the sample is derived can be detected. Further, by using the amount of fucosylated hemopexin measured in the above measurement step as an index, in addition to detecting the presence or absence of the cancer, information about the degree (progression or abundance) of the cancer in the subject from whom the sample is derived can be obtained. Such a cancer detection method can be used in the cancer inspection method of the present invention in addition to research purposes such as drug discovery.

[0081] [Cancer inspection method] In the cancer inspection method of the present invention, fucosylated hemopexin in a sample derived from a subject is measured, and using this as an index, the presence or absence of pancreatic cancer and / or bile duct cancer is detected, or information about the degree of the cancer is obtained, whereby in the subject, it is possible to predict whether or not the subject has pancreatic cancer and / or bile duct cancer, predict the risk of developing the cancer (i.e., the risk of developing cancer in the future), discriminate the presence or absence or the possibility of developing the cancer, or evaluate the progression or severity of the cancer.

[0082] That is, as embodiments of the cancer inspection method of the present invention, specifically, for example, the following embodiments: A screening method for screening a subject predicted to have at least one type of cancer selected from the group consisting of pancreatic cancer and bile duct cancer, the method comprising a measurement step of measuring fucosylated hemopexin in a sample derived from the subject, and a selection step of selecting the subject using the measured fucosylated hemopexin as an index. A method for predicting the risk that a subject will develop at least one type of cancer selected from the group consisting of pancreatic cancer and bile duct cancer, the method comprising a measurement step of measuring fucosylated hemopexin in a sample derived from the subject, and a prediction step of predicting the risk that the subject will develop the cancer using the measured fucosylated hemopexin as an index. A method for discriminating the presence or absence of at least one cancer selected from the group consisting of pancreatic cancer and bile duct cancer in a subject, comprising a measuring step of measuring fucosylated hemopexin in a sample derived from the subject, and a discriminating step of discriminating the subject using the measured fucosylated hemopexin as an index; A method for evaluating the progression or severity of at least one cancer selected from the group consisting of pancreatic cancer and bile duct cancer in a subject, comprising a measuring step of measuring fucosylated hemopexin in a sample derived from the subject, and an evaluating step of evaluating the subject using the measured fucosylated hemopexin as an index; include;

[0083] [Screening method] In the present invention, "screening" means selecting a subject who may be suffering from the cancer (pancreatic cancer and / or bile duct cancer). Specifically, in the present invention, the screening method includes a method of selecting a subject from a group with no or low possibility of suffering from (including recurrence) pancreatic cancer and / or bile duct cancer, predicting that the subject has a high possibility of suffering from the cancer. In the screening method, according to the purpose, for example, the subject may be selected at a level where a medium possibility can be expected.

[0084] More specifically, for example, the subjects are divided into two groups: a group consisting of those who are truly suffering from the cancer (positive group) and a group consisting of those who are not suffering from the cancer (normal group: negative group), and the subject is selected from the normal group (negative group) predicting that the subject is included in the positive group. As a prediction criterion in this case, for example, it is preferable that the negative agreement rate (the ratio of the subjects predicted to be in the normal group by the screening who are truly in the normal group) is 95% or more.

[0085] [Cancer risk prediction method] In the present invention, "predicting the risk" means predicting whether or not the subject is likely to develop (including recurrence) the cancer (pancreatic cancer and / or bile duct cancer, preferably pancreatic cancer) in the future, or evaluating the degree thereof (such as high / moderate / low evaluation) if possible. Further, as a result of predicting the risk, screening may be included to select a subject predicted to have the possibility or a high possibility from a group having no possibility or a low possibility. Since fucosylated haptoglobin is considered to be significantly increased even in chronic pancreatitis with a high risk of developing pancreatic cancer in the future, fucosylated haptoglobin can be an index for predicting the risk of developing pancreatic cancer in particular.

[0086] More specifically, for example, it is divided into two groups: a group (positive group) consisting of those who will truly develop the cancer in the future within a certain period, and a group (normal group: negative group) consisting of those who will not develop the cancer in the same future, and it is determined whether the subject is included in the positive group or the negative group. As a discrimination criterion in this case, for example, it is preferable that the negative agreement rate (the ratio of subjects discriminated as the normal group by the method who are truly in the normal group) is 95% or more.

[0087] 〔Discrimination method〕 In the present invention, "discrimination" means distinguishing pancreatic cancer and / or bile duct cancer from other diseases or conditions and determining whether the subject has the cancer. Further, it includes not only the determination of the presence or absence of the disease, but also the evaluation of the degree thereof (such as high / moderate / low evaluation) if there is a possibility of the disease. In the present invention, the discrimination method specifically includes a method for determining whether or not the subject has pancreatic cancer or bile duct cancer or the possibility thereof is high, regardless of the presence or absence of symptoms. For example, a method for determining whether the cancer the subject has is pancreatic cancer or bile duct cancer, or a method for determining that the possibility of the cancer is high; a method for determining whether pancreatic cancer or bile duct cancer the subject had in the past has recurred, or a method for determining that the possibility of the cancer recurring is high is also included.

[0088] More specifically, for example, it is divided into two groups: a group consisting of those who are truly suffering from the cancer (positive group) and a group consisting of those who are not suffering from the cancer (normal group: negative group), and it is determined whether the subject is included in the positive group or the negative group. Such discrimination can be applied to assist in the diagnosis of pancreatic cancer and / or bile duct cancer by a doctor or the like. As the discrimination criterion in this case, for example, it is preferable that the negative coincidence rate (the ratio of the subjects discriminated as the normal group by the discrimination who are truly in the normal group) is 95% or more.

[0089] 〔Evaluation method〕 In the present invention, the evaluation method includes, for example, a method for evaluating the progression degree or severity of pancreatic cancer and / or bile duct cancer that the subject is suffering from; a method for providing an index for determining the treatment policy for the cancer that the subject is suffering from; a method for determining the treatment effect of the cancer.

[0090] More specifically, for example, the amount of fucose - added hemopexin in a group consisting of those who are not suffering from the cancer or those with a low progression degree or severity of the cancer is compared with the amount of fucose - added hemopexin of the subject. If the amount in the subject is larger, it can be evaluated that the progression degree or severity is high, and if it is smaller, it can be evaluated that the progression degree or severity is low. Also, the amount of fucose - added hemopexin at the time of the initial discovery or before treatment of the cancer of the subject is compared with the current amount of fucose - added hemopexin. If the amount has increased, it can be evaluated that the treatment effect is low, and if it has decreased, it can be evaluated that the treatment effect is high.

[0091] (Cut - off value) In the screening method's selection step, the prediction step of the cancer susceptibility prediction method, and the discrimination step of the discrimination method, if even a small amount of fucose - added hemopexin is detected in the measurement step, the detected subject may be predicted or discriminated as having (or having a high possibility of having) pancreatic cancer and / or bile duct cancer, or may be predicted as having a possibility (or a high possibility) of suffering from the cancer. However, it is preferable to predict or discriminate according to the measured amount of fucose - added hemopexin.

[0092] For example, the amount of fucosylated haptoglobin measured in the measurement step is compared with a predetermined cut-off value, and a subject with an amount of fucosylated haptoglobin higher than the cut-off value is predicted or determined to have pancreatic cancer and / or bile duct cancer (or is likely to have it or is highly likely to have it).

[0093] In the present invention, the "cut-off value" is a predetermined value that serves as a criterion for determination based on the amount of fucosylated haptoglobin, and indicates the boundary value for determining the above-mentioned positive group and negative group. Such a cut-off value is not particularly limited because it is appropriately set according to the purpose of the cancer detection method of the present invention, the method for measuring the amount of fucosylated haptoglobin, the nature of the subject and the sample, the dilution conditions, etc. For example, by setting the cut-off value to a relatively low value, the detection sensitivity can be increased, that is, patients in the early stage of the cancer can be collected more widely to some extent, enabling early detection. On the other hand, by setting the cut-off value to a relatively high value, it becomes possible to perform the screening and the discrimination with higher accuracy.

[0094] As an example of the cut-off value of the screening method or the discrimination method, for example, when the sample is a serum specimen, diluted 1 / 500 by volume ratio, anti-haptoglobin antibody-immobilized particles are used as the capture body, and blocked labeled AOL is used as the label, and the amount of fucosylated haptoglobin measured by a sandwich immunoassay is expressed as the luminescence intensity (count) of light having a maximum absorption at a wavelength of 463 nm with the labeling substance being ALP and the substrate being AMPPD (in the case of Example 1), examples include 1,500,000 to 2,500,000 counts, preferably 1,600,000 to 2,000,000 counts, but it is not limited thereto. The cut-off value determined within the above range is selected and applied from within the range according to the purpose of the cancer detection method of the present invention, the method for measuring fucosylated haptoglobin, the nature of the subject and the sample, etc.

[0095] According to such a cancer detection method of the present invention, it is possible to provide information for determining a treatment policy specific to pancreatic cancer and / or bile duct cancer, etc., by distinguishing it from other cancers. Further, it is possible to identify a subject who is highly likely to develop (including recurrence) pancreatic cancer and / or bile duct cancer, or a subject who has developed (including recurrence) or is highly likely to develop the above cancer, and by performing further examinations and definitive diagnoses, early detection and early treatment intervention of pancreatic cancer and / or bile duct 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, it is also possible to obtain information on the malignancy and prognosis of cancer by the cancer detection method of the present invention. Note that the method of the present invention is also a method for assisting the diagnosis of pancreatic cancer and / or bile duct cancer by a doctor or the like, or a method for providing information for the diagnosis of pancreatic cancer and / or bile duct cancer by a doctor or the like.

[0096] Furthermore, the method of the present invention is also suitable as a method for combining with a conventional measurement method of other monitoring markers, and thereby it becomes possible to further improve the detection sensitivity and diagnostic accuracy of pancreatic cancer and / or bile duct cancer.

[0097] <Kit> The kit of the present invention is a kit for use in the above cancer detection method or cancer inspection method of the present invention, and is a kit comprising a first probe molecule capable of specifically binding to hemopexin and a second probe molecule capable of specifically binding to fucose. The first probe molecule and the second probe molecule are as described above, including their preferred embodiments.

[0098] Further, as the kit of the present invention, it is also preferable to include a capturer comprising a water-insoluble carrier and any one of the first probe molecule and the second probe molecule immobilized on the water-insoluble carrier, and a labeler comprising a labeling substance and the other of the first probe molecule and the second probe molecule. The capturer and the labeler are as described above, including their preferred embodiments.

[0099] In the kit of the present invention, the first probe molecule, the second probe molecule, the capturer, and the label may each independently be in a solid (powder) form or in a liquid form dissolved in a buffer solution. When in a liquid form, the concentrations of the first probe molecule, the second probe molecule, the capturer, and the label in each solution are not particularly limited, but are each independently preferably, for example, 0.01 to 10 μg / mL, more preferably 0.1 to 5.0 μg / mL, and even more preferably 0.5 to 3.0 μg / mL.

[0100] In addition to the above, the kit of the present invention may further include components to be provided in ordinary immunological measurement methods such as ELISA, CLEIA, immunochromatography, and methods analogous thereto. For example, when the above sandwich method is used as the principle of the measurement method according to the present invention, at least one selected from the group consisting of magnetic beads, plates, sensor chips for immobilizing the probe molecule, the standard sample (each concentration), control reagents, the particle suspension medium, the reaction buffer, the washing solution, the second water-soluble polymer, and free lectin may be further included. Further, when the labeling substance is an enzyme, a substrate, a reaction stop solution, etc. necessary for detecting and quantifying the labeling substance may be further included.

[0101] Furthermore, the kit of the present invention may optionally include the diluent, a pretreatment solution for pretreating the sample; a dilution cartridge; a reaction stop solution or neutralizing solution for pretreatment. When immunochromatography is adopted as the sandwich method, a device including a zone carrying the capturer and / or the label may be further included. The device may include other components suitable for immunochromatography, such as a developing solution pad and an absorption pad. Furthermore, the kit of the present invention may further include an instruction manual for using the kit.

Examples

[0102] Hereinafter, the present invention will be described more specifically based on Examples and Comparative Examples, but the present invention is not limited to the following Examples. In each of the Examples and Comparative Examples, the display of “%” indicates weight / volume (w / v: g / mL) percent unless otherwise specified.

[0103] (Example 1) Measurement of AOL-bound glycated hemopexin (fucosylated hemopexin) contained in a serum specimen using blocked-labeled lectin (AOL) (1) Preparation of hydrazinated dextran To 4.8 mL of 0.1 M phosphate buffer (pH 7.0), 240.0 mg of dextran with a molecular weight of 250k (manufactured by CarboMer) was added and dissolved by stirring for 30 minutes in the dark at 25°C. Next, 2.664 mL of 150 mM NaIO4 and 0.536 mL of ion-exchanged water were added, and the mixture was stirred for 30 minutes in the dark at 25°C. Then, using a PD-10 column (manufactured by GE Healthcare, Sephadex G-25 packed column: hereinafter simply referred to as “Sephadex G-25”), buffer exchange was performed with 0.1 M sodium phosphate buffer (pH 6.0) to obtain 20.0 mL of a solution. To the obtained solution, 5.04 g of NH2NH2·HCl was added, and the mixture was stirred for 2 hours in the dark at 25°C. 800 mg of DMAB (dimethylamine borane) was added, and the mixture was further stirred for 2 hours in the dark at 25°C. After dialysis with 4 L of ion-exchanged water using an RC50K (regenerated cellulose with a molecular weight of 50,000) dialysis membrane for 3 hours in the dark, the mixture was left standing 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 a solution. The concentration of dextran in the obtained solution was adjusted to 1.0 mg / mL to obtain a solution of hydrazinated dextran.

[0104] (2) Preparation of dextran-enzyme conjugate For 30.0 mL of 10 mg / mL alkaline phosphatase (manufactured by Oriental Yeast Co., Ltd., ALP-50), buffer exchange was performed by gel filtration (Sephadex G-25) using 0.1 M sodium phosphate buffer (pH 6.0) to prepare 90.6 mL of a 3.0 mg / mL solution. Then, 45.3 mL of 27 mM NaIO4 was added and stirred in the dark at 25 °C for 3 minutes. Next, 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 hydrazinated dextran prepared in (1) of Example 1 was added so that the concentration of hydrazide groups (amino groups) became 25 μM, and stirred in the dark at 25 °C 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 in the dark at 25 °C for 2 hours. An ultrafiltration module (Pellicon XL50, manufactured by Merck Millipore) was attached to a Labscale TFF System (manufactured by Merck Millipore) and concentrated to 15 mL, and gel filtration (Superdex 200pg) was performed using 0.1 M sodium phosphate buffer (pH 7.0) to obtain 14 mL of a 3.0 mg / mL solution of dextran-enzyme conjugate.

[0105] (3) Maleimide-PEGylation of Dextran-Enzyme Conjugate To the dextran-enzyme conjugate prepared in (2) of Example 1, 0.1 M sodium phosphate buffer (pH 7.0) was added to prepare 750 μL of a 2 mg / mL dextran-enzyme conjugate. To this, 8.35 μL of 250 mM SM(PEG)4 (manufactured by Thermo Fisher Scientific, SM(PEG)4) dissolved in DMSO was added and mixed, and the mixture was inverted and mixed in the dark at 25 °C for 1 hour. After the reaction, buffer exchange was performed using a PD-10 column (Sephadex G-25) with 0.1 M sodium phosphate buffer (pH 6.3) containing 20 mM EDTA·2Na and 0.5% CHAPS. After buffer exchange, the maleimide-PEGylated dextran-enzyme conjugate was concentrated using a centrifugal filter (Amicon Ultra 50K, manufactured by Merck), and the final concentration was adjusted to 2 mg / mL.

[0106] (4) Thiolation of lectin To 1 mL of a 5 mg / mL solution of Galanthus nivalis agglutinin (AOL; manufactured by Tokyo Chemical Industry Co., Ltd.), 1.5 mL of 0.1 M sodium phosphate buffer (pH 7.0) was added to obtain 2.5 mL of a 2 mg / mL AOL solution. To 2.5 mL of the AOL solution, 100 μL of 0.5 M EDTA·2Na (pH 8.0) was added and mixed, and then 75 μL of a 10 mg / mL 2-iminothiolane hydrochloride solution was added, and the mixture was inverted and mixed in the dark at 25 °C for 1 hour. After the reaction, buffer exchange was performed using a PD-10 column (Sephadex G-25) with 0.1 M sodium phosphate buffer (pH 6.3) containing 20 mM EDTA·2Na and 0.5% CHAPS. The AOL solution after buffer exchange was adjusted to 650 μg / mL.

[0107] (5) Coupling To 2 mL of the thiolated AOL solution adjusted to 650 μg / mL obtained in (4) of Example 1, 500 μL of the solution of maleimide-PEGylated dextran-enzyme conjugate (2 mg / mL) obtained in (3) of Example 1 was added, and the mixture was inverted and mixed for 1 hour in the dark at 25°C to couple AOL with the dextran-enzyme conjugate. After the reaction, 25 μL of 200 mM 3-Mercapto-1,2-propanediol was added, and the mixture was inverted and mixed for 30 minutes in the dark at 25°C. Thereafter, 50 μL of 200 mM 2-Iodoacetamide was added, and the mixture was inverted and mixed for 30 minutes in the dark at 25°C. The solution after the reaction was concentrated using a centrifugal filter (Amicon Ultra 50K, manufactured by Merck), passed through a φ0.22 μm filter, and purified by gel filtration chromatography (column: Superose 6 Increase 10 / 300 GL, buffer: 0.1M MES, 0.5M NaCl, 1mM MgCl2, 0.1mM ZnCl2, 5mM Glucose, 0.05% CHAPS, pH 6.8), and finally 3.5 mL of a solution of blocked labeled lectin (AOL) (dextran-enzyme-AOL conjugate) at 151.1 μg / mL was obtained.

[0108] (6) Preparation of measurement reagent 0.6 mg of anti-hempexin antibody 32 (manufactured by Abcam) was buffer-exchanged to 50 mM MES (pH 5.5) using a PD-10 column (Sephadex G-25) to obtain 0.5 mL of an anti-hempexin antibody solution at 0.837 mg / mL. The obtained anti-hempexin antibody solution was mixed with carboxylated magnetic particles (manufactured by Fujirebio) previously activated with EDC (N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride, manufactured by Merck) and Sulfo-NHS (N-hydroxysulfosuccinimide, manufactured by ThermoFisher), and inverted and mixed at 25 °C for 1 hour to bind the anti-hempexin antibody to the magnetic particles. After the reaction with the antibody, the particles were quenched with 1 M Tris-HCl, 10% BSA, 0.1% NaN3, pH 7.0, and then washed and masked with a masking buffer (50 mM MES, 1 mM EDTA·2Na, 150 mM NaCl, 2% BSA, 0.1% ProClin 300, pH 6.0), finally obtaining 13 mg of anti-hempexin antibody-conjugated magnetic particles.

[0109] To 13 mg of anti-hempexin antibody-conjugated magnetic particles (hereinafter simply referred to as "antibody-conjugated particles"), 3 mL of a 20 mM sodium periodate solution (20 mM NaIO4, 100 mM NaOAc, 150 mM NaCl, pH 5.5) was added and mixed, and the sugar chains of the antibody conjugated to the particles were oxidized by performing inversion mixing at 4 °C in the dark for 30 minutes. The antibody-conjugated particles after the oxidation treatment were washed three times with 3 mL of 0.1 M sodium phosphate buffer (pH 6.0). The washed antibody-conjugated particles were replaced with a 0.1 M sodium phosphate buffer (pH 6.0) containing 10 mM glycine, and inversion mixing was performed at 25 °C in the dark for 1 hour to block the aldehyde groups generated by the oxidation of the sugar chains of the antibody with glycine. Further, 50 μL of 10 mg / mL DMAB was added to the antibody-conjugated particle solution after the reaction, and inversion mixing was performed at 25 °C in the dark for 30 minutes to stabilize the unstable bond between the aldehyde group derived from the antibody sugar chain and glycine. The antibody-conjugated particles after stabilization were washed three times with 1.5 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), and BSA was physically adsorbed by performing inversion mixing under the conditions of the same buffer and 37 °C for 16 hours. The antibody-conjugated magnetic particles with physically adsorbed BSA 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 at 4 °C in the same buffer. The obtained oxidized anti-hempexin antibody-conjugated magnetic particles were diluted in a 50 mM Tris-based solution so that the concentration of the antibody-conjugated particles became 0.005% to prepare an oxidized anti-hempexin antibody-conjugated particle solution.

[0110] Also, the blocked labeled lectin (AOL) obtained in (5) of Example 1 was diluted in a 50 mM MES-based solution so that the concentration became 0.5 μg / mL to prepare a labeled body fluid.

[0111] (7) Measurement of AOL-conjugated sugar chain-added hempexin (fucosylated hempexin) contained in a serum specimen Eleven serum specimens collected from healthy individuals (healthy group: healthy individuals 1 - 11), ten serum specimens collected from pancreatic cancer patients (pancreatic cancer group: pancreatic cancer 1 - 10), and five serum specimens collected from cholangiocarcinoma patients (cholangiocarcinoma group: cholangiocarcinoma 1 - 5) were each diluted to a concentration of 1 / 500 by volume using a specimen diluent (manufactured by Fujirebio Inc.).

[0112] For each diluted specimen, the measurement of AOL-bound glycosylated hemopexin (fucosylated hemopexin) contained in the serum specimen was performed using Lumipulse® L-2400 (manufactured by Fujirebio Inc.). That is, 50 μL of each diluted specimen solution was mixed with 50 μL of the oxidized anti-hemopexin antibody-conjugated particle solution prepared in (6) of Example 1 and reacted at 37°C for 8 minutes. Subsequently, the magnetic particles were magnetically collected and washed 5 times with a Lumipulse® washing solution (manufactured by Fujirebio Inc.). Then, 50 μL of the labeled body fluid prepared in (6) of Example 1 was added to each and reacted at 37°C for 8 minutes. Subsequently, the magnetic particles were magnetically collected, washed 5 times, and then 50 μL of a Lumipulse® substrate solution (manufactured by Fujirebio Inc.) containing AMPPD (3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoryloxy)phenyl-1,2-dioxetane disodium salt) was added and reacted at 37°C for 4 minutes. The luminescence intensity (count) of the light having a maximum absorption at a wavelength of 463 nm, which is released by the catalytic action of the alkaline phosphatase of the blocked labeled lectin (AOL) bound to the magnetic particles and the decomposition of AMPPD, was measured using Lumipulse® L-2400 (manufactured by Fujirebio Inc.) and used as the measurement result. The measurement results for each specimen are shown in Table 1 below. The results shown are the average values of duplicate measurements.

[0113]

Table 1

[0114] (8) Comparison of measurement values among the groups of healthy individuals, pancreatic cancer, and cholangiocarcinoma Regarding the results in Table 1, it was examined whether there was a statistically significant difference between the healthy population group and the pancreatic cancer group. Figure 1 shows the measurement results of fucosylated hemopexin in the healthy population group and the pancreatic cancer group. When the test was performed by the Wilcoxon test, a significant difference was found in the measured values of fucosylated hemopexin between the healthy population group and the pancreatic cancer group (Figure 1, p < 0.0001). Furthermore, a cut-off value was calculated from the measured values of the healthy population group, and the detection ability of pancreatic cancer patients was tested when specimens exceeding the cut-off value were regarded as positive. The cut-off value was calculated to be 1,911,499 counts or less by adding the value obtained by multiplying the standard deviation value of the measured values of the healthy population group by 2 to the average of the measured values of the healthy population group. As a result, 9 out of 10 specimens in the pancreatic cancer group were determined to be positive at this cut-off value, which was 90.9%. Also, all 11 specimens in the healthy population group were determined to be negative. From the above, it was shown that the measurement of fucosylated hemopexin becomes a powerful new index for discriminating between healthy individuals and pancreatic cancer patients.

[0115] Similarly, regarding the results in Table 1, it was examined whether there was a statistically significant difference between the healthy population group and the cholangiocarcinoma group. Figure 2 shows the measurement results of fucosylated hemopexin in the healthy population group and the cholangiocarcinoma group. When the test was performed by the Wilcoxon test, a significant difference was found in the measured values of fucosylated hemopexin between the healthy population group and the cholangiocarcinoma group (Figure 2, p = 0.0022). Furthermore, using the cut-off value calculated in the same manner as above, the detection ability of cholangiocarcinoma patients by fucosylated hemopexin was tested, and all 5 specimens measured this time were determined to be positive. From the above, it was shown that the measurement of fucosylated hemopexin becomes a powerful new index for discriminating between healthy individuals and cholangiocarcinoma patients.

[0116] (Comparative Example 1) Verification of the reactivity of fucosylated hemopexin against cancers other than pancreatic cancer and cholangiocarcinoma As a comparison with Non-Patent Document 2, 50 serum specimens collected from hepatocellular carcinoma patients (hepatocellular carcinoma group: hepatocellular carcinoma 1 to 50) were measured for fucosylated hemopexin in the same manner as in (7) of Example 1. The measurement results for each specimen are shown in Table 2 below.

[0117] [Table 2]

[0118] Regarding the measurement results of the hepatocellular carcinoma group shown in Table 2 and the measurement results of the healthy population group shown in Table 1, it was examined whether there was a statistically significant difference between the healthy population group and the hepatocellular carcinoma group. Figure 3 shows the measurement results of fucosylated hemopexin in the healthy population group and the hepatocellular carcinoma group. When the test was performed by the Wilcoxon test, a significant difference was also found in the measured values of fucosylated hemopexin between the healthy population group and the hepatocellular carcinoma group (Figure 3, p = 0.0009). However, only 10 out of 50 specimens in the hepatocellular carcinoma group were determined to be positive exceeding the cut-off value calculated in the same manner as in (8) of Example 1, and the detection frequency was clearly lower compared to the pancreatic cancer group and the cholangiocarcinoma group.

[0119] Furthermore, regarding the measurement results of the hepatocellular carcinoma group shown in Table 2 and the measurement results of the pancreatic cancer group and the cholangiocarcinoma group shown in Table 1, it was also examined whether there was a statistically significant difference between each of the pancreatic cancer group and the hepatocellular carcinoma group or the cholangiocarcinoma group and the hepatocellular carcinoma group. Figure 4 shows the measurement results of fucosylated hemopexin in the pancreatic cancer group and the hepatocellular carcinoma group, and Figure 5 shows the measurement results of fucosylated hemopexin in the cholangiocarcinoma group and the hepatocellular carcinoma group, respectively. When the test was performed by the Wilcoxon test, clearly, the measured values in the pancreatic cancer group and the measured values in the cholangiocarcinoma group were higher than the measured values in the hepatocellular carcinoma group (Figure 4, pancreatic cancer group vs hepatocellular carcinoma group, p ≤ 0.0001; Figure 5, cholangiocarcinoma group vs hepatocellular carcinoma group, p = 0.0003).

[0120] From the above results, the measured values of fucosylated hemopexin do not show uniformly high values for all cancers, but vary depending on the type of cancer, and in particular, it was shown that they are specifically high in pancreatic cancer and cholangiocarcinoma.

Industrial Applicability

[0121] According to the present invention, it becomes possible to provide a cancer detection method and a cancer test method that can specifically detect pancreatic cancer and bile duct cancer with high sensitivity using a new biomarker as an index, and a kit used for these methods.

Claims

1. A method for detecting pancreatic cancer, comprising a measurement step of measuring fucose - added hemopexin in a sample, wherein the sample is serum, and the measurement step is a step of contacting the sample with a first probe molecule capable of specifically binding to hemopexin and a second probe molecule capable of specifically binding to fucose.

2. A method for examining pancreatic cancer, comprising a measurement step of measuring fucose - added hemopexin in a sample derived from a subject, wherein the sample is serum, and the measurement step is a step of contacting the sample with a first probe molecule capable of specifically binding to hemopexin and a second probe molecule capable of specifically binding to fucose.

3. The method according to claim 2, which is a method for providing information for screening a subject predicted to have pancreatic cancer using fucose - added hemopexin as an index.

4. The method according to claim 2, which is a method for providing information for predicting the risk that a subject has pancreatic cancer using fucose - added hemopexin as an index.

5. The method according to any one of claims 1 to 4, wherein in the fucose - added hemopexin, the fucose is at least one selected from the group consisting of α1 - 6 fucose and α1 - 2 fucose.

6. The method according to any one of claims 1 to 5, wherein in the fucose - added hemopexin, the fucose is at least one selected from the group consisting of an AOL - binding sugar chain that binds to AOL and an AAL - binding sugar chain that binds to AAL.

7. The method according to any one of claims 1 to 6, wherein the first probe molecule is an antibody capable of specifically binding to hemopexin.

8. The method according to any one of claims 1 to 7, wherein the second probe molecule is a lectin capable of specifically binding to fucose.

9. The measurement step is a step of contacting the sample with a capture body and a label body, the capture body comprises a water - insoluble carrier and either one of the first probe molecule and the second probe molecule immobilized on the water - insoluble carrier, and the label body comprises a labeling substance and the other of the first probe molecule and the second probe molecule. The method according to any one of claims 1 to 8, characterized in that...

10. The capture body includes a water-insoluble carrier and a first probe molecule immobilized on the water-insoluble carrier, and The labeling body includes 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 blocked labeling lectin which is a lectin capable of specifically binding to fucose. The method according to claim 9, characterized in that...

11. A kit for use in the method according to any one of claims 1 to 10, characterized by comprising a first probe molecule capable of specifically binding to hemopexin and a second probe molecule capable of specifically binding to fucose.

12. The kit according to claim 11, characterized in that the first probe molecule is an antibody capable of specifically binding to hemopexin.

13. The kit according to claim 11 or 12, characterized in that the second probe molecule is a lectin capable of specifically binding to fucose.

14. 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 labeling body comprising a labeling substance and the other of a first probe molecule and a second probe molecule. The kit according to any one of claims 11 to 13, characterized by comprising the above.

15. The capture body includes a water-insoluble carrier and a first probe molecule immobilized on the water-insoluble carrier, and The labeling body includes 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 blocked labeling lectin which is a lectin capable of specifically binding to fucose. The kit according to claim 14, characterized in that...

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