JP7770666B2 - Cancer detection method, cancer testing method, and kits used therefor - Google Patents

Description

The present invention relates to a cancer detection method, a cancer testing method, and kits used therein; more specifically, to a method for detecting at least one type of cancer selected from the group consisting of pancreatic cancer and bile duct cancer, a method for testing said cancer, and kits used therein.

Biomarkers such as tumor markers are useful for detecting diseases such as malignant tumors, as guidelines for determining treatment plans, and as monitoring markers for assessing treatment effectiveness, and have been the subject of intensive research in recent years. Examples of such biomarkers include CA19-9 and CEA. For example, CA19-9 is known to have elevated blood concentrations in patients with gastrointestinal cancers, particularly colorectal cancer, pancreatic cancer, bile duct cancer, and gallbladder cancer, and has traditionally been used to detect these cancers, as guidelines for determining treatment plans, and as a monitoring marker for assessing treatment effectiveness.

In recent years, attention has been focused on changes in the sugar chain structure of glycoproteins in cancer. For example, Non-Patent Document 1 reports that fucosylated haptoglobin (Fuc-Hpt) is significantly increased in pancreatic cancer patients compared to healthy individuals. Furthermore, Non-Patent Document 2 describes that fucosylated transferrin is significantly elevated in hepatocellular carcinoma patients.

Eiji Miyoshi et al., Cancer Science, Vol. 107, No. 10, 2016, p. 1357-1362 Yokota Tsuyoshi, "Study on transferrin sugar chain mutations in serum of hepatocellular carcinoma patients, especially in relation to alpha-fetoprotein sugar chains", Liver, Vol. 35, No. 8, 1994, pp. 587-595

However, the above-mentioned conventional monitoring markers still lack sufficient specificity for each cancer type, and while pancreatic cancer and bile duct cancer are particularly difficult to diagnose early, they have not yet been sufficient to distinguish with high accuracy between pancreatic cancer patients and healthy individuals, or between bile duct cancer patients and healthy individuals. Furthermore, as mentioned above, structural changes in the glycans of glycoproteins in cancer have attracted attention, but have not yet been fully elucidated. These glycans hold promise as new biomarkers for cancer detection.

The present invention was made in consideration of the above-mentioned problems, and aims to provide a cancer detection method and cancer testing method that use new biomarkers as indicators and are capable of specifically detecting pancreatic cancer and bile duct cancer with high accuracy, as well as kits for use in these methods.

With the aim of searching for glycans as new tumor markers that could not be measured with existing antibodies, the inventors conducted screening using not only anti-transferrin antibodies but also a complex (blocked labeled lectin) comprising a water-soluble carrier made of a water-soluble polymer and a labeling substance and lectin immobilized on the water-soluble carrier. As a result, they found that the amount of molecules detected when AOL was used as the lectin, i.e., transferrin with lectin-binding fucose attached (fucosylated transferrin), was significantly increased in pancreatic cancer patients and bile duct cancer patients compared to healthy subjects. Therefore, they discovered that measuring fucose-attached transferrin makes it possible to specifically detect pancreatic cancer and bile duct cancer and to distinguish pancreatic cancer patients from healthy subjects, or bile duct cancer patients from healthy subjects, with high accuracy, leading to the completion of the present invention.

The present invention has been made based on these findings and has the following aspects.
[1]
A method for detecting 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 fucose-added transferrin in a sample.
[2]
A method for testing for 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 fucose-added transferrin in a sample derived from a subject.
[3]
The method according to [2], which is a 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, comprising: a measuring step of measuring fucose-linked transferrin in a sample derived from the subject; and a selecting step of selecting a subject using the measured fucose-linked transferrin as an indicator.
[4]
The method according to [2], which is a method for predicting a risk of a subject developing at least one type of cancer selected from the group consisting of pancreatic cancer and bile duct cancer, comprises: a measuring step of measuring fucose-linked transferrin in a sample derived from the subject; and a predicting step of predicting the risk of the subject developing the cancer using the measured fucose-linked transferrin as an indicator.
[5]
The method according to any one of [1] to [4], wherein the fucose in the fucosylated transferrin 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 transferrin, fucose is at least one selected from the group consisting of an AOL-linked glycan that binds to AOL and an AAL-linked glycan that binds to AAL.
[7]
The method according to any one of [1] to [6], wherein the measuring step is a step of contacting the sample with a first probe molecule capable of specifically binding to transferrin and a second probe molecule capable of specifically binding to fucose.
[8]
The method according to [7], wherein the first probe molecule is an antibody capable of specifically binding to transferrin.
[9]
The method according to [7] or [8], wherein the second probe molecule is a lectin capable of specifically binding to fucose.
[10]
the measuring step is a step of contacting the sample with a capture body and a label,
the capture body comprises a water-insoluble carrier and either a first probe molecule or a second probe molecule immobilized on the water-insoluble carrier; and
the label comprises a labeling substance and the other of the first probe molecule and the second probe molecule;
The method according to any one of [7] to [9],
[11]
The capture body comprises a water-insoluble carrier and a first probe molecule immobilized on the water-insoluble carrier, and
the labeled body comprises a water-soluble carrier, and a labeling substance and a second probe molecule immobilized on the water-soluble carrier, and the second probe molecule is a blocked labeled lectin that is a lectin capable of specifically binding to fucose;
The method according to [10],
[12]
A kit for use in the method according to any one of [7] to [11], the kit comprising a first probe molecule capable of specifically binding to transferrin 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 transferrin.
[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 body comprising a water-insoluble carrier and either a first probe molecule or a second probe molecule immobilized on the water-insoluble carrier; and
a label comprising a labeling substance and the other of the first probe molecule and the second probe molecule;
The kit according to any one of [12] to [14], comprising:
[16]
The capture body comprises a water-insoluble carrier and a first probe molecule immobilized on the water-insoluble carrier, and
the labeled body comprises a water-soluble carrier, and a labeling substance and a second probe molecule immobilized on the water-soluble carrier, and the second probe molecule is a blocked labeled lectin that is a lectin capable of specifically binding to fucose;
The kit according to [15],

The present invention makes it possible to provide a cancer detection method and cancer testing method that use new biomarkers as indicators and are capable of specifically detecting pancreatic cancer and bile duct cancer with high accuracy, as well as kits for use in these methods.

1 is a graph showing the results of measuring fucosylated transferrin in a healthy subject group and a pancreatic cancer group. 1 is a graph showing the results of measuring fucosylated transferrin in a healthy subject group and a bile duct cancer group. 1 is a graph showing the results of measuring fucosylated transferrin in a healthy subject group and a hepatocellular carcinoma group. 1 is a graph showing the measurement results of fucosylated transferrin in a pancreatic cancer group and a hepatocellular carcinoma group. 1 is a graph showing the results of measuring fucosylated transferrin in a bile duct cancer group and a hepatocellular carcinoma group.

The present invention will now be described in detail with reference to preferred embodiments.

<Cancer detection methods, cancer testing methods>
The cancer detection method of the present invention is a method for detecting at least one type of cancer selected from the group consisting of pancreatic cancer and bile duct cancer, and includes a measurement step of measuring fucose-linked transferrin in a sample. Also, the cancer testing method of the present invention is a method for testing at least one type of cancer selected from the group consisting of pancreatic cancer and bile duct cancer, and includes a measurement step of measuring fucose-linked transferrin in a sample derived from a subject.

[cancer]
In the present invention, "cancer" includes epithelial malignant tumors (carcinomas) and non-epithelial malignant tumors (sarcomas). The cancer to be detected or examined in the method of the present invention is at least one type of cancer selected from the group consisting of pancreatic cancer and bile duct cancer. Pancreatic cancer in the present invention refers to cancer occurring in the pancreas, including, for example, invasive pancreatic ductal carcinoma (conventional pancreatic cancer), pancreatic endocrine tumor, intraductal papillary mucinous tumor, mucinous cystic tumor, acinar cell carcinoma, undifferentiated carcinoma, serous cystadenocarcinoma, and metastatic pancreatic cancer. Among these, invasive pancreatic ductal carcinoma is a representative pancreatic cancer, and is preferred in the present invention. Furthermore, bile duct cancer in the present invention refers to cancer occurring in the bile duct (either within the liver or outside the liver), including, for example, intrahepatic cholangiocarcinoma, extrahepatic cholangiocarcinoma (hilar cholangiocarcinoma, upper bile duct carcinoma, middle bile duct carcinoma, and lower bile duct carcinoma), ampullary carcinoma, and cystic duct carcinoma in the bile duct region. Among these, the bile duct cancer according to the present invention is preferably extrahepatic bile duct cancer, which occurs in the bile duct outside the liver.

[Fucose-added transferrin]
In the present invention, "fucosylated transferrin (sometimes referred to herein as "fucosylated transferrin")" refers to a molecule comprising transferrin and fucose attached and bound to transferrin. Multiple fucose may be attached to one transferrin molecule.

"Transferrin" is a glycoprotein that has a specific affinity for heme or iron and is involved in the transport of hemoglobin. It has traditionally been used as a monitoring marker for diagnosing iron deficiency anemia and liver damage. Transferrin typically consists of a protein portion consisting of approximately 700 amino acids and a glycan portion consisting of multiple glycans attached to it. It has a molecular weight of approximately 800,000, of which approximately 10% is the glycan portion. Note that when simply referring to "transferrin" in the present invention, the glycan portion of such transferrin does not include "fucose," as described below.

"Fucose" is a monosaccharide with the IUPAC name "(3S,4R,5R,6S)-6-methyloxane-2,3,4,5-tetrol," and is preferably in the L form. Fucose may be directly bound to the protein portion of transferrin, but it is preferably attached by binding to the sugar chain that makes up the sugar chain portion of transferrin.

Fucosylated transferrin according to the present invention preferably contains at least one type of fucose selected from the group consisting of α1-6 fucose and α1-2 fucose. In the present invention, "α1-6 fucose" refers to a glycan in which fucose is attached via an α1,6 bond to N-acetylglucosamine (GlcNAc) present at the reducing end (base) of an N-glycan, and is also referred to as "core fucose." In the present invention, "α1-2 fucose" refers to a glycan in which fucose is attached via an α1,2 bond to the monosaccharide galactose (Gal), and is preferably a glycan in which the terminal fucose residue is α1,2-linked to the second galactose residue from the end of the glycan (Fucα1-2Gal). In this case, the structure of the other part of the sugar chain to which fucose is bound is not particularly limited, but the fucose-added transferrin of the present invention preferably contains fucose as fucose constituting at least one sugar chain selected from the group consisting of AOL-binding sugar chains that bind to AOL and AAL-binding sugar chains that bind to AAL, and more preferably contains fucose as fucose constituting the AOL-binding sugar chain that binds to AOL.

The average mass of such fucose-added transferrin (average mass measured by gel filtration chromatography (GFC) using a marker; the same applies hereinafter) is not particularly limited, but is preferably 70,000 to 90,000 Da, and more preferably 75,000 to 85,000 Da.

[Subject]
In the present invention, the term "subject" refers to the subject on whom the cancer testing method of the present invention is performed, and is preferably a human. The subject of the present invention may be a healthy individual or a person with pancreatic cancer or bile duct cancer but asymptomatic, for the purpose of screening or risk assessment. The subject may also be a patient already known to be afflicted with the cancer or a patient who has previously been afflicted with the cancer, for the purpose of determining prognosis, deciding on a treatment plan, confirming the effectiveness of treatment, or confirming recurrence. Furthermore, in the present invention, the term "healthy individual" refers to a person who does not have the cancer to be tested (i.e., pancreatic cancer and/or bile duct cancer). Whether a subject truly has pancreatic cancer and/or bile duct cancer is determined (definitively diagnosed) by a biopsy of pancreatic tissue and/or bile duct tissue collected from the subject.

[sample]
The "sample" used in the cancer detection method and cancer testing method of the present invention (sometimes collectively referred to simply as the "method of the present invention" herein) is not particularly limited as long as it is a sample in which fucose-modified transferrin can be present. Typical examples include blood samples such as serum, plasma, and whole blood collected from subjects for cancer testing, such as the subject; body fluid samples other than blood, such as urine, sputum, saliva, sweat, cerebrospinal fluid, digestive fluid, semen, lymph, and ascites; mucosal samples such as oral mucosa, pharyngeal mucosa, and intestinal mucosa; and various biopsy samples. The sample may also be cultured cells or cell culture medium. A blood sample is preferred as the sample of the present invention, and serum (also referred to as a "serum sample") is more preferred.

These samples may be diluted or suspended in a diluent as needed. Examples of diluents include buffers such as phosphate buffer, Tris buffer, Good's buffer, borate buffer, acetate buffer, citrate buffer, glycine buffer, succinate buffer, and phthalate buffer. Since transferrin is typically found in high concentrations in blood (e.g., 190-320 mg/dL), the sample used in the present invention may be highly diluted (e.g., 1/10 to 1/1,000,000 by volume for a blood specimen). This allows measurement even with a small amount of sample, and also reduces the influence of contaminants, further improving detection accuracy.

The sample of the present invention may also be subjected to appropriate pretreatment, if necessary. Examples of pretreatment include grinding, freezing, heating, concentration, fractionation, desalting, and the like; addition of pH adjusters, stabilizers, preservatives, antiseptics, surfactants, and the like; and purification, which may be performed alone or in combination of two or more. The purification is not particularly limited, and examples include column treatment; treatment in which impurities are adsorbed by a substance, such as an antibody, immobilized on a water-insoluble carrier and removed from a sample containing fucose-modified transferrin; and treatment in which transferrin containing fucose-modified transferrin is captured by an anti-transferrin antibody immobilized on a water-insoluble carrier, and impurities are removed by washing or the like, followed by release.

[Measurement process]
The method of the present invention includes a measurement step of measuring fucose-linked transferrin in a sample. In the present invention, "measurement" includes detection in which the presence or absence and amount of fucose-linked transferrin in a sample are extracted as a signal, and quantification or semi-quantification of the amount of fucose-linked transferrin according to the amount of the signal.

The method for measuring fucose-modified transferrin is not particularly limited as long as it is a method that can measure fucose-modified transferrin, and conventionally known methods, methods based thereon, or combinations thereof can be appropriately employed. Examples include, but are not limited to, methods using probe molecules that can specifically bind to fucose-modified transferrin; 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 absorptiometry; flow cytometry; mass spectrometry; and combinations thereof.

Among these, the measurement step according to the present invention is preferably a method using a probe molecule capable of specifically binding to fucose-added transferrin. Examples of such probe molecules include antibodies; binding proteins such as protein A, protein G, and protein L; avidin D, streptavidin, and other avidins; lectins; galectins, sugar chain receptors, and immunoreceptors. In the present invention, the term "antibody" includes not only complete antibodies, but also antibody fragments (e.g., Fab, Fab', F(ab') ₂ , Fv, single-chain antibodies, diabodies, and the like) and minibodies formed by binding antibody variable regions.

A probe molecule capable of specifically binding to fucose-added transferrin is preferably a combination of a probe molecule capable of specifically binding to transferrin (referred to in the present invention as a "first probe molecule") and a probe molecule capable of specifically binding to fucose (referred to in the present invention as a "second probe molecule").

[First probe molecule]
Examples of the first probe molecule capable of specifically binding to transferrin include antibodies capable of specifically binding to transferrin (including anti-protein antibodies that recognize the protein portion of transferrin, anti-glycan antibodies that recognize the glycan portion of transferrin, and antibodies that recognize the protein portion and glycan portion of transferrin; sometimes referred to herein as "anti-transferrin antibodies"), and among these, anti-transferrin protein antibodies that recognize at least a portion of the protein portion of transferrin are preferred, and antibodies that do not interfere with the binding of fucose to the second probe molecule are also preferred.

The anti-transferrin antibody is not particularly limited as long as it has the ability to bind to transferrin, and may be a polyclonal or monoclonal antibody, although monoclonal antibodies are preferred from the standpoints of homogeneity and stability. Anti-transferrin antibodies can be produced by appropriately adopting and improving conventionally known production methods, and commonly available antibodies may also be used as appropriate.

When the following lectins are used as second probe molecules, for example, the anti-transferrin antibodies of the present invention are preferably antibodies in which the lectin-binding glycans have been removed or destroyed by oxidation treatment, glycosidase treatment, protease treatment, or the like to prevent the lectin from also recognizing antibody glycans and reducing detection sensitivity; antibodies in which the region containing glycosylated amino acids in the antibody molecule has been removed; or antibodies produced under conditions in which glycosylation does not occur, such as by using genetically modified E. coli or cells expressing antibody genes or by restricting the nutritional conditions for culturing antibody-producing cells.

[Second probe molecule]
Examples of the second probe molecule capable of specifically binding to fucose include an antibody capable of specifically binding to fucose (sometimes referred to as an "anti-fucose antibody" in this specification) and a lectin capable of specifically binding to fucose (for example, Aspergillus oryzae lectin (AOL), Atractylodes macrocarpa lectin (AAL), Lens lectin (LCA), Winged bean lectin (LTL), Urea lectin (UEA-I), Coryloides sugitake lectin (PhoSL), and European eel lectin (AAA)). Among these, lectins capable of specifically binding to fucose are preferred, lectins capable of specifically binding to at least one selected from the group consisting of α1-6 fucose and α1-2 fucose are more preferred, and lectins capable of specifically binding to α1-6 fucose are even more preferred. More specifically, such lectins are preferably Aspergillus oryzae lectin (AOL) and/or Aspergillus oryzae lectin (AAL), with Aspergillus oryzae lectin (AOL) being particularly preferred.

Aspergillus oryzae lectin (AOL: Aspergillus oryzae lectin) is a protein that exhibits binding activity by recognizing mainly α1-6 fucose (core fucose) sugar chain structures. Aleuria aurantia lectin (AAL: Aleuria aurantia lectin) is a protein that exhibits binding activity by recognizing mainly α1-6 fucose (core fucose) and α1-2 fucose sugar chain structures. These lectins may be modified lectins into which mutations have been introduced, or artificially synthesized lectins, with the aim of increasing the specificity of sugar chain recognition activity. Generally available lectins may also be used as appropriate.

When a first probe molecule and a second probe molecule are used, the measurement step involves contacting the sample with the first probe molecule and the second probe molecule. This allows both transferrin and fucose to be recognized, and fucose-added transferrin containing both can be detected and measured. The contact of the sample with the first probe molecule and the contact of the sample with the second probe molecule may be simultaneous or at different times, and if at different times, either contact can occur first.

[Labeling substance]
In the present invention, fucose-added transferrin is preferably measured by detecting a signal generated by a labeling substance attached to a probe molecule (preferably a first probe molecule and/or a second probe molecule) capable of specifically binding to fucose-added transferrin, or attached to a molecule that recognizes these molecules (e.g., a secondary antibody or protein A). The amount of fucose-added transferrin can be determined by measuring the amount of the detected signal and, as necessary, semi-quantitating or quantifying it. The "signal" includes color development (color development), quenching, reflected light, luminescence, fluorescence, radiation from a radioisotope, and the like, and includes signals that can be confirmed with the naked eye as well as signals that can be confirmed using a measurement method or device appropriate for the type of signal. In the present invention, the amount of fucose-added transferrin may be calibrated using a calibration curve using a standard sample, or the like. However, from the viewpoint of simpler and faster measurement, the signal amount may be directly used as the amount of fucose-added transferrin of the present invention.

The labeling substance of the present invention can be any substance used as a labeling substance in known immunological assays or methods similar thereto, without particular limitation. Examples include enzymes; luminescent substances such as acridinium derivatives; fluorescent substances such as europium; fluorescent proteins such as allophycocyanin (APC) and phycoerythrin (R-PE); radioactive substances such as ¹²⁵ I; low-molecular-weight labeling substances such as fluorescein isothiocyanate (FITC) and rhodamine isothiocyanate (RITC); gold particles; avidin; biotin; latex; dinitrophenyl phosphate (DNP); and digoxigenin (DIG). 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 depending on the substrate by adding a chromogenic substrate, a fluorescent substrate, a chemiluminescent substrate, or the like. Examples of the enzyme include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (ALP), β-galactosidase (β-gal), glucose oxidase, and luciferase.

[Sandwich method]
Measurement methods using the first and second probe molecules include, but are not limited to, immunological measurement methods such as sandwich methods, competitive methods, and immunoturbidimetry, as well as measurement methods based on these principles. Examples of such measurement methods include, in general, methods using microplates or particles as a carrier, such as ELISA, digital ELISA, CLEIA (chemiluminescent enzyme immunoassay), CLIA (chemiluminescent immunoassay), ECLIA (electrochemical immunoassay), and RIA (radioimmunoassay); immunochromatography; surface plasmon resonance analysis; and detection methods using fluorescence resonance energy transfer.

The sandwich method is preferred as a measurement method according to the present invention, as it tends to enable the construction of a measurement system with higher sensitivity and specificity. Below, a more specific embodiment of the measurement process according to the present invention will be explained using the sandwich method as an example.

In the case of using the sandwich method, the following embodiments are possible:
the measuring step is a step of contacting the sample with a capture body and a label,
the capture body comprises a water-insoluble carrier and either a first probe molecule or a second probe molecule immobilized on the water-insoluble carrier; and
The label comprises a labeling substance and the other of the first probe molecule and the second probe molecule.
In the first aspect, the first probe molecule and the second probe molecule may be provided in either the capture body or the label, respectively, but one is contained in the capture body and the other is contained in the label. This allows both fucose and transferrin to be captured, and fucose-added transferrin to be captured and detected with high accuracy and ease.

Other embodiments of the sandwich method are not limited to the above, and may include, for example, an embodiment in which the label is a first label comprising a first labeling substance and either a first probe molecule or a second probe molecule, and a second label comprising a second labeling substance and the other of the first probe molecule or the second probe molecule, and the capture body is a capture body comprising a water-insoluble carrier and a probe molecule immobilized on the water-insoluble carrier (hereinafter sometimes referred to as the "second embodiment"). In the second embodiment, the first labeling substance and the second labeling substance generate signals that are different from each other. In addition, examples of the probe molecule provided in the capture body in this case include probe molecules (including first and second probe molecules) that can specifically bind to fucose-added transferrin; and probe molecules that can specifically bind to the first and/or second probe molecules.

Such sandwich methods include the two-step forward sandwich method (a method in which the capture agent reacts with fucose-modified transferrin in the sample, and the fucose-modified transferrin bound to the capture agent reacts with the labeled agent sequentially), the reverse sandwich method (a method in which the labeled agent is reacted with fucose-modified transferrin in the sample in advance, and the resulting complex is reacted with the capture agent), and the one-step method (a method in which the fucose-modified transferrin in the sample, the capture agent, and the labeled agent react simultaneously in one step), and any of these can be used.

For example, in the forward sandwich method, the sample is first contacted with the capture body, and the fucose-added transferrin is captured by the capture body via binding between the probe molecule of the capture body and the fucose-added transferrin (e.g., binding between a first probe molecule and transferrin) (primary reaction: capture step). Next, the fucose-added transferrin captured by the capture body is contacted with the label, and is labeled via binding between the probe molecule of the label and the fucose-added transferrin (e.g., binding between a second probe molecule and fucose) (secondary reaction: labeling step). This reaction forms a complex containing the capture body and the fucose-added transferrin label. Unbound sample and label are removed by washing as necessary (washing step), and then the signal derived from the label is measured using a predetermined method depending on the label (measurement step).

(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 transferrin immobilized on the water-insoluble carrier, the probe molecule being directly or indirectly bound to the water-insoluble carrier. The probe molecule provided in the capture body according to the first aspect is preferably either a first probe molecule or a second probe molecule. In this case, the probe molecule provided in the capture body may be either the first probe molecule or the second probe molecule. However, when a lectin is used as the second probe molecule, from the viewpoint of higher capture ability, the probe molecule provided in the capture body is preferably the first probe molecule capable of specifically binding to transferrin, and is more preferably an anti-transferrin antibody.

<Non-water-soluble carrier>
The water-insoluble carrier contained in the capture body is a water-insoluble substance that mainly functions as a carrier for supporting and immobilizing the probe molecule. In the present invention, the term "water-insoluble substance" refers to a substance that is insoluble in water (having a solubility in water of 0.001 g/mL or less, preferably 0.0001 g/mL or less, the same applies hereinafter) at room temperature and normal pressure.

Materials for such water-insoluble carriers can be those commonly used in immunoassays and similar assays, and are not particularly limited. Examples include at least one selected from the group consisting of high molecular weight polymers (polystyrene, (meth)acrylic acid esters, polymethyl methacrylate, polyimide, nylon, etc.), gelatin, glass, latex, silica, metals (gold, platinum, etc.), and metal compounds (iron oxide, cobalt oxide, nickel ferrite, etc.). The water-insoluble carrier material may also be a composite of these materials or a composite of these materials with another material. For example, it may be an organic-inorganic composite consisting of at least one organic polymer selected from the group consisting of high molecular weight 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. Furthermore, the water-insoluble carrier may be surface-modified with an active group such as a carboxy group, an epoxy group, a tosyl group, an amino group, a hydroxy group, an isothiocyanate group, an isocyanate group, an azide group, an aldehyde group, a carbonate group, an allyl group, an aminooxy group, a maleimide group, or a thiol group.

In addition, in the present invention, the shape of the water-insoluble carrier is not particularly limited and may be, for example, a plate, fiber, membrane, particle, etc., but from the viewpoint of reaction efficiency, particles are preferred, and from the viewpoint of automation and shortening the reaction time, magnetic particles are more preferred. As such a water-insoluble carrier, conventionally known carriers can be used as appropriate, or commercially available carriers can also be used as appropriate.

<Configuration of Capture Body and Manufacturing Method>
The content of the probe molecule in the capture body is not particularly limited and can be adjusted as appropriate depending on the ease of binding of the probe molecule to fucose-added transferrin, etc.; for example, the mass of the probe molecule (when two or more types of probe molecules are combined, the total mass) relative to 100 parts by mass of the water-insoluble carrier (preferably particles) (when two or more types of water-insoluble carriers are combined, the total mass) is preferably 0.1 to 10 parts by mass, and more preferably 1 to 5 parts by mass.

The capture body can be produced by immobilizing the probe molecule on the water-insoluble carrier. This production method can be any conventionally known method or a method based thereon, and the probe molecule can be immobilized directly or indirectly on the water-insoluble carrier.

When directly immobilizing, for example, the water-insoluble carrier and/or the probe molecule may contain an active group such as a carboxyl group, epoxy group, tosyl group, amino group, hydroxyl group, isothiocyanate group, isocyanate group, azide group, aldehyde group, carbonate group, allyl group, aminooxy group, maleimide group, or thiol group, or the active group may be added as needed and bonded to the probe molecule, thereby directly immobilizing the probe molecule to the water-insoluble carrier.

Indirect immobilization can be achieved by immobilizing a linker that binds to the probe molecule to the water-insoluble carrier, and then binding the probe molecule to the linker, thereby indirectly immobilizing the probe molecule to the water-insoluble carrier. The linker is not particularly limited, and examples include secondary antibodies that can bind to the probe molecule, protein G, protein A, photocleavable photocleavable linkers, and linker molecules having an active group (e.g., hydrazine salts and hydrazides). Alternatively, the probe molecule may be modified in some way, and a substance that captures the modified portion may be immobilized to the water-insoluble carrier, thereby immobilizing the probe molecule on the water-insoluble carrier. For example, a typical example of the modified portion is biotin, and a typical example of the substance that captures the modified portion is streptavidin, but these examples are not limited thereto.

The ratio of the water-insoluble carrier and probe molecule used in these reactions can be selected appropriately to achieve the preferred ratio range for the capture body described above. If necessary, the water-insoluble carrier may be blocked with an appropriate blocking agent (e.g., bovine serum albumin or gelatin) to prevent nonspecific adsorption to the probe molecule or water-insoluble carrier. Furthermore, commercially available capture bodies may also be used as appropriate.

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

(Blocked labeled lectin)
In the present invention, the labeled entity may be a labeled antibody comprising the labeled substance and the antibody (e.g., anti-transferrin antibody, anti-fucose antibody), a labeled lectin comprising the labeled substance and a lectin, or a blocked labeled lectin. These may be used alone or in combination of two or more. Among these, the labeled entity in the present invention, particularly in the first aspect, is preferably a blocked labeled lectin. In the present invention, the "blocked labeled lectin" refers to a complex comprising a water-soluble carrier made of a water-soluble polymer, and a labeled substance and a lectin immobilized on the water-soluble carrier, in which the water-soluble carrier, the labeled 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 a second probe molecule. In this case, the probe molecule provided in the capture body is preferably a first probe molecule. The lectin is as described above, preferably AOL and/or AAL, more preferably AOL. The labeled substance, including its preferred embodiments, is as described above.

In the blocked labeled lectin, the labeling substance and lectin may be supported on the water-soluble carrier. The labeling substance and lectin may be bound to the water-soluble carrier independently of each other; the water-soluble carrier, labeling substance, and lectin may all be bound to the other two; the lectin may be bound to the water-soluble carrier via the labeling substance; or the labeling substance may be bound to the water-soluble carrier via the lectin. Generally, the affinity at each binding point between a lectin and a lectin-binding sugar chain structure is weak. However, in such blocked labeled lectins, multiple lectins form a complex connected by the water-soluble carrier, which is a polymer. Therefore, by using this, multiple binding points are generated in a single complex, improving overall affinity and enabling highly sensitive detection of the target fucose.

<Water-soluble carrier>
The water-soluble carrier contained in the blocked labeled lectin mainly functions as a carrier for supporting the labeling substance and lectin, and is made of a water-soluble polymer. The water-soluble polymer constituting the water-soluble carrier of the present invention (hereinafter referred to as a "first water-soluble polymer") is not particularly limited as long as it is a water-soluble polymer that can immobilize and support the labeling substance and 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 room temperature and normal pressure.

The weight-average molecular weight (weight-average molecular weight calculated as polystyrene by gel permeation chromatography (GPC); the same applies hereinafter) of the first water-soluble polymer according to the present invention is preferably 6,000 to 4,000,000, and more preferably 20,000 to 1,000,000, from the standpoints of measurement sensitivity and water solubility.

Furthermore, from the viewpoint of tending to obtain blocked labeled antibodies with a more preferable average particle size, the average mass of the first water-soluble polymer according to the present invention is preferably 70,000 to 1,000,000 Da, and more preferably 150,000 to 700,000 Da.

The blocked labeled lectin may also contain, as the first water-soluble polymer, multiple types of water-soluble polymers with different weight-average molecular weights. Furthermore, the blocked labeled lectin is preferably a combination of a high-molecular-weight blocked labeled lectin in which the weight-average molecular weight of the first water-soluble polymer is 200,000 or more and a low-molecular-weight blocked labeled lectin in which the weight-average molecular weight of the first water-soluble polymer is less than 100,000 (more preferably 100,000 or less). It is even more preferable to use a combination of a high-molecular-weight blocked labeled lectin in which the weight-average molecular weight of the first water-soluble polymer is 200,000 to 700,000 (more preferably 250,000 to 500,000) and a low-molecular-weight blocked labeled lectin in which the weight-average molecular weight of the first water-soluble polymer is 20,000 to 100,000 (more preferably 50,000 to 70,000). When the high-molecular-weight blocked labeled lectin and the low-molecular-weight blocked labeled lectin are combined 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.

Examples of the first water-soluble polymer of 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; polypeptides; DNA; and modified forms thereof (e.g., diethylaminoethyldextran, sodium dextran sulfate, etc.). These may be used alone or in combination of two or more. Among these, the first water-soluble polymer of the present invention is preferably at least one selected from the group consisting of polysaccharides and modified forms thereof, more preferably at least one selected from the group consisting of dextran, aminodextran, and modified forms thereof, and even more preferably dextran, because they are inexpensive and available in large quantities and are relatively easy to chemically process, such as by adding functional groups or through coupling reactions.

<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 adjusted appropriately depending on the measurement mechanism, etc.; however, in order to further improve the measurement sensitivity, it is preferable to set the number of molecules of the labeling substance bound to one molecule of the first water-soluble polymer to be as large as possible. For example, when the labeling substance is an enzyme, the mass of the labeling substance (when the first water-soluble polymer is a combination of two or more types, the total mass of the first water-soluble polymers; the same applies hereinafter) per 100 parts by mass of the first water-soluble polymer (when the first water-soluble polymer is a combination of two or more types, the total mass of the first water-soluble polymers) is preferably 100 to 1,000 parts by mass, more preferably 300 to 800 parts by mass.

The amount of lectin contained in the blocked labeled lectin is not particularly limited, but in order to further improve measurement sensitivity, it is preferable to set the number of lectin molecules that bind to one molecule of the first water-soluble polymer as large as possible. For example, the mass of lectin (total mass when two or more types of lectin are used) per 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.

Furthermore, 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. A weight-average molecular weight of 1,000,000 or more tends to result in higher measurement sensitivity, while a weight-average molecular weight of 10,000,000 or less tends to more sufficiently suppress aggregation and other problems in aqueous solution.

The blocked labeled lectin can be produced by immobilizing the labeled substance and lectin on the water-soluble carrier. This production method can be any conventionally known method or a method based thereon, as appropriate. The labeled substance and lectin (hereinafter sometimes collectively referred to as "supported substance") can be immobilized directly or indirectly on the water-soluble carrier.

Methods for directly immobilizing the supported substance on the water-soluble support include, for example, attaching active groups such as carboxyl groups, epoxy groups, tosyl groups, amino groups, hydroxyl groups, isothiocyanate groups, isocyanate groups, azide groups, aldehyde groups, carbonate groups, allyl groups, aminooxy groups, maleimide groups, thiol groups, and pyridyl disulfide groups to the supported substance and/or the first water-soluble polymer constituting the water-soluble support, or using water-soluble polymers containing these active groups as the supported substance and/or the water-soluble support and bonding them to the supported substance and/or the water-soluble support. The supported substance and first water-soluble polymer to which the active groups have been attached may be commercially available products, or they may be prepared by introducing the active groups onto the surfaces of the supported substance and the water-soluble polymer under appropriate reaction conditions. For example, thiol groups can be introduced using commercially available reagents such as S-acetylmercaptosuccinic anhydride and 2-iminothiolane hydrochloride. Furthermore, introduction of a maleimide group into an amino group on the first water-soluble polymer constituting the supported substance and/or the water-soluble carrier can be carried out using commercially available reagents such as N-(6-maleimidocaproyloxy)succinimide, N-(4-maleimidobutylyloxy)succinimide, etc. Introduction of a pyridyl disulfide 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. Alternatively, a pyridyl disulfide group can be introduced and then reduced to form a thiol group.

Examples of methods for indirectly immobilizing the supported substance to the water-soluble carrier include immobilization via a linker such as polyhistidine, polyethylene glycol, an oligopeptide containing cysteine and/or lysine, or a linker molecule having an active group (for example, one of those listed in the above-mentioned method for producing a capture body). The selection and size of the linker can be appropriately determined taking into account the strength of the bond with the supported substance and steric hindrance caused by immobilizing the supported substance to the water-soluble carrier.

In the method for producing the blocked labeled lectin, the labeling substance and lectin may be immobilized on the water-soluble carrier at the same time, or separately and sequentially. However, from the standpoint of ease of production and ease of control of the amounts of labeling substance and lectin, it is preferable to immobilize one of them on the water-soluble carrier before immobilizing the other. The blocked labeled lectin can also be produced by immobilizing the labeling substance and lectin on separate water-soluble carriers, and then linking the labeling substance immobilized on one water-soluble carrier (blocked labeling substance) with the lectin immobilized on the other water-soluble carrier, either directly or via the linker, etc.

The method for producing the blocked labeled lectin is not particularly limited. For example, as described in the Examples below, when the labeling substance is an enzyme and the first water-soluble polymer is a polysaccharide or glycoprotein, the first water-soluble polymer is first oxidized with an oxidizing agent such as sodium periodate to provide an aldehyde group, reacted with hydrazine hydrochloride, and then reacted with a reducing agent such as dimethylamine borane (DMAB) to hydrazinate. Meanwhile, the enzyme is also oxidized with an oxidizing agent such as sodium periodate to provide an aldehyde group to its sugar chain. The hydrazine residue provided above is then reacted with the aldehyde group to form a hydrazone bond, yielding a first water-soluble polymer-enzyme conjugate. The resulting first water-soluble polymer-enzyme conjugate is then treated with a crosslinker (e.g., SM(PEG) ₄ , SMCC, etc.) having N-hydroxysuccinimide and maleimide groups at each end to introduce the maleimide group. Alternatively, the lectin can be thiolated with a thiolation reagent to provide a thiol group, or in the case of a lectin having a disulfide bond within the molecule, the thiol group can be obtained by reduction. Finally, the maleimide group introduced into the first water-soluble polymer-enzyme conjugate can be bound to the thiol group provided on the lectin, thereby forming a covalent bond between the first water-soluble polymer (water-soluble carrier), enzyme, and lectin. This method yields a product in which two or more molecules of the first water-soluble polymer are bound via the enzyme, and the lectin is bound to the first water-soluble polymer. The ratios of the first water-soluble polymer, labeling substance, and lectin used in these reactions can be appropriately selected so as to achieve the preferred ranges for the respective contents in the blocked, labeled lectin.

(Capture step)
In the capture step, the method for contacting the sample with the capture body is not particularly limited, and any conventionally known method or a method based thereon can be used as appropriate. For example, if the water-insoluble carrier is a plate, the sample can be injected into the plate (probe molecule-immobilized plate), or if the water-insoluble carrier is particles, the capture body (probe molecule-immobilized particles) can be added to the sample.

In the reaction between the sample and the capture agent in the capture step, the content (final concentration) of the capture agent in the reaction solution containing the capture agent and the sample (if two or more capture agents are combined, the total of these; the same applies below) is not particularly limited and is adjusted appropriately depending on the type and concentration of the sample, but from the perspective of efficient capture in a short period of time, 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.

The conditions for the capture step are not particularly limited and can be adjusted as appropriate. For example, the capture step can be performed at room temperature to 45°C, preferably 20 to 37°C, at a pH of approximately 6 to 9, preferably 7 to 8, for approximately 5 seconds to 10 minutes, preferably 30 seconds to 5 minutes, but is not limited to these conditions.

(Labeling step)
In the reaction between the labeled body and fucose-added transferrin in the labeling step, the content (final concentration) of the labeled body (when two or more types of labeled bodies are combined, the total of the labels; the same applies hereinafter) in the reaction solution containing the labeled body and fucose-added transferrin is not particularly limited and is adjusted appropriately depending on the type, concentration, etc. of the sample. However, from the viewpoint that use of an excessive amount may cause a high background signal, the content 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, for example.

Furthermore, other conditions for the labeling step are not particularly limited and can be adjusted as appropriate. For example, the labeling step can be performed 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 approximately 3 to 120 minutes, preferably 5 to 10 minutes, but is not limited to these conditions.

(Washing step)
The sandwich method preferably further includes a washing step, following the capture step and/or labeling step, in which the fucose-conjugated transferrin bound to the capture body is separated from other contaminants not bound (captured) by the capture body and the contaminants are removed. The method for removing the contaminants is not particularly limited and may be a conventionally known method or a method based thereon. For example, if the capture body is a probe molecule-immobilized plate, the method may involve removing the liquid phase (supernatant) from the plate. If the capture body is probe molecule-immobilized particles, the method may involve recovering the particles by centrifugation or magnetic collection and then removing the liquid phase (supernatant). Furthermore, in the washing step, injection and removal of a washing solution may be repeated as necessary. Examples of the washing solution include known neutral (preferably pH 6-9) buffer solutions (e.g., sodium phosphate buffer, MES, Tris, CFB, MOPS, PIPES, HEPES, tricine buffer, bicine buffer, glycine buffer, etc.), and may also contain a stabilizing protein such as BSA or a surfactant.

When the sandwich method is used as the measurement step of the present invention, the sample may be diluted with a diluent as described above. However, if the capturer is a probe molecule-immobilized particle or the like, the sample may be suspended in a particle suspension medium (particle liquid). Furthermore, other reaction buffers may be added as appropriate to the reaction system between the sample and the capturer and/or the label. These particle suspension mediums and reaction buffers are not particularly limited, and examples thereof include, but are not limited to, known buffer solutions (sodium phosphate buffer, MES, Tris, CFB, MOPS, PIPES, HEPES, tricine buffer, bicine buffer, glycine buffer, etc.). Furthermore, each solution may independently contain a stabilizing protein such as BSA, serum, etc.

Furthermore, when the blocked labeled lectin is used as the label, in order to further improve measurement sensitivity, the reaction system between the sample and the label may also contain other substances such as a water-soluble polymer (hereinafter referred to as a "second water-soluble polymer"; this is different from the first water-soluble polymer constituting the water-soluble carrier in that it is a free water-soluble polymer that does not carry the labeling substance or lectin), a free lectin (which is different from the lectin contained in the blocked labeled lectin, etc., in that it is not immobilized to either the water-soluble carrier or the labeling substance), etc.

The second water-soluble polymer may be the same as those listed as the first water-soluble polymer, and may be one of these alone or a combination of two or more. It may also be the same type of polymer as the first water-soluble polymer. Among these, from the viewpoint of tending to further improve measurement sensitivity, the second water-soluble polymer is preferably at least one selected from the group consisting of polysaccharides and modified products thereof, more preferably at least one selected from the group consisting of dextran, aminodextran, and modified products thereof, and even more preferably dextran.

Furthermore, from the viewpoint of tending to further improve measurement sensitivity, 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.

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

The free lectin may be the same as those listed above as lectins, and may be one of these alone or a combination of two or more. It may also be the same type of lectin contained in the blocked labeled lectin. Among these, from the standpoint of improving reactivity and suppressing background, it is particularly preferable that the free lectin be the same type of lectin contained in the blocked labeled lectin.

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

(Measurement step)
In the measuring step, a signal is measured according to the labeling substance. For example, when the labeling substance is an enzyme, a chromogenic or luminescent substrate corresponding to the enzyme is added and reacted, and the resulting signal (e.g., color development or luminescence) is measured. This allows the presence or absence of fucose-modified transferrin in the sample to be detected based on the presence or absence of a signal. Furthermore, when fucose-modified transferrin is present, the amount of fucose-modified transferrin in the sample can be obtained as the amount of signal, and the amount of fucose-modified transferrin in the sample can be calibrated by comparing it with the measured value for a standard sample, as necessary.

[Cancer detection method]
According to the cancer detection method of the present invention, the presence or absence of pancreatic cancer and/or bile duct cancer in the sample or the subject from whom the sample was derived can be detected by using the presence or absence of fucose-modified transferrin measured in the above-mentioned measurement step as an indicator. Furthermore, by using the amount of fucose-modified transferrin measured in the above-mentioned measurement step as an indicator, in addition to detecting the presence or absence of the cancer, information on the degree (progression and abundance) of the cancer in the sample or the subject from whom the sample was derived can be obtained. Such a cancer detection method can be used for research purposes such as drug discovery, as well as for the cancer testing method of the present invention.

[Cancer testing method]
In the cancer testing method of the present invention, fucose-added transferrin is measured in a sample derived from a subject, and the presence or absence of pancreatic cancer and/or bile duct cancer is detected using the measured value as an index, or information on the severity of the cancer is obtained, thereby making it 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), determine the presence or absence of the cancer or the possibility of it, or evaluate the progression or severity of the cancer.

That is, specific embodiments of the cancer testing method of the present invention include, for example, the following embodiments:
A method for screening a subject who is predicted to have at least one type of cancer selected from the group consisting of pancreatic cancer and bile duct cancer, comprising: a measuring step of measuring fucose-linked transferrin in a sample derived from the subject; and a selecting step of selecting a subject using the measured fucose-linked transferrin as an indicator;
A method for predicting the risk of a subject developing at least one type of cancer selected from the group consisting of pancreatic cancer and bile duct cancer, comprising: a measuring step of measuring fucose-added transferrin in a sample derived from the subject; and a predicting step of predicting the risk of the subject developing the cancer using the measured fucose-added transferrin as an indicator;
A method for distinguishing the presence or absence of at least one type of cancer selected from the group consisting of pancreatic cancer and bile duct cancer in a subject, the method comprising: a measuring step of measuring fucose-added transferrin in a sample derived from the subject; and a distinguishing step of distinguishing the subject using the measured fucose-added transferrin as an indicator;
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, the method comprising: a measuring step of measuring fucose-modified transferrin in a sample derived from the subject; and an evaluation step of evaluating the subject using the measured fucose-modified transferrin as an index;
Examples include:

[Screening method]
In the present invention, "screening" refers to selecting subjects who may be affected by the cancer (pancreatic cancer and/or bile duct cancer). Specifically, the screening method of the present invention includes a method of predicting a subject's high probability of having pancreatic cancer and/or bile duct cancer (including recurrence) and selecting the subject from a group with no or low probability. In the screening method, subjects may be selected at a level where a medium probability can be expected, depending on the purpose.

More specifically, for example, subjects are divided into two groups: a group consisting of those who truly have the cancer (positive group) and a group consisting of those who do not have the cancer (normal group: negative group), and subjects are predicted to be included in the positive group and selected from the normal group (negative group). In this case, the prediction standard is preferably, for example, a negative agreement rate (the proportion of subjects predicted to be in the normal group by the screening who are actually in the normal group) of 95% or more.

[Cancer risk prediction method]
In the present invention, "predicting risk" refers to predicting whether or not there is a possibility of a subject developing (including recurrence of) the cancer (pancreatic cancer and/or bile duct cancer, preferably pancreatic cancer) in the future, or, if there is a possibility, assessing the degree of the possibility (evaluation such as high/moderate/low). Furthermore, this may also include screening, in which subjects predicted to have a high or high possibility as a result of the risk prediction are separated from a group of subjects with no or low possibility. Fucosylated transferrin is also thought to be significantly increased in patients with chronic pancreatitis, which carries a high risk of developing pancreatic cancer in the future, and thus fucose-modified transferrin can be an index for predicting the risk of developing pancreatic cancer in particular.

More specifically, for example, subjects are divided into two groups: a group consisting of those who will truly develop the cancer within a certain period of time in the future (positive group), and a group consisting of those who will not develop the cancer in the same future period (normal group: negative group), and the subject is determined to be in the positive group or the negative group. In this case, the discrimination criterion is preferably, for example, a negative agreement rate (the proportion of subjects who are determined to be in the normal group by the method but are actually in the normal group) of 95% or higher.

[Identification method]
In the present invention, "differentiation" refers to distinguishing pancreatic cancer and/or bile duct cancer from other diseases or conditions and determining whether a subject has the cancer. It also includes not only determining whether a subject has the cancer, but also assessing the degree of the cancer (e.g., high, medium, low) if there is a possibility of the cancer. The differentiation method in the present invention specifically includes a method for determining whether a subject has pancreatic cancer or bile duct cancer, or whether there is a high possibility of the cancer, regardless of the presence or absence of symptoms. For example, it also includes a method for determining whether the cancer a subject has is pancreatic cancer or bile duct cancer, or a method for determining that there is a high possibility of the cancer; a method for determining whether a subject has previously had pancreatic cancer or bile duct cancer, or a method for determining that there is a high possibility of the cancer recurring; and a method for determining whether a subject has previously had pancreatic cancer or bile duct cancer, or a method for determining that there is a high possibility of the cancer recurring.

More specifically, for example, subjects are divided into two groups: a group consisting of those who truly have the cancer (positive group) and a group consisting of those who do not have the cancer (normal group: negative group), and the subject is determined to be in the positive group or the negative group. Such discrimination can be applied to assist doctors and others in diagnosing pancreatic cancer and/or bile duct cancer. In this case, the discrimination criterion is preferably, for example, a negative agreement rate (the proportion of subjects who are classified as being in the normal group by this discrimination but are actually in the normal group) of 95% or more.

[Evaluation method]
In the present invention, the evaluation method includes, for example, a method for evaluating the progression or severity of pancreatic cancer and/or bile duct cancer in a subject; a method for providing an index for determining a treatment policy for the cancer in a subject; and a method for determining the effectiveness of a treatment for the cancer.

More specifically, for example, the amount of fucose-coupled transferrin in a group of people who are not affected by the cancer or who have a low stage or severity of the cancer can be compared with the amount of fucose-coupled transferrin in the subject, and if the amount in the subject is higher, the stage or severity can be evaluated as high, and if the amount is lower, the stage or severity can be evaluated as low. Furthermore, the amount of fucose-coupled transferrin in the subject at the time the cancer was first discovered or before treatment can be compared with the current amount of fucose-coupled transferrin, and if the amount has increased, the therapeutic effect can be evaluated as low, and if the amount has decreased, the therapeutic effect can be evaluated as high.

(Cutoff value)
In the selection step of the screening method, the prediction step of the cancer incidence risk prediction method, and the discrimination step of the differentiation method, if even a small amount of fucose-added transferrin is detected in the measurement step, the detected subject may be predicted or discriminated as having (or having a high probability of having) pancreatic cancer and/or bile duct cancer, or may be predicted as having (or having a high probability of having) the cancer, but it is preferable to make the prediction or discrimination based on the measured amount of fucose-added transferrin.

For example, it is preferable to compare the amount of fucose-modified transferrin measured in the measurement step with a predetermined cutoff value, and predict or identify subjects whose amount of fucose-modified transferrin is higher than the cutoff value as having (or having the potential to have or the potential to have) pancreatic cancer and/or bile duct cancer.

In the present invention, the term "cutoff value" refers to a predetermined value that serves as a standard for making a judgment based on the amount of fucose-modified transferrin, and refers to the boundary value for distinguishing between the positive and negative groups. Such a cutoff value is not particularly limited, and is set appropriately depending on the purpose of the cancer testing method of the present invention, the method for measuring the amount of fucose-modified transferrin, the properties of the subject and sample, dilution conditions, etc. For example, setting the cutoff value to a relatively low value increases the detection sensitivity, i.e., makes it possible to collect a relatively wide range of patients in the early stages of cancer, enabling early detection. On the other hand, setting the cutoff value to a relatively high value enables the screening and differentiation to be performed with higher accuracy.

An example of a cutoff value for the screening method or the differentiation method is, for example, a serum specimen diluted to 1/500 by volume, anti-transferrin antibody-immobilized particles as the capture body, and the amount of fucose-modified transferrin measured by a sandwich immunoassay using blocked labeled AOL as the label, with ALP as the label and AMPPD as the substrate. When the amount is expressed as the emission intensity (counts) of light having a maximum absorption at a wavelength of 463 nm (in the case of Example 1), the cutoff value is 50,000 to 90,000 counts, preferably 60,000 to 80,000 counts, but is not limited to these. Note that the cutoff value determined within the above range is selected and applied depending on the purpose of the cancer testing method of the present invention, the method for measuring fucose-modified transferrin, the properties of the subject and sample, etc.

The cancer testing method of the present invention makes it possible to distinguish pancreatic cancer and/or bile duct cancer from other cancers and provide information for determining treatment strategies specific to pancreatic cancer and/or bile duct cancer. Furthermore, by identifying subjects who are likely to suffer from pancreatic cancer and/or bile duct cancer (including recurrence), or who have or are likely to suffer from such cancer (including recurrence), and conducting further testing or a definitive diagnosis, early detection and early therapeutic intervention for pancreatic cancer and/or bile duct cancer become possible. Furthermore, since information on cancer-derived glycan structures has been reported to be an indicator of cancer invasiveness, the cancer testing method of the present invention also makes it possible to obtain information on the malignancy and prognosis of cancer. The method of the present invention also serves as a method for assisting physicians in diagnosing pancreatic cancer and/or bile duct cancer, or a method for providing physicians with information for diagnosing pancreatic cancer and/or bile duct cancer.

Furthermore, the method of the present invention is also suitable for combination with other conventional methods for measuring monitoring markers, thereby making it possible to further improve the detection sensitivity and diagnostic accuracy of pancreatic cancer and/or bile duct cancer.

<Kit>
The kit of the present invention is a kit for use in the cancer detection method or cancer testing method of the present invention, and includes a first probe molecule capable of specifically binding to transferrin 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 preferred embodiments thereof.

The kit of the present invention also preferably includes a capture body comprising a water-insoluble carrier and either a first probe molecule or a second probe molecule immobilized on the water-insoluble carrier, and a label comprising a labeling substance and the other of the first probe molecule and the second probe molecule. The capture body and label are as described above, including their preferred embodiments.

In the kit of the present invention, the first probe molecule, the second probe molecule, the capture body, and the label may each independently be in solid (powder) form or in liquid form dissolved in a buffer solution. When in liquid form, the concentrations of the first probe molecule, the second probe molecule, the capture body, and the label in each solution are not particularly limited, but are each independently, for example, preferably 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.

The kit of the present invention may further include components that are required for conventional immunological measurement methods such as ELISA, CLEIA, and immunochromatography, as well as methods similar thereto. For example, when the measurement method of the present invention uses the sandwich method described above as the principle, the kit may further include at least one component selected from the group consisting of magnetic beads or plates for immobilizing the probe molecules, a sensor chip, the standard sample (each concentration), a control reagent, the particle suspension medium, the reaction buffer, the washing solution, a second water-soluble polymer, and a free lectin. Furthermore, when the labeled substance is an enzyme, the kit may further include a substrate, a reaction stop solution, etc., necessary for detecting and quantifying the labeled substance.

Furthermore, the kit of the present invention may include, as necessary, the diluent, a pretreatment solution for pretreatment of the sample, a dilution cartridge, and a pretreatment reaction stop solution or neutralization solution. Furthermore, when immunochromatography is used as the sandwich method, the kit may further include a device including a zone carrying the capture body and/or the label. The device may also include other components suitable for immunochromatography, such as a developer pad or an absorbent pad. Furthermore, the kit of the present invention may further include instructions for use of the kit.

The present invention will be explained in more detail below based on examples and comparative examples, but the present invention is not limited to the following examples. In each example and comparative example, "%" indicates weight/volume (w/v: g/mL) percentage unless otherwise specified.

Example 1 Measurement of AOL-Bound Glycosylated Transferrin (Fucosylated Transferrin) in Serum Samples Using Blocked Labeled Lectin (AOL) (1) Preparation of Hydrazinated Dextran 240.0 mg of dextran with a molecular weight of 250 kJ (CarboMer) was added to 4.8 mL of 0.1 M phosphate buffer (pH 7.0) and dissolved by stirring for 30 minutes in a dark place 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 a dark place at 25°C. Next, using a PD-10 column (GE Healthcare, Sephadex G-25 packed column: hereinafter simply "Sephadex G-25"), buffer exchange was performed with 0.1 M sodium phosphate buffer (pH 6.0), and 20.0 mL of solution was obtained. To the resulting solution, 5.04 g of NH ₂ NH ₂ ·HCl was added and stirred for 2 hours in the dark at 25 ° C. 800 mg of DMAB (dimethylamine borane) was added and further stirred for 2 hours in the dark at 25 ° C. Dialysis with 4 L of ion-exchanged water using a RC50K (regenerated cellulose with a molecular weight of 50,000) dialysis membrane was performed in the dark for 3 hours, and then the mixture was left to stand overnight at 4 ° C. Buffer exchange was performed by gel filtration (Sephadex G-25) using 0.1 M sodium phosphate buffer (pH 6.0), and 85.0 mL of solution was obtained. The concentration of dextran in the resulting solution was adjusted to 1.0 mg/mL to obtain a solution of hydrazinated dextran.

(2) Preparation of Dextran-Enzyme Conjugate 30.0 mL of 10 mg/mL alkaline phosphatase (ALP-50, Oriental Yeast Co., Ltd.) was buffer-exchanged 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. Next, 45.3 mL of 27 mM _NaIO4 was added, and the mixture was 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. The 1.0 mg/mL hydrazinolated dextran prepared in (1) of Example 1 was added to the mixture to adjust the hydrazide group (amino group) concentration to 25 μM, and the mixture was stirred in the dark at 25°C for 16 hours. 85 mg of DMAB was added, and the mixture was stirred in the dark at 25°C for 2 hours. Next, 10.1 mL of 1.5 M Tris buffer (pH 9.0) was added, and the mixture was stirred in the dark at 25°C for 2 hours. An ultrafiltration module (Pellicon XL50, Merck Millipore) was attached to a Labscale TFF System (Merck Millipore), and the mixture was concentrated to 15 mL. Gel filtration (Superdex 200 pg) using 0.1 M sodium phosphate buffer (pH 7.0) was performed to obtain 14 mL of a 3.0 mg/mL dextran-enzyme conjugate solution.

(3) Maleimide-PEGylation of Dextran-Enzyme Conjugate 750 μL of 2 mg/mL dextran-enzyme conjugate was prepared by adding 0.1 M sodium phosphate buffer (pH 7.0) to the dextran-enzyme conjugate prepared in Example 1(2). To this was added 8.35 μL of 250 mM SM(PEG) ₄ (manufactured by Thermo Fisher Scientific, SM(PEG) ₄ ) dissolved in DMSO, followed by mixing by inversion in a dark place at 25°C for 1 hour. After the reaction, the buffer was exchanged 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 (Merck, Amicon Ultra 50K) to adjust the final concentration to 2 mg/mL.

(4) Lectin Thiolation: 1.5 mL of 0.1 M sodium phosphate buffer (pH 7.0) was added to 1 mL of 5 mg/mL Aspergillus oryzae lectin solution (AOL; manufactured by Tokyo Chemical Industry Co., Ltd.) to obtain 2.5 mL of 2 mg/mL AOL solution. 100 μL of 0.5 M EDTA 2Na (pH 8.0) was added to the 2.5 mL AOL solution and mixed. 75 μL of 10 mg/mL 2-iminothiolane hydrochloride solution was then added and mixed by inversion in a dark place 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.

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

(6) Preparation of Measurement Reagent 1 mg of anti-transferrin antibody F2H8G6 (manufactured by ThermoFisher) was buffer-exchanged into 50 mM MES (pH 5.5) using a PD-10 column (Sephadex G-25) to obtain 0.5 mL of a 1.5 mg/mL anti-transferrin antibody solution. The obtained anti-transferrin antibody solution was mixed with carboxylated magnetic particles (manufactured by Fujirebio Inc.) that had been activated in advance with EDC (N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, manufactured by Merck) and Sulfo-NHS (N-hydroxysulfosuccinimide, manufactured by ThermoFisher), and the mixture was mixed by inversion at 25°C for 1 hour, thereby binding the anti-transferrin antibody to the magnetic particles. After the antibody binding reaction, the particles were stopped with 1 M Tris-HCl, 10% BSA, 0.1% NaN ₃ , pH 7.0, and then washed and masked with masking buffer (50 mM MES, 1 mM EDTA·2Na, 150 mM NaCl, 2% BSA, 0.1% ProClin 300, pH 6.0), finally yielding 24 mg of anti-transferrin antibody-bound magnetic particles.

6 mL of 20 mM sodium periodate solution (20 mM _NaIO4 , 100 mM NaOAc, 150 mM NaCl, pH 5.5) was added to 24 mg of anti-transferrin antibody-bound magnetic particles (hereinafter simply referred to as "antibody-bound particles") and mixed. The mixture was then mixed by end-over-end mixing at 4°C in the dark for 30 minutes to oxidize the sugar chains of the antibody bound to the particles. After oxidation, the antibody-bound particles were washed three times with 6 mL of 0.1 M sodium phosphate buffer (pH 6.0). The washed antibody-bound particles were then replaced with 0.1 M sodium phosphate buffer (pH 6.0) containing 10 mM glycine, and the mixture was mixed by end-over-end mixing at 25°C in the dark for 1 hour to block the aldehyde groups generated by oxidation of the sugar chains of the antibody with glycine. After the reaction, 100 μL of 10 mg/mL DMAB was added to the antibody-bound particle solution and mixed by end-over-end mixing at 25°C for 30 minutes in the dark to stabilize the unstable bond between the aldehyde group derived from the antibody sugar chain and glycine. The stabilized antibody-bound particles were washed three times with 3 mL of a 2% BSA-containing buffer (50 mM MES, 1 mM EDTA, 150 mM NaCl, 2% BSA, 0.1% ProClin 300, pH 6.0) and then mixed by end-over-end mixing in the same buffer at 37°C for 16 hours to physically adsorb BSA. The antibody-bound 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 in the same buffer at 4°C. The obtained oxidized anti-transferrin antibody-bound magnetic particles were diluted with a 50 mM Tris-based solution so that the antibody-bound particle concentration was 0.005%, thereby preparing an oxidized anti-transferrin antibody-bound particle liquid.

Furthermore, a labeled body fluid was prepared by diluting the blocked labeled lectin (AOL) obtained in Example 1 (5) into a 50 mM MES-based solution to a concentration of 0.5 μg/mL.

(7) Measurement of AOL-linked glycosylated transferrin (fucosylated transferrin) contained in serum samples Eleven serum samples collected from healthy subjects (healthy subject group: healthy subjects 1 to 11), 10 serum samples collected from pancreatic cancer patients (pancreatic cancer group: pancreatic cancer 1 to 10), and 5 serum samples collected from bile duct cancer patients (bile duct cancer group: bile duct cancer 1 to 5) were each diluted to a volume ratio of 1/500 using sample diluent (manufactured by Fujirebio Inc.).

AOL-linked glycosylated transferrin (fucosylated transferrin) contained in each diluted sample was measured using a Lumipulse® L-2400 (Fujirebio Inc.). Specifically, 50 μL of each diluted sample solution was mixed with 50 μL of the oxidized anti-transferrin antibody-bound particle solution prepared in Example 1 (6), and the mixture was allowed to react at 37°C for 8 minutes. The magnetic particles were then collected and washed five times with Lumipulse® cleaning solution (Fujirebio Inc.). Next, 50 μL of the labeled body fluid prepared in Example 1 (6) was added, and the mixture was allowed to react at 37°C for 8 minutes. The magnetic particles were then magnetically collected and washed five times. 50 μL of Lumipulse® substrate solution (Fujirebio) containing AMPPD (3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoryloxy)phenyl-1,2-dioxetane disodium salt) was then added, and the mixture was allowed to react at 37°C for 4 minutes. AMPPD was decomposed by the catalytic action of alkaline phosphatase in the blocked labeled lectin (AOL) bound to the magnetic particles, and the emission intensity (counts) of light with a maximum absorption at a wavelength of 463 nm was measured using a Lumipulse® L-2400 (Fujirebio) to obtain the measurement results. The measurement results for each sample are shown in Table 1 below. The results shown are the average of duplicate measurements.

(8) Comparison of Measurement Values Between Healthy Subjects, Pancreatic Cancer, and Bile Duct Cancer Groups The results in Table 1 were examined to determine whether there was a statistically significant difference between the healthy subject group and the pancreatic cancer group. Figure 1 shows the measurement results of fucosylated transferrin in the healthy subject group and the pancreatic cancer group. A Wilcoxon test revealed a significant difference in the fucosylated transferrin measurements between the healthy subject group and the pancreatic cancer group (Figure 1, p = 0.002). Furthermore, a cutoff value was calculated from the measurements in the healthy subject group, and the ability to detect pancreatic cancer patients was tested when samples exceeding the cutoff value were considered positive. The cutoff value was calculated as 72,257 counts or less, calculated by adding the mean measurement value of the healthy subject group to the standard deviation of the measurements in the healthy subject group multiplied by 2. As a result, 9 out of 10 samples in the pancreatic cancer group were determined to be positive at this cutoff value, accounting for 90.9%. Furthermore, in the healthy subject group, only 1 out of 11 samples was determined to be positive (i.e., false positive) using the cutoff value, resulting in a specificity of 90.9%. These results demonstrate that measurement of fucosylated transferrin can be a novel and effective indicator for distinguishing between healthy subjects and pancreatic cancer patients.

Similarly, the results in Table 1 were examined to determine whether a statistically significant difference was observed between the healthy subject group and the bile duct cancer group. Figure 2 shows the measurement results for fucosylated transferrin in the healthy subject group and the bile duct cancer group. A Wilcoxon test revealed a significant difference in the measured fucosylated transferrin levels between the healthy subject group and the bile duct cancer group (Figure 2, p = 0.0032). Furthermore, when the cutoff value calculated in the same manner as above was used to test the ability of fucosylated transferrin to detect bile duct cancer patients, all five samples measured in this study were determined to be positive. These results demonstrate that measurement of fucosylated transferrin can be a powerful new indicator for distinguishing between healthy subjects and bile duct cancer patients.

(Comparative Example 1) Verification of the reactivity of fucosylated transferrin against cancers other than pancreatic cancer and bile duct cancer For comparison with Non-Patent Document 2, fucosylated transferrin was measured for 50 serum samples collected from hepatocellular carcinoma patients (hepatocellular carcinoma group: hepatocellular carcinomas 1 to 50) in the same manner as in (7) of Example 1. The measurement results for each sample are shown in Table 2 below.

The measurement results for the hepatocellular carcinoma group shown in Table 2 and the measurement results for the healthy subject group shown in Table 1 were examined to determine whether a statistically significant difference was observed between the healthy subject group and the hepatocellular carcinoma group. Figure 3 shows the measurement results for fucosylated transferrin in the healthy subject group and the hepatocellular carcinoma group. When tested using the Wilcoxon test, no significant difference was observed in the measured values of fucosylated transferrin between the healthy subject group and the hepatocellular carcinoma group (Figure 3, p = 0.0545), and not a single sample in the hepatocellular carcinoma group was determined to be positive because it exceeded the cutoff value calculated in the same manner as in Example 1 (8).

Furthermore, the measurement results for the hepatocellular carcinoma group shown in Table 2 and the measurement results for the pancreatic cancer group and cholangiocarcinoma group shown in Table 1 were also examined to determine whether statistically significant differences were observed between the pancreatic cancer group and the hepatocellular carcinoma group, or between the cholangiocarcinoma group and the hepatocellular carcinoma group. Figure 4 shows the measurement results for fucosylated transferrin in the pancreatic cancer group and the hepatocellular carcinoma group, and Figure 5 shows the measurement results for fucosylated transferrin in the cholangiocarcinoma group and the hepatocellular carcinoma group. A Wilcoxon test clearly showed that the measured values in the pancreatic cancer group and the cholangiocarcinoma group were higher than those 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).

These results show that fucosylated transferrin levels are not indiscriminately high in all cancers, but vary depending on the type of cancer, with levels being specifically high in pancreatic and bile duct cancers.

The present invention makes it possible to provide a cancer detection method and cancer testing method that use new biomarkers as indicators and are capable of specifically detecting pancreatic cancer and bile duct cancer with high sensitivity, as well as kits for use in these methods.

Claims (15)

A method for detecting pancreatic cancer, comprising a measuring step of measuring fucose-added transferrin in a sample, wherein the sample is serum, and the measuring step is a step of contacting the sample with a first probe molecule capable of specifically binding to transferrin and a second probe molecule capable of specifically binding to fucose . A method for testing for pancreatic cancer, comprising a measuring step of measuring fucose-added transferrin in a sample derived from a subject, wherein the sample is serum, and the measuring step is a step of contacting the sample with a first probe molecule capable of specifically binding to transferrin and a second probe molecule capable of specifically binding to fucose . The method according to claim 2, which provides information for screening subjects suspected of having pancreatic cancer using fucose-added transferrin as an indicator. The method according to claim 2, which provides information for predicting a subject's risk of developing pancreatic cancer using fucose-added transferrin as an indicator. The method according to any one of claims 1 to 4, wherein the fucose in the fucose-added transferrin is at least one type selected from the group consisting of α1-6 fucose and α1-2 fucose. The method according to any one of claims 1 to 5, wherein in the fucose-added transferrin, the fucose is at least one selected from the group consisting of an AOL-linked glycan that binds to AOL and an AAL-linked glycan that binds to AAL. The method according to any one of claims 1 to 6 , wherein the first probe molecule is an antibody capable of specifically binding to transferrin. 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. the measuring step is a step of contacting the sample with a capture body and a label,
the capture body comprises a water-insoluble carrier and either a first probe molecule or a second probe molecule immobilized on the water-insoluble carrier; and
the label comprises a labeling substance and the other of the first probe molecule and the second probe molecule;
The method according to any one of claims 1 to 8 , characterized in that The capture body comprises a water-insoluble carrier and a first probe molecule immobilized on the water-insoluble carrier, and
the labeled body comprises a water-soluble carrier, and a labeling substance and a second probe molecule immobilized on the water-soluble carrier, and the second probe molecule is a blocked labeled lectin that is a lectin capable of specifically binding to fucose;
10. The method according to claim 9 . A kit for use in the method according to any one of claims 1 to 10 , comprising a first probe molecule capable of specifically binding to transferrin and a second probe molecule capable of specifically binding to fucose. 12. The kit according to claim 11 , wherein the first probe molecule is an antibody capable of specifically binding to transferrin. 13. The kit according to claim 11 or 12 , wherein the second probe molecule is a lectin capable of specifically binding to fucose. A capture body comprising a water-insoluble carrier and either a first probe molecule or a second probe molecule immobilized on the water-insoluble carrier; and
a label comprising a labeling substance and the other of the first probe molecule and the second probe molecule;
The kit according to any one of claims 11 to 13 , characterized in that it comprises: The capture body comprises a water-insoluble carrier and a first probe molecule immobilized on the water-insoluble carrier, and
the labeled body comprises a water-soluble carrier, and a labeling substance and a second probe molecule immobilized on the water-soluble carrier, and the second probe molecule is a blocked labeled lectin that is a lectin capable of specifically binding to fucose;
The kit according to claim 14 , characterized in that