Cancer detection method, cancer testing method, and kits used therefor

Abstract

To provide a cancer detection method which allows for specifically and accurately detecting prostate cancer and colorectal cancer using a novel biomarker as an indicator.SOLUTION: A method of detecting at least one cancer selected from a group consisting of prostate cancer and colorectal cancer is provided, the method comprising a measurement step of measuring the level of GalNAc-added CA19-9 in a sample.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a cancer detection method, a cancer testing method, and a kit used therein, and more specifically, to a method for detecting at least one type of cancer selected from the group consisting of prostate cancer and colorectal cancer, a method for testing said cancer, and a kit used therein. [Background technology]

[0002] Biomarkers such as tumor markers are useful as a monitoring marker for detecting diseases such as malignant tumors, determining treatment plans, and assessing the effectiveness of treatment, and have been actively studied in recent years. One example of such a biomarker is CA19-9. CA19-9 is a type I sugar chain antigen recognized by a monoclonal antibody (NS19-9) obtained in 1979 by Koprowski et al. using the human colon cancer cultured cell line SW-1116 as an immunogen. The antigenic determinant is the Lewis A antigen (Le a ) to which sialic acid has been added. CA19-9 exists in the blood as a mucin-type glycoprotein (Non-Patent Document 1, etc.) and is detected in the blood of healthy individuals (meaning those without cancer, the same applies below), but its concentration is known to be elevated in patients with cancer of the digestive system, particularly cancer of the colon, pancreas, bile duct, gallbladder, etc. (Non-Patent Document 1).

[0003] For this reason, CA19-9 has traditionally been used as a monitoring marker for detecting these cancers, as a guide for determining treatment plans, and for assessing the effectiveness of treatment. For example, Fujirebio Inc. manufactures and sells "Lumipulse Presto (registered trademark) CA19-9" as a kit for measuring CA19-9 in a sample by the sandwich method, in which a solid-phase anti-CA19-9 antibody (capturer) is used to capture CA19-9 present in a sample, and then a labeled anti-CA19-9 antibody (labeled body) is bound to the captured CA19-9.

[0004] In addition, for example, the amount of PSA (prostate specific antigen) in blood has been used as an index for the detection of prostate cancer, for guidance in determining a treatment policy, and for evaluating the effectiveness of treatment. Furthermore, it has been reported that GalNAc (N-acetylgalactosamine) is significantly increased in the PSA of prostate cancer patients compared to healthy subjects (Non-Patent Document 2, etc.). In addition, for example, Patent Document 1 describes a method for determining prostate cancer based on the ratio between the amount of free PSA in a sample and the amount of PSA having a sugar chain (α2,3-linked sialic acid) in which a terminal sialic acid residue of the sugar chain is α2,3-linked to the second galactose residue from the terminal of the sugar chain. However, since the amount of PSA increases not only in prostate cancer patients but also in patients with prostatic hyperplasia, when the cutoff value is set in the low concentration range, the hit rate for prostate cancer tends to be low, which is an issue. In addition, there is also an issue that it cannot be used as a monitoring marker in prostate cancer patients in whom the amount of PSA has not increased due to hormone therapy, etc. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2019 / 221279 [Non-patent literature]

[0006] [Non-Patent Document 1] John L. Magnani et al., Cancer Research 43, 1983, p.5489-5492 [Non-Patent Document 2] Fukushima et al., Glycobiology, 2010, vol. 20, no. 4, p. 452-460 Summary of the Invention [Problem to be solved by the invention]

[0007] It has been reported that CA19-9 detected in the blood of healthy individuals exists as part of low- to medium-molecular-weight mucin of approximately 200,000 to 1 million Da, whereas CA19-9 detected in the blood of cancer patients exists as part of high-molecular-weight mucin of approximately 5 to 10 million Da. It is therefore anticipated that glycans other than CA19-9 may also serve as new biomarkers for cancer detection.

[0008] In addition, as described above, a small amount of CA19-9 is also detected in the blood of healthy individuals, so in conventional methods for measuring CA19-9, a cutoff value is usually set and used as an index to predict or distinguish between negative and positive results for the above cancers, etc. However, the present inventors have found that such conventional methods for measuring CA19-9 use only one type of anti-CA19-9 antibody specific to sialyl Lewis A, and therefore the specificity for each type of cancer is still insufficient, and the present inventors have found that such methods are still insufficient to distinguish between prostate cancer patients and healthy individuals or prostate hyperplasia patients, or between colon cancer patients and healthy individuals with high accuracy.

[0009] The present invention has been made in consideration of the above-mentioned problems, and aims to provide a cancer detection method and a cancer testing method that use a new biomarker as an indicator and are capable of specifically detecting prostate cancer and colorectal cancer with high accuracy, as well as kits for use in these methods. [Means for solving the problem]

[0010] In order to search for sugar chains as new tumor markers that could not be measured by existing antibodies, the present inventors performed screening using anti-CA19-9 antibodies as well as a complex (blocked labeled lectin) comprising a water-soluble carrier made of a water-soluble polymer and a labeling substance and a lectin fixed to the water-soluble carrier. As a result, it was found that the amount of molecules detected when WFA was used as the lectin, that is, the amount of CA19-9 with lectin-binding GalNAc (N-acetylgalactosamine) added (GalNAc-added CA19-9) was significantly increased in prostate cancer patients and colon cancer patients. Therefore, it was found that by measuring the amount of GalNAc-added CA19-9, it is possible to specifically detect prostate cancer and colon cancer, and to distinguish prostate cancer patients from healthy individuals or prostate hyperplasia patients, or colon cancer patients from healthy individuals with a higher accuracy than when only CA19-9 was measured, and thus the present invention was completed.

[0011] The present invention, based on these findings, has the following features. [1] A method for detecting at least one type of cancer selected from the group consisting of prostate cancer and colorectal cancer, comprising a measurement step of measuring the amount of GalNAc-added CA19-9 in a sample. [2] A method for testing for at least one type of cancer selected from the group consisting of prostate cancer and colorectal cancer, comprising a measurement step of measuring the amount of GalNAc-added CA19-9 in a sample derived from a subject. [3] The method described in [2] is a method for screening subjects who are predicted to have at least one type of cancer selected from the group consisting of prostate cancer and colorectal cancer, and is characterized in that it comprises a measurement step of measuring the amount of GalNAc-attached CA19-9 in a sample derived from the subject, and a selection step of selecting subjects using the measured amount of GalNAc-attached CA19-9 as an indicator. [4] The method according to any one of [1] to [3], wherein in GalNAc-added CA19-9, GalNAc is a WFA-binding glycan that binds to WFA. [5] The method according to any one of [1] to [4], characterized in that the measuring step is a step of contacting the sample with a first probe molecule capable of specifically binding to CA19-9 and a second probe molecule capable of specifically binding to GalNAc. [6] The method according to [5], wherein the first probe molecule is an antibody capable of specifically binding to CA19-9. [7] The method according to [5] or [6], wherein the second probe molecule is a lectin capable of specifically binding to GalNAc. [8] the measuring step is a step of contacting the sample with a 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 [5] to [7], [9] 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, the second probe molecule being a blocked labeled lectin that is a lectin capable of specifically binding to GalNAc; The method according to [8],

[10] A kit for use in the method according to any one of [5] to [9], comprising a first probe molecule capable of specifically binding to CA19-9 and a second probe molecule capable of specifically binding to GalNAc.

[11] The kit according to

[10] , wherein the first probe molecule is an antibody capable of specifically binding to CA19-9.

[12] The kit according to

[10] or

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

[13] A capture body comprising a water-insoluble carrier and either 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

[10] to

[12] , comprising:

[14] 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, the second probe molecule being a blocked labeled lectin that is a lectin capable of specifically binding to GalNAc; The kit according to

[13] , Effect of the Invention

[0012] According to the present invention, it is possible to provide a cancer detection method and a cancer testing method that use a new biomarker as an indicator and are capable of specifically detecting prostate cancer and colorectal cancer with high accuracy, as well as kits used in these methods. [Brief description of the drawings]

[0013] [Figure 1] 1 is a graph showing the results of measuring CA19-9 in a healthy subject group and a prostate cancer group. [Diagram 2] 1 is a graph showing the results of measuring CA19-9 / WFA in a healthy subject group and a prostate cancer group. [Diagram 3] 1 is a graph showing the results of CA19-9 measurement in a prostate hyperplasia group and a prostate cancer group. [Figure 4] 1 is a graph showing the results of CA19-9 / WFA measurement in a prostate hyperplasia group and a prostate cancer group. [Diagram 5] 1 is a graph showing the relationship between CA19-9 measurement values ​​and CA19-9 / WFA measurement values. [Figure 6] 1 is a graph showing the relationship between PSA measurement values ​​and CA19-9 / WFA measurement values. [Figure 7] 1 is a graph showing the results of measuring CA19-9 in a healthy subject group and a colon cancer group. [Figure 8] 1 is a graph showing the results of measuring CA19-9 / WFA in a healthy subject group and a colon cancer group. [Figure 9] 1 is a graph showing the relationship between CA19-9 measurement values ​​and CA19-9 / WFA measurement values. [Figure 10] 1 is a graph showing the results of measuring CA19-9 / WFA in a healthy subject group and a breast cancer group. [Figure 11] 1 is a graph showing the results of measuring CA19-9 / MAM in a healthy subject group and a prostate cancer group. [Figure 12] 1 is a graph showing the results of measuring CA19-9 / MAM in a healthy subject group and a colon cancer group. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present invention will be described in detail below based on preferred embodiments thereof.

[0015] <Cancer detection methods, cancer testing methods> The cancer detection method of the present invention is a method for detecting at least one cancer selected from the group consisting of prostate cancer and colon cancer, and includes a measurement step of measuring the amount of GalNAc-added CA19-9 in a sample. The cancer testing method of the present invention is a method for testing at least one cancer selected from the group consisting of prostate cancer and colon cancer, and includes a measurement step of measuring the amount of GalNAc-added CA19-9 in a sample derived from a subject.

[0016] [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 prostate cancer and colon cancer. Prostate cancer according to the present invention refers to cancer occurring in the prostate, and examples thereof include prostate cancer, prostate small cell carcinoma, prostate ductal carcinoma, prostate sarcoma, prostate squamous cell carcinoma, prostate adenosquamous cell carcinoma, prostate basal cell carcinoma, prostate mucinous carcinoma, and prostate signet ring cell carcinoma. Among these, prostate cancer according to the present invention is preferably prostate cancer. Furthermore, colon cancer according to the present invention refers to cancer occurring in the large intestine (particularly the colon or rectum), and examples thereof include adenocarcinoma, endocrine cell carcinoma, adenosquamous cell carcinoma, squamous cell carcinoma, carcinoid tumor, non-epithelial malignant tumor, and lymphoma in the region. Among these, colon adenocarcinoma is preferable as colon cancer according to the present invention.

[0017] [GalNAc-added CA19-9] In the present invention, "GalNAc-added CA19-9" refers to a molecule comprising CA19-9 and GalNAc (N-acetylgalactosamine) added and bound to CA19-9.

[0018] In the present invention, "CA19-9" refers to a type I sugar chain antigen recognized by a monoclonal antibody (NS19-9) obtained by using the human colon cancer cultured cell line SW-1116 as an immunogen. CA19-9 is characterized by the Lewis A antigen (LeA antigen) of the Lewis blood group antigens as an antigenic determinant for the anti-CA19-9 antibody described below. a) has sialic acid attached thereto, forming sialyl Lewis A (Koprowski et al., Somat. Cell Genet., 5, 957, 1979).

[0019] "GalNAc (N-acetylgalactosamine)" is a monosaccharide with the IUPAC name "2-(acetylamino)-2-deoxy-D-galactose". The GalNAc-added CA19-9 of the present invention preferably contains GalNAc as a WFA-binding sugar chain that binds to WFA, and preferably contains GalNAc as a terminal GalNAc residue.

[0020] In one molecule of GalNAc-added CA19-9, there may be multiple CA19-9 and GalNAc, and CA19-9 and GalNAc may be directly bound or may be bound via a core protein or other sugar chain. Examples of the core protein include mucin, apolipoprotein, kininogen, and ARCVF. In this case, the average mass of GalNAc-added CA19-9 (average mass measured by calibration with a marker by gel filtration chromatography (GFC), the same applies below) is not particularly limited, but is preferably 25,000 to 10,000,000 Da, and more preferably 1,000,000 to 10,000,000 Da.

[0021] CA19-9 and GalNAc may be in a form that exists on particles larger than the core protein, such as extracellular vesicles such as exosomes or viruses. In this case, the particle size of GalNAc-added CA19-9 (including the particles) is not particularly limited, but is about 50 to 500 nm.

[0022] [Subject] In the present invention, the term "subject" refers to a subject to which the cancer testing method of the present invention is performed, and is preferably a human. The subject according to the present invention may be a healthy individual or a person who is afflicted with prostate cancer or colon cancer but has no subjective symptoms, for the purpose of screening, etc. Also, the subject may be a patient who is already known to be afflicted with the cancer or a patient who has been afflicted with the cancer in the past, for the purpose of determining the prognosis, determining a treatment plan, confirming the effectiveness of treatment, confirming recurrence, etc. Also, in the present invention, the term "healthy individual" refers to a person who is not afflicted with the cancer to be tested (i.e., prostate cancer and / or colon cancer). The fact that the subject is truly afflicted with prostate cancer and / or colon cancer is determined (definitively diagnosed) by a biopsy of prostate tissue and / or colon tissue taken from the subject.

[0023] [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" in this specification) is not particularly limited as long as it is a sample in which GalNAc-tagged CA19-9 can be present. In general, examples of the sample include blood samples such as serum, plasma, and whole blood collected from subjects for cancer testing such as the subjects; 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 fluid. The sample according to the present invention is preferably a blood sample, and more preferably serum (also referred to as a "serum sample").

[0024] These samples may be diluted or suspended in a diluent as necessary, or may be pretreated as appropriate. Examples of the diluent include buffers such as phosphate buffer, Tris buffer, Good's buffer, borate buffer, acetate buffer, citrate buffer, glycine buffer, succinate buffer, and phthalate buffer. Examples of the pretreatment include treatments such as pulverization, freezing, heating, concentration, fractionation, and desalting; treatments for adding pH adjusters, stabilizers, preservatives, antiseptics, surfactants, and the like; and purification treatments, which may be performed alone or in combination of two or more of these. Examples of the purification treatments are not particularly limited, but include treatments using a column; treatments for adsorbing impurities in a sample fixed to a water-insoluble carrier using a substance such as an antibody that adsorbs impurities, and removing the impurities from a sample containing GalNAc-added CA19-9; treatments for capturing CA19-9 containing GalNAc-added CA19-9 using an anti-CA19-9 antibody fixed to a water-insoluble carrier, removing impurities by washing, and then releasing the impurities.

[0025] [Measurement process] The method of the present invention includes a measuring step for measuring the amount of GalNAc-added CA19-9 in a sample. The method for measuring the amount of GalNAc-added CA19-9 is not particularly limited as long as it is a method capable of measuring the amount of GalNAc-added CA19-9, and a conventionally known method or a method based thereon, or a combination thereof, can be appropriately adopted. For example, a method using a probe molecule capable of specifically binding to GalNAc-added CA19-9; 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 a combination thereof can be mentioned, but are not limited thereto.

[0026] In particular, the measurement step according to the present invention is preferably a method using a probe molecule capable of specifically binding to GalNAc-labeled CA19-9. Examples of such probe molecules include antibodies; binding proteins such as protein A, protein G, and protein L; avidins such as avidin D and streptavidin; lectins; galectins, sugar chain receptors, and immunoreceptors. In the present invention, the term "antibody" includes complete antibodies as well as antibody fragments (e.g., Fab, Fab', F(ab')). 2 , Fv, single-chain antibodies, diabodies, etc.), and minibodies in which the variable regions of antibodies are combined.

[0027] A probe molecule capable of specifically binding to GalNAc-attached CA19-9 is preferably a combination of a probe molecule capable of specifically binding to CA19-9 (referred to in the present invention as a "first probe molecule") and a probe molecule capable of specifically binding to GalNAc (referred to in the present invention as a "second probe molecule").

[0028] [First probe molecule] Examples of the first probe molecule capable of specifically binding to CA19-9 include an antibody capable of specifically binding to CA19-9 (sometimes referred to as an "anti-CA19-9 antibody" in this specification) and an antibody capable of specifically binding to a protein to which CA19-9 binds (such as the core protein), and among these, an anti-CA19-9 antibody is preferred.

[0029] In the present invention, the term "anti-CA19-9 antibody" refers to an antibody capable of specifically recognizing and binding to sialyl Lewis A of CA19-9. Such an anti-CA19-9 antibody is not particularly limited as long as it has the ability to bind to CA19-9, and may be a polyclonal or monoclonal antibody, but is preferably a monoclonal antibody from the viewpoint of homogeneity and stability. The anti-CA19-9 antibody can be produced by appropriately adopting and improving a conventionally known production method, and a commonly available antibody may be used as appropriate.

[0030] As the anti-CA19-9 antibody of the present invention, for example, when the following lectin is used as a second probe molecule, in order to prevent the lectin from also recognizing the antibody glycan and reducing the detection sensitivity, it is preferable that the lectin-binding glycan has been removed or destroyed by oxidation treatment, glycosidase treatment, protease treatment, etc.; the region containing glycated amino acids in the antibody molecule has been removed; or the antibody is produced under conditions in which glycan addition does not occur, such as by using genetically modified E. coli or cells expressing an antibody gene or by restricting the nutritional conditions in the culture of antibody-producing cells.

[0031] [Second probe molecule] Examples of the second probe molecule capable of specifically binding to GalNAc include antibodies capable of specifically binding to GalNAc (sometimes referred to as "anti-GalNAc antibodies" herein), lectins capable of specifically binding to GalNAc (e.g., Wisteria floribunda lectin (WFA), soybean lectin (SBA), Wisteria moniliforme lectin (VVA), Bacillus subhirtella lectin (DBA), Bacillus subhirtella lectin (BPL), and Bacillus moniliforme lectin (HPA)), and galectins capable of specifically binding to GalNAc. Among these, lectins capable of specifically binding to GalNAc are preferred, and Wisteria floribunda lectin (WFA) is particularly preferred. Wisteria floribunda lectin (WFA: Wisteria floribunda agglutinin) is a protein that exhibits binding activity by recognizing the sugar chain structure of GalNAc, and is a legume lectin derived from the plant Wisteria floribunda. The lectin may be a modified lectin into which a mutation has been introduced or an artificially synthesized lectin for the purpose of increasing the specificity of the sugar chain recognition activity, etc. In addition, a lectin that is generally available may be appropriately used.

[0032] When the first and second probe molecules are used, the measurement step involves contacting the sample with the first and second probe molecules. This allows both CA19-9 and GalNAc to be recognized, and allows GalNAc-added CA19-9 containing both to 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 may occur first.

[0033] [Labeling substance] In the present invention, the amount of GalNAc-added CA19-9 is preferably measured by detecting a signal generated by a labeling substance added to a probe molecule capable of specifically binding to GalNAc-added CA19-9 (preferably the first probe molecule and / or the second probe molecule) or added to a molecule that recognizes these molecules (e.g., a secondary antibody or protein A). The amount of GalNAc-added CA19-9 can be measured by measuring the amount of the detected signal and, if necessary, semi-quantitating or quantifying it. The "signal" includes color development (coloring), quenching, reflected light, luminescence, fluorescence, radiation from a radioisotope, and the like, and includes signals that can be confirmed by a measurement method or device according to the type of signal as well as signals that can be confirmed by the naked eye. In the present invention, the amount of GalNAc-added CA19-9 to be measured may be semi-quantitative or quantitative using a standard sample or the like, but from the viewpoint of simpler and faster measurement, the amount of the signal may be directly used as the amount of GalNAc-added CA19-9 of the present invention.

[0034] The labeling substance according to the present invention can be any labeling substance used in known immunological assay methods or methods based thereon, without any particular limitation. For example, enzymes, luminescent substances such as acridinium derivatives, fluorescent substances such as europium, fluorescent proteins such as allophycocyanin (APC) and phycoerythrin (R-PE), etc. 125Examples of the labeling substance include radioactive substances such as I; low molecular weight labeling substances such as fluorescein isothiocyanate (FITC) and rhodamine isothiocyanate (RITC); gold particles; avidin; biotin; latex; dinitrophenyl (DNP); and digoxigenin (DIG), and 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 as a substrate. Examples of the enzyme include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (ALP), β-galactosidase (β-gal), glucose oxidase, and luciferase.

[0035] [Sandwich method] The measurement method using the first probe molecule and the second probe molecule includes, but is not limited to, immunological measurement methods such as the sandwich method, the competitive method, and the turbidimetric immunoassay, and measurement methods based on these principles. For example, generally, the measurement method may employ methods using a microplate 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.

[0036] As the measurement method according to the present invention, the sandwich method is preferred from the viewpoint of being able to construct a measurement system with higher sensitivity and specificity. Hereinafter, the measurement step according to the present invention will be described in more detail with reference to the sandwich method as an example.

[0037] In the case of using the sandwich method, the following embodiment is adopted: 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 embodiment, the first probe molecule and the second probe molecule may be provided in either the capture body or the label, respectively, but one is included in the capture body and the other is included in the label. In this way, both GalNAc and CA19-9 are captured, and GalNAc-added CA19-9 can be captured and detected with high accuracy and ease.

[0038] Other aspects of the sandwich method are not limited to the above, and may be, for example, a first label comprising a first label and one of the first and second probe molecules, and a second label comprising a second label and the other of the first and second probe molecules, and the capture body may be a capture body comprising a water-insoluble carrier and a probe molecule immobilized on the water-insoluble carrier (hereinafter, sometimes referred to as "second aspect"). In the second aspect, the first label and the second label generate signals different from each other. In this case, the probe molecule provided in the capture body may be a probe molecule (including the first and second probe molecules) capable of specifically binding to GalNAc-added CA19-9; or a probe molecule capable of specifically binding to the first and / or second probe molecules.

[0039] Examples of such sandwich methods include the two-step forward sandwich method (a method in which the capture body is reacted with GalNAc-attached CA19-9 in the sample and the GalNAc-attached CA19-9 bound to the capture body is reacted with the labeled body sequentially), the reverse sandwich method (a method in which the labeled body is reacted with GalNAc-attached CA19-9 in the sample in advance and the resulting complex is reacted with the capture body), and the one-step method (a method in which the GalNAc-attached CA19-9 in the sample, the capture body, and the labeled body are reacted simultaneously in one step), and any of these can be used.

[0040] For example, in the forward sandwich method, the sample is first contacted with the capture body, and the capture body captures GalNAc-added CA19-9 through the binding between the probe molecule of the capture body and GalNAc-added CA19-9 (e.g., the binding between the first probe molecule and CA19-9) (primary reaction: capture step). Next, the label is contacted with the GalNAc-added CA19-9 captured by the capture body, and the label is labeled through the binding between the probe molecule of the label and GalNAc-added CA19-9 (e.g., the binding between the second probe molecule and GalNAc) (secondary reaction: label step). This reaction forms a complex containing the capture body-GalNAc-added CA19-9-label. The unbound sample and label are washed and removed as necessary (washing step), and then the signal derived from the label is measured by a predetermined method according to the label (measurement step).

[0041] (Capture body) The "capture body" according to the present invention is a complex comprising a water-insoluble carrier and a probe molecule capable of specifically binding to GalNAc-labeled CA19-9 immobilized on the water-insoluble carrier, and is a conjugate in which the water-insoluble carrier and the probe molecule are directly or indirectly bound to each other. The probe molecule provided in the capture body according to the first embodiment is preferably either a first probe molecule or a second probe molecule, and 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 CA19-9, and more preferably an anti-CA19-9 antibody.

[0042] <Non-water-soluble carrier> The water-insoluble carrier contained in the capture body mainly functions as a carrier for supporting and immobilizing the probe molecule, and is a water-insoluble substance. 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 below) at room temperature and normal pressure.

[0043] The material of such a water-insoluble carrier can be any material generally used in immunological assays and assays similar thereto, and is not particularly limited. For example, 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.) can be mentioned. In addition, the material of the water-insoluble carrier may be a composite of these materials or a composite of these materials with other materials, and may be, for example, an organic-inorganic composite material 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.

[0044] In the present invention, the shape of the water-insoluble carrier is not particularly limited, and may be, for example, any of a plate, a fiber, a membrane, a particle, etc., but from the viewpoint of reaction efficiency, it is preferable that it is a particle, and from the viewpoint of automation and shortening the time, it is more preferable that it is a magnetic particle. As such a water-insoluble carrier, a conventionally known carrier can be appropriately used, and a commercially available carrier can also be appropriately used.

[0045] <Configuration and manufacturing method of the capture body> 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 between the probe molecule and GalNAc-attached CA19-9, etc. For example, the mass of the probe molecule (the total mass if the probe molecule is a combination of two or more types) per 100 parts by mass of the water-insoluble carrier (preferably particles) (the total mass if the water-insoluble carrier is a combination of two or more types) is preferably 0.1 to 10 parts by mass, and more preferably 1 to 5 parts by mass.

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

[0047] In the case of direct immobilization, for example, the water-insoluble carrier and / or the probe molecule may have 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, or the active group may be added as necessary and bonded to the probe molecule, thereby allowing the probe molecule to be directly immobilized on the water-insoluble carrier.

[0048] In the case of indirect immobilization, for example, a linker that binds to the probe molecule is immobilized on the water-insoluble carrier, and the probe molecule is bound to the linker, thereby indirectly immobilizing the probe molecule on the water-insoluble carrier. The linker is not particularly limited, and examples thereof include a secondary antibody that can bind to the probe molecule, protein G, protein A, a photocleavable photocleavable linker, and a linker molecule having the active group (e.g., hydrazine salt, hydrazide). In addition, the probe molecule may be modified in some way, and a substance that captures the modified portion may be immobilized on the water-insoluble carrier to immobilize the probe molecule on the water-insoluble carrier. For example, a representative example of the modified portion is biotin, and a representative example of the substance that captures the modified portion is streptavidin, but the present invention is not limited thereto.

[0049] The ratio of the water-insoluble carrier and the probe molecule to be subjected to these reactions can be appropriately selected so as to achieve the preferred range of the ratio in the capture body. If necessary, the water-insoluble carrier may be blocked with a suitable blocking agent (e.g., bovine serum albumin, gelatin, etc.) to prevent non-specific adsorption to the probe molecule or the water-insoluble carrier. Furthermore, commercially available products may be used as such capture bodies.

[0050] (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 GalNAc-added CA19-9, and is a conjugate in which the labeling substance and the probe molecule are directly or indirectly bound to each other. The probe molecule provided in the labeled body according to the first embodiment is preferably the other of the first and second probe molecules 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, but from the viewpoint of providing a molecule such as an anti-CA19-9 antibody with high affinity for an antigen as the first probe molecule in the capture body, the second probe molecule capable of specifically binding to GalNAc is preferred, a lectin capable of specifically binding to GalNAc is more preferred, and WFA is particularly preferred.

[0051] (Blocked labeled lectin) In the present invention, the label may be a labeled antibody comprising the label and the antibody (anti-CA19-9 antibody, anti-GalNAc antibody, etc.), a labeled lectin comprising the label and a lectin, or a blocked labeled lectin, and may be one of these or a combination of two or more of these. Among these, the label in the present invention, particularly in the first aspect, is preferably a blocked labeled lectin. In the present invention, the "blocked labeled lectin" is a complex comprising a water-soluble carrier made of a water-soluble polymer, a label and a lectin immobilized on the water-soluble carrier, and is a conjugate in which the water-soluble carrier, the label, and the lectin are directly or indirectly bound. In the present invention, the blocked labeled lectin comprises a lectin capable of specifically binding to GalNAc 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, and is preferably WFA. The label is as described above, including its preferred embodiments.

[0052] In the blocked labeled lectin, it is sufficient that the labeling substance and the lectin are supported on the water-soluble carrier, and the labeling substance and the lectin may be bound to the water-soluble carrier independently of each other, or the water-soluble carrier, the labeling substance, and the lectin may all be bound to the other two, or 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 the lectin and the lectin-binding sugar chain structure is weak, but in such a blocked labeled lectin, a complex is formed in which multiple lectins are connected by the water-soluble carrier, which is a polymer. Therefore, by using this, multiple binding points are generated in one complex, so that the overall affinity is improved and the target GalNAc can be detected with high sensitivity.

[0053] <Water-soluble carrier> The water-soluble carrier contained in the blocked labeled lectin mainly functions as a carrier for carrying the labeled substance and lectin, and is made of a water-soluble polymer. The water-soluble polymer constituting the water-soluble carrier according to the present invention (hereinafter referred to as "first water-soluble polymer") is not particularly limited as long as it is a water-soluble polymer that can immobilize and carry the labeled 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, more preferably 0.1 g / mL or more at room temperature and normal pressure.

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

[0055] Furthermore, the first water-soluble polymer according to the present invention preferably has an average mass of 70,000 to 1,000,000 Da, and more preferably 150,000 to 700,000 Da, from the viewpoint that a blocked labeled antibody with a more preferable average particle size tends to be obtained.

[0056] The blocked labeled lectin may contain, as the first water-soluble polymer, a plurality of water-soluble polymers having different weight-average molecular weights. The blocked labeled lectin is preferably a combination of a high-molecular-weight blocked labeled lectin having a first water-soluble polymer with a weight-average molecular weight of 200,000 or more and a low-molecular-weight blocked labeled lectin having a first water-soluble polymer with a weight-average molecular weight of less than 100,000 (more preferably 100,000 or less), and more preferably a combination of a high-molecular-weight blocked labeled lectin having a first water-soluble polymer with a weight-average molecular weight of 200,000 to 700,000 (more preferably 250,000 to 500,000) and a low-molecular-weight blocked labeled lectin having a first water-soluble polymer with a weight-average molecular weight of 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 the high molecular weight blocked labeled lectin:mass of the low molecular weight blocked labeled lectin) is preferably 10:1 to 1:10, more preferably 5:1 to 1:5, and even more preferably 3:1 to 1:3.

[0057] Examples of the first water-soluble polymer according to the present invention include polysaccharides such as dextran, aminodextran, Ficoll (trade name), dextrin, agarose, pullulan, various celluloses (e.g., hemicellulose, ligulin, etc.), chitin, and chitosan; β-galactosidase; thyroglobulin; hemocyanin; polylysine; polypeptide; DNA; and modified forms thereof (e.g., diethylaminoethyl dextran, sodium dextran sulfate, etc.), and one of these may be used alone or two or more may be used in combination. Among these, the first water-soluble polymer according to 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, from the viewpoints of being inexpensive and available in large quantities, and being relatively easy to chemically process, such as by adding functional groups or performing coupling reactions.

[0058] (Constitution and Production Method of Blocked Labeled Lectin) In the blocked labeled lectin, the content of the labeling substance is not particularly limited and can be appropriately adjusted 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 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 of the first water-soluble polymers; the same applies below) per 100 parts by mass of the first water-soluble polymer is preferably 100 to 1,000 parts by mass, more preferably 300 to 800 parts by mass.

[0059] In the blocked labeled lectin, the content of the lectin is not particularly limited, but in order to further improve the measurement sensitivity, it is preferable to set the number of lectin molecules binding to one molecule of the first water-soluble polymer as large as possible. For example, the mass of the lectin (when two or more types of lectins are combined, the total mass of the lectins) 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.

[0060] Moreover, the blocked labeled lectin preferably has a weight-average molecular weight per molecule of 1,000,000 to 10,000,000, and more preferably 1,500,000 to 5,000,000. When the weight-average molecular weight is 1,000,000 or more, the measurement sensitivity tends to be higher, while when it is 10,000,000 or less, aggregation in an aqueous solution tends to be more sufficiently suppressed.

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

[0062] Examples of the method for directly immobilizing the supported substance on the water-soluble carrier include a method of immobilizing the supported substance and / or the first water-soluble polymer constituting the water-soluble carrier by imparting 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, a thiol group, or a pyridyl disulfide group, or by using a water-soluble polymer having such an active group as the supported substance and / or the water-soluble carrier and binding them. The supported substance and the first water-soluble polymer to which the active group has been imparted may be commercially available as they are, or may be prepared by introducing the active group into the surface of the supported substance and the water-soluble polymer under appropriate reaction conditions. As an example, the introduction of a thiol group can be carried out using a commercially available reagent such as S-acetylmercaptosuccinic anhydride or 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 a commercially available reagent such as N-(6-maleimidocaproyloxy)succinimide or N-(4-maleimidobutyryloxy)succinimide. Introduction of a pyridyl disulfide group can be carried out using a commercially available reagent such as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-{6-[3-(2-pyridyldithio)propionamido]hexanoyloxy}sulfosuccinimide, sodium salt (Sulfo-AC5-SPDP). Furthermore, the pyridyl disulfide group can also be introduced by reducing it after introduction to form a thiol group.

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

[0064] In the method for producing the blocked labeled lectin, the labeling substance and the lectin may be immobilized on the water-soluble carrier at the same time, or may be immobilized separately and sequentially, but from the viewpoint of ease of production and ease of control of the amounts of the labeling substance and the 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 the lectin on separate water-soluble carriers, and binding the labeling substance immobilized on one water-soluble carrier (blocked labeling substance) to the lectin immobilized on the other water-soluble carrier directly or via the linker or the like.

[0065] The method for producing the blocked labeled lectin is not particularly limited. For example, as described in the following Examples, when the labeled 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 give an aldehyde group, reacted with hydrazine hydrochloride, and then reacted with a reducing agent such as dimethylamine borane (DMAB) to convert it into a hydrazide. Meanwhile, the enzyme is also oxidized with an oxidizing agent such as sodium periodate to give an aldehyde group to its sugar chain. Next, the hydrazine residue given above is reacted with the aldehyde group to form a hydrazone bond, thereby obtaining a first water-soluble polymer-enzyme conjugate. The obtained first water-soluble polymer-enzyme conjugate is crosslinked with a crosslinker (e.g., SM (PEG)) having N-hydroxysuccinimide and maleimide groups at each end. 4, SMCC, etc.) to introduce a maleimide group. Meanwhile, the lectin is thiolated with a thiolation reagent to give a thiol group, or in the case of a lectin having a disulfide bond in the molecule, the thiol group is obtained by reduction. Finally, the maleimide group introduced into the first water-soluble polymer-enzyme conjugate is bonded to the thiol group given to the lectin, thereby covalently bonding the first water-soluble polymer (water-soluble carrier)-enzyme-lectin. According to this method, a product is obtained in which two or more molecules of the first water-soluble polymer are bonded via the enzyme, and the lectin is bonded to the product. The ratio of the first water-soluble polymer, the labeling substance, and the lectin to be subjected to these reactions can be appropriately selected so as to achieve the preferred range of each content in the above-mentioned blocked labeled lectin.

[0066] (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 similar thereto can be appropriately adopted. For example, when the water-insoluble carrier is a plate, the sample can be injected into the plate (probe molecule immobilized plate), and when the water-insoluble carrier is particles, the capture body (probe molecule immobilized particles) can be added to the sample.

[0067] In the reaction between the sample and the capture body in the capture step, the content (final concentration) of the capture body in the reaction solution containing the capture body and the sample (when the capture body is a combination of two or more types, the total of the capture bodies, the same applies below) is not particularly limited and is adjusted appropriately depending on the type, concentration, etc. of the sample, but from the viewpoint of efficient capture in a short period of time, it is preferably, for example, 0.01 to 1.5 mass%, more preferably 0.05 to 1 mass%, and even more preferably 0.1 to 0.5 mass%.

[0068] 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 about 6 to 9, preferably 7 to 8, for about 5 seconds to 10 minutes, preferably 30 seconds to 5 minutes, but are not limited to these conditions.

[0069] (Label Step) In the reaction between the labeled form and GalNAc-attached CA19-9 in the labeling step, the content (final concentration) of the labeled form in the reaction solution containing the labeled form and GalNAc-attached CA19-9 (when the labeled form is a combination of two or more types, the total of the labels, the same applies below) is not particularly limited and is adjusted appropriately depending on the type, concentration, etc. of the sample, but 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, from the viewpoint that use of an excess amount may cause a high background signal.

[0070] Other conditions for the labeling step are also 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 pH 5.0 to 7.0, preferably 5.5 to 6.5, for about 3 to 120 minutes, preferably about 5 to 10 minutes, but is not limited to these conditions.

[0071] (Washing step) The sandwich method preferably further includes a washing step for separating the GalNAc-added CA19-9 bound to the capture body from other impurities not bound to (captured by) the capture body and removing the impurities after the capture step and / or the label step. The method for removing the impurities is not particularly limited, and a conventionally known method or a method similar thereto can be appropriately adopted. For example, when the capture body is the probe molecule immobilized plate, a method for removing the liquid phase (supernatant) from the plate, and when the capture body is the probe molecule immobilized particle, a method for recovering the particle by centrifugation or magnetic collection and removing the liquid phase (supernatant) can be mentioned. In addition, 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 to 9) buffer solutions (sodium phosphate buffer, MES, Tris, CFB, MOPS, PIPES, HEPES, tricine buffer, bicine buffer, glycine buffer, etc.), and may also be those to which a stabilizing protein such as BSA or a surfactant has been added.

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

[0073] In addition, when the blocked labeled lectin is used as the label, from the viewpoint of further improving the measurement sensitivity, a water-soluble polymer (hereinafter referred to as a "second water-soluble polymer"; which 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 and lectin), a free lectin (which is different from the lectin contained in the blocked labeled lectin, etc., in that it is not fixed to either the water-soluble carrier or the labeling substance) or the like may be allowed to coexist in the reaction system between the sample and the label.

[0074] The second water-soluble polymer may be the same as the first water-soluble polymer, and may be one of them 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, 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, from the viewpoint of a tendency to further improve the measurement sensitivity.

[0075] 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.

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

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

[0078] The amount of the free lectin is not particularly limited, but in a reaction solution containing the sample, the blocked labeled lectin, and the free lectin, the content of the free lectin (when the free lectin is a combination of two or more types, the total content of the free lectins) 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.

[0079] (Measurement step) In the measurement step, a signal is measured according to the labeling substance. For example, when the labeling substance is an enzyme, a chromogenic or luminogenic substrate corresponding to the enzyme is added and reacted to measure a signal (e.g., color or luminescence). This allows the amount of GalNAc-added CA19-9 in the sample to be detected as a signal amount, and the amount of GalNAc-added CA19-9 in the sample can be quantified by comparing with the measured value in a standard sample, if necessary.

[0080] [Cancer detection methods] According to the cancer detection method of the present invention, the amount of GalNAc-added CA19-9 measured by the above-mentioned measurement step is used as an index to detect the presence or absence of prostate cancer and / or colon cancer in the sample or the subject from which the sample is derived. 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.

[0081] [Cancer testing method] In the cancer testing method of the present invention, the amount of GalNAc-attached CA19-9 in a sample derived from a subject is measured and used as an indicator to detect the presence or absence of prostate cancer and / or colon cancer, thereby making it possible to predict whether or not the subject is suffering from prostate cancer and / or colon cancer, determine the presence or absence or possibility of the subject being suffering from said cancer, or evaluate the progression or severity of said cancer.

[0082] 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 prostate cancer and colorectal cancer, comprising: a measuring step of measuring the amount of GalNAc-added CA19-9 in a sample derived from the subject; and a selecting step of selecting a subject using the measured amount of GalNAc-added CA19-9 as an index; A method for distinguishing the presence or absence of at least one type of cancer selected from the group consisting of prostate cancer and colorectal cancer in a subject, comprising: a measuring step of measuring the amount of GalNAc-added CA19-9 in a sample derived from the subject; and a distinguishing step of distinguishing the subject using the measured amount of GalNAc-added CA19-9 as an indicator; A method for evaluating the progress or severity of at least one cancer selected from the group consisting of prostate cancer and colorectal cancer in a subject, comprising: a measuring step of measuring the amount of GalNAc-added CA19-9 in a sample derived from the subject; and an evaluation step of evaluating the subject using the measured amount of GalNAc-added CA19-9 as an index; Examples include:

[0083] [Screening method] In the present invention, "screening" refers to selecting a subject who may be affected by the cancer (prostate cancer and / or colon cancer). In the present invention, the screening method specifically includes a method of predicting a subject's high possibility of being affected by prostate cancer and / or colon cancer (including recurrence) and selecting the subject from a group with no or low possibility. In the screening method, subjects may be selected at a level where a medium possibility is expected, for example, depending on the purpose.

[0084] More specifically, for example, subjects are divided into two groups, a group consisting of those truly affected by the cancer (positive group) and a group consisting of those not affected by 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.

[0085] [Identification method] In the present invention, "differentiation" refers to distinguishing prostate cancer and / or colon cancer from other diseases or conditions and determining whether a subject is affected with the cancer. In addition, it includes not only the determination of the presence or absence of the cancer, but also the evaluation of the degree of the possibility of the cancer (evaluation of high / medium / low, etc.). In the present invention, the differentiation method specifically includes a method for determining whether a subject is affected with prostate cancer or colon cancer or whether there is a high possibility of the cancer, regardless of the presence or absence of symptoms, and includes, for example, a method for determining whether the cancer that a subject is affected with is prostate cancer or colon cancer, or a method for determining that there is a high possibility of the cancer; a method for determining whether the prostate cancer or colon cancer that a subject has previously suffered from has recurred, or a method for determining that there is a high possibility of the cancer recurring.

[0086] More specifically, for example, subjects are divided into two groups, a group consisting of those truly affected by the cancer (positive group) and a group consisting of those not affected by the cancer (normal group: negative group), and it is determined whether the subject is included in the positive group or the negative group. Such discrimination can be applied to assist doctors in diagnosing prostate cancer and / or colon cancer. In this case, the discrimination criterion is preferably, for example, a negative agreement rate (the proportion of subjects who are discriminated as being in the normal group by the discrimination but are actually in the normal group) of 95% or more.

[0087] [Evaluation method] In the present invention, the evaluation methods include, for example, a method for evaluating the progression or severity of prostate cancer and / or colorectal cancer from which a subject is afflicted; a method for providing an index for determining a treatment policy for the cancer from which a subject is afflicted; and a method for assessing the effectiveness of treatment for the cancer.

[0088] More specifically, for example, the amount of GalNAc-added CA19-9 in a group of subjects not affected by the cancer or those with a low stage or severity of the cancer is compared with the amount of GalNAc-added CA19-9 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. Also, the amount of GalNAc-added CA19-9 in the subject at the time of discovery of the cancer or before treatment can be compared with the current amount of GalNAc-added CA19-9, 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.

[0089] (Cutoff value) In the selection step of the screening method and the discrimination step of the differentiation method, for example, the amount of GalNAc-attached CA19-9 measured in the measurement step is compared with a predetermined cutoff value, and subjects whose amount of GalNAc-attached CA19-9 is higher than the cutoff value are predicted or discriminated as suffering from (or likely to suffer from) prostate cancer and / or colon cancer.

[0090] In the present invention, the "cut-off value" is a predetermined value that serves as a standard for determining the amount of GalNAc-tagged CA19-9, and refers to a boundary value for determining the positive group and the negative group. Such a cut-off value is not particularly limited, since it is appropriately set depending on the purpose of the cancer testing method of the present invention, the method for measuring the amount of GalNAc-tagged CA19-9, the properties of the subject and sample, dilution conditions, etc. For example, by setting the cut-off value to a relatively low value, the detection sensitivity is increased, that is, patients in the early stage of the cancer can be collected to a certain extent, and early detection is possible. On the other hand, since a small amount of GalNAc-tagged CA19-9 is detected even in samples derived from healthy individuals, the screening and differentiation can be performed with higher accuracy by setting the cut-off value to a relatively high value.

[0091] An example of the cut-off value is, for example, when the amount of GalNAc-added CA19-9 measured by sandwich immunoassay using a serum specimen diluted to 1 / 10 in volume ratio, anti-CA19-9 antibody immobilized particles as the capture body, and blocked labeled WFA as the label, and the amount is expressed as the emission intensity (count) of light having a maximum absorption at a wavelength of 463 nm with ALP as the label and AMPPD as the substrate (in the case of Example 1), the cut-off value is 50,000 to 80,000 counts, preferably 60,000 to 70,000 counts, but is not limited thereto. Note that the cut-off value determined in the above range is selected and applied according to the purpose of the cancer testing method of the present invention, the method for measuring the amount of GalNAc-added CA19-9, and the properties of the subject and sample.

[0092] According to such a cancer testing method of the present invention, it is possible to provide information for determining a treatment policy specific to prostate cancer and / or colon cancer, by distinguishing it from other cancers. In addition, by identifying a subject who is afflicted with prostate cancer and / or colon cancer (including recurrence) or who is likely to be afflicted with prostate cancer and / or colon cancer, and performing further tests and definitive diagnosis, it is possible to detect prostate cancer and / or colon cancer early and to intervene in the treatment early. Furthermore, since it has been reported that information on the sugar chain structure derived from cancer is an index of the invasiveness of cancer, it is also possible to obtain information on the malignancy and prognosis of cancer by the cancer testing method of the present invention. The method of the present invention is also a method for assisting a doctor or the like in diagnosing prostate cancer and / or colon cancer, or a method for providing information for a doctor or the like to diagnose prostate cancer and / or colon cancer.

[0093] Furthermore, the method of the present invention is also suitable as a method for combining with conventional CA19-9 measurement methods or measurement methods for other monitoring markers, thereby making it possible to further improve the detection sensitivity and diagnostic accuracy of prostate cancer and / or colorectal cancer.

[0094] <Kit> The kit of the present invention is a kit for use in the above-mentioned cancer detection method or cancer testing method of the present invention, and includes a first probe molecule capable of specifically binding to CA19-9 and a second probe molecule capable of specifically binding to GalNAc. The first probe molecule and the second probe molecule are as described above, including preferred embodiments thereof.

[0095] The kit of the present invention also preferably includes a capture body comprising a water-insoluble carrier and either one of 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 the label are as described above, including their preferred embodiments.

[0096] In the kit of the present invention, the first probe molecule, the second probe molecule, the capturer, and the label may each be independently in a solid (powder) form or in a liquid form dissolved in a buffer solution. When in a liquid form, the concentrations of the first probe molecule, the second probe molecule, the capturer, and the label in each solution are not particularly limited, but are each independently, for example, preferably 0.01 to 10 μg / mL, more preferably 0.1 to 5.0 μg / mL, and further preferably 0.5 to 3.0 μg / mL.

[0097] The kit of the present invention may further include components that should be included in ordinary immunological measurement methods such as ELISA, CLEIA, immunochromatography, and methods similar thereto. For example, when the above-mentioned sandwich method is the principle of the measurement method of the present invention, the kit may further include at least one 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. In addition, when the labeling substance is an enzyme, the kit may further include a substrate, a reaction stop solution, and the like necessary for detection and quantification of the labeling substance.

[0098] Furthermore, the kit of the present invention may further comprise, as necessary, the diluent, a pretreatment solution for pretreatment of the sample, a dilution cartridge, and a pretreatment reaction stop solution or neutralization solution. When immunochromatography is employed as the sandwich method, the kit may further comprise a device including a zone carrying the capture body and / or the label. The device may comprise other components suitable for immunochromatography, such as a developer pad or an absorbent pad. Furthermore, the kit of the present invention may further comprise an instruction manual for use of the kit. EXAMPLES

[0099] The present invention will be described in more detail below with reference to 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.

[0100] Example 1: Measurement of WFA-bound glycan-added CA19-9 (CA19-9 / WFA) in serum samples using blocked labeled lectin (WFA) (1) Preparation of hydrazide dextran 240.0 mg of dextran (CarboMer) with a molecular weight of 250 k 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. Then, 150 mM NaIO 4 2.664 mL of the above and 0.536 mL of ion-exchanged water were added, and the mixture was stirred in the dark at 25° C. for 30 minutes. Next, buffer exchange was performed with 0.1 M sodium phosphate buffer (pH 6.0) using a PD-10 column (GE Healthcare, Sephadex G-25 packed column: hereinafter simply referred to as "Sephadex G-25") to obtain 20.0 mL of solution. 5.04 g of NH 2 NH 2 HCl was added and stirred in the dark at 25°C for 2 hours. 800mg of DMAB (dimethylamine borane) was added and stirred in the dark at 25°C for another 2 hours. Dialysis was performed against 4L of ion-exchanged water using a RC50K (regenerated cellulose with a molecular weight of 50,000) dialysis membrane 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 0.1M sodium phosphate buffer (pH 6.0) to obtain 85.0mL of solution. The concentration of dextran in the obtained solution was adjusted to 1.0mg / mL, and a solution of hydrazide dextran was obtained.

[0101] (2) Preparation of dextran-enzyme conjugates 30.0 mL of 10 mg / mL alkaline phosphatase (ALP-50, manufactured by Oriental Yeast Co., Ltd.) was subjected to buffer exchange 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. 4 45.3 mL of was added and stirred in the dark at 25°C for 3 minutes. Next, buffer exchange was performed by gel filtration (Sephadex G-25) using 0.1 M sodium phosphate buffer (pH 6.0) to prepare a 0.5 mg / mL solution. 1.0 mg / mL hydrazide dextran prepared in (1) of Example 1 was added so that the concentration of hydrazide groups (amino groups) became 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 concentrate was concentrated to 15 mL, followed by gel filtration (Superdex 200 pg) using 0.1 M sodium phosphate buffer (pH 7.0) to obtain 14 mL of a 3.0 mg / mL dextran-enzyme conjugate solution.

[0102] (3) Maleimide-PEGylation of dextran-enzyme conjugates 0.1M sodium phosphate buffer (pH 7.0) was added to the dextran-enzyme conjugate prepared in Example 1(2) to prepare 750 μL of 2 mg / mL dextran-enzyme conjugate. 4 (Thermo Fisher Scientific, SM(PEG) 4) was added, mixed, and mixed by inversion in the dark at 25℃ for 1 hour. After the reaction, the buffer was exchanged using a PD-10 column (Sephadex G-25) with 0.1M sodium phosphate buffer (pH 6.3) containing 20mM EDTA·2Na and 0.5% CHAPS. After the buffer exchange, the maleimide-PEGylated dextran-enzyme conjugate was concentrated using a centrifugal filter (Merck, Amicon Ultra 50K) to adjust the final concentration to 2mg / mL.

[0103] (4) Thiolation of lectins 5 mg of W. nodani lectin (WFA; VECTOR) was dissolved in 2.5 mL of 0.1 M sodium phosphate buffer (pH 7.0) to obtain a 2 mg / mL WFA solution. 100 μL of 0.5 M EDTA·2Na (pH 8.0) was added to the 2.5 mL WFA solution and mixed, then 75 μL of 10 mg / mL 2-iminothiolane hydrochloride solution was added and mixed 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. The WFA concentration after the buffer exchange was adjusted to 650 μg / mL.

[0104] (5) Coupling To 2 mL of WFA thiolated to 650 μg / mL obtained in (4) of Example 1, 500 μL of the maleimide-PEGylated dextran-enzyme conjugate solution (2 mg / mL) obtained in (3) of Example 1 was added, and the mixture was mixed by inversion in a dark place at 25° C. for 1 hour to couple the WFA 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 inversion in a dark place at 25° C. for 30 minutes. After that, 50 μL of 200 mM 2-Iodoacetamide was added, and the mixture was mixed by inversion in a dark place at 25° C. for 30 minutes. After the reaction, the solution was concentrated using a centrifugal filter (Merck, Amicon Ultra 50K) and then passed through a φ0.22 μm filter, and subjected to gel filtration chromatography (column: Superose 6 Increase 10 / 300 GL, buffer: 0.1 M MES, 0.5 M NaCl, 1 mM MgCl 2 , 0.1 mM ZnCl 2 , 5 mM Glucose, 0.05% CHAPS, pH 6.8) to finally obtain 2.0 mL of a solution of 373.9 μg / mL blocked labeled lectin (WFA) (dextran-enzyme-WFA conjugate).

[0105] (6) Preparation of measurement reagents 24 mg of anti-CA19-9 antibody-bound magnetic particles (manufactured by Fujirebio Inc., hereafter simply referred to as "antibody-bound particles") was mixed with 6 mL of 20 mM sodium periodate solution (20 mM NaIO 4, 100 mM NaOAc, 150 mM NaCl, pH 5.5) was added and mixed, and the mixture was mixed by inversion at 4°C for 30 minutes in the dark to oxidize the sugar chains of the antibodies bound to the particles. The antibody-bound particles after oxidation were washed three times with 6 mL of 0.1 M sodium phosphate buffer (pH 6.0). The washed antibody-bound particles were replaced with 0.1 M sodium phosphate buffer (pH 6.0) containing 10 mM glycine, and the mixture was mixed by inversion at 25°C for 1 hour in the dark to block the aldehyde groups generated by the oxidation of the sugar chains of the antibodies with glycine. Furthermore, 100 μL of 10 mg / mL DMAB was added to the antibody-bound particle solution after the reaction, and the mixture was mixed by inversion at 25°C for 30 minutes in the dark to stabilize the unstable bond between the aldehyde groups derived from the sugar chains of the antibodies and glycine. The stabilized antibody-bound particles were washed three times with 0.6 mL of a buffer containing 2% BSA (50 mM MES, 1 mM EDTA, 150 mM NaCl, 2% BSA, 0.1% ProClin 300, pH 6.0), and the particles were mixed by inversion in the same buffer at 37°C for 16 hours to physically adsorb BSA. The antibody-bound particles to which BSA was physically adsorbed were washed three times with a storage buffer (50 mM Tris, 2% BSA, 150 mM NaCl, 1 mM EDTA, 0.1% ProClin 300, pH 7.2), and stored in the same buffer at 4°C. The obtained oxidized anti-CA19-9 antibody-bound magnetic particles were diluted in a 50 mM Tris-based solution so that the concentration of the antibody-bound particles was 0.005%, to prepare an oxidized anti-CA19-9 antibody-bound particle solution.

[0106] In addition, the blocked labeled lectin (WFA) obtained in Example 1(5) was diluted in a 50 mM MES-based solution to a concentration of 0.5 μg / mL to prepare a labeled body fluid.

[0107] (7) Measurement of CA19-9 and WFA-linked glycosylated CA19-9 (CA19-9 / WFA) in serum samples Ten serum samples collected from healthy individuals (healthy individual group: healthy individuals 1-10), ten serum samples collected from benign prostatic hyperplasia patients (benign prostatic hyperplasia group: benign prostatic hyperplasia 1-10), fifteen serum samples collected from prostate cancer patients (prostate cancer group: prostate cancer 1-15), and fifteen serum samples collected from colon cancer patients (colon cancer group: colon cancer 1-15) were each diluted to a concentration of 1 / 10 by volume using sample diluent (Fujirebio).

[0108] For each diluted sample, the WFA-linked glycan-added CA19-9 (CA19-9 / WFA) contained in the sample was measured using Lumipulse (registered trademark) L-2400 (manufactured by Fujirebio Inc.). That is, 50 μL of each diluted sample solution was mixed with 50 μL of the oxidized anti-CA19-9 antibody-bound particle solution prepared in (6) of Example 1, and reacted at 37° C. for 8 minutes. Next, the magnetic particles were collected and washed five times with Lumipulse (registered trademark) washing solution (manufactured by Fujirebio Inc.). Next, 50 μL of the labeled body fluid prepared in (6) of Example 1 was added to each sample, and reacted at 37° C. for 8 minutes. Next, the magnetic particles were collected and washed five times, after which 50 μL of Lumipulse (registered trademark) substrate solution (manufactured by Fujirebio Inc.) containing AMPPD (3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoryloxy)phenyl-1,2-dioxetane disodium salt) was added and reacted at 37°C for 4 minutes. The luminescence intensity (counts) of light having a maximum absorption at a wavelength of 463 nm, which is released as a result of decomposition of AMPPD by the catalytic action of alkaline phosphatase of the blocked labeled lectin (WFA) bound to the magnetic particles, was measured using Lumipulse (registered trademark) L-2400 (manufactured by Fujirebio Inc.) to obtain the measurement results.

[0109] The CA19-9 contained in each diluted sample was measured by Lumipulse (registered trademark) L-2400 (manufactured by Fujirebio Inc.) using Lumipulse Presto (registered trademark) CA19-9 (manufactured by Fujirebio Inc.) which is equipped with anti-CA19-9 antibody-bound particles and alkaline phosphatase (ALP)-labeled anti-CA19-9 antibody. The measurement results were output as the luminescence intensity (counts) of the substrate (AMPPD) as described above. The measurement results for each sample are shown in Table 1 below. The results shown are the average values ​​of duplicate measurements.

[0110] [Table 1]

[0111] (8) Comparison of measurements between healthy subjects, benign prostatic hyperplasia, and prostate cancer groups The results in Table 1 were examined to see whether there was a statistically significant difference between the healthy group and the prostate cancer group. Figure 1 shows the CA19-9 measurement results for the healthy group and the prostate cancer group, Figure 2 shows the CA19-9 / WFA measurement results for the healthy group and the prostate cancer group, Figure 3 shows the CA19-9 measurement results for the prostate hyperplasia group and the prostate cancer group, and Figure 4 shows the CA19-9 / WFA measurement results for the prostate hyperplasia group and the prostate cancer group.

[0112] When the Wilcoxon test was performed, no significant difference was observed between the healthy group and the prostate cancer group in the measured values ​​of CA19-9 (Figure 1, p = 0.7603), whereas a significant difference was observed in the measured values ​​of CA19-9 / WFA (Figure 2, p = 0.0010). Similarly, no significant difference was observed between the prostate hyperplasia group and the prostate cancer group in the measured values ​​of CA19-9 (Figure 3, p = 0.6774), whereas a significant difference was observed in the measured values ​​of CA19-9 / WFA (Figure 4, p = 0.0017).

[0113] FIG. 5 shows the relationship between the measured values ​​of CA19-9 and CA19-9 / WFA. As shown in FIG. 5, the results of this study did not show a clear correlation between the measured values ​​of CA19-9 and CA19-9 / WFA (regression line: CA19-9 / WFA (count) = 66238.78 - 1.6997236 × CA19-9 (count), R 2 = 0.009369, p-value of analysis of variance = 0.5802). This indicates that the measured value of CA19-9 / WFA does not simply indicate the amount of CA19-9 present, but is a biomarker distinct from CA19-9.

[0114] These results suggest that the measurement of CA19-9 / WFA can be a novel indicator for distinguishing between healthy individuals and prostate cancer patients, or between patients with benign prostatic hyperplasia and patients with prostate cancer.

[0115] (9) Comparison of CA19-9 / WFA and PSA measurements First, the amount of PSA contained in 10 serum samples taken from patients with prostatic hyperplasia and 15 serum samples taken from patients with prostate cancer, similar to those used in Example 1 (7), was measured. Each sample was not diluted, and was measured using Lumipulse Presto (registered trademark) PSA (manufactured by Fujirebio Inc.) equipped with anti-PSA antibody-bound particles and alkaline phosphatase (ALP)-labeled anti-PSA antibody, using Lumipulse (registered trademark) L-2400 (manufactured by Fujirebio Inc.). The measurement results were output as PSA concentration (ng / mL). The measurement results for each sample are shown in Table 2 below.

[0116] [Table 2]

[0117] Next, the results of Table 2 were used to test the ability to detect prostate cancer patients when the cutoff value of PSA measurement is 4ng / mL, and samples above the cutoff value are considered positive.As a result, in the prostate cancer group, 4 out of 15 samples were determined to be positive at the cutoff value, which is 26.7%.In addition, in the prostate hyperplasia group, 1 out of 10 samples was determined to be positive at the cutoff value, which was a false positive.

[0118] On the other hand, for the CA19-9 / WFA measurement values ​​(Table 1), a cutoff value was calculated from the measurements of the healthy group, and the ability to detect prostate cancer patients was tested when samples with values ​​above the cutoff value were deemed positive. The cutoff value was calculated by adding the average measurement value of the healthy group to the standard deviation of the measurements of the healthy group multiplied by 2, resulting in a count of 67,659 or less. As a result, 10 out of 15 samples in the prostate cancer group were determined to be positive at this cutoff value, which was 66.7%. In addition, one out of 20 samples in the healthy group and the prostatic hyperplasia group was determined to be false positive, resulting in a specificity of 95.0%.

[0119] Furthermore, FIG. 6 shows the relationship between the PSA measurement value and the CA19-9 / WFA measurement value. As shown in FIG. 6, the results of this study did not show a clear correlation between the PSA measurement value and the CA19-9 / WFA measurement value (regression line: CA19-9 / WFA (count) = 67163.799-1433.0774 × PSA (ng / mL), R 2 = 0.01667, ANOVA p-value = 0.5384).

[0120] These results suggest that the detection ability (sensitivity, specificity) of CA19-9 / WFA for prostate cancer is sufficiently high compared with PSA, and that it can be used as a diagnostic index independent of PSA.

[0121] (10) Comparison of measurements between healthy subjects and colon cancer subjects The results in Table 1 were examined to see whether there were any statistically significant differences between the healthy subject group and the colon cancer group. Figure 7 shows the CA19-9 measurement results for the healthy subject group and the colon cancer group, Figure 8 shows the CA19-9 / WFA measurement results for the healthy subject group and the colon cancer group, and Figure 9 shows the relationship between the CA19-9 measurement values ​​and the CA19-9 / WFA measurement values.

[0122] When the results were tested by Wilcoxon test, no significant difference was observed between the healthy group and the colon cancer group in the measured values ​​of CA19-9 (FIG. 7, p=0.1714), whereas a significant difference was observed in the measured values ​​of CA19-9 / WFA (FIG. 8, p=0.0184). In addition, when the ability to detect prostate cancer patients was tested when samples with CA19-9 / WFA values ​​exceeding the cutoff value calculated in (9) of Example 1 were determined to be positive, 11 out of 15 samples in the colon cancer group were determined to be positive at the cutoff value, which was 73.3%. On the other hand, none of the healthy group were determined to be false positive. This shows that the ability (sensitivity, specificity) of CA19-9 / WFA measurement to detect colon cancer is sufficiently high.

[0123] Furthermore, as shown in FIG. 9, the results of this study did not show a clear correlation between the measured values ​​of CA19-9 and CA19-9 / WFA (regression line: CA19-9 / WFA (counts) = 70068.689 + 1.8730954 × CA19-9 (counts), R 2 = 0.041392, p-value of analysis of variance = 0.3293). This indicates that the measured value of CA19-9 / WFA does not simply indicate the amount of CA19-9 present, but is a biomarker distinct from CA19-9.

[0124] These results suggest that the measurement of CA19-9 / WFA can be a novel indicator for distinguishing between healthy individuals and colorectal cancer patients.

[0125] (Comparative Example 1) Verification of reactivity of CA19-9 / WFA to cancers other than prostate cancer and colon cancer As an example of cancer other than prostate cancer and colon cancer in which the measured value of CA19-9 / WFA was high, 15 serum samples collected from breast cancer patients (breast cancer group: breast cancer 1-15) were measured for CA19-9 / WFA in the same manner as in (7) of Example 1. The measurement results for each sample are shown in Table 3 below.

[0126] [Table 3]

[0127] The measurement results of the breast cancer group shown in Table 3 and the measurement results of the healthy control group shown in Table 1 were examined to see whether there was a statistically significant difference between the healthy control group and the breast cancer group. Figure 10 shows the measurement results of CA19-9 / WFA in the healthy control group and the breast cancer group. When the results were tested by Wilcoxon test, no significant difference was observed in the measurement values ​​of CA19-9 / WFA between the healthy control group and the breast cancer group (Figure 10, p = 0.0806). This result showed that the measurement values ​​of CA19-9 / WFA do not show high values ​​indiscriminately for all cancers, but show high values ​​specific to the type of cancer.

[0128] (Comparative Example 2) Measurement of Glycosylated CA19-9 by Blocked Labeled Lectin Using Lectin Other Than WFA A blocked labeled lectin (MAM) was prepared by binding canine endemic lectin (MAM, J-Chemical Co., Ltd.), which recognizes α2,3-linked sialic acid, instead of WFA. That is, 2.0 mL of a solution of 382.1 μg / mL blocked labeled lectin (MAM) (dextran-enzyme-MAM conjugate) was finally obtained by the same method as in (1) to (5) of Example 1, except that MAM was used instead of WFA and dextran was changed to one with a molecular weight of 500 k (Fluka Co., Ltd.). Note that the size of the dextran does not affect the sugar chain type specificity of the lectin.

[0129] Except for using the obtained blocked enzyme lectin (MAM) instead of the blocked labeled lectin (WFA), the same method as in (6) to (7) of Example 1 was used to measure MAM-linked glycan-added CA19-9 (CA19-9 / MAM) contained in 10 serum samples collected from healthy subjects (healthy subjects group: healthy subjects 1 to 10), 15 serum samples collected from prostate cancer patients (prostate cancer group: prostate cancer 1 to 15), and 15 serum samples collected from colon cancer patients (colon cancer group: colon cancer 1 to 15). The measurement results for each sample are shown in Table 4 below.

[0130] [Table 4]

[0131] The results of Table 4 were examined to see whether there was a statistically significant difference between the healthy group and the prostate cancer group or colon cancer group. Figure 11 shows the measurement results of CA19-9 / MAM for the healthy group and the prostate cancer group, and Figure 12 shows the measurement results of CA19-9 / MAM for the healthy group and the colon cancer group. When the results were tested by Wilcoxon test, no significant difference was observed in the measured values ​​of CA19-9 / MAM between the healthy group and the prostate cancer group (Figure 11, p = 0.1924). Similarly, no significant difference was observed in the measured values ​​of CA19-9 / MAM between the healthy group and the colon cancer group (Figure 12, p = 0.3340). From the above, it was shown that the measurement of CA19-9 / WFA, which measures the combination of CA19-9 and WFA-bound glycans, is a new indicator specific to distinguishing between healthy people and prostate cancer patients or colon cancer patients. [Industrial Applicability]

[0132] According to the present invention, it is possible to provide a cancer detection method and a cancer testing method that use a new biomarker as an indicator and are capable of specifically detecting prostate cancer and colorectal cancer with high sensitivity, as well as kits used in these methods.

Claims

1. A method for detecting at least one type of cancer selected from the group consisting of prostate cancer and colorectal cancer, comprising a measuring step of measuring the amount of GalNAc-added CA19-9 in a sample.

2. A method for testing for at least one type of cancer selected from the group consisting of prostate cancer and colorectal cancer, comprising a measurement step of measuring the amount of GalNAc-added CA19-9 in a sample derived from a subject.

3. The method according to claim 2, which is a method for screening a subject predicted to have at least one type of cancer selected from the group consisting of prostate cancer and colorectal cancer, and comprises a measuring step of measuring the amount of GalNAc-added CA19-9 in a sample derived from the subject, and a selecting step of selecting the subject using the measured amount of GalNAc-added CA19-9 as an index.

4. The method according to any one of claims 1 to 3, wherein in GalNAc-added CA19-9, GalNAc is a WFA-linked sugar chain that binds to WFA.

5. The method according to any one of claims 1 to 4, characterized in that the measuring step is a step of contacting the sample with a first probe molecule capable of specifically binding to CA19-9 and a second probe molecule capable of specifically binding to GalNAc.

6. The method according to claim 5, wherein the first probe molecule is an antibody capable of specifically binding to CA19-9.

7. The method according to claim 5 or 6, wherein the second probe molecule is a lectin capable of specifically binding to GalNAc.

8. the measuring step is a step of contacting the sample with a 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. Method according to any one of claims 5 to 7, characterized in that

9. 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, the second probe molecule being a blocked labeled lectin that is a lectin capable of specifically binding to GalNAc; 9. The method according to claim 8.

10. A kit for use in the method according to any one of claims 5 to 9, comprising a first probe molecule capable of specifically binding to CA19-9 and a second probe molecule capable of specifically binding to GalNAc.

11. The kit according to claim 10, wherein the first probe molecule is an antibody capable of specifically binding to CA19-9.

12. The kit according to claim 10 or 11, wherein the second probe molecule is a lectin capable of specifically binding to GalNAc.

13. A capture body comprising a water-insoluble carrier and either 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 10 to 12, characterized in that it comprises:

14. 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, the second probe molecule being a blocked labeled lectin that is a lectin capable of specifically binding to GalNAc; The kit according to claim 13 .

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