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 using a novel biomarker as an indicator.SOLUTION: A method of detecting prostate cancer is provided, the method comprising a measurement step of measuring the level of α2-3 sialylated CEA 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 therefor, and more particularly to a prostate cancer detection method, a prostate cancer testing method, and a kit used therefor. [Background technology]

[0002] Biomarkers such as tumor markers are useful as a monitoring marker for detecting diseases such as malignant tumors, a guide for determining a treatment policy, and an evaluation of the effectiveness of treatment, and have been actively studied in recent years. An example of such a biomarker is CEA (carcinoembryonic antigen). CEA is a glycoprotein with a molecular weight of about 200,000, which was discovered in 1965 by Gold P. and Freedman SO from a human colon cancer tissue extract, and is also known as CD66e or CEACAM5. The protein portion of CEA is a single-chain polypeptide consisting of about 700 amino acids, and it has been reported that one molecule of CEA has 28 N-glycosylation sites, most of which are bound to complex glycans, and contain 50% or more of glycans (Non-Patent Document 1). CEA can be 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 gastrointestinal cancer, particularly colorectal cancer, pancreatic cancer, stomach cancer, etc., and there have also been reported cases where high blood concentrations were observed in patients with lung cancer, uterine cancer, ovarian cancer, embryonal cancer, etc.

[0003] For this reason, CEA 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) CEA," a kit for measuring CEA in a sample using the sandwich method in which CEA present in a sample is captured with a solid-phase anti-CEA antibody (capture body) and then a labeled anti-CEA antibody (labeled body) is bound to it.

[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 individuals (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 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] Katsuko Yamashita et al., Journal of the Japanese Society for Molecular Tumor Marker Research, Vol. 19, 2003, p. 1-2 [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] As mentioned above, although the glycan content of CEA reaches 50%, the glycan structure has not yet been fully elucidated. These glycans are expected to have the potential to serve as new biomarkers for cancer detection.

[0008] In addition, as mentioned above, a small amount of CEA is detected in the blood of healthy people, so in the conventional method of measuring CEA, a cutoff value is usually set, and the cutoff value is used as an index to predict or distinguish between the above-mentioned cancers and the like as negative or positive. However, in such conventional CEA measuring methods, since only one type of anti-CEA antibody is used, the specificity for each cancer type is still insufficient. Furthermore, in prostate cancer patients, the measured CEA value rarely increases due to the onset of prostate cancer, and the present inventors have found that the conventional method of measuring CEA is insufficient to distinguish prostate cancer patients from healthy people or patients with prostate hyperplasia 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 can specifically detect prostate cancer with high accuracy using a new biomarker as an indicator, 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 with existing antibodies, the present inventors performed screening using not only anti-CEA antibodies but also a complex (blocked labeled lectin) comprising a water-soluble carrier made of a water-soluble polymer and a labeling substance and lectin immobilized on the water-soluble carrier. As a result, they found that the amount of molecules detected when MAM was used as the lectin, i.e., the amount of CEA with α2-3 sialic acid (α2-3 sialylated CEA), which is a lectin-binding sugar chain, was significantly increased in prostate cancer patients compared to healthy subjects. Therefore, they found that by measuring the amount of α2-3 sialylated CEA, it is possible to specifically detect prostate cancer and to distinguish prostate cancer patients from healthy subjects or patients with prostatic hyperplasia with higher accuracy than when CEA is measured with anti-CEA antibodies alone, and thus completed the present invention.

[0011] The present invention, based on these findings, has the following features. [1] A method for detecting prostate cancer, comprising a measuring step for measuring the amount of α2-3 sialylated CEA in a sample. [2] A method for testing for prostate cancer, comprising a measuring step of measuring the amount of α2-3 sialylated CEA in a sample derived from a subject. [3] The method described in [2] is a method for screening subjects suspected of having prostate cancer, characterized in that it comprises a measurement step of measuring the amount of α2-3 sialylated CEA in a sample derived from the subject, and a selection step of selecting subjects using the measured amount of α2-3 sialylated CEA as an indicator. [4] The method according to any one of [1] to [3], wherein in the α2-3 sialic acid-added CEA, the α2-3 sialic acid is a MAM-binding glycan that binds to MAM. [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 CEA and a second probe molecule capable of specifically binding to α2-3 sialic acid. [6] The method according to [5], wherein the first probe molecule is an antibody capable of specifically binding to CEA. [7] The method according to [5] or [6], wherein the second probe molecule is a lectin capable of specifically binding to α2-3 sialic acid. [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 α2-3 sialic acid; 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 CEA and a second probe molecule capable of specifically binding to α2-3 sialic acid.

[11] The kit according to

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

[12] The kit according to

[10] or

[11] , wherein the second probe molecule is a lectin capable of specifically binding to α2-3 sialic acid.

[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 α2-3 sialic acid; 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 capable of specifically detecting prostate cancer with high accuracy using a new biomarker as an indicator, 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 CEA in a healthy subject group and a prostate cancer group. [Diagram 2] 1 is a graph showing the results of measuring CEA / MAM in a healthy subject group and a prostate cancer group. [Diagram 3] 1 is a graph showing the results of measuring CEA in a prostate hyperplasia group and a prostate cancer group. [Figure 4] 1 is a graph showing the results of measuring CEA / MAM in a prostate hyperplasia group and a prostate cancer group. [Diagram 5]1 is a graph showing the relationship between CEA measurement values ​​and CEA / MAM measurement values. [Figure 6] 1 is a graph showing the relationship between PSA measurement values ​​and CEA / MAM measurement values. [Figure 7] 1 is a graph showing the results of measuring CEA / WFA in a healthy subject group and a prostate 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 prostate cancer, which includes a measuring step of measuring the amount of α2-3 sialylated CEA in a sample. Also, the cancer testing method of the present invention is a method for testing prostate cancer, which includes a measuring step of measuring the amount of α2-3 sialylated CEA 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 prostate 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.

[0017] [α2-3 sialylated CEA] In the present invention, "α2-3 sialic acid-added CEA" refers to a CEA molecule containing α2-3 sialic acid (α2,3-linked sialic acid) that is a sugar chain, and is also called "α2-3 sialic acid-containing CEA." In the present invention, "CEA" refers to carcinoembryonic antigen, which is a glycoprotein. CEA is one of the molecules that constitute the CEACAM (carcinoembryonic antigen-related adhesion molecule) family that is involved in various processes such as cell adhesion, proliferation, differentiation, and tumor suppression, and is also known as "CD66e" or "CEACAM5 (carcinoembryonic antigen related cell adhesion molecule 5)." In the present specification, CD66e or CEACAM5 and CEA are used synonymously. CEA typically consists of a protein portion that is a single-chain polypeptide consisting of about 700 amino acids, and a sugar chain portion consisting of multiple sugar chains bound to it.

[0018] The α2-3 sialic acid added CEA of the present invention contains at least α2-3 sialic acid as the sugar chain portion. In the present invention, "α2-3 sialic acid" refers to a sugar chain (sialic acid α2-3Gal) in which the 2-position carbon of sialic acid is glycosidically bonded to the 3-position carbon of monosaccharide galactose (Gal). In one molecule of α2-3 sialic acid added CEA, multiple α2-3 sialic acids may be present, and the protein portion of CEA and α2-3 sialic acid may be bonded directly or via another sugar chain. The α2-3 sialic acid added CEA of the present invention preferably contains α2-3 sialic acid as a MAM-bound glycan that binds to MAM, more preferably contains a terminal sialic acid residue as a glycan (sialic acid α2-3Gal) in which the terminal sialic acid residue is α2,3-linked to the second galactose residue from the end of the glycan, and even more preferably contains terminal α2-3 sialic acid (sialic acid α2-3Galβ1-4GlcNAc) at the N-glycosylation site in which N-acetylglucosamine (GlcNAc) is β-linked to the amide moiety of the protein portion of CEA.

[0019] The average mass of such α2-3 sialylated CEA (average mass as determined by calibration with a marker in gel filtration chromatography (GFC), hereinafter the same) is not particularly limited, but is preferably 100,000 to 300,000 Da, and more preferably 180,000 to 200,000 Da. Furthermore, the content of the sugar chain moiety in one molecule of α2-3 sialylated CEA is not particularly limited, but is preferably 40 to 60% by mass.

[0020] [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 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). Whether the subject is truly afflicted with prostate cancer is determined (definitively diagnosed) by a biopsy of prostate tissue taken from the subject.

[0021] [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 α2-3 sialylated CEA 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").

[0022] These samples may be diluted or suspended in a diluent as necessary, or may be appropriately pretreated. 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 crushing, 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. Examples of the purification treatments are not particularly limited, and include treatments using a column; treatments for adsorbing impurities in a sample that is fixed to a water-insoluble carrier, such as an antibody that adsorbs the impurities, and removing the impurities from a sample that contains α2-3 sialylated CEA; treatments for capturing CEA containing α2-3 sialylated CEA using an anti-CEA antibody fixed to a water-insoluble carrier, removing the impurities by washing, and then releasing the CEA.

[0023] [Measurement process] The method of the present invention includes a measuring step of measuring the amount of α2-3 sialylated CEA in a sample. The method for measuring the amount of α2-3 sialylated CEA is not particularly limited as long as it is a method capable of measuring the amount of α2-3 sialylated CEA, and a conventionally known method, a method based thereon, or a combination thereof may be appropriately adopted. For example, a method using a probe molecule capable of specifically binding to α2-3 sialylated CEA; 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 may be appropriately adopted. Examples of the method include, but are not limited to, a method using a probe molecule capable of specifically binding to α2-3 sialylated CEA; high performance liquid chromatography isotope dilution mass spectrometry (LC-IDMS); inductively coupled plasma optical emission spectrometry (ICP-MS); atomic absorption spectrometry (AAS); HPLC; FPLC; NMR; IR; FTIR; UV-VIS absorptiometry; flow cytometry; mass spectrometry; and a combination thereof.

[0024] Among them, the measurement step according to the present invention is preferably a method using a probe molecule capable of specifically binding to α2-3 sialylated CEA. 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, and the like) and minibodies formed by binding the variable regions of antibodies.

[0025] A probe molecule capable of specifically binding to α2-3 sialic acid-added CEA is preferably a combination of a probe molecule capable of specifically binding to CEA (referred to in the present invention as a "first probe molecule") and a probe molecule capable of specifically binding to α2-3 sialic acid (referred to in the present invention as a "second probe molecule").

[0026] [First probe molecule] In the present invention, unless otherwise specified, "capable of specifically binding to CEA" refers to being capable of specifically binding to a site of CEA other than α2-3 sialic acid (more preferably, sialic acid α2-3Galβ1-4GlcNAc sugar chain). The "site other than α2-3 sialic acid" may be the whole or a part of the protein portion, the whole or a part of the sugar chain portion other than α2-3 sialic acid, or a combination of two or more of these. Examples of the first probe molecule capable of specifically binding to CEA include antibodies capable of specifically binding to CEA (including anti-protein antibodies whose recognition site is the protein portion of CEA, anti-glycan antibodies whose recognition site is the glycan portion of CEA, and antibodies whose recognition sites are the protein portion and glycan portion of CEA; sometimes referred to as "anti-CEA antibodies" in this specification), and anti-CEACAM family antibodies (antibodies whose recognition sites are CEA and other CEACAM family molecules). Among these, anti-CEA antibodies are preferred, and anti-CEA protein antibodies whose recognition site is at least a portion of the protein portion of CEA are more preferred, and antibodies that do not interfere with the binding between α2-3 sialic acid and the second probe molecule are also preferred.

[0027] The anti-CEA antibody is not particularly limited as long as it has the ability to bind to CEA, and may be a polyclonal or monoclonal antibody, but is preferably a monoclonal antibody from the viewpoint of homogeneity and stability. The anti-CEA antibody can be produced by appropriately adopting and improving a conventionally known production method, and a commonly available antibody may be used as appropriate.

[0028] As the anti-CEA 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 sugar chain and reducing the detection sensitivity, it is preferable that the lectin-binding sugar chain has been removed or destroyed by oxidation treatment, glycosidase treatment, protease treatment, etc.; the region containing the sugar chain-added amino acid in the antibody molecule has been removed; or the antibody is produced under conditions in which sugar chain 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.

[0029] [Second probe molecule] Examples of the second probe molecule capable of specifically binding to α2-3 sialic acid include antibodies capable of specifically binding to α2-3 sialic acid (sometimes referred to as "anti-α2-3 sialic acid antibodies" in this specification), artificial proteins that bind to α2-3 sialic acid such as Lectenz (registered trademark), and lectins capable of specifically binding to α2-3 sialic acid (e.g., Corylus arbutus lectin such as Corylus arbutus lectin (MAM / MAL / MAA-II) and Corylus arbutus lectin (MAH / MAA-I); Matsutake mushroom lectin (ACG); and jackfruit lectin (Jacalin)). Among these, lectins capable of specifically binding to α2-3 sialic acid are preferred, Corylus arbutus lectin is more preferred, and MAM is particularly preferred. MAM (Maackia amurensis Lectin; also called MAL or MAA-II) is a protein that recognizes and exhibits binding activity to the sugar chain structure of α2-3 sialic acid (particularly sialic acid α2-3Galβ1-4GlcNAc), and is a legume lectin derived from the seeds of the plant Maackia amurensis. 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. Furthermore, a lectin that is generally available may be used as appropriate.

[0030] 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 α2-3 sialic acid and the portion of CEA other than α2-3 sialic acid to be recognized, and allows α2-3 sialylated CEA 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 performed simultaneously or at different times, and if they are performed at different times, either contact may occur first.

[0031] [Labeling substance] In the present invention, the amount of α2-3 sialylated CEA is preferably measured by detecting a signal generated by a labeling substance added to a probe molecule capable of specifically binding to α2-3 sialylated CEA (preferably the first probe molecule and / or the second probe molecule) or added to a molecule recognizing these molecules (e.g., a secondary antibody or protein A). The amount of α2-3 sialylated CEA can be measured by measuring the amount of the detected signal and, if necessary, semi-quantitatively or quantitatively determining the amount. 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 the naked eye as well as signals that can be confirmed by a measuring method or device depending on the type of signal. In the present invention, the amount of α2-3 sialylated CEA to be measured may be semi-quantitative or quantitative using a standard sample, etc., but from the viewpoint of simpler and faster measurement, the amount of the signal may be directly used as the amount of α2-3 sialylated CEA of the present invention.

[0032] 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.

[0033] [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.

[0034] 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.

[0035] In the case of using the sandwich method, 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 α2-3 sialic acid and CEA are captured, and α2-3 sialylated CEA can be captured and detected with high accuracy and ease.

[0036] 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 either 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 comprises 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 α2-3 sialylated CEA; or a probe molecule capable of specifically binding to the first and / or second probe molecules.

[0037] Examples of such sandwich methods include the two-step forward sandwich method (a method in which the capture body is reacted with α2-3 sialylated CEA in the sample and the α2-3 sialylated CEA 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 α2-3 sialylated CEA 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 reaction of α2-3 sialylated CEA in the sample, the capture body, and the labeled body is carried out simultaneously in one step), and any of these can be used.

[0038] For example, in the forward sandwich method, the sample is first contacted with the capture body, and α2-3 sialylated CEA is captured by the capture body via the bond between the probe molecule of the capture body and α2-3 sialylated CEA (e.g., the bond between the first probe molecule and CEA) (primary reaction: capture step). Next, the α2-3 sialylated CEA captured by the capture body is contacted with the label, and labeled via the bond between the probe molecule of the label and α2-3 sialylated CEA (e.g., the bond between the second probe molecule and α2-3 sialic acid) (secondary reaction: label step). This reaction forms a complex containing the capture body-α2-3 sialylated CEA-label. Unbound sample and label are washed and removed as necessary (washing step), and then a signal derived from the label is measured by a predetermined method depending on the label (measurement step).

[0039] (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 α2-3 sialylated CEA 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 aspect 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 CEA, and more preferably an anti-CEA antibody.

[0040] <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.

[0041] 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.

[0042] 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.

[0043] <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 α2-3 sialylated CEA, for example. However, 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] (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 α2-3 sialylated CEA, and is a conjugate in which the labeling substance and the probe molecule are directly or indirectly bound to each other. The probe molecule in the labeled body according to the first embodiment is preferably the other of the first and second probe molecules, which is the molecule in the capture body. In this case, the probe molecule in the labeled body may be either the first or second probe molecule, but from the viewpoint of using a molecule such as an anti-CEA antibody having high affinity for an antigen as the first probe molecule in the capture body, the second probe molecule capable of specifically binding to α2-3 sialic acid is preferred, a lectin capable of specifically binding to α2-3 sialic acid is more preferred, and MAM is particularly preferred.

[0049] (Blocked labeled lectin) In the present invention, the label may be a labeled antibody comprising the label and the antibody (anti-CEA antibody, anti-α2-3 sialic acid 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 α2-3 sialic acid as a second probe molecule. In this case, the probe molecule provided in the capturer is preferably a first probe molecule. The lectin is as described above, and is preferably MAM. The label is as described above, including its preferred embodiments.

[0050] 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 α2-3 sialic acid can be detected with high sensitivity.

[0051] <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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] (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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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 treated with a crosslinker having N-hydroxysuccinimide and maleimide groups at each end (e.g., SM(PEG)4, SMCC, etc.) to introduce a maleimide group. On the other hand, 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 bound to the thiol group given to the lectin, thereby covalently binding 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 bound via an enzyme, and the lectin is bound to the first water-soluble polymer. The ratio of the first water-soluble polymer, labeling substance, and 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.

[0064] (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.

[0065] 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%.

[0066] 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.

[0067] (Label Step) In the reaction between the labeled substance and α2-3 sialylated CEA in the labeling step, the content (final concentration) of the labeled substance in the reaction solution containing the labeled substance and α2-3 sialylated CEA (when the labeled substance is a combination of two or more types, the total of the labels, the same applies below) is not particularly limited and is appropriately adjusted 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 excessive use may cause a high background signal.

[0068] 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.

[0069] (Washing step) The sandwich method preferably further includes a washing step after the capture step and / or the label step, in which the α2-3 sialylated CEA bound to the capture body is separated from other impurities not bound to (captured by) the capture body, and the impurities are removed. 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, the method includes removing the liquid phase (supernatant) from the plate, and when the capture body is the probe molecule immobilized particle, the method includes recovering the particle by centrifugation or magnetic collection and removing the liquid phase (supernatant). 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 include those to which a stabilizing protein such as BSA or a surfactant has been added.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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).

[0075] 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.

[0076] 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.

[0077] (Measurement step) In the measuring step, a signal is measured according to the labeling substance. For example, when the labeling substance is an enzyme, a chromogenic or luminogenic substrate corresponding to the enzyme is added and reacted to measure a signal (e.g., color or luminescence). This allows the amount of α2-3 sialylated CEA in the sample to be detected as a signal amount, and the amount of α2-3 sialylated CEA in the sample can be quantified by comparing with the measured value in a standard sample, if necessary.

[0078] [Cancer detection methods] According to the cancer detection method of the present invention, the amount of α2-3 sialylated CEA measured by the above-mentioned measurement step is used as an index to detect the presence or absence of prostate cancer in the sample or the subject from whom the sample is derived. Such a cancer detection method can be used for research purposes such as drug discovery, as well as the cancer testing method of the present invention.

[0079] [Cancer testing method] In the cancer testing method of the present invention, the amount of α2-3 sialylated CEA in a sample derived from a subject is measured and used as an indicator to detect the presence or absence of prostate cancer, thereby making it possible to predict whether or not the subject is suffering from prostate cancer, determine the presence or absence or possibility of the subject being suffering from the cancer, or evaluate the progression or severity of the cancer.

[0080] That is, specific embodiments of the cancer testing method of the present invention include, for example, the following embodiments: A method for screening subjects who are suspected to have prostate cancer, comprising: a measuring step of measuring the amount of α2-3 sialylated CEA in a sample derived from the subject; and a selecting step of selecting subjects using the measured amount of α2-3 sialylated CEA as an index; A method for distinguishing the presence or absence of prostate cancer in a subject, comprising: a measuring step of measuring the amount of α2-3 sialylated CEA in a sample derived from the subject; and a distinguishing step of distinguishing the subject using the measured amount of α2-3 sialylated CEA as an indicator; A method for evaluating the progression or severity of prostate cancer in a subject, comprising: a measuring step of measuring the amount of α2-3 sialylated CEA in a sample derived from the subject; and an evaluation step of evaluating the subject using the measured amount of α2-3 sialylated CEA as an index; Examples include:

[0081] [Screening method] In the present invention, "screening" refers to selecting subjects who may be affected by prostate 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 (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.

[0082] 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.

[0083] [Identification method] In the present invention, "differentiation" refers to distinguishing prostate cancer from other diseases or conditions and determining whether a subject is affected with the cancer. It also 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 not, 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 not, or a method for determining that there is a high possibility of the cancer; a method for determining whether the prostate 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.

[0084] More specifically, for example, subjects are divided into two groups, a group consisting of those who are truly affected by the cancer (positive group) and a group consisting of those who are 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 and others in diagnosing prostate 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.

[0085] [Evaluation method] In the present invention, the evaluation method includes, for example, a method for evaluating the progression or severity of prostate cancer in a subject; a method for providing an index for determining a treatment policy for the cancer in a subject; and a method for assessing the effectiveness of treatment for the cancer.

[0086] More specifically, for example, the amount of α2-3 sialylated CEA in a group of subjects not affected by the cancer or those with a low stage or severity of the cancer can be compared with the amount of α2-3 sialylated CEA 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 α2-3 sialylated CEA in the subject at the time of discovery of the cancer or before treatment can be compared with the current amount of α2-3 sialylated CEA, 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.

[0087] (Cutoff value) In the selection step of the screening method and the discrimination step of the differentiation method, for example, the amount of α2-3 sialylated CEA measured in the measurement step is compared with a predetermined cutoff value, and subjects whose amount of α2-3 sialylated CEA is higher than the cutoff value are predicted or discriminated as suffering from (or likely to suffer from) prostate cancer.

[0088] In the present invention, the term "cut-off value" refers to a predetermined value that serves as a standard for determining the amount of α2-3 sialylated CEA, and indicates 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 α2-3 sialylated CEA, 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 can be increased, that is, patients in the early stage of the cancer can be collected to a certain extent, and early detection can be made possible. On the other hand, since a small amount of α2-3 sialylated CEA 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.

[0089] An example of the cut-off value is, for example, when the amount of α2-3 sialylated CEA measured by sandwich immunoassay using a serum specimen diluted to 1 / 10 in volume ratio, anti-CEA antibody immobilized particles as the capture body, and blocked labeled MAM 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 using ALP as the label and AMPPD as the substrate (in the case of Example 1), the cut-off value is 210,000 to 320,000 counts, preferably 270,000 to 300,000 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 α2-3 sialylated CEA, and the properties of the subject and sample.

[0090] 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 by distinguishing it from other cancers. In addition, by identifying subjects who are suffering from prostate cancer (including recurrence) or who are likely to suffer from prostate cancer and performing further tests or definitive diagnosis, early detection and early therapeutic intervention of prostate cancer are possible. 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. Note that the method of the present invention is also a method for assisting a doctor or the like in diagnosing prostate cancer, or a method for providing information for a doctor or the like to diagnose prostate cancer.

[0091] Furthermore, the method of the present invention is also suitable as a method for combining with conventional CEA 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.

[0092] <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 CEA and a second probe molecule capable of specifically binding to α2-3 sialic acid. The first probe molecule and the second probe molecule are as described above, including preferred embodiments thereof.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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

[0097] 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.

[0098] Example 1: Measurement of MAM-linked glycan-added CEA (CEA / MAM) in serum samples using blocked labeled lectin (MAM) (1) Preparation of hydrazide dextran 240.0 mg of dextran (CarboMer) with a molecular weight of 500k was added to 4.8 mL of 0.1 M phosphate buffer (pH 7.0) and stirred for 30 minutes in a dark place at 25 °C to dissolve. Then, 2.664 mL of 150 mM NaIO4 and 0.536 mL of ion-exchanged water were added and stirred for 30 minutes in a dark place at 25 °C. 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 "Sephadex G-25") to obtain 20.0 mL of solution. 5.04 g of NH2NH2·HCl was added to the obtained solution and stirred for 2 hours in a dark place at 25 °C. 800 mg of DMAB (dimethylamine borane) was added and further stirred for 2 hours in a dark place at 25 °C. The mixture was dialyzed against 4 L 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 allowed to stand overnight at 4°C. Buffer exchange was performed by gel filtration (Sephadex G-25) using 0.1 M sodium phosphate buffer (pH 6.0) to obtain 85.0 mL of solution. The concentration of dextran was adjusted to 1.0 mg / mL to obtain a solution of hydrazide dextran.

[0099] (2) Preparation of dextran-enzyme conjugates 30.0 mL of 10 mg / mL alkaline phosphatase (Oriental Yeast Co., Ltd., ALP-50) 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. Then, 45.3 mL of 27 mM NaIO4 was added and stirred for 3 minutes in the dark at 25 ° C. Then, buffer exchange was performed by gel filtration (Sephadex G-25) using 0.1 M sodium phosphate buffer (pH 6.0) to prepare a 0.5 mg / mL solution. 1.0 mg / mL hydrazide dextran prepared in (1) of Example 1 was added so that the concentration of hydrazide groups (amino groups) was 25 μM, and the mixture was stirred for 16 hours in the dark at 25 ° C. 85 mg of DMAB was added and the mixture was stirred for 2 hours in the dark at 25 ° C. Next, 10.1 mL of 1.5 M Tris buffer (pH 9.0) was added, and the mixture was stirred for 2 hours in the dark at 25° C. An ultrafiltration module (Pellicon XL50, Merck Millipore) was attached to a Labscale TFF System (Merck Millipore), and the mixture was concentrated to 15 mL, and gel filtration (Superdex 200 pg) was performed using 0.1 M sodium phosphate buffer (pH 7.0) to obtain 14 mL of a 3.0 mg / mL solution of the dextran-enzyme conjugate.

[0100] (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 750μL of 2mg / mL dextran-enzyme conjugate. 8.35μL of 250mM SM(PEG)4 (Thermo Fisher Scientific, SM(PEG)4) dissolved in DMSO was added and mixed, and the mixture was mixed by inversion in a dark place at 25℃ for 1 hour. After the reaction, the buffer was exchanged with 0.1M sodium phosphate buffer (pH 6.3) containing 20mM EDTA·2Na and 0.5% CHAPS using a PD-10 column (Sephadex G-25). 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.

[0101] (4) Thiolation of lectins 6 mg of canine endemic lectin (MAM; J-Chemical) was dissolved in 3 mL of 0.1 M sodium phosphate buffer (pH 7.0) to obtain a 2 mg / mL MAM solution. 100 μL of 0.5 M EDTA·2Na (pH 8.0) was added to 2.5 mL of the MAM solution, mixed, and 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 MAM concentration after the buffer exchange was adjusted to 650 μg / mL.

[0102] (5) Coupling To 2 mL of MAM obtained in (4) of Example 1 and adjusted to 650 μg / mL by thiolation, 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 MAM 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. The reaction solution was concentrated using a centrifugal filter (Merck, Amicon Ultra 50K) and then passed through a φ0.22 μm filter and purified by gel filtration chromatography (column: Superose 6 Increase 10 / 300 GL, buffer: 0.1 M MES, 0.5 M NaCl, 1 mM MgCl2, 0.1 mM ZnCl2, 5 mM Glucose, 0.05% CHAPS, pH 6.8), finally obtaining 2.0 mL of a solution of 382.1 μg / mL blocked labeled lectin (MAM) (dextran-enzyme-MAM conjugate).

[0103] (6) Preparation of measurement reagents 24 mg of anti-CEA antibody-bound magnetic particles (Fujirebio) were mixed with 6 mL of 20 mM sodium periodate solution (20 mM NaIO4, 100 mM NaOAc, 150 mM NaCl, pH 5.5) and then mixed at 4°C for 30 minutes in the dark to oxidize the sugar chains of the antibodies bound to the particles. After oxidation, the particles were washed three times with 6 mL of 0.1 M sodium phosphate buffer (pH 6.0). The washed particles were replaced with 0.1 M sodium phosphate buffer (pH 6.0) containing 10 mM glycine, and mixed 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, 100μL of 10mg / mL DMAB was added to the antibody-bound particle solution after the reaction, and the solution was mixed by inversion at 25℃ for 30 minutes in the dark to stabilize the unstable bond between the aldehyde group derived from the antibody sugar chain and glycine. After the reaction, the particles were washed three times with 0.6mL of a buffer containing 2% BSA (50mM MES, 1mM EDTA, 150mM NaCl, 2% BSA, 0.1% ProClin 300, pH6.0), and the particles were physically adsorbed with BSA by inversion mixing in the same buffer at 37℃ for 16 hours. The antibody-bound particles with physically adsorbed BSA were washed three times with a storage buffer (50mM Tris, 2% BSA, 150mM NaCl, 1mM EDTA, 0.1% ProClin 300, pH7.2) and stored in the same buffer at 4℃. The obtained oxidized anti-CEA antibody-bound magnetic particles were diluted with a 50 mM Tris-based solution so that the antibody-bound particle concentration was 0.005%, to prepare an oxidized anti-CEA antibody-bound particle liquid.

[0104] In addition, the blocked labeled lectin (MAM) 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.

[0105] (7) Measurement of CEA and MAM-linked glycosylated CEA (CEA / MAM) in serum samples Ten serum samples collected from healthy individuals (healthy individual group: healthy individuals 1-10), ten serum samples collected from patients with benign prostatic hyperplasia (benign prostatic hyperplasia group: benign prostatic hyperplasia 1-10), and fifteen serum samples collected from patients with prostate cancer (prostate cancer group: prostate cancer 1-15) were each diluted to a volume ratio of 1 / 10 using sample diluent (Fujirebio).

[0106] For each diluted sample, MAM-linked glycan-added CEA (CEA / MAM) contained in the serum 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-CEA 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, 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 (MAM) bound to the magnetic particles, was measured using Lumipulse (registered trademark) L-2400 (manufactured by Fujirebio Inc.) to obtain the measurement results.

[0107] The CEA contained in each diluted sample was measured by Lumipulse (registered trademark) L-2400 (manufactured by Fujirebio) in the same manner as above, using Lumipulse Presto (registered trademark) CEA (manufactured by Fujirebio) equipped with anti-CEA antibody-bound particles and alkaline phosphatase (ALP)-labeled anti-CEA antibody. The measurement results were output as the luminescence intensity (counts) of the substrate (AMPPD) in the same manner as above. The measurement results for each sample are shown in Table 1 below. The results shown are the average values ​​of duplicate measurements.

[0108] [Table 1]

[0109] (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 were any statistically significant differences between the healthy group and the prostate cancer group. Figure 1 shows the CEA measurement results for the healthy group and the prostate cancer group, Figure 2 shows the CEA / MAM measurement results for the healthy group and the prostate cancer group, Figure 3 shows the CEA measurement results for the prostate hyperplasia group and the prostate cancer group, and Figure 4 shows the CEA / MAM measurement results for the prostate hyperplasia group and the prostate cancer group.

[0110] When the Wilcoxon test was performed, no significant difference was observed between the healthy group and the prostate cancer group in terms of the CEA measurement value (Figure 1, p = 0.1018), whereas a significant difference was observed in terms of the CEA / MAM measurement value (Figure 2, p = 0.0247).Similarly, no significant difference was observed between the prostate hyperplasia group and the prostate cancer group in terms of the CEA measurement value (Figure 3, p = 0.9337), whereas a significant difference was observed in terms of the CEA / MAM measurement value (Figure 4, p = 0.0006).

[0111] FIG. 5 shows the relationship between the CEA measurement value and the CEA / MAM measurement value. As shown in FIG. 5, no clear correlation was observed between the CEA measurement value and the CEA / MAM measurement value (regression line: CEA / MAM (count)=257173.58-10.643388×CEA (count), R 2 = 0.015112, p-value of analysis of variance = 0.4817). This indicates that the measured value of CEA / MAM does not simply represent the amount of CEA present, but is a biomarker different from CEA (which is recognized by anti-CEA antibodies that can specifically bind to sites other than MAM-bound glycans).

[0112] These results suggest that the measurement of CEA / MAM 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.

[0113] (9) Comparison of CEA / MAM 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.

[0114] [Table 2]

[0115] 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.

[0116] On the other hand, for the CEA / MAM measurements (Table 1), a cutoff value was calculated from the measurements of healthy subjects, 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 subject group to the standard deviation of the measurements of the healthy subject group multiplied by 2, resulting in a count of 299,216 or less. As a result, 8 out of 15 samples in the prostate cancer group were determined to be positive at this cutoff value, accounting for 53.3%. In addition, all 20 samples from the healthy subject group and the prostate hyperplasia group combined were determined to be negative.

[0117] Furthermore, FIG. 6 shows the relationship between the PSA measurement value and the CEA / MAM 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 CEA / MAM measurement value (regression line: CEA / MAM (count) = 233853.68-4056.1952 × PSA (ng / mL), R 2 = 0.008851, ANOVA p-value = 0.6546).

[0118] These results indicate that the detection ability (sensitivity, specificity) of prostate cancer by CEA / MAM measurement is sufficiently high compared to PSA, and furthermore, that it can be used as a diagnostic indicator independent of PSA.

[0119] Comparative Example 1 Measurement of Glycosylated CEA by Blocked Labeled Lectin Using Lectin Other Than MAM A blocked labeled lectin (WFA) was prepared by binding Wisteria nodalis lectin (WFA, VECTOR) that recognizes N-acetylgalactosamine instead of MAM. That is, except that WFA was used instead of MAM and dextran was changed to one with a molecular weight of 250k (CarboMer), 2.0 mL of a solution of 373.9 μg / mL blocked labeled lectin (WFA) (dextran-enzyme-WFA conjugate) was finally obtained in the same manner as in (1) to (5) of Example 1. Note that the size of the dextran does not affect the sugar chain type specificity of the lectin.

[0120] Except for using the obtained blocked enzyme lectin (WFA) instead of the blocked enzyme lectin (MAM), the WFA-linked glycosylated CEA (CEA / WFA) contained in each sample was measured for 10 serum samples taken from healthy subjects (healthy subjects group: healthy subjects 1-10) and 15 serum samples taken from prostate cancer patients (prostate cancer group: prostate cancer patients 1-15) similar to those used in (7) of Example 1 in the same manner as in (6)-(7) of Example 1. The measurement results for each sample are shown in Table 3 below.

[0121] [Table 3]

[0122] The results in Table 3 were examined to see whether there was a statistically significant difference between the healthy group and the prostate cancer group. Figure 7 shows the CEA / WFA measurement results for the healthy group and the prostate cancer group. When the results were tested using the Wilcoxon test, no significant difference was observed in the CEA / WFA measurement values ​​between the healthy group and the prostate cancer group (Figure 7, p = 0.4540). From the above, it was demonstrated that the measurement of CEA / MAM, which measures the combination of CEA and MAM-bound glycans, is a new indicator that is specific for distinguishing between healthy individuals and prostate cancer patients. [Industrial Applicability]

[0123] According to the present invention, it is possible to provide a cancer detection method and a cancer testing method capable of specifically detecting prostate cancer with high sensitivity using a new biomarker as an indicator, as well as kits used in these methods.

Claims

1. A method for detecting prostate cancer, comprising a measuring step of measuring the amount of α2-3 sialylated CEA in a sample.

2. A method for testing for prostate cancer, comprising a measuring step of measuring the amount of α2-3 sialylated CEA in a sample derived from a subject.

3. The method according to claim 2, which is a method for screening subjects who are predicted to have prostate cancer, comprising: a measuring step of measuring the amount of α2-3 sialylated CEA in a sample derived from the subject; and a selecting step of selecting subjects using the measured amount of α2-3 sialylated CEA as an indicator.

4. The method according to any one of claims 1 to 3, wherein in the α2-3 sialic acid-added CEA, the α2-3 sialic acid is a MAM-binding sugar chain that binds to MAM.

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 CEA and a second probe molecule capable of specifically binding to α2-3 sialic acid.

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

7. The method according to claim 5 or 6, wherein the second probe molecule is a lectin capable of specifically binding to α2-3 sialic acid.

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 α2-3 sialic acid; 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 CEA and a second probe molecule capable of specifically binding to α2-3 sialic acid.

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

12. The kit according to claim 10 or 11, wherein the second probe molecule is a lectin capable of specifically binding to α2-3 sialic acid.

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 α2-3 sialic acid; The kit according to claim 13 , characterized in that

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