Cancer screening methods

Abstract

The present invention relates to a method for detecting a specific component in a biological sample (e.g., a blood sample) obtained from a subject in order to predict whether the subject has cancer. The present invention provides a method comprising isolating one or more components selected from the group consisting of SerpinA3 and Gal3BP from the biological sample. The present invention also provides a method comprising detecting a glycoprotein in a biological sample (e.g., a blood sample) obtained from a subject by performing an assay using a first molecule selected from the group consisting of a molecule that binds to a BC2LCN-binding sugar chain, a molecule that binds to SerpinA3, and a molecule that binds to Gal3BP, and a second molecule that binds to a BC2LCN-binding sugar chain.

Description

Technical Field

[0001] The present invention relates to a cancer detection method and a detection kit thereof. Background Art

[0002] BC2LCN is a sugar-binding protein, a type of lectin. According to previous reports, it has been shown to have affinity for H-type 1 (Fucα1-2Galβ1-3GlcNAc) and H-type 3 (Fucα1-2Galβ1-3GalNAc), which are glycoprotein sugar chains, and can be used as a probe for detecting undifferentiated markers in human ES cells or iPS cells that show podocyte marker proteins (podocalyxin) with H-type 3 sugar chains (Patent Document 1). In addition, according to previous reports, it has been shown that BC2LCN binds firmly to cancer tissue and can be used as a probe for detecting cancer markers (Patent Document 2). According to Patent Document 2, a sandwich assay using BC2LCN and an antibody that binds to keratin sulfate confirmed the presence of proteins recognized by BC2LCN and keratin sulfate in the blood components of some cancer patients.

[0003] SerpinA3, also known as α1-antichymotrypsin, is known to be associated with several diseases such as tumors. SerpinA3 is currently being developed as a blood test marker (Non-Patent Document 1).

[0004] Gal3BP, also known as LGAL3SBP or MAC-2-BP, is found at high concentrations in the serum of patients with breast cancer, lung cancer, colorectal cancer, ovarian cancer, and endometrial cancer (Non-Patent Document 2).

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: WO2014 / 126146

[0008] Patent Document 2: WO2017 / 061449

[0009] Non-patent literature

[0010] Non-patent document 1: Nie, S. et al., J. Proteome Res. 13: 1873-1884, 2014

[0011] Non-patent document 2: Koths, K. et al., J. Biol. Chem. 268: 14245-14249, 1993 Summary of the Invention

[0012] The present invention relates to a cancer detection method and a detection kit thereof.

[0013] According to the present invention, the following inventions are provided.

[0014] (1) A method for analyzing the interior of a blood sample obtained from a subject, the method comprising:

[0015] Blood components contained in a blood sample are analyzed by a sandwich assay or immunochromatographic assay using a first molecule selected from the group consisting of BC2LCN, an antibody that binds to SerpinA3, and an antibody that binds to Gal3BP, and a second molecule that is BC2LCN.

[0016] (2) The method according to (1) above, wherein the first molecule is an antibody that binds to SerpinA3.

[0017] (3) The method according to (1) above, wherein the first molecule is an antibody that binds to Gal3BP.

[0018] (4) The method according to (1) above, wherein the first molecule is BC2LCN.

[0019] (5) The method according to any one of (1) to (4) above, wherein the subject has cancer or is at risk of having cancer.

[0020] (6) The method according to (5) above, wherein the subject suffers from or is likely to suffer from pancreatic cancer.

[0021] (7) The method according to (6) above, wherein the subject has or is likely to have stage 1 or stage 2 pancreatic cancer.

[0022] (8) A sandwich assay or immunochromatographic assay kit comprising a first molecule selected from the group consisting of BC2LCN, an antibody that binds to SerpinA3, and an antibody that binds to Gal3BP, and a second molecule that is BC2LCN, and capable of measuring the amount of glycoprotein in a subject's blood sample using the first molecule and the second molecule.

[0023] (9) The kit according to (8) above, wherein the first molecule is an antibody that binds to SerpinA3, and the protein portion of the glycoprotein is SerpinA3.

[0024] (10) The kit according to (8) above, wherein the first molecule is an antibody that binds to Gal3BP, and the protein portion of the glycoprotein is Gal3BP.

[0025] (11) The kit according to (8) above, comprising: a second molecule which is solid-phased BC2LCN; and a labeled first molecule selected from the group consisting of labeled BC2LCN, an antibody that binds to labeled SerpinA3, and an antibody that binds to labeled Gal3BP.

[0026] (12) The kit according to any one of (8) to (11) above, which is used for detecting cancer.

[0027] (13) The kit according to (12) above, wherein the cancer is a cancer selected from the group consisting of pancreatic cancer and colorectal cancer.

[0028] (14) The kit according to (13) above, wherein the cancer is stage 1 or stage 2 pancreatic cancer.

[0029] (15) A method for predicting whether a subject has cancer, the method comprising performing the method according to any one of (1) to (7) above.

[0030] (16) A method of analyzing the interior of a blood sample obtained from a subject,

[0031] The method includes analyzing a blood sample for the presence or concentration of a blood component capable of simultaneously binding to two BC2LCNs and a component selected from the group consisting of BC2LCN-binding SerpinA3 and BC2LCN-binding Gal3BP.

[0032] According to the present invention, the following inventions can also be provided.

[0033] (1A) A method for analyzing a biological sample obtained from a subject, the method comprising:

[0034] The components contained in the biological sample are analyzed by performing an assay using a first molecule selected from the group consisting of molecules binding to BC2LCN-binding sugar chains, molecules binding to SerpinA3, and molecules binding to Gal3BP, and a second molecule being BC2LCN.

[0035] (2A) The method according to (1A) above, wherein the first molecule is an antibody that binds to SerpinA3.

[0036] (3A) The method according to (1A) above, wherein the first molecule is an antibody that binds to Gal3BP.

[0037] (4A) The method according to (1A) above, wherein the first molecule is BC2LCN.

[0038] (5A) The method according to any one of (1A) to (4A) above, wherein the subject has or is likely to have cancer.

[0039] (6A) The method according to (5A) above, wherein the subject has pancreatic cancer or is at risk of having pancreatic cancer.

[0040] (7A) The method according to (6A) above, wherein the subject has or is likely to have stage 1 or stage 2 pancreatic cancer.

[0041] (8A) A sandwich assay kit, an immunochromatographic assay kit, a lectin electrophoresis kit, a mass spectrometry analysis kit, a lectin affinity chromatography kit, or a μTAS kit, comprising a first molecule selected from the group consisting of BC2LCN, a molecule that binds to SerpinA3, and a molecule that binds to Gal3BP, and a second molecule that is a molecule that binds to a BC2LCN-binding sugar chain, and capable of using the first molecule and the second molecule to measure the amount of glycoprotein in a biological sample of a subject.

[0042] (9A) The kit according to (8A) above, wherein the first molecule is an antibody that binds to SerpinA3, and the protein portion of the glycoprotein is SerpinA3.

[0043] (10A) The kit according to (8A) above, wherein the first molecule is an antibody that binds to Gal3BP, and the protein portion of the glycoprotein is Gal3BP.

[0044] (11A) The kit according to (8A) above, comprising: a second molecule which is solid-phased BC2LCN; and a labeled first molecule selected from the group consisting of labeled BC2LCN, an antibody that binds to labeled SerpinA3, and an antibody that binds to labeled Gal3BP.

[0045] (12A) The kit according to any one of (8A) to (11A) above, which is used for detecting cancer.

[0046] (13A) The kit according to (12A) above, wherein the cancer is pancreatic cancer.

[0047] (14A) The kit according to (13A) above, wherein the cancer is stage 1 or stage 2 pancreatic cancer.

[0048] (15A) A method for predicting whether a subject has cancer, the method comprising performing the method according to any one of (1A) to (7A) above.

[0049] (16A) A method of detecting a specific component in a biological sample obtained from a subject,

[0050] The specific component is a component capable of binding to two BC2LCNs simultaneously, and a component selected from the group consisting of BC2LCN-binding SerpinA3 and BC2LCN-binding Gal3BP.

[0051] (17A) A method comprising isolating a component capable of simultaneously binding to two BC2LCNs and one or more components selected from the group consisting of SerpinA3 and Gal3BP from a biological sample obtained from a subject.

[0052] (18A) The method according to (17A) above, wherein the subject is a subject suffering from or likely to suffer from pancreatic cancer.

[0053] (19A) The method according to (17A) above, wherein the pancreatic cancer is stage I or stage II pancreatic cancer.

[0054] (20A) The method according to any one of (17A) to (19A) above, further comprising measuring the amount of the separated component.

[0055] (21A) The method according to (20A) above, further comprising comparing a cutoff value with the measured amount of the component.

[0056] (22A) The method according to (21A) above, wherein the cutoff value is a value greater than the average value of the amount of the component corresponding to the measured component in healthy persons, and is a value less than the average value of the amount of the component corresponding to the measured component in pancreatic cancer patients.

[0057] (23A) The method according to any one of (17A) to (19A) above, further comprising detecting the separated component. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 These are the results of a sandwich assay that measured the concentrations of blood factors that can be simultaneously recognized by two molecules of BC2LCN in blood samples from patients with various cancers and healthy individuals.

[0059] Figure 2 The results of precipitating blood components bound to BC2LCN from a blood sample using BC2LCN, performing gel electrophoresis, and immunoblotting using BC2LCN (left) and silver staining (right) are shown.

[0060] Figure 3The graph shows the results of precipitating blood components bound to BC2LCN from blood samples using BC2LCN, performing gel electrophoresis, and immunoblotting using antibodies against SeprinA3 (left) and Gal3BP (right).

[0061] Figure 4 The figures show the results of sandwich assays using BC2LCN and an antibody binding to SerpinA3 in blood samples from patients with various cancers and healthy subjects.

[0062] Figure 5 The figures show the results of sandwich assays using BC2LCN and an antibody binding to Gal3BP in blood samples from patients with various cancers and healthy subjects.

[0063] Figure 6 The results show the concentrations of blood factors that can be simultaneously recognized by two molecules of BC2LCN, the concentration of BC2LCN-binding SerpinA3, the concentration of BC2LCN-binding Gal3BP, and the concentration of SerpinA3 measured using two different anti-SerpinA3 antibodies in blood samples from pancreatic cancer patients and healthy subjects.

[0064] Figure 7 This is an ROC curve that shows the relationship between sensitivity (Sensitivity) and specificity (Specificity) in an evaluation system for detecting pancreatic cancer using the concentration of a blood factor that can be simultaneously recognized by two molecules of BC2LCN in blood samples from pancreatic cancer patients and healthy subjects, the concentration of BC2LCN-binding SerpinA3, the concentration of BC2LCN-binding Gal3BP, and the concentration of SerpinA3 measured using two different anti-SerpinA3 antibodies as indicators.

[0065] Figure 8A The results show the concentrations of blood factors that can be simultaneously recognized by two molecules of BC2LCN in blood samples from pancreatic cancer patients, measured for each stage of the UICC TMN classification.

[0066] Figure 8B The graph shows the results of measuring the concentration of BC2LCN-binding SerpinA3 in blood samples of pancreatic cancer patients according to the UICCTMN classification stages.

[0067] Figure 8C The graph shows the results of measuring the concentration of BC2LCN-binding Gal3BP in blood samples of pancreatic cancer patients according to the UICC TMN classification stage.

[0068] Figure 8DThe figures show the results of measuring SerpinA3 concentrations in blood samples of pancreatic cancer patients using two different anti-SerpinA3 antibodies according to the UICC TMN classification stages.

[0069] Figure 9 The figures show the results of histological staining of rBC2LCN (sometimes referred to as "rBC2" in the figure) and immunohistological staining for SerpinA3 and Gal3BP using tissue sections of normal and pancreatic pancreas including pancreatic cancer.

[0070] Figure 10 The results of analysis of blood samples from healthy individuals, chronic pancreatitis patients, and pancreatic cancer patients using a sandwich assay using two molecules of BC2LCN, a sandwich assay using BC2LCN and an anti-SerpinA3 antibody, a sandwich assay using BC2LCN and an anti-Gal3BP antibody, an ELISA using an anti-SerpinA3 antibody, and an ELISA using an anti-Gal3BP antibody are shown.

[0071] Figure 11 ROC curves for detecting pancreatic cancer in healthy individuals and patients with chronic pancreatitis are shown using a sandwich assay using two BC2LCN molecules, a sandwich assay using BC2LCN and an anti-SerpinA3 antibody, a sandwich assay using BC2LCN and an anti-Gal3BP antibody, an ELISA using an anti-SerpinA3 antibody, and an ELISA using an anti-Gal3BP antibody. Blood samples were used for analysis.

[0072] Figure 12 The ROC curves for detecting stage I pancreatic cancer in healthy subjects are shown using a sandwich assay using two BC2LCN molecules, a sandwich assay using BC2LCN and an anti-SerpinA3 antibody, a sandwich assay using BC2LCN and an anti-Gal3BP antibody, an ELISA using an anti-SerpinA3 antibody, and an ELISA using an anti-Gal3BP antibody. Analysis was performed using blood samples.

[0073] Figure 13 ROC curves are shown for detecting stage II pancreatic cancer in healthy subjects using a sandwich assay using two molecules of BC2LCN, a sandwich assay using BC2LCN and an anti-SerpinA3 antibody, a sandwich assay using BC2LCN and an anti-Gal3BP antibody, an ELISA using an anti-SerpinA3 antibody, and an ELISA using an anti-Gal3BP antibody. Analysis was performed using blood samples.

[0074] Figure 14The ROC curves for detecting stage III pancreatic cancer in healthy subjects using a sandwich assay using two molecules of BC2LCN, a sandwich assay using BC2LCN and an anti-SerpinA3 antibody, and a sandwich assay using BC2LCN and an anti-Gal3BP antibody are shown. Analyses were performed using blood samples.

[0075] Figure 15 The ROC curves for detecting stage IV pancreatic cancer in healthy subjects using a sandwich assay using two molecules of BC2LCN, a sandwich assay using BC2LCN and an anti-SerpinA3 antibody, and a sandwich assay using BC2LCN and an anti-Gal3BP antibody are shown. Analyses were performed using blood samples.

[0076] Figure 16 The figures show the results of analysis of blood samples from patients before pancreatic cancer resection surgery, 30 days after the surgery (30POD), and 3 months after the surgery (3m) by a sandwich assay using BC2LCN and anti-SerpinA3 antibody.

[0077] Figure 17 The figures show the results of analysis of blood samples from patients before pancreatic cancer resection surgery, 30 days after the surgery (30POD), and 3 months after the surgery (3m) by a sandwich assay using BC2LCN and anti-Gal3BP antibody.

[0078] Figure 18 The figures show the results of analysis of blood samples from patients before pancreatic cancer resection surgery, 30 days after the surgery (30POD), and 3 months after the surgery (3m) by ELISA using CA19-9.

[0079] Figure 19 The figures show the results of analysis of blood samples from a patient (patient A) before pancreatic cancer resection, 1 day after surgery (1POD), 7 days after surgery (7POD), 30 days after surgery (30POD), and after recurrence by a sandwich assay using BC2LCN and anti-SerpinA3 antibody. DETAILED DESCRIPTION

[0080] In this specification, "subject" means a mammal, and particularly means mammals including humans. In this specification, a "subject" may be a healthy person, a patient suffering from cancer, or a patient who may suffer from cancer.

[0081] In this specification, "tumor" means a cell group that proliferates independently. Independent proliferation means the property of cells that break away from the normal cell proliferation control in the body and continue to proliferate autonomously. Tumors are roughly divided into benign tumors and malignant tumors (cancer). Benign tumors are tumors that proliferate independently but do not cause metastasis, infiltration and cachexia. Malignant tumors, in addition to autonomous proliferation, can also cause infiltration into normal tissues, metastasis and cachexia.

[0082] In this specification, "biological sample" means a sample obtained from an object (e.g., tissue, cell, extracellular fluid and body fluid). As body fluid, blood, urine, ascites, pleural effusion and secretions (e.g., endocrine fluid and exocrine fluid, for example, digestive fluids such as pancreatic juice) can be mentioned. In this specification, a "blood sample" is blood obtained from an object (e.g., peripheral blood) or a sample derived from blood (e.g., serum and plasma). In this specification, a "blood component" is a component contained in blood, especially a liquid component contained in blood. In this specification, as described later, when the biological sample is a blood sample, the component is a blood component.

[0083] As used herein, "antibody" means immunoglobulin. An antibody is a living molecule that has the ability to bind to an antigen. An antibody can be specific for an antigen. Antibodies can be monoclonal or polyclonal. Antibodies can include bispecific antibodies. Antibodies can be full-length antibodies or antigen-binding fragments thereof.

[0084] In this specification, "BC2LCN" is derived from Burkholderia cenocepacia The N-terminal domain of BC2LCN (Sulak, O., et al., Structure, 18(1): 59-72, 2010) is a lectin. Previous reports have shown that BC2LCN has affinity for H-type 1 (Fucα1-2Galβ1-3GlcNAc) and H-type 3 (Fucα1-2Galβ1-3GalNAc), glycoprotein sugar chains, and that BC2LCN can be used as an undifferentiated marker for human ES cells or iPS cells that express podocyte marker proteins with H-type 3 sugar chains (WO2014 / 126146). Furthermore, previous reports have shown that BC2LCN is specifically expressed in cancer tissues and can be used to detect cancer (WO2017 / 061449). According to WO2017 / 061449, a sandwich assay using BC2LCN and an antibody that binds to keratin sulfate confirmed the presence of proteins recognized by BC2LCN and keratin sulfate in the blood components of some cancer patients. B. cenocepaciaBC2LCN produced by a host other than a host cell is referred to as recombinant BC2LCN or rBC2LCN. In this specification, the sugar chain or protein bound to BC2LCN is referred to as BC2LCN-binding sugar chain or protein. BC2LCN-binding protein is modified by BC2LCN-binding sugar chain. BC2LCN-binding sugar chain can be an H-type1 sugar chain and / or an H-type3 sugar chain. As BC2LCN, for example, the lectin registered as YP_002232818 in the National Center for Biotechnology Information (NCBI) can be cited. As BC2LCN, any BC2LCN can be used as long as it has the ability to bind to sugar chains, but as BC2LCN, BC2LCN that has changed (for example, improved) the binding ability to the sugar chains disclosed in JP2020-146027 can also be used. Variants of BC2LCN can be used. Examples of BC2LCN include proteins (BC2LCN) having an amino acid sequence that is 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% identical to the amino acid sequence of the lectin registered as YP_002232818. BC2LCN binds to at least one selected from the group consisting of H-type 3 sugar chains and H-type 1 sugar chains.

[0085] In this specification, "SerpinA3" refers to a serine protease inhibitor (serine), also known as α1 antichymotrypsin (AACT). In humans, for example, it can be a protein translated from an mRNA having a base sequence registered as GenBank Accession No. K01500.1. In humans, the SerpinA3 protein is produced by cleaving a signal peptide consisting of the 1st to 23rd amino acids from a 423-amino acid precursor (e.g., NCBI Reference No. NP_001076.2).

[0086] In this specification, "Gal3BP" refers to galectin-3 binding protein. Gal3BP can be a protein obtained by translation from an mRNA having a base sequence registered with the National Center for Biotechnology Information (NCBI) as reference number NM_005567.4. In humans, the Gal3BP protein can be produced by cleavage of a signal peptide consisting of the 1st to 18th amino acid sequence from a 585-amino acid precursor (e.g., having an amino acid sequence registered with NCBI as reference number NP_005558.1). Gal3BP has a molecular weight of 90 kDa in serum and is found at high concentrations in the serum of patients with breast, lung, colorectal, ovarian, and endometrial cancers (Koths, K. et al., J. Biol. Chem. 268:14245-14249, 1993).

[0087] In this specification, a "sandwich assay" is an assay for detecting a target component contained in a sample. A person skilled in the art can perform a sandwich assay by conventional methods. A sandwich assay is generally an assay in which a target component is brought into contact with a capture molecule A that is bound to the target component solid-phased on a support (e.g., a substrate and beads), the target component is captured on the support, the unbound target component is removed (or separated), and then the capture molecule B that is bound to the labeled target component is brought into contact with the target component to detect the label of the target component captured on the support as an indicator. As a support that can be used in the present invention, any support (especially an insoluble support) used in conventional immunoassays can be used, for example, polystyrene, polyacrylic acid, polymethacrylic acid, polymethyl methacrylate, polyacrylamide, polyglycidyl methacrylate, polypropylene, polyolefin, polyimide, polyurethane, polyester, polyvinyl chloride, polyethylene, polychlorocarbonate, silicone resin, silicone rubber, agarose, dextran, ethylene-maleic anhydride copolymer and other organic substances; glass, silicon oxide, diatom, porous glass, frosted glass, aluminum oxide, silica gel, metal oxide and other inorganic substances; iron, cobalt, nickel, magnetite, chromite and other magnetic substances; and supports prepared from alloys of these magnetic substances. Solid phase treatment can be performed on the solid phase (for example, all surfaces of beads, magnetic beads, membranes, plates, etc.). Various commercially available magnetic beads can be used. As magnetic beads, for example, the magnetic beads disclosed in WO2012 / 173002A can be used. In a sandwich assay, the solid-phased capture molecule A and the labeled capture molecule B typically do not compete with each other for binding to the target component and must bind to the target component simultaneously. Capture molecule A can be the first molecule described below, and capture molecule B can be the second molecule. Capture molecule A can be the second molecule described below, and capture molecule B can be the first molecule.

[0088] Sandwich assays can also be performed using microfluidic chips (micro-total analysis systems; μTAS). The microfluidic chip has a flow path, and the flow path has a sample inlet. For example, in the sample inlet, a capture molecule X bound to a polycation or polyanion (e.g., DNA) is brought into contact with the sample to form a complex of the capture molecule X and the target component. An electric field is applied to move the complex. If a labeled capture molecule Y present in the middle of the flow path comes into contact with the complex, a complex of the target component, the capture molecule X, and the capture molecule Y is formed. Furthermore, the complex moves in the flow path by the electric field and reaches the detection portion on the flow path, so it can be detected based on the label bound to the capture molecule Y. For example, detection can be performed optically using a laser or the like. The capture molecule X can be the first molecule described below, and the capture molecule Y can be the second molecule. The capture molecule X can be the second molecule described below, and the capture molecule Y can be the first molecule. μTAS can also be used to detect the presence of multiple biomarkers in parallel.

[0089] Another example of a sandwich assay (e.g., AlphaLISA) is an assay in which capture molecules N and M are immobilized on separate beads A and B, respectively. When a target molecule is present, the target molecule, capture molecule N, and bead A form a complex with capture molecule M and bead B. Bead A, which includes a photosensitizer that releases singlet oxygen upon excitation, is irradiated with excitation light. Singlet oxygen reaches bead B only when the complex is formed (when beads A and B are in proximity), causing bead B to emit chemiluminescence. The presence of the target molecule is detected by detecting the light from bead B. Thus, the present invention provides an AlphaLISA assay kit comprising beads immobilized with BC2LCN and beads immobilized with an antibody that binds to SerpinA3 or Gal3BP. In the present invention, a test kit can also be used to detect the presence of a target molecule using fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET) between BC2LCN labeled with a first fluorescent molecule and an antibody that binds to SerpinA3 or Gal3BP labeled with a second fluorescent molecule. In this case, beads or a support for solidification are not required. The present invention can also provide such a test kit.

[0090] In this specification, "immunochromatographic assay" is an assay for detecting a target component contained in a sample, and is generally performed as follows: a capture molecule C labeled with a marker such as colloidal gold is brought into contact with a target component contained in a liquid sample to form a complex of the labeled capture molecule C and the target component, and then the complex is moved on a chromatographic membrane and brought into contact with a capture molecule D solid-phased on a test line to capture the complex of the labeled capture molecule C and the target component onto the test line, and the marker on the test line is detected as an indicator. In an immunochromatographic assay, generally, the labeled capture molecule C and the capture molecule D solid-phased on the test line do not compete with each other for the target component, but need to bind to the target component at the same time. In an immunochromatographic assay, in addition to the test line, a control line is also provided, on which a molecule bound to the capture molecule C (an antibody, etc., or an anti-IgG antibody when the capture molecule C is an IgG antibody) is solid-phased and can capture the labeled capture component C that is not bound to the target component. In an immunochromatographic assay, a strip is provided, which sequentially comprises a sample pad containing a sample, a conjugate pad containing a labeled capture molecule C, a membrane (a mobile layer) through which a complex between the target component in the sample and the capture molecule C migrates, and an absorbent pad (an absorbent pad) that absorbs moisture from the sample. On the membrane, a test line (where capture molecule D is immobilized) and a control line (where an antibody against capture molecule C is immobilized) are present. If the sample is contained in the sample pad, it can migrate along the strip toward the absorbent pad if the sample contains the target component. Upon migration to the conjugate pad, the labeled capture molecule C forms a complex with the target component, which is captured by the immobilized capture molecule D on the test line and also on the control line. If the sample does not contain the target component, the sample migrates toward the absorbent pad, but the labeled capture molecule C does not form a complex and is not captured on the test line, but only on the control line. Consequently, if both the control line and the test line develop color, it indicates that the sample contains the target component. If the control line develops color and the test line does not, it indicates that the sample does not contain the target component. If the control line does not develop color, it can be determined that the immunochromatographic evaluation system is not functioning correctly. The capture molecule C can be the first molecule described below, and the capture molecule D can be the second molecule. The capture molecule C can be the second molecule described below, and the capture molecule D can be the first molecule.

[0091] As used herein, "lectin electrophoresis" refers to electrophoresis using a gel containing lectins. It is a method for separating lectin-binding molecules, taking advantage of the fact that molecules that interact with lectins migrate more slowly during electrophoresis than molecules that do not interact. Lectins are either free in the gel or immobilized on the gel. Lectins rarely migrate during electrophoresis, so simply mixing them with a gel material to create a gel can reduce the electrophoretic speed of glycoproteins. Alternatively, lectins can be bound to the gel by non-covalent or covalent bonds. When separating BC2LCN-binding molecules from BC2LCN-non-binding molecules, lectin electrophoresis using BC2LCN as a lectin can separate biological samples. Molecules with BC2LCN-binding sugar chains repeatedly interact with the immobilized lectin on the gel during electrophoresis, resulting in delayed electrophoresis. On the other hand, BC2LCN-non-binding molecules do not interact with the immobilized lectin on the gel, resulting in faster electrophoresis. By utilizing this principle, molecules having BC2LCN-binding sugar chains and BC2LCN-non-binding molecules can be separated by electrophoresis. Lectin electrophoresis can be appropriately performed by those skilled in the art.

[0092] As used herein, "lectin affinity chromatography" refers to chromatography that utilizes the property of lectins to specifically bind to sugar chains. In lectin affinity chromatography, for example, affinity chromatography is performed on a sample using a column containing an insoluble support on which the lectin is immobilized. If a component in the sample has an affinity for the lectin, that component is separated from other components. In the present invention, for example, BC2LCN can be used as the lectin.

[0093] According to the present invention, when analyzing the serum of patients with various cancers, in a sandwich assay using solid-phase BC2LCN and labeled BC2LCN, it was found that there is a blood component that can simultaneously bind to two BC2LCNs, and that the amount of this blood component in the serum of patients with cancer tends to be higher than that of healthy people.

[0094] According to the present invention, when the serum of patients with various cancers was analyzed, a sandwich assay using BC2LCN and an antibody that binds to SerpinA3 revealed that BC2LCN-binding SerpinA3 exists as a blood component, and that the amount of BC2LCN-binding SerpinA3 in the serum of patients with cancer tends to be higher than that of healthy people.

[0095] According to the present invention, when the serum of patients with various cancers was further analyzed, in a sandwich assay using BC2LCN and an antibody binding to Gal3BP, it was also found that BC2LCN-binding Gal3BP exists as a blood component, and that the amount of BC2LCN-binding Gal3BP in the serum of patients with cancer tends to be higher than that of healthy people.

[0096] According to the present invention, a method is provided for analyzing a biological sample obtained from a subject, the method comprising detecting one or more components (sometimes referred to as "target components") selected from the group consisting of SerpinA3 (e.g., BC2LCN-binding SerpinA3) and Gal3BP (e.g., BC2LCN-binding Gal3BP) in the biological sample obtained from the subject.

[0097] The method of the present invention may include preparing a biological sample. The method of the present invention may also include determining the amount of the above-mentioned component.

[0098] According to the present invention, one or more components selected from the group consisting of SerpinA3 (eg, BC2LCN-binding SerpinA3) and Gal3BP (eg, BC2LCN-binding Gal3BP) can be detected using a molecule that binds to the component.

[0099] In one embodiment, the one or more components can be detected after being separated from other components.

[0100] For example, SerpinA3 can be detected using a molecule that binds to SerpinA3. Alternatively, two different molecules capable of simultaneously binding to SerpinA3 can be used. Using two different molecules capable of simultaneously binding to SerpinA3 allows detection of SerpinA3 using immunochromatographic or sandwich assays. SerpinA3 can also be detected by mass spectrometry. In either case, a calibration curve can be generated using a SerpinA3 standard substance. The resulting calibration curve can then be used to quantify SerpinA3 using the measured values ​​associated with SerpinA3.

[0101] Furthermore, BC2LCN-binding SerpinA3 can be detected using molecules that bind to BC2LCN-binding sugar chains and molecules that bind to SerpinA3. Detecting molecules that bind to BC2LCN-binding sugar chains can include separating the BC2LCN-binding molecule from other components using the molecules that bind to BC2LCN-binding sugar chains. Alternatively, if the molecule that binds to BC2LCN-binding sugar chains is BC2LCN, separating the BC2LCN-binding molecule from other components using lectin affinity chromatography or lectin electrophoresis can also include separating SerpinA3 from other components. Either the separation of SerpinA3 or the separation of the BC2LCN-binding components can be performed first.

[0102] For example, Gal3BP can be detected using a molecule that binds to Gal3BP. Alternatively, two different molecules that can simultaneously bind to Gal3BP can be used. If two different molecules that can simultaneously bind to Gal3BP are used, Gal3BP can be detected by immunochromatographic assay or sandwich assay. Gal3BP can also be detected by mass spectrometry. In any case, a calibration curve can be generated using a Gal3BP standard substance. Furthermore, the resulting calibration curve can be used to quantify Gal3BP using the measured values ​​associated with Gal3BP.

[0103] Furthermore, BC2LCN-binding Gal3BP can be detected using molecules that bind to BC2LCN-binding sugar chains and molecules that bind to Gal3BP. The molecules that bind to BC2LCN-binding sugar chains can include separating the BC2LCN-binding molecules from other components using the molecules that bind to BC2LCN-binding sugar chains, or can include separating the BC2LCN-binding molecules from other components using lectin electrophoresis when the molecules that bind to BC2LCN-binding sugar chains are BC2LCN. Furthermore, the method can also include separating Gal3BP from other components. Either separation of Gal3BP or separation of BC2LCN-binding components can be performed first.

[0104] According to the present invention, a method for analyzing a biological sample obtained from a subject is provided, the method comprising analyzing a component (sometimes referred to as a "target component") contained in the biological sample by performing an assay using a first molecule selected from the group consisting of a molecule that binds to a BC2LCN-binding sugar chain, a molecule that binds to SerpinA3, and a molecule that binds to Gal3BP, and a second molecule that is BC2LCN.

[0105] The biological sample is a biological sample obtained from the above-mentioned object. The object can be an object suffering from cancer or possibly suffering from cancer. Cancer can be, for example, pancreatic cancer. Pancreatic cancer can be, for example, a pancreatic cancer of a stage selected from the group consisting of stages I, II, III and IV, for example, pancreatic cancer of stage I, or pancreatic cancer of stage II. As biological samples, for example, tissues, cells and body fluids can be mentioned. As body fluids, blood (for example, plasma and serum), sweat, saliva, urine, ascites, pleural effusion and secretions (for example, endocrine fluid and exocrine fluid, for example, digestive fluids such as pancreatic juice) can be mentioned. Tissues and cells can be, for example, cancer or suspected cancer tissues and cells, respectively. Tissues and cells can include, for example, cancer or suspected cancer parts, respectively.

[0106] As molecules that bind to BC2LCN-binding sugar chains, antibodies or antigen-binding fragments thereof that bind to BC2LCN-binding sugar chains can be cited. As molecules that bind to BC2LCN-binding sugar chains, aptamers that bind to BC2LCN-binding sugar chains (for example, nucleic acid aptamers selected from the group consisting of DNA and RNA) can also be cited. As molecules that bind to BC2LCN-binding sugar chains, medium molecules such as cyclic peptides can also be cited. Molecules that bind to these BC2LCN-binding sugar chains can be obtained by methods well known to those skilled in the art. For example, antibodies that bind to BC2LCN-binding sugar chains can be obtained from non-human mammals immunized with BC2LCN-binding sugar chains. Aptamers that bind to BC2LCN-binding sugar chains can be obtained from a nucleic acid pool using affinity for BC2LCN-binding sugar chains. Cyclic peptides that bind to BC2LCN-binding sugar chains can be obtained from a cyclic peptide pool using affinity for BC2LCN-binding sugar chains. The molecule that binds to the BC2LCN-binding sugar chain may be, for example, BC2LCN.

[0107] Examples of molecules that bind to SerpinA3 include antibodies or antigen-binding fragments thereof that bind to SerpinA3. Examples of molecules that bind to SerpinA3 include aptamers (e.g., nucleic acid aptamers selected from the group consisting of DNA and RNA) that bind to SerpinA3. Examples of molecules that bind to SerpinA3 include intermediate molecules such as cyclic peptides. These molecules that bind to SerpinA3 can be obtained by methods known to those skilled in the art. For example, antibodies that bind to SerpinA3 can be obtained from non-human mammals immunized with SerpinA3. Aptamers that bind to SerpinA3 can be obtained from a pool of nucleic acids using their affinity for SerpinA3. Cyclic peptides that bind to SerpinA3 can be obtained from a pool of cyclic peptides using their affinity for SerpinA3. SerpinA3 sometimes forms complexes with other components in biological samples. In the present invention, such complexes can be detected. For example, PSA is an example of another component that forms a complex with SerpinA3. Therefore, Serpin A3 can be detected using molecules that bind to PSA and molecules that bind to Serpin A3. Alternatively, Serpin A3 can be detected using labeled recombinant PSA and utilizing the property of Serpin A3 binding to PSA.

[0108] Examples of molecules that bind to Gal3BP include antibodies or antigen-binding fragments thereof that bind to Gal3BP. Examples of molecules that bind to Gal3BP include aptamers (e.g., nucleic acid aptamers selected from the group consisting of DNA and RNA) that bind to Gal3BP. Examples of molecules that bind to Gal3BP include intermediate molecules such as cyclic peptides. These molecules that bind to Gal3BP can be obtained by methods known to those skilled in the art. For example, antibodies that bind to Gal3BP can be obtained from non-human mammals immunized with Gal3BP. Aptamers that bind to Gal3BP can be obtained from a pool of nucleic acids using their affinity for Gal3BP. Cyclic peptides that bind to Gal3BP can be obtained from a pool of cyclic peptides using their affinity for Gal3BP. Gal3BP sometimes forms complexes with other components in biological samples. In the present invention, such complexes can be detected. For example, other components that form complexes with Gal3BP include galectins (particularly galectin 3). Therefore, Gal3BP can be detected using molecules that bind to galectins (especially galectin 3) and molecules that bind to Gal3BP. Alternatively, Gal3BP can be detected using labeled recombinant galectins (especially galectin 3) and utilizing the binding properties of Gal3BP to galectins (especially galectin 3).

[0109] According to the present invention, a method is provided for analyzing a biological sample (e.g., a blood sample) obtained from a subject, the method comprising analyzing the presence or concentration of a component (a specific component, e.g., a blood component) in the biological sample (e.g., a blood sample), wherein the component (e.g., a blood component) is a component capable of simultaneously binding to two BC2LCNs, and a component selected from the group consisting of SerpinA3 (e.g., BC2LCN-binding SerpinA3) and Gal3BP (e.g., BC2LCN-binding Gal3BP) (sometimes referred to as a target component (e.g., a target blood component)). In the analysis method of the present invention, if one, two, or all of the above components are detected, it indicates that the subject may have cancer. In the analysis method of the present invention, if the concentration of one of the above components is greater than a cutoff value, it indicates that the subject may have cancer.

[0110] In the analysis method of the present invention, the subject may be a human, for example, a healthy person, a patient with cancer, or a patient who may have cancer.

[0111] In one embodiment, the cancer may be one or more cancers selected from the group consisting of pancreatic cancer, colorectal cancer, rectal adenocarcinoma, bladder cancer, breast cancer, non-small cell lung cancer, stomach cancer, cervical cancer, uterine corpus cancer, hepatocellular carcinoma, thyroid cancer, prostate cancer, melanoma, esophageal cancer, ovarian cancer, bile duct cancer, non-Hodgkin's lymphoma, and chronic myeloid leukemia. In one embodiment, the cancer may be pancreatic cancer. In one embodiment, the pancreatic cancer may be of a stage selected from the group consisting of stage I and stage II. In one embodiment, the pancreatic cancer may be stage I. In one embodiment, the pancreatic cancer may be stage II.

[0112] In one embodiment, the target component or target blood component is a component capable of simultaneously binding to two BC2LCNs. In one embodiment, the target component or target blood component is BC2LCN-binding SerpinA3. In one embodiment, the target component or target blood component is BC2LCN-binding Gal3BP. In these embodiments, the cancer may be any of the above-described cancers, but may also be, for example, one or more cancers selected from the group consisting of pancreatic cancer, colorectal cancer, rectal adenocarcinoma, bladder cancer, breast cancer, uterine corpus cancer, hepatocellular carcinoma, thyroid cancer, melanoma, esophageal cancer, bile duct cancer, ovarian cancer, non-Hodgkin's lymphoma, and chronic myeloid leukemia. In one embodiment, the cancer may be pancreatic cancer. In one embodiment, the pancreatic cancer may be of a stage selected from the group consisting of stage I and stage II. In one embodiment, the pancreatic cancer may be stage I. In one embodiment, the pancreatic cancer may be stage II. In one embodiment, the cutoff value may be, for example, 0, the first quartile, the average, the third quartile, or the maximum value of the target component (e.g., target blood component) concentration in a healthy group, or a value between any two of these values ​​(e.g., the third quartile or the maximum value, or a value therebetween). In one embodiment, the cutoff value may be, for example, 0, the first quartile, the average, the third quartile, or the maximum value of the target component (e.g., target blood component) concentration in a cancer patient group, or a value therebetween.

[0113] In one embodiment, the cutoff value may be the cutoff value at the point closest to the point where sensitivity is 1 and specificity is 1 on the ROC curve, a value greater than the cutoff value, or a value less than the cutoff value. In another embodiment, the cutoff value may be the cutoff value at the point where YoudenIndex is maximum, a value greater than the cutoff value, or a value less than the cutoff value.

[0114] The cutoff value can be determined by balancing sensitivity and false-positive rate. If the goal is to detect as many cancer patients as possible even if there are many false-positives (in the case of increasing sensitivity), the cutoff value can be set lower. If the goal is to reduce false positives (conversely, in the case of decreasing sensitivity), the cutoff value can be set higher. A person skilled in the art can appropriately set the cutoff value based on the purpose of the examination.

[0115] In the analysis method of the present invention, when the concentration of a target component (e.g., a target blood component) in a biological sample (e.g., a blood sample) obtained from a subject is greater than a cutoff value, information indicating that the subject may have cancer can be obtained. Thus, the analysis method of the present invention may include providing information indicating that the subject may have cancer when the concentration of a target component (e.g., a target blood component) in a biological sample (e.g., a blood sample) obtained from a subject is greater than a cutoff value.

[0116] In the analysis method of the present invention, when the concentration of a target component (e.g., a target blood component) in a biological sample (e.g., a blood sample) obtained from a subject is greater than a cutoff value, information indicating that the subject may have pancreatic cancer can be obtained. Thus, the analysis method of the present invention may include providing information indicating that the subject may have pancreatic cancer when the concentration of a target component (e.g., a target blood component) in a biological sample (e.g., a blood sample) obtained from a subject is greater than a cutoff value.

[0117] In the analysis method of the present invention, the target component (e.g., target blood component) can be detected even in a biological sample (e.g., blood sample) of a patient with early-stage cancer (stage I and stage II). Thus, the analysis method of the present invention may include providing information indicating that the subject may have early-stage cancer when the concentration of the target component (e.g., target blood component) in a biological sample (e.g., blood sample) obtained from the subject is greater than a cutoff value. The early-stage cancer may be early-stage pancreatic cancer. The stage classification of pancreatic cancer can be determined by a doctor according to the UICC TNM classification (UICC: TNM Classification of Malignant Tumours, 8th Edn. Wiley-Blackwell; 2017.94-95). Stage I includes stage 1A and stage 1B, and stage II includes stage 2A and stage 2B. Stage III and stage IV have the same meanings as stage 3 and stage 4, respectively.

[0118] [Table 1]

[0119] Table 1: Stage classification of pancreatic cancer (UICC TMN classification, 8th edition)

[0120]

[0121] The provision of information can be performed by sending or outputting the information. The output of information can be performed on a display or printed material.

[0122] The analysis methods of the present invention may further include determining that the subject is likely to have cancer.

[0123] According to the present invention, a method for treating cancer is provided, the method comprising providing at least a portion of an object determined to be likely to have cancer in the analysis method of the present invention to cancer therapy. As cancer therapy, one or more of the group consisting of radiotherapy, chemotherapy and surgical therapy can be cited. Cancer therapy can be standard therapy. Radiotherapy can include irradiating the cancer with radiation. Chemotherapy can include administering an anticancer agent to the object. Surgical therapy can include resection of the cancer. As chemotherapy for cancer (e.g., pancreatic cancer), for example, FOLFIRINOX therapy, gemcitabine / nab-paclitaxel therapy (GnP therapy), gemcitabine / S1 therapy (GS therapy), liposome-irinotecan / 5FU / LV therapy (Nal-IRI / FL therapy), gemcitabine therapy and S1 therapy, etc., can be cited, and the object can be treated by any one or more of these therapies. Examples of the anticancer agent include one or more selected from the group consisting of platinum preparations such as oxaliplatin, irinotecan, liposomal irinotecan, Revo Folinate, gemcitabine, nab-paclitaxel, 5-fluorouracil (5-FU), and S-1.

[0124] According to the present invention, a pharmaceutical composition comprising a therapeutically effective amount of one or more agents selected from the group consisting of the aforementioned anticancer agents can be provided, wherein the pharmaceutical composition is used to treat cancer in a subject determined to be likely to have cancer in the analytical method of the present invention. The pharmaceutical composition further comprises, in addition to the pharmaceutically active ingredient, a pharmaceutically acceptable excipient. A person skilled in the art can appropriately select a pharmaceutically acceptable excipient.

[0125] In one embodiment of the analysis method of the present invention, a method is provided for analyzing the interior of a biological sample (e.g., a blood sample) obtained from a subject, the method comprising: analyzing components (e.g., blood components) included in the biological sample (e.g., a blood sample) by performing a sandwich assay or an immunochromatographic assay using a first molecule selected from the group consisting of BC2LCN, an antibody that binds to SerpinA3, and an antibody that binds to Gal3BP, and a second molecule that is BC2LCN.

[0126] In one embodiment, the first molecule is solid-phased on a support (eg, a substrate), and the second molecule may be labeled. In one embodiment, the first molecule may be labeled, and the second molecule may be solid-phased on a support.

[0127] Solid-phase can utilize known solid-phase methods such as direct method, polystyrene tag method (WO2018 / 190357), biotin-streptavidin method, HaloTag method, amine coupling method, and V5 tag method. In the direct method, the solid-phased capture molecule is directly in contact with the support, thereby being solid-phased on the support. In the polystyrene tag method, the capture molecule is solid-phased on a polystyrene plate via a polystyrene tag fused with the capture molecule. In the biotin-streptavidin method, the capture molecule modified with biotin is bound to a support coated with streptavidin, thereby capturing the molecule on the support. In the HaloTag method, the capture molecule is solid-phased on a support coated with HaloTag via a HaloTag protein fused with the capture molecule. In the amine coupling method, the carboxyl group formed on the solid phase surface can be actively esterified, and the protein can be solid-phased by a chemical bond via an amino group. In the V5 tag method, a capture molecule to which a V5 tag is linked is bound to a support coated with an anti-V5 tag antibody, whereby the capture molecule is immobilized on the support.

[0128] As a label, in the sandwich assay, a matrix (chromogenic matrix, fluorescent matrix and luminescent matrix) and an enzyme used in the enzyme antibody method (for example, peroxidase, glucose oxidase and alkaline phosphatase) can be used. If it is a person skilled in the art, conventional methods can be used for labeling. For example, it is possible to label by covalent bonding. For example, a biotin-linked antibody can be linked to an avidin label reaction and labeled. As a matrix, for example, a fluorescent matrix can be mentioned. As a peroxidase, for example, horseradish peroxidase can be used. Horseradish peroxidase produces color development, fluorescence or chemiluminescence by adding a chromogenic matrix, a fluorescent matrix or a luminescent matrix. Thus, the presence of a capture molecule labeled with color development, fluorescence or chemiluminescence as an indicator can be detected. As a colorimetric substrate for horseradish peroxidase, for example, tetramethylbenzidine (TMB), o-phenylenediamine (OPD), 2,2'-azinobis[3-ethylbenzo-thiazoline-6-sulfonic acid (ABTS) and Amplex (trademark) Red can be mentioned, which can be used for the detection of labeled molecules in the presence of hydrogen peroxide. As a luminescent substrate for alkaline phosphatase, p-nitrophenyl phosphate (pNPP), 4-methylumbelliferyl phosphate (4-MUP) and AttoPhos (trademark) can be mentioned, which can be used for the detection of capture molecules. Glucose oxidase oxidizes glucose to produce gluconic acid and hydrogen peroxide. Hydrogen peroxide can be easily detected using a colorimetric probe (e.g., peroxidase) for hydrogen peroxide detection. Hydrogen peroxide can develop color, for example, in the presence of peroxidase and its colorimetric substrate.

[0129] As a label, a pigment (eg, a colorant such as colloidal gold) can be used in immunochromatographic assays.

[0130] In a certain embodiment, the first molecule is BC2LCN. In this embodiment, recombinant BC2LCN can be used as BC2LCN. BC2LCN can be directly solid-phased, or, for example, solid-phased on a support that is biotin-labeled and coated with streptavidin (or neutravidin). In addition, on the basis of fusing BC2LCN with HaloTag protein, it can be solid-phased on a support coated with HaloTag ligand. And, on the basis of fusing a polystyrene tag to BC2LCN, it can be directly solid-phased on a polystyrene plate. Whether in a sandwich assay or in an immunochromatographic assay, when the first molecule is BC2LCN, the target component simultaneously binds to both BC2LCN as the first molecule and BC2LCN as the second molecule. Thus, the target component detected by a sandwich assay and an immunochromatographic assay in which the first molecule is BC2LCN is a component that can bind to multiple BC2LCNs simultaneously. In the immunochromatographic assay, an antibody against BC2LCN or a sugar chain antigen that binds to BC2LCN, such as an H-type 1 sugar chain antigen or an H-type 3 sugar chain antigen, may be immobilized on a control line.

[0131] In one embodiment, the first molecule is an antibody that binds to SerpinA3. In this embodiment, SerpinA3 having BC2LCN-binding sugar chains is considered to be detected. Antibodies that bind to SerpinA3 can be prepared by conventional methods using binding to SerpinA3 as an indicator.

[0132] Antibodies that bind to SerpinA3 can be further confirmed by their binding to SerpinA3 with BC2LCN-binding sugar chains. As used in the Examples, SerpinA3 with BC2LCN-binding sugar chains can be purified as a component that reacts with immobilized BC2LCN and a SerpinA3-binding antibody. Antibodies that bind to SerpinA3 can be selected based on whether they bind to this purified component.

[0133] In an embodiment where the first molecule is an antibody that binds to SerpinA3, the first molecule is immobilized on a support (eg, a substrate or beads) and the second molecule may be labeled, or the first molecule may be labeled and the second molecule may be immobilized on a support.

[0134] In one embodiment, the first molecule is an antibody that binds to Gal3BP. In this embodiment, it is believed that Gal3BP having a BC2LCN-binding sugar chain is detected. Antibodies that bind to Gal3BP can be prepared by conventional methods using binding to Gal3BP as an indicator.

[0135] Antibodies that bind to Gal3BP can be further confirmed by their binding to Gal3BP with BC2LCN-binding sugar chains. As used in the Examples, Gal3BP with BC2LCN-binding sugar chains can be purified as a component that reacts with immobilized BC2LCN and a Gal3BP-binding antibody. Antibodies that bind to Gal3BP can be selected based on whether they bind to this purified component.

[0136] In an embodiment where the first molecule is an antibody that binds to Gal3BP, the first molecule is immobilized on a support (eg, a substrate or beads) and the second molecule may be labeled, or the first molecule may be labeled and the second molecule may be immobilized on a support.

[0137] According to the present invention, a method is provided, which is a method for predicting whether a subject has cancer, a method for obtaining preliminary information on whether a subject has cancer, a method for determining whether a subject has cancer, a method or preliminary method for predicting whether a subject has cancer, a method for detecting cancer in a subject, a method for detecting cancer cells in a subject, a method for detecting a biomarker for cancer in a subject (the biomarker is a component capable of simultaneously binding to two BC2LCNs, a component selected from the group consisting of BC2LCN-binding SerpinA3 and BC2LCN-binding Gal3BP), or a method for analyzing whether a subject has cancer.

[0138] The method includes: analyzing the presence or concentration of a component (e.g., a blood component) in a biological sample (e.g., a blood sample), whether the component (e.g., a blood component) is a component capable of simultaneously binding to two BC2LCNs, or a component selected from the group consisting of BC2LCN-binding SerpinA3 and BC2LCN-binding Gal3BP; or analyzing the component (e.g., a blood component) contained in a biological sample (e.g., a blood sample) by performing a sandwich assay or immunochromatographic assay using a first molecule selected from the group consisting of BC2LCN, an antibody binding to SerpinA3, and an antibody binding to Gal3BP, and a second molecule being BC2LCN.

[0139] According to the present invention, a method is provided for detecting a component (e.g., a blood component) in a biological sample (e.g., a blood sample) of a subject. The component (e.g., a blood component) is a component capable of simultaneously binding to two BC2LCNs, and is selected from the group consisting of BC2LCN-binding SerpinA3 and BC2LCN-binding Gal3BP. The method may include contacting a first molecule selected from the group consisting of BC2LCN, an antibody that binds to SerpinA3, and an antibody that binds to Gal3BP, and a second molecule that is BC2LCN with the biological sample (e.g., a blood sample), and detecting the resulting complex of the component (e.g., a blood component), the first molecule, and the second molecule. The method may include detecting the component (e.g., a blood component) using a sandwich assay or an immunochromatographic assay using the first molecule selected from the group consisting of BC2LCN, an antibody that binds to SerpinA3, and an antibody that binds to Gal3BP, and the second molecule that is BC2LCN.

[0140] According to the present invention, a sandwich assay or immunochromatographic assay kit is provided, comprising a first molecule selected from the group consisting of BC2LCN, an antibody that binds to SerpinA3, and an antibody that binds to Gal3BP, and a second molecule that is BC2LCN, and the first molecule and the second molecule can be used to measure the amount of glycoprotein in the biological sample (e.g., a blood sample) of the object. In the kit of the present invention, in a certain embodiment, the first molecule is solid-phased on a substrate or a strip, and the second molecule is labeled. In the kit of the present invention, in a certain embodiment, the first molecule is labeled, and the second molecule is solid-phased on a substrate or a strip. In a certain embodiment, the first molecule is labeled, and the second molecule is contained in a gel or solid-phased on a gel. In the case where the label is an enzyme used in the enzyme antibody method, the kit of the present invention may also include a chromogenic matrix. In the case where the biological sample is a blood sample, the component is a blood component.

[0141] In one embodiment of the kit of the present invention, the first molecule is BC2LCN. In one embodiment of the present invention, the first molecule is an antibody that binds to SerpinA3. In one embodiment of the present invention, the first molecule is an antibody that binds to Gal3BP.

[0142] The kit of the present invention can be used for detection of target molecules or detection, determination, prediction, or diagnosis of cancer.

[0143] According to the present invention, a combination of a first molecule selected from the group consisting of BC2LCN, an antibody that binds to SerpinA3, and an antibody that binds to Gal3BP, and a second molecule that is BC2LCN is provided. This combination of the present invention can be used to detect components capable of simultaneously binding to two BC2LCNs, as well as components selected from the group consisting of BC2LCN-binding SerpinA3 and BC2LCN-binding Gal3BP, in a sample (e.g., a biological sample (e.g., a blood sample)). In this combination, one of the first and second molecules can be immobilized on a support, and the other can be labeled for detection. This combination can be used to diagnose cancer using a biological sample (e.g., a blood sample). According to the present invention, a kit for diagnosing cancer using a biological sample (e.g., a blood sample) comprising this combination is provided. This combination can be a sandwich assay kit, an immunochromatographic assay kit, a lectin electrophoresis kit, a mass spectrometry analysis kit, a lectin affinity chromatography kit, or a μTAS kit. The lectin electrophoresis kit can include a gel containing a lectin. Lectin affinity chromatography kits can include a column containing a support on which a lectin is immobilized. These kits can also include purified SerpinA3, purified BC2LCN-binding SerpinA3, purified Gal3BP, or purified BC2LCN-binding Gal3BP as standard substances. μTAS kits can include a microfluidic chip. The microfluidic chip has a detection section in which molecules bound to one or more selected from the group consisting of SerpinA3, BC2LCN-binding SerpinA3, Gal3BP, BC2LCN-binding Gal3BP, and protein complexes comprising these can be immobilized.

[0144] The method of the present invention is for analyzing a biological sample obtained from a subject, comprising: analyzing a component (sometimes referred to as a "target component") contained in the biological sample by performing an assay using a first molecule selected from the group consisting of a molecule that binds to a BC2LCN-binding sugar chain, a molecule that binds to SerpinA3, and a molecule that binds to Gal3BP, and a second molecule that is BC2LCN. In the method, the signal obtained by the assay can decrease due to a decrease in cancer in the body (for example, cancer removal). Thus, in the method of the present invention, the assay signal can indicate the amount of cancer in the body. Furthermore, in the above method, the signal obtained by the assay can increase due to recurrence. Thus, in the method of the present invention, an increase in the signal can indicate cancer recurrence.

[0145] According to the present invention, a method is provided, comprising isolating, from a biological sample obtained from a subject, a component capable of simultaneously binding to two BC2LCNs and one or more components selected from the group consisting of SerpinA3 and Gal3BP. According to the present invention, a method is provided, comprising detecting, from a biological sample obtained from a subject, a component capable of simultaneously binding to two BC2LCNs and one or more components selected from the group consisting of SerpinA3 and Gal3BP. According to the present invention, a method is also provided for analyzing a biological sample obtained from a subject, comprising isolating, from a biological sample obtained from a subject, a component capable of simultaneously binding to two BC2LCNs and one or more components selected from the group consisting of SerpinA3 and Gal3BP.

[0146] Separation means separating the target component from at least one other component. Separation can include concentration, enrichment, and purification of the target component. Concentration means increasing the concentration. Enrichment and purification mean selectively increasing the abundance ratio of the target component relative to other components. Enrichment and purification can be accompanied by concentration of the target component. Separation can also include binding to a solid-phase molecule. In this case, the target component is separated from other unbound components by binding to the solid-phase molecule. In addition to adsorption of the target component to the solid-phase surface, separation can also include removal of unadsorbed components. Removal of unadsorbed components can be performed by washing the surface. Removal of unadsorbed components can also be performed by removing the liquid phase. In the case where the solid phase is present on the surface of the beads, removal of unadsorbed components can also be performed by recovering the beads. In this manner, the present invention can also include measuring the amount of the separated component. The measurement can be performed by various well-known assays by those skilled in the art, thereby obtaining an assay signal. The measured amount can be compared with other quantities (e.g., cutoff values) that serve as indicators. The cutoff value can be as described above.

[0147] In the method of the present invention, when the measured amount is greater than other amounts serving as indicators, it can indicate that the subject from which the biological sample was derived may have cancer. Thus, the method of the present invention can be a method for detecting cancer, a method for detecting cancer cells, a method for diagnosing cancer, a non-diagnostic method for diagnosing cancer, a method for obtaining preliminary information for diagnosing cancer, a method for predicting cancer, or a non-diagnostic method for predicting cancer. In the method of the present invention, the signal obtained by the assay can decrease due to a reduction in cancer in the body (e.g., cancer removal). Thus, in the method of the present invention, the assay signal can indicate the amount of cancer in the body. Furthermore, in the above method, the signal obtained by the assay can increase due to recurrence. Thus, in the method of the present invention, an increase in the signal can indicate cancer recurrence. The method of the present invention can be performed on two or more biological samples recovered at different times. Thus, in one embodiment, the biological samples can be two or more biological samples recovered at different times. In one embodiment, the method of the present invention can further include comparing the signal obtained by the assay (corresponding to the measured amount) with a signal obtained in another sample recovered at the same time. Thus, the present invention can be used to monitor the course of treatment and / or recurrence of cancer in a patient.

[0148] The method of the present invention can be implemented in combination with existing methods. For example, as an existing method, there is a known method that includes measuring the amount of CA19-9 in a body fluid sample. The concentration of CA19-9 increases in body fluid samples of people with cancer. Moreover, if the amount of cancer increases, the concentration increases, and if the amount of cancer decreases, its concentration decreases. In this way, by combining CA19-9 with the method of the present invention, the accuracy and / or specificity of detecting cancer by analyzing body fluid samples can be improved. CA19-9 can be an indicator of pancreatic cancer, especially pancreatic cancer in stages III and IV. Therefore, the present invention can also include measuring the concentration of CA19-9 in a biological sample.

[0149] One or more components selected from the group consisting of SerpinA3 and Gal3BP being measured are further analyzed using the presence of BC2LCN-binding sugar chain modifications as an indicator. Whether one or more components selected from the group consisting of SerpinA3 and Gal3BP have BC2LCN-binding sugar chain modifications can be determined using a factor that binds to the sugar modification (sugar chain) as described above. Alternatively, one or more components selected from the group consisting of SerpinA3 and Gal3BP can be detected after separation using a factor that binds to the sugar modification (sugar chain). One or more components selected from the group consisting of SerpinA3 and Gal3BP can be detected after separation using a molecule that binds to SerpinA3 and a molecule that binds to Gal3BP.

[0150] Example

[0151] Example 1: Detection of glycoproteins in the serum of cancer patients using rBC2LCN

[0152] Glycoproteins were detected in the sera of patients with various cancers by an rBC2LCN-based sandwich assay.

[0153] First, biotinylated rBC2LCN was diluted with PBS to 0.3 μg / mL and added to an avidin plate (non-adhesive type) (Sumitomo Bakelite Co., Ltd., BS-X7603) at 50 μL / well. The plate was incubated at room temperature for 1 hour. The resulting rBC2LCN-immobilized plate was washed five times with PBS / 0.1% Tween 20 and then reacted with 50 μL / well (n=3) of diluted serum from various cancer patients diluted 100-fold with PBS. After reacting at room temperature for 1 hour, the plate was washed five times with PBS / 0.1% Tween 20, and HRP-labeled rBC2LCN (1 μg / mL) was added at 50 μL / well and reacted at room temperature for 1 hour. After washing five times with PBS / 0.1% Tween 20, 50 μL / well of TMB solution (FUJIFILM Wako Pure Chemical Corporation) was added and color was developed at room temperature for 30 minutes. The color reaction was stopped by adding 50 μL / well of 1N HCl, and the OD450 / 620 value was measured. The average of the obtained values ​​is shown. In this rBC2LCN sandwich assay, a higher OD450 / 620 value indicates a greater amount of glycoprotein bound to the rBC2LCN.

[0154] The results are as follows Figure 1 As shown. Figure 1 As shown, it was clarified that the glycoprotein detected by the rBC2LCN-based sandwich assay had a concentration higher than the maximum value observed in the healthy group in patients with pancreatic cancer, colon cancer, bladder cancer, breast cancer, non-Hodgkin's lymphoma, uterine cancer, ovarian cancer, and chronic myeloid leukemia.

[0155] The detected glycoprotein must simultaneously bind to both the immobilized rBC2LCN and the detection rBC2LCN. The results of this example clearly demonstrate that components (e.g., blood components) that can simultaneously bind to two molecules of rBC2LCN are generally found at higher concentrations in cancer patients than in healthy individuals, making this method useful for component (e.g., blood component) analysis.

[0156] Example 2: Identification of glycoproteins recognized by rBC2LCN

[0157] In this example, rBC2LCN-bound beads were used to precipitate glycoproteins in the serum of pancreatic cancer patients and healthy subjects, respectively. The precipitated glycoproteins were eluted with rBC2LCN, the resulting eluate was subjected to electrophoresis, and glycoproteins inherent to pancreatic cancer were identified by mass spectrometry.

[0158] Biotinylated rBC2LCN was bound to Dynabeads M280 Streptavidin (Invitrogen). It was then incubated with pancreatic cancer and healthy serum at 4°C overnight, washed, and 0.2M fucose was added. The glycoproteins bound to the rBC2LCN were eluted by incubation at room temperature for 1 hour. The resulting eluate was subjected to electrophoresis, transferred to a PVDF membrane, and then blotted with HRP-labeled rBC2LCN. As a result, no reactivity was found on the rBC2LCN blot in healthy serum, but reactivity was observed around 75-150 kDa in pancreatic cancer serum. Therefore, the portion surrounded by red at 100-150 kDa was cut out from the electrophoresed and silver-stained gel. Each of the cut gel slices was hydrolyzed with trypsin to obtain a peptide mixture. The peptide mixture was provided to LC-MS / MS to obtain MS / MS data. The amino acid sequence database search results of the MS / MS data were output as a peptide identification list. As a result, SerpinA3 and Gal3BP were identified as candidate proteins detected by rBC2LCN in cancer patients.

[0159] Therefore, glycoproteins that showed binding to rBC2LCN immobilized beads were electrophoresed from the sera of pancreatic cancer and healthy subjects, and Western blotting was performed using antibodies against SerpinA3 and Gal3BP and an HRP-labeled secondary antibody. Figure 3 As shown, SerpinA3 and Gal3BP were detected significantly more strongly in the sera of pancreatic cancer patients compared to healthy controls. SerpinA3 and Gal3BP were obtained as proteins adsorbed to beads by rBC2LCN, indicating that these glycoproteins are reactive with rBC2LCN.

[0160] Normal pancreatic tissue and pancreatic cancer tissue were obtained from the patient and provided for immunohistochemical staining. Immunohistochemical staining was performed according to conventional methods. As the primary antibody, for SerpinA3 staining, anti-SerpinA3 antibody (manufactured by R&D Systems, product number: MAB12945, dilution ratio: 200 times) was used, and for Gal3BP staining, anti-Gal3BP antibody (manufactured by R&D Systems, product number: AF2226, 1 μg / mL) was used. In addition, as the secondary antibody, biotinylated anti-mouse IgG (manufactured by NICHIREI CORPORATION., product number: 424021, dilution ratio: stock solution) and biotinylated anti-goat IgG (manufactured by NICHIREI CORPORATION., product number: 414011, dilution ratio: stock solution) were used, respectively. The results are as follows. Figure 9 As shown. Figure 9 As shown, it was found that almost no staining images were found in normal pancreatic tissues, whereas SerpinA3 and Gal3BP were strongly expressed in the pancreatic ducts in pancreatic cancer tissues.

[0161] This indicates that SerpinA3 and Gal3BP detected in the blood originate from the pancreatic ducts of pancreatic cancer tissues.

[0162] Example 3: Analysis of cancer patients using rBC2LCN and SerpinA3 antibody or Gal3BP antibody in a sandwich assay serum

[0163] Therefore, a sandwich assay using rBC2LCN and SerpinA3 antibodies was constructed and analyzed using sera from healthy individuals and patients with various cancers, including pancreatic cancer. Biotinylated rBC2LCN was diluted to 0.3 μg / mL in PBS and added to an avidin plate (non-adhesive type) (Sumitomo Bakelite Co., Ltd., BS-X7603) at 50 μL / well. The plate was incubated at room temperature for 1 hour. The resulting rBC2LCN-immobilized plate was washed five times with PBS / 0.1% Tween 20 and then reacted with 50 μL / well (n=3) of diluted sera from patients with various cancers diluted 100-fold in PBS. After a one-hour reaction at room temperature, the plate was washed five times with PBS / 0.1% Tween 20 and HRP-labeled SerpinA3 antibody (1 μg / mL; R&D Systems, Cat#: AF1295) was added at 50 μL / well and allowed to react at room temperature for 1 hour. After washing five times with PBS / 0.1% Tween 20, 50 μL / well of TMB solution (FUJIFILM Wako Pure Chemical Corporation) was added and color development was allowed to proceed at room temperature for 30 minutes. The color development reaction was stopped by adding 50 μL / well of 1N HCl, and OD450 / 620 was measured. The average of the obtained values ​​is shown.

[0164] The results are as follows Figure 4 As shown. Figure 4 As shown, the glycoprotein detected by the sandwich assay of rBC2LCN and SerpinA3 antibodies (i.e., SerpinA3 having a specific sugar chain that binds to the BC2LCN lectin) was hardly detected in healthy subjects, but was strongly detected in all cancers (all examined cancers) including colon cancer, pancreatic cancer, bladder cancer, gastric cancer, small cell lung cancer, breast cancer, non-Hodgkin's lymphoma, cervical cancer, rectal adenocarcinoma, hepatocellular carcinoma, thyroid cancer, prostate cancer, uterine corpus cancer, melanoma, esophageal cancer, ovarian cancer, bile duct cancer, and chronic myeloid leukemia.

[0165] Next, a sandwich assay using rBC2LCN and Gal3BP antibodies was constructed and analyzed using sera from healthy individuals and patients with various cancers, including pancreatic cancer. Biotinylated rBC2LCN was diluted to 0.3 μg / mL in PBS and added to an avidin plate (non-adhesive type) (Sumitomo Bakelite Co., Ltd., BS-X7603) at 50 μL / well. The plate was incubated at room temperature for 1 hour. The resulting rBC2LCN-immobilized plate was washed five times with PBS / 0.1% Tween 20 and then reacted with 50 μL / well (n=3) of diluted sera from patients with various cancers diluted 100-fold in PBS. After incubation at room temperature for 1 hour, the plate was washed five times with PBS / 0.1% Tween 20. An HRP-labeled Gal3BP antibody (1 μg / mL; R&D Systems, Cat#: AF2226) was added at 50 μL / well and allowed to react at room temperature for 1 hour. After washing five times with PBS / 0.1% Tween 20, 50 μL / well of TMB solution (FUJIFILM Wako Pure Chemical Corporation) was added and color was developed at room temperature for 30 minutes. The color reaction was stopped by adding 50 μL / well of 1N HCl, and the OD450 / 620 was measured. A graph was created from the average of the obtained values. The average of the obtained values ​​is shown.

[0166] The results are as follows Figure 5 As shown. Figure 5 As shown, the glycoprotein detected by the sandwich assay of rBC2LCN and Gal3BP antibodies (i.e., Gal3BP having a specific sugar chain that binds to the BC2LCN lectin) was almost undetectable in healthy subjects, but on the other hand, was strongly detected in all cancers (all examined cancers) including colon cancer, pancreatic cancer, bladder cancer, gastric cancer, small cell lung cancer, breast cancer, non-Hodgkin's lymphoma, cervical cancer, rectal adenocarcinoma, hepatocellular carcinoma, thyroid cancer, prostate cancer, uterine corpus cancer, melanoma, esophageal cancer, ovarian cancer, bile duct cancer, and chronic myeloid leukemia.

[0167] And, in Figure 10 In the study, healthy subjects, pancreatitis patients, and pancreatic cancer patients were compared. The results showed that pancreatic cancer patients showed stronger signals compared to healthy subjects or pancreatitis patients in sandwich assays of rBC2LCN and rBC2LCN, sandwich assays of rBC2LCN and anti-SerpinA3 antibodies, sandwich assays of rBC2LCN and anti-Gal3BP antibodies, and sandwich assays of anti-SerpinA3 antibodies and anti-SerpinA3 antibodies. This demonstrates that these assays can distinguish pancreatic cancer patients from healthy subjects or pancreatitis patients.

[0168] This revealed that SerpinA3 having a specific sugar chain that binds to the BC2LCN lectin and Gal3BP having a specific sugar chain that binds to the BC2LCN lectin are widely present in the sera of cancer patients.

[0169] In addition, using serum specimens from other healthy subjects and serum specimens from pancreatic cancer patients, sandwich assays based on rBC2LCN and rBC2LCN, sandwich assays based on rBC2LCN and anti-SerpinA3 antibodies, sandwich assays based on rBC2LCN and anti-Gal3BP antibodies, and sandwich assays based on anti-SerpinA3 antibodies (R&D Systems, Cat#: AF1295) and anti-SerpinA3 antibodies (R&D Systems, Cat#: AF1295) were performed by the above-described method.

[0170] The results are as follows Figure 6 As shown. Figure 6 As shown, in the sandwich assays using rBC2LCN and rBC2LCN, the sandwich assays using rBC2LCN and anti-SerpinA3 antibodies, and the sandwich assays using rBC2LCN and anti-Gal3BP antibodies, higher assay intensities were obtained in the serum of pancreatic cancer patients compared to the serum of healthy subjects. In contrast, in the sandwich assays using anti-SerpinA3 antibodies and anti-Serpin A3 antibodies, the assay intensities were equivalent in the serum of healthy subjects and pancreatic cancer patients. This result indicates that SerpinA3 is present in both the serum of healthy subjects and the serum of pancreatic cancer patients, but in pancreatic cancer patients, SerpinA3 is specifically modified with a sugar that is recognized by rBC2LCN.

[0171] The above results were analyzed and ROC curves were drawn with specificity as the horizontal axis and sensitivity as the vertical axis. Figure 7 shown.

[0172] like Figure 7 As shown in the upper left panel, in the sandwich assay based on rBC2LCN and rBC2LCN (rBC2-rBC2), the area under the ROC curve (AUC) was 0.679, and the cutoff value in the ROC curve (calculated from the ROC curve point at the shortest distance from sensitivity 1 and specificity 1) was 0.329. At this time, the specificity was 0.820 and the sensitivity was 0.575.

[0173] like Figure 7As shown in the upper right panel, in the sandwich assay (rBC2-SerpinA3) based on rBC2LCN and anti-SerpinA3 antibody, the area under the ROC curve (AUC) was 0.892, and the cutoff value in the ROC curve (calculated from the ROC curve point at the shortest distance from sensitivity 1 and specificity 1) was 0.014. At this time, the specificity was 0.840 and the sensitivity was 0.825.

[0174] like Figure 7 As shown in the lower left panel, in the sandwich assay based on rBC2LCN and anti-Gal3BP antibody (rBC2-Gal3BP), the area under the ROC curve (AUC) was 0.743, and the cutoff value in the ROC curve (calculated from the ROC curve point at the shortest distance from sensitivity 1 and specificity 1) was 0.01. At this time, the specificity was 0.900 and the sensitivity was 0.575.

[0175] In addition, if Figure 7 As shown in the lower right panel, in the sandwich assay (SerpinA3-SerpinA3) based on anti-SerpinA3 antibodies and anti-SerpinA3 antibodies, the area under the ROC curve (AUC) was 0.718, and the cutoff value in the ROC curve (calculated from the ROC curve point at the shortest distance from sensitivity 1 and specificity 1) was 1.056, at which point the specificity was 0.8 and the sensitivity was 0.65.

[0176] These results clearly demonstrate that the sandwich assay using rBC2LCN and an anti-SerpinA3 antibody (rBC2-SerpinA3) exhibits the highest accuracy, while the sandwich assay using rBC2LCN and an anti-Gal3BP antibody (rBC2-Gal3BP) demonstrates the highest specificity. Furthermore, it was confirmed that the sandwich assay using rBC2LCN and rBC2LCN (rBC2-rBC2) or the sandwich assay using an anti-SerpinA3 antibody and an anti-SerpinA3 antibody (SerpinA3-SerpinA3) exhibited somewhat useful accuracy.

[0177] In addition, ROC curves were created to investigate whether it was possible to distinguish between healthy individuals (n=49) and patients with pancreatitis (n=9) and pancreatic cancer (n=85). Figure 11As shown, it was confirmed that the sandwich assays based on rBC2LCN and anti-SerpinA3 antibodies (rBC2-SerpinA3) and the sandwich assays based on rBC2LCN and anti-Gal3BP antibodies (rBC2-Gal3BP) showed the best AUCs. Furthermore, it was confirmed that the assays have certain usefulness in the sandwich assays based on rBC2LCN and rBC2LCN (rBC2-rBC2) or the sandwich assays based on anti-SerpinA3 antibodies and anti-SerpinA3 antibodies (SerpinA3-SerpinA3).

[0178] Example 5: Serum analysis of pancreatic cancer patients at various stages

[0179] In this example, serum samples from patients with various pancreatic cancers ranging from stage 1A to stage 4 were evaluated by sandwich assay.

[0180] The stage of cancer is determined by doctors according to the UICC TNM classification (UICC: TNM Classification of Malignant Tumors, 8th Edn. Wiley-Blackwell; 2017.94-95). As described in Examples 1 and 3, the serum of cancer patients was subjected to a sandwich assay based on rBC2LCN and rBC2LCN, a sandwich assay based on rBC2LCN and anti-SerpinA3 antibody, a sandwich assay based on rBC2LCN and anti-Gal3BP antibody, and a sandwich assay based on anti-SerpinA3 antibody and anti-SerpinA3 antibody, respectively. The results are as follows. Figures 8A to 8D shown.

[0181] like Figures 8A to 8C As shown, samples showing high assay strength were found in all stages 1 to 4 in the sandwich assay based on rBC2LCN and rBC2LCN, the sandwich assay based on rBC2LCN and anti-SerpinA3 antibody, and the sandwich assay based on rBC2LCN and anti-Gal3BP antibody.

[0182] like Figure 8D As shown, in a sandwich assay based on anti-SerpinA3 antibody and anti-SerpinA3 antibody, Figure 6 As shown, high assay intensities were observed in all samples.

[0183] ROC curves were constructed between stage I pancreatic cancer patients (n=9) and healthy controls (n=49). Figure 12 As shown. Figure 12As shown in Figure 2, stage I pancreatic cancer patients were well detected in any sandwich assay. ROC curves were constructed between stage II pancreatic cancer patients (n=51) and healthy subjects (n=49). Figure 13 As shown. Figure 13 As shown in Figure 2, stage II pancreatic cancer patients were well detected in any sandwich assay. ROC curves were constructed between stage III pancreatic cancer patients (n=8) and healthy subjects (n=49). Figure 14 As shown. Figure 14 As shown, in any of the sandwich assays shown, patients with stage III pancreatic cancer were well detected. ROC curves were created between patients with stage IV pancreatic cancer (n=17) and healthy subjects (n=49). Figure 15 As shown. Figure 15 As shown, stage IV pancreatic cancer patients were well detected in any of the sandwich assays shown.

[0184] Pancreatic cancer patients underwent pancreatic resection surgery. Blood samples (n=38) before and after surgery were analyzed by sandwich assay. The results of the sandwich assay using rBC2LCN and anti-SerpinA3 antibody are shown in Figure 16 The results of the sandwich assay between rBC2LCN and anti-Gal3BP antibody are shown in Figure 17 The results of CA19-9 assay are shown in Figure 18 In each figure, 30POD means 30 days after surgery, and 3m means 3 months after surgery. Figures 16-18 As shown, the signals of each sandwich assay were reduced by cancer resection. Therefore, it can be understood that the results of cancer resection affect the levels of components in the blood in these assays.

[0185] In addition, blood samples from relapsed patients were assayed over time. The assay was a sandwich assay using BC2LCN and anti-SerpinA3 antibodies. Figure 19 As shown. Figure 19 As shown, the postoperative assay signal decreased over time in patient A. However, when the blood of relapsed patient A was analyzed, the signal increased significantly. This demonstrates that the assay system of the present invention can be used to detect relapse.

[0186] Therefore, by appropriately setting the cutoff value in the sandwich assay based on rBC2LCN and rBC2LCN, the sandwich assay based on rBC2LCN and anti-SerpinA3 antibody, and the sandwich assay based on rBC2LCN and anti-Gal3BP antibody, it is possible to detect a wide range of pancreatic cancer, such as early stage 1 and stage 2, to stage 4 pancreatic cancer.

Claims

1. Use of a first molecule selected from the group consisting of molecules binding to SerpinA3 and molecules binding to Gal3BP and a second molecule being BC2LCN in the manufacture of a kit for analyzing a biological sample obtained from a subject.

2. The use according to claim 1, wherein The first molecule is an antibody that binds to SerpinA3.

3. The use according to claim 1, wherein The first molecule is an antibody that binds to Gal3BP.

4. The use according to any one of claims 1 to 3, wherein The kit is for use in a subject having or having a likelihood of having cancer.

5. The use according to claim 4, wherein The subject has or is at risk for pancreatic cancer.

6. The use according to claim 5, wherein The subject has or is at risk for stage 1 or stage 2 pancreatic cancer.

7. The use according to any one of claims 1 to 3, wherein The kit is used to predict whether a subject has cancer.

8. The use according to any one of claims 1 to 3, wherein The kit is used for detecting a specific component in a biological sample obtained from a subject, wherein the specific component is a component selected from the group consisting of BC2LCN-binding SerpinA3 and BC2LCN-binding Gal3BP.

9. A sandwich assay kit comprising a first molecule selected from the group consisting of molecules binding to SerpinA3 and molecules binding to Gal3BP and a second molecule being BC2LCN, wherein the first and second molecules are used to measure the amount of glycoprotein in a subject's biological sample.

10. A kit for immunochromatographic assay, a kit for lectin electrophoresis, a kit for mass spectrometry analysis, a kit for lectin affinity chromatography, or a kit for μTAS, comprising a first molecule selected from the group consisting of a molecule that binds to SerpinA3 and a molecule that binds to Gal3BP, and a second molecule that is BC2LCN, and capable of measuring the amount of glycoprotein in a biological sample of a subject using the first molecule and the second molecule.

11. The kit according to claim 9 or 10, wherein The first molecule is an antibody that binds to SerpinA3, and the protein portion of the glycoprotein is SerpinA3.

12. The kit according to claim 9 or 10, wherein The first molecule is an antibody that binds to Gal3BP, and the protein portion of the glycoprotein is Gal3BP.

13. The kit according to claim 9 or 10, comprising: a second molecule which is solid-phased BC2LCN; and a labeled first molecule selected from the group consisting of a labeled antibody that binds to SerpinA3 and a labeled antibody that binds to Gal3BP. The kit according to claim 9 or 10, which is used for detecting cancer.

15. The kit according to claim 14, wherein The cancer is pancreatic cancer.

16. The kit according to claim 15, wherein The cancer is stage 1 or stage 2 pancreatic cancer.

17. A method comprising isolating one or more components selected from the group consisting of BC2LCN-binding SerpinA3 and BC2LCN-binding Gal3BP from a biological sample obtained from a subject.

18. The method according to claim 17, wherein The subject is a subject having or at risk of having pancreatic cancer.

19. The method according to claim 17, wherein Pancreatic cancer is stage I or stage II pancreatic cancer.

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