Cancer Screening Methods

Abstract

The present invention pertains to a method for detecting a specific component in a biological sample (for example, a blood sample) obtained from a target to predict whether or not the target suffers from cancer. The present invention provides a method that comprises separating one or more components selected from the group consisting of Serpin 3 and Gal 3BP in a biological sample. The present invention also provides a method that comprises detecting a glycoprotein contained in a biological sample (for example, a blood sample) obtained from a target by an assay, the assay being carried out by using a first molecule, which is selected from the group consisting of a molecule binding to a sugar chain having a BC2LCN-binding activity, a molecule binding to Serpin A3 and a molecule binding to Gal 3BP, and a second molecule binding to a sugar chain having a BC2LCN-binding activity.

Description

[Technical field]

[0001] The present invention relates to a cancer testing method and a test kit therefor. [Background technology]

[0002] BC2LCN is a carbohydrate-binding protein, i.e., a type of lectin, and according to previous reports, it has affinity for the glycoprotein glycans H-type1 (Fucα1-2Galβ1-3GlcNAc) and H-type3 (Fucα1-2Galβ1-3GalNAc), and it has been revealed that it can be used as a probe for detecting undifferentiated markers of human ES cells and iPS cells expressing podocalyxin having an H-type3 glycan (Patent Document 1). In addition, according to previous reports, it has been revealed that BC2LCN strongly binds to cancer tissues and can be used as a probe for detecting cancer markers (Patent Document 2). According to Patent Document 2, the presence of proteins recognized by BC2LCN and keratan sulfate has been confirmed in blood components of some cancer patients by a sandwich assay using BC2LCN and an antibody that binds to keratan sulfate.

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

[0004] Gal3BP, also called 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). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO2014 / 126146 [Patent Document 2] WO2017 / 061449 [Non-patent literature]

[0006] [Non-Patent Document 1] Nie, S. et al., J. Proteome Res. 13:1873-1884, 2014 [Non-Patent Document 2] Koths, K. et al., J. Biol. Chem. 268:14245-14249, 1993 Summary of the Invention

[0007] The present invention relates to a cancer testing method and a test kit therefor.

[0008] According to the present invention, the following inventions are provided. (1) A method for analyzing a blood sample obtained from a subject, comprising the steps of: A method comprising analyzing a blood component contained in a blood sample by a sandwich assay or immunochromatographic assay performed 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 which is BC2LCN. (2) The method according to (1) above, wherein the first molecule is an antibody that binds to SerpinA3. (3) The method according to (1) above, wherein the first molecule is an antibody that binds to Gal3BP. (4) The method according to (1) above, wherein the first molecule is BC2LCN. (5) The method according to any one of (1) to (4) above, wherein the subject has cancer or is at risk of having cancer. (6) The method according to (5) above, wherein the subject has or is likely to have pancreatic cancer. (7) The method according to (6) above, wherein the subject has or is likely to have stage 1 or 2 pancreatic cancer. (8) A kit for sandwich assay or immunochromatographic assay, 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 which is BC2LCN, and capable of measuring the amount of glycoprotein in a subject's blood sample using the first molecule and the second molecule. (9) The kit according to (8) above, wherein the first molecule is an antibody that binds to SerpinA3, and the protein portion in the glycoprotein is SerpinA3. (10) The kit according to (8) above, wherein the first molecule is an antibody that binds to Gal3BP, and the protein moiety in the glycoprotein is Gal3BP. (11) The kit described in (8) above, comprising a second molecule which is immobilized 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. (12) The kit according to any one of (8) to (11) above, which is used for detecting cancer. (13) The kit according to (12) above, wherein the cancer is selected from the group consisting of pancreatic cancer and colorectal cancer. (14) The kit according to (13) above, wherein the cancer is stage 1 or 2 pancreatic cancer. (15) A method for predicting whether or not a subject has cancer, the method comprising carrying out the method according to any one of (1) to (7) above. (16) A method for analyzing a blood sample obtained from a subject, comprising: A method comprising: analyzing the presence or concentration of a blood component in a blood sample, wherein the blood component is a component selected from the group consisting of a component capable of simultaneously binding two BC2LCNs, BC2LCN-binding SerpinA3, and BC2LCN-binding Gal3BP. According to the present invention, the following inventions can also be provided. (1A) A method for analyzing a biological sample obtained from a subject, comprising: A method comprising analyzing a component contained in a biological sample by an assay performed using a first molecule selected from the group consisting of a molecule that binds to a BC2LCN-binding glycan, a molecule that binds to SerpinA3, and a molecule that binds to Gal3BP, and a second molecule which is BC2LCN. (2A) The method according to (1A) above, wherein the first molecule is an antibody that binds to SerpinA3. (3A) The method described in (1A) above, wherein the first molecule is an antibody that binds to Gal3BP. (4A) The method according to (1A) above, wherein the first molecule is BC2LCN. (5A) The method according to any one of (1A) to (4A) above, wherein the subject has cancer or is at risk of having cancer. (6A) The method according to (5A) above, wherein the subject has or is likely to have pancreatic cancer. (7A) The method according to (6A) above, wherein the subject has or is likely to have stage 1 or 2 pancreatic cancer. (8A) A kit for a sandwich assay, a kit for immunochromatography assay, a kit for lectin electrophoresis, a kit for mass spectrometry, a kit for lectin affinity chromatography, or a kit for μTAS, which comprises 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 glycan, and which can measure the amount of glycoprotein in a subject biological sample using the first molecule and the second molecule. (9A) The kit according to (8A) above, wherein the first molecule is an antibody that binds to SerpinA3, and the protein moiety in the glycoprotein is SerpinA3. (10A) The kit according to (8A) above, wherein the first molecule is an antibody that binds to Gal3BP, and the protein moiety in the glycoprotein is Gal3BP. (11A) The kit described in (8A) above, comprising a second molecule which is immobilized 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. (12A) The kit according to any one of (8A) to (11A) above, which is used for detecting cancer. (13A) The kit according to (12A) above, wherein the cancer is pancreatic cancer. (14A) The kit according to (13A) above, wherein the cancer is stage 1 or 2 pancreatic cancer. (15A) A method for predicting whether or not a subject has cancer, the method comprising carrying out any of the methods described in (1A) to (7A) above. (16A) A method for detecting a specific component in a biological sample obtained from a subject, comprising: A method wherein the specific component is a component selected from the group consisting of a component capable of simultaneously binding two BC2LCNs, a BC2LCN-binding SerpinA3, and a BC2LCN-binding Gal3BP. (17A) A method comprising isolating one or more components selected from the group consisting of components capable of simultaneously binding to two BC2LCNs, SerpinA3 and Gal3BP, from a biological sample obtained from a subject. (18A) The method according to (17A) above, wherein the subject has pancreatic cancer or is likely to have pancreatic cancer. (19A) The method according to (17A) above, wherein the pancreatic cancer is stage I or II pancreatic cancer. (20A) The method according to any one of (17A) to (19A) above, further comprising measuring the amount of the separated components. (21A) The method according to (20A) above, further comprising comparing the amount of the measured component with a cutoff value. (22A) The method according to (21A) above, wherein the cutoff value is a value equal to or greater than the average amount of a component corresponding to the measured component in healthy subjects and equal to or less than the average amount of a component corresponding to the measured component in pancreatic cancer patients. (23A) The method according to any one of (17A) to (19A) above, further comprising detecting the separated components. [Brief description of the drawings]

[0009] [Figure 1] Figure 1 shows 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. [Diagram 2] Figure 2 shows the results of Western blotting with BC2LCN (left) and silver staining (right) of blood components that bind to BC2LCN precipitated from a blood sample using BC2LCN, followed by gel electrophoresis. [Diagram 3] Figure 3 shows the results of precipitating blood components that bind to BC2LCN from a blood sample using BC2LCN, followed by gel electrophoresis and Western blotting with an antibody against SeprinA3 (left) and an antibody against Gal3BP (right). [Figure 4] Figure 4 shows the results of a sandwich assay performed using antibodies that bind to BC2LCN and SerpinA3 in blood samples from patients with various cancers and healthy individuals. [Diagram 5] FIG. 5 shows the results of a sandwich assay performed using antibodies that bind to BC2LCN and Gal3BP in blood samples from patients with various cancers and healthy individuals. [Figure 6] Figure 6 shows the concentrations of blood factors that can be simultaneously recognized by two BC2LCN molecules, 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 individuals. [Figure 7]Figure 7 shows the ROC curves showing the relationship between the concentrations of blood factors that can be simultaneously recognized by two molecules of BC2LCN in blood samples from pancreatic cancer patients and healthy individuals, the concentration of BC2LCN-binding SerpinA3, the concentration of BC2LCN-binding Gal3BP, and the concentration of SerpinA3 measured using two different types of anti-SerpinA3 antibodies, as indicators, and the sensitivity and specificity of an evaluation system for detecting pancreatic cancer. [Figure 8A] FIG. 8A shows the results of measuring the concentrations of blood factors that can be simultaneously recognized by two molecules of BC2LCN in blood samples from pancreatic cancer patients according to the stage of the UICC TMN classification. [Figure 8B] FIG. 8B shows the results of measuring the concentration of BC2LCN-binding SerpinA3 in blood samples from pancreatic cancer patients according to stage according to the UICC TMN classification. [Figure 8C] FIG. 8C shows the results of measuring the concentration of BC2LCN-binding Gal3BP in blood samples from pancreatic cancer patients according to the UICC TMN classification stage. [Figure 8D] FIG. 8D shows the results of measuring the concentration of Serpin A3 in blood samples from pancreatic cancer patients according to the UICC TMN classification, using two different types of anti-Serpin A3 antibodies. [Figure 9] Figure 9 shows the results of histological staining with rBC2LCN (sometimes simply referred to as "rBC2" in the figures) of pancreatic tissue sections, including normal and pancreatic cancer, as well as immunohistological staining for SerpinA3 and Gal3BP. [Figure 10] Figure 10 shows the results of analysis of blood samples from healthy subjects, patients with chronic pancreatitis, and patients with pancreatic cancer in a sandwich assay using two BC2LCN molecules, a sandwich assay using BC2LCN and an anti-Serpin A3 antibody, a sandwich assay using BC2LCN and an anti-Gal3BP antibody, an ELISA using an anti-Serpin A3 antibody, and an ELISA using an anti-Gal3BP antibody. [Figure 11]Figure 11 shows the ROC curves for detecting pancreatic cancer patients from healthy subjects and patients with chronic pancreatitis in the following systems: sandwich assay using two molecules of BC2LCN, sandwich assay using BC2LCN and anti-SerpinA3 antibody, sandwich assay using BC2LCN and anti-Gal3BP antibody, ELISA using anti-SerpinA3 antibody, and ELISA using anti-Gal3BP antibody. Analysis was performed using blood samples. [Figure 12] Figure 12 shows ROC curves for detecting stage I pancreatic cancer patients from healthy subjects in the following systems: sandwich assay using two molecules of BC2LCN, sandwich assay using BC2LCN and anti-SerpinA3 antibody, sandwich assay using BC2LCN and anti-Gal3BP antibody, ELISA using anti-SerpinA3 antibody, and ELISA using anti-Gal3BP antibody. Analysis was performed using blood samples. [Figure 13] Figure 13 shows the ROC curves for detecting stage II pancreatic cancer patients from healthy subjects in the following systems: sandwich assay using two molecules of BC2LCN, sandwich assay using BC2LCN and anti-SerpinA3 antibody, sandwich assay using BC2LCN and anti-Gal3BP antibody, ELISA using anti-SerpinA3 antibody, and ELISA using anti-Gal3BP antibody. Analysis was performed using blood samples. [Figure 14] Figure 14 shows the ROC curves for detecting stage III pancreatic cancer patients from healthy subjects in 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. The analysis was performed using blood samples. [Figure 15] Figure 15 shows the ROC curves for detecting stage IV pancreatic cancer patients from healthy subjects in 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. The analysis was performed using blood samples. [Figure 16] FIG. 16 shows the results of analyzing blood samples from a patient before pancreatic cancer removal surgery, 30 days (30POD) after the surgery, and 3 months (3m) after the surgery by a sandwich assay using BC2LCN and anti-SerpinA3 antibody. [Figure 17] FIG. 17 shows the results of analyzing blood samples from a patient before pancreatic cancer removal surgery, 30 days (30POD) after the surgery, and 3 months (3m) after the surgery by a sandwich assay using BC2LCN and anti-Gal3BP antibody. [Figure 18] FIG. 18 shows the results of analyzing blood samples from a patient before pancreatic cancer removal surgery, 30 days (30POD) after the surgery, and 3 months (3m) after the surgery by ELISA using CA19-9. [Figure 19] FIG. 19 shows the results of analysis of blood samples from a patient (Patient A) before pancreatic cancer resection, 1 day (1POD) after the surgery, 7 days (7POD) after the surgery, 30 days (30POD) after the surgery, and after recurrence, using a sandwich assay with BC2LCN and anti-Serpin A3 antibody. Detailed Description of the Invention

[0010] As used herein, the term "subject" refers to a mammal, particularly a mammal including a human. As used herein, the term "subject" may be a healthy individual, or may be a patient with cancer or a patient at risk of having cancer.

[0011] As used herein, "tumor" refers to a cell population that exhibits autonomous growth. Autonomous growth refers to the property of cells that escape normal cell growth control in the body and continue to grow autonomously. Tumors are broadly classified into benign tumors and malignant tumors (cancer). Benign tumors are tumors that grow autonomously but do not metastasize or invade, or cause cachexia. Malignant tumors, in addition to autonomous growth, cause one or more of invasion into normal tissues, metastasis, and cachexia.

[0012] As used herein, a "biological sample" refers to a sample (e.g., tissue, cell, extracellular fluid, and bodily fluid) obtained from a subject. Bodily fluids include blood, urine, peritoneal fluid, pleural fluid, and secretions (e.g., endocrine and exocrine fluids, e.g., digestive fluids such as pancreatic juice). As used herein, a "blood sample" refers to blood (e.g., peripheral blood) or blood-derived samples (e.g., serum and plasma) obtained from a subject. As used herein, a "blood component" refers to a component contained in blood, particularly a liquid component contained in blood. As used herein, when the biological sample is a blood sample, the component is a blood component, as described below.

[0013] As used herein, "antibody" refers to an immunoglobulin. An antibody is a biological molecule that has the property of binding to an antigen. An antibody can have specificity for an antigen. An antibody can be a monoclonal or polyclonal antibody. An antibody can include bispecific antibodies. An antibody can be a full-length antibody or a fragment thereof that has binding ability for an antigen.

[0014] As used herein, "BC2LCN" refers to Burkholderia CenocepaciaIt is the N-terminal domain of BC2LCN, a lectin derived from the fibronectin β-lactamase (Sulak, O., et al., Structure, 18(1):59-72, 2010). Previous reports have revealed that BC2LCN has affinity for the glycoprotein glycans H-type1 (Fucα1-2Galβ1-3GlcNAc) and H-type3 (Fucα1-2Galβ1-3GalNAc), and that BC2LCN can be used as an undifferentiated marker for human ES cells and iPS cells expressing podocalyxin with H-type3 glycan (WO2014 / 1236146). Previous reports have also revealed that BC2LCN is specifically expressed in cancer tissues and can be used to detect cancer (WO2017 / 061449). According to WO2017 / 061449, the presence of proteins recognized by BC2LCN and keratan sulfate has been confirmed in blood components of some cancer patients by a sandwich assay using an antibody that binds to BC2LCN and keratan sulfate. B. CenocepaciaBC2LCN produced using a host other than the above may be referred to as recombinant BC2LCN or rBC2LCN. In this specification, the glycan or protein to which BC2LCN binds is referred to as a BC2LCN-binding glycan or protein. The BC2LCN-binding protein is modified with a BC2LCN-binding glycan. The BC2LCN-binding glycan may be an H-type 1 glycan and / or an H-type 3 glycan. Examples of BC2LCN include lectins registered as YP_002232818 at the National Center for Biotechnology Information (NCBI). BC2LCN can be used as long as it has the ability to bind to glycans, but BC2LCN can also be used that has been modified (e.g., improved) in its ability to bind to glycans as disclosed in JP2020-146027. Modified versions of BC2LCN may also be used. BC2LCN includes a protein (BC2LCN) having an amino acid sequence that is 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identical to the amino acid sequence of the lectin registered under YP_002232818. BC2LCN binds to at least one selected from the group consisting of H-type3 glycan and H-type1 glycan.

[0015] As used herein, "SerpinA3" is a serine protease inhibitor (serpin) also known as α1 antichymotrypsin (AACT). In humans, it can be a protein obtained by translation from an mRNA having a nucleotide sequence registered under GenBank accession number K01500.1. In humans, SerpinA3 protein can be generated by cleaving a signal peptide consisting of the 1st to 23rd amino acid sequence from a 423 amino acid long precursor (for example, NCBI reference number: NP_001076.2).

[0016] As used herein, "Gal3BP" refers to a galectin-3-binding protein. Gal3BP may be a protein obtained by translation from an mRNA having a base sequence registered under the US National Center for Biotechnology Information (NCBI) reference number NM_005567.4. In humans, Gal3BP protein may be generated by, for example, cleaving a signal peptide consisting of the 1st to 18th amino acid sequence from a 585 amino acid long precursor (e.g., having an amino acid sequence registered under NCBI 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 cancer, lung cancer, colorectal cancer, ovarian cancer, and endometrial cancer (Koths, K. et al., J. Biol. Chem. 268: 14245-14249, 1993).

[0017] As used herein, a "sandwich assay" is an assay used to detect a target component contained in a sample. A sandwich assay can be performed by a person skilled in the art using a conventional method. A sandwich assay is an assay in which a target component is usually brought into contact with a capture molecule A that binds to the target component immobilized on a support (e.g., a substrate or a bead) to capture the target component on the support, and then, after removing (or separating) the unbound target component, a capture molecule B that binds to the labeled target component is brought into contact, and the target component captured on the support is detected using the label as an indicator. The support that can be used in the present invention can be any support (especially an insoluble support) used in a typical immunoassay, and examples of such supports include organic substances such as 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, and ethylene-maleic anhydride copolymer; inorganic substances such as glass, silicon oxide, diatom, porous glass, ground glass, alumina, silica gel, and metal oxide; magnetic substances such as iron, cobalt, nickel, magnetite, and chromite; and alloys of these magnetic substances. The solid phase can be immobilized on a solid phase (for example, any surface such as beads, magnetic beads, membranes, and plates). Various commercially available magnetic beads can be used. As the magnetic beads, for example, beads disclosed in WO2012 / 173002A may be used. In a sandwich assay, the above-mentioned immobilized capture molecule A and the above-mentioned labeled capture molecule B usually need to bind to the target component simultaneously without competing with each other for binding to the target component. The capture molecule A may be a first molecule described below, and the capture molecule B may be a second molecule. The capture molecule A may be a second molecule described below, and the capture molecule B may be a first molecule.

[0018] The sandwich assay can also be performed by a microfluidic chip (micro-total analysis systems; μTAS). The microfluidic chip includes a flow path, and the flow path has a sample inlet. For example, at 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 between the capture molecule X and the target component, and an electric field is applied to move the complex. When the complex comes into contact with a labeled capture molecule Y present in the middle of the flow path, a complex of the target component, the capture molecule X, and the capture molecule Y is formed, and the complex is further moved through the flow path by the electric field and reaches a detection section on the flow path, where it can be detected by the label bound to the capture molecule Y. Detection can be performed optically, for example, by a laser or the like. The capture molecule X can be a first molecule described below, and the capture molecule Y can be a second molecule. The capture molecule X can be a second molecule described below, and the capture molecule Y can be a first molecule. The presence of multiple biomarkers can also be detected in parallel using μTAS. Other sandwich assays include an assay (e.g., AlphaLISA) in which a capture molecule N and a capture molecule M are immobilized on separate beads A and B, respectively, and when a target molecule is present, a complex is formed between the target molecule, the capture molecule N, and the bead A, and between the capture molecule M and the bead B. By irradiating excitation light to bead A, which contains a photosensitizer that releases singlet oxygen upon excitation, singlet oxygen reaches bead B only when a complex is formed (when beads A and B are in close proximity), causing bead B to emit chemiluminescence, and the presence of the target molecule is detected by detecting the light from bead B. Thus, in the present invention, an AlphaLISA assay kit can be provided that includes beads on which BC2LCN is immobilized and beads on which an antibody that binds to SerpinA3 or Gal3BP is immobilized. In the present invention, an assay kit for detecting the presence of a target molecule using fluorescence resonance energy transfer (FRET) and 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 may also be used. In this case, beads or supports for immobilization are not required. In the present invention, such an assay kit may be provided.

[0019] In this specification, the term "immunochromatographic assay" refers to an assay used to detect a target component contained in a sample, and is usually an assay in which a capture molecule C labeled with a label such as gold colloid is brought into contact with a target component contained in a liquid sample to form a complex between 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 immobilized on a test line to capture the complex between the labeled capture molecule C and the target component on the test line, and the label on the test line is used as an indicator for detection. In an immunochromatographic assay, the labeled capture molecule C and the capture molecule D immobilized on the test line usually do not compete with each other for binding to the target component, and must bind to the target component at the same time. In an immunochromatographic assay, a control line is provided in addition to the test line, and a molecule (such as an antibody, which may be an anti-IgG antibody if the capture molecule C is an IgG antibody) that binds to the capture molecule C is immobilized on the control line, and the labeled capture component C that has not bound to the target component can be captured. In the immunochromatographic assay, a strip is provided that includes, in this order, a sample pad for containing a sample, a conjugate pad in which a labeled capture molecule C is present, a membrane that is a mobile layer in which a complex between the target component in the sample and the capture molecule C moves, and an absorbent pad that absorbs moisture in the sample. On the membrane, there are a test line on which a capture molecule D is immobilized and a control line on which an antibody against the capture molecule C is immobilized. When a sample is contained in the sample pad, if the sample contains the target component, it moves on the strip toward the absorbent pad. When it moves to the conjugate pad, the labeled capture molecule C and the target component form a complex, and the complex is captured by the capture molecule D immobilized on the test line and also captured on the control line. If the sample does not contain the target component, the sample moves toward the absorbent pad, and the labeled capture molecule C does not form a complex and is not captured on the test line, but only on the control line. As a result, if the control line and the test line develop color, it indicates that the sample contains the target component, and if the control line and the test line do not develop color, 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 working properly. The capture molecule C may be a first molecule described below, and the capture molecule D may be a second molecule. The capture molecule C may be a second molecule described below, and the capture molecule D may be a first molecule.

[0020] In this specification, "lectin electrophoresis" refers to electrophoresis using a gel containing lectin, and is a method for separating lectin-binding molecules that utilizes the fact that molecules that interact with lectin during electrophoresis move slower than molecules that do not interact with lectin. The lectin is free in the gel or immobilized on the gel. Since lectin hardly moves by electrophoresis, the migration speed of glycoproteins can be reduced by simply mixing it with the gel material to prepare a gel. Alternatively, the lectin is non-covalently or covalently linked to the gel. When separating BC2LCN-binding molecules from molecules that do not bind to BC2LCN, biological samples can be separated by lectin electrophoresis using BC2LCN as the lectin. Molecules that have BC2LCN-binding glycans repeatedly interact with the lectin immobilized on the gel during electrophoresis, so their migration is delayed. On the other hand, molecules that do not bind to BC2LCN do not interact with the lectin immobilized on the gel and migrate quickly. Using this principle, molecules having BC2LCN-binding glycans can be separated from molecules not capable of binding to BC2LCN by electrophoresis. Lectin electrophoresis can be performed appropriately by those skilled in the art.

[0021] In this specification, "lectin affinity chromatography" refers to chromatography that utilizes the property of lectin to specifically bind to glycans. In lectin affinity chromatography, for example, affinity chromatography is performed on a sample using a column containing an insoluble support on which lectin has been immobilized. When a component in a sample has affinity for lectin, the component is separated from other components. In the present invention, for example, BC2LCN can be used as the lectin.

[0022] According to the present invention, the sera of patients with various cancers were analyzed and it was found that in a sandwich assay using solid-phase BC2LCN and labeled BC2LCN, there exist blood components that can simultaneously bind to two BC2LCNs, and that the amount of these blood components in the serum of patients with cancer tends to be higher than that of healthy individuals. Furthermore, according to the present invention, the sera of patients with various cancers were analyzed, and it was found that in a sandwich assay using BC2LCN and an antibody that binds to SerpinA3, BC2LCN-binding SerpinA3 exists as a blood component, and that the amount of BC2LCN-binding SerpinA3 in the serum of cancer patients tends to be higher than that of healthy individuals. Further, according to the present invention, the sera of patients with various cancers were analyzed, and it was found that in a sandwich assay using BC2LCN and an antibody that binds to Gal3BP, BC2LCN-binding Gal3BP exists as a blood component, and that the amount of BC2LCN-binding Gal3BP in the serum of cancer patients tends to be higher than that of healthy individuals.

[0023] According to the present invention, 1. A method for analyzing a biological sample obtained from a subject, 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 a biological sample obtained from a subject; method The method of the present invention may include providing a biological sample. The method of the present invention may further include measuring the amount of the component.

[0024] According to the present invention, one or more components selected from the group consisting of SerpinA3 (e.g., BC2LCN-binding SerpinA3) and Gal3BP (e.g., BC2LCN-binding Gal3BP) can be detected using a molecule that binds to the component. In some embodiments, the one or more components can be separated from the other components prior to detection of the one or more components. For example, SerpinA3 can be detected using a molecule that binds to SerpinA3. Two different molecules that can bind to SerpinA3 simultaneously can also be used. When two different molecules that can bind to SerpinA3 simultaneously are used, SerpinA3 can be detected by immunochromatographic assay or sandwich assay. SerpinA3 can also be detected by mass spectrometry. In either case, a calibration curve can be obtained using a standard substance for SerpinA3. Also, SerpinA3 can be quantified by applying the measured values ​​for SerpinA3 to the obtained verification line. In addition, BC2LCN-binding SerpinA3 can be detected using a molecule that binds to a BC2LCN-binding glycan and a molecule that binds to SerpinA3. The molecule that binds to a BC2LCN-binding glycan may include separating the BC2LCN-binding molecule from the others using a molecule that binds to a BC2LCN-binding glycan, or, when the molecule that binds to a BC2LCN-binding glycan is BC2LCN, may include separating the BC2LCN-binding molecule from the others using lectin affinity chromatography or lectin electrophoresis. In addition, it may include separating SerpinA3 from the other components. Either the separation of SerpinA3 or the separation of the BC2LCN-binding component may be performed first. For example, Gal3BP can be detected using a molecule that binds to Gal3BP. Two different molecules that can simultaneously bind to Gal3BP can also be used. When 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 either case, a calibration curve can be obtained using a standard substance for Gal3BP. Moreover, Gal3BP can be quantified by applying the measured values ​​for Gal3BP to the obtained verification line. Furthermore, BC2LCN-binding Gal3BP can be detected using a molecule that binds to a BC2LCN-binding glycan and a molecule that binds to Gal3BP. The molecule that binds to a BC2LCN-binding glycan may include separating the BC2LCN-binding molecule from other molecules using a molecule that binds to a BC2LCN-binding glycan, or, when the molecule that binds to a BC2LCN-binding glycan is BC2LCN, may include separating the BC2LCN-binding molecule from other molecules using lectin electrophoresis. Also, it may include separating Gal3BP from other components. Either the separation of Gal3BP or the separation of the BC2LCN-binding component may be performed first.

[0025] According to the present invention, 1. A method for analyzing a biological sample obtained from a subject, comprising: A method comprising: analyzing a component (sometimes referred to as a "target component") contained in a biological sample by an assay carried out using a first molecule selected from the group consisting of a molecule that binds to a BC2LCN-binding glycan, a molecule that binds to SerpinA3, and a molecule that binds to Gal3BP, and a second molecule that is BC2LCN. is provided.

[0026] The biological sample is a biological sample obtained from the subject. The subject may have cancer or may have the possibility of having cancer. The cancer may be, for example, pancreatic cancer. The pancreatic cancer may be, for example, pancreatic cancer at a stage selected from the group consisting of stages I, II, III, and IV, for example, pancreatic cancer at stage I, for example, pancreatic cancer at stage II. The biological sample may be, for example, tissue, cell, and body fluid. The body fluid may be blood (for example, plasma and serum), sweat, saliva, urine, ascites, pleural fluid, and secretions (for example, endocrine and exocrine fluids, for example, digestive fluids such as pancreatic juice). The tissue and cell may be, for example, a tissue and cell that is cancerous or suspected to be cancerous, respectively. The tissue and cell may be, for example, a part that is cancerous or suspected to be cancerous, respectively.

[0027] Examples of molecules that bind to BC2LCN-binding glycan include antibodies or antigen-binding fragments thereof that bind to BC2LCN-binding glycan. Examples of molecules that bind to BC2LCN-binding glycan include aptamers (e.g., nucleic acid aptamers selected from the group consisting of DNA and RNA) that bind to BC2LCN-binding glycan. Examples of molecules that bind to BC2LCN-binding glycan further include medium-sized molecules such as cyclic peptides. These molecules that bind to BC2LCN-binding glycan can be obtained by methods well known to those skilled in the art. For example, antibodies that bind to BC2LCN-binding glycan can be obtained from non-human mammals immunized with BC2LCN-binding glycan. Aptamers that bind to BC2LCN-binding glycan can be obtained from a nucleic acid pool using affinity for BC2LCN-binding glycan. Cyclic peptides that bind to BC2LCN-binding glycan can be obtained from a cyclic peptide pool using affinity for BC2LCN-binding glycan. The molecule that binds to BC2LCN-binding glycan can be, for example, BC2LCN.

[0028] The molecule that binds to SerpinA3 includes an antibody that binds to SerpinA3 or an antigen-binding fragment thereof. The molecule that binds to SerpinA3 also includes an aptamer that binds to SerpinA3 (e.g., a nucleic acid aptamer selected from the group consisting of DNA and RNA). The molecule that binds to SerpinA3 further includes a medium molecule such as a cyclic peptide. These molecules that bind to SerpinA3 can be obtained by methods well known to those skilled in the art. For example, an antibody that binds to SerpinA3 can be obtained from a non-human mammal immunized with SerpinA3. An aptamer that binds to SerpinA3 can be obtained from a nucleic acid pool using its affinity to SerpinA3. A cyclic peptide that binds to SerpinA3 can be obtained from a cyclic peptide pool using its affinity to SerpinA3. SerpinA3 may form a complex with other components in a biological sample. In the present invention, such a complex may be detected. For example, another component that forms a complex with SerpinA3 includes PSA. Therefore, SerpinA3 can also be detected using a molecule that binds to PSA and a molecule that binds to SerpinA3. Alternatively, SerpinA3 can be detected by using labeled recombinant PSA, taking advantage of the property of SerpinA3 to bind to PSA.

[0029] The molecule that binds to Gal3BP includes an antibody that binds to Gal3BP or an antigen-binding fragment thereof. The molecule that binds to Gal3BP also includes an aptamer that binds to Gal3BP (e.g., a nucleic acid aptamer selected from the group consisting of DNA and RNA). The molecule that binds to Gal3BP further includes a medium molecule such as a cyclic peptide. These molecules that bind to Gal3BP can be obtained by methods well known to those skilled in the art. For example, an antibody that binds to Gal3BP can be obtained from a non-human mammal immunized with Gal3BP. An aptamer that binds to Gal3BP can be obtained from a nucleic acid pool using its affinity for Gal3BP. A cyclic peptide that binds to Gal3BP can be obtained from a cyclic peptide pool using its affinity for Gal3BP. Gal3BP may form a complex with other components in a biological sample. In the present invention, such a complex may be detected. For example, another component that forms a complex with Gal3BP includes galectin (particularly, galectin 3). Therefore, Gal3BP can also be detected using a molecule that binds to galectin (particularly, galectin 3) and a molecule that binds to Gal3BP. Alternatively, Gal3BP can be detected by using a labeled recombinant galectin (particularly galectin 3) and taking advantage of the property of Gal3BP binding to galectins (particularly galectin 3).

[0030] According to the present invention there is provided a method for analyzing a biological sample (e.g. a blood sample) obtained from a subject, comprising: A method is provided which comprises analyzing the presence or concentration of a component (a specific component, e.g., a blood component) in a biological sample (e.g., a blood sample), where the component (e.g., blood component) is a component (sometimes referred to as a target component (e.g., a target blood component)) selected from the group consisting of components capable of simultaneously binding two BC2LCNs, SerpinA3 (e.g., BC2LCN-binding SerpinA3), and Gal3BP (e.g., BC2LCN-binding Gal3BP). In the analytical method of the present invention, detection of one, two, or all of the above components indicates a possibility that the subject has cancer. In the analytical method of the present invention, detection of a concentration of one of the above components greater than a cutoff value indicates a possibility that the subject has cancer.

[0031] In the analysis method of the present invention, the subject may be a human, for example, a healthy individual, a patient with cancer, or a patient who may have cancer.

[0032] In some embodiments, the cancer may be one or more cancers selected from the group consisting of pancreatic cancer, colon cancer, rectal adenocarcinoma, bladder cancer, breast cancer, non-small cell lung cancer, gastric cancer, cervical cancer, uterine cancer, hepatocellular carcinoma, thyroid cancer, prostate cancer, melanoma, esophageal cancer, ovarian cancer, bile duct cancer, non-Hodgkin's lymphoma, and chronic myeloid leukemia. In some embodiments, the cancer may be pancreatic cancer. In some embodiments, the pancreatic cancer may be at a stage selected from the group consisting of stage I and II. In some embodiments, the pancreatic cancer may be at stage I. In some embodiments, the pancreatic cancer may be at stage II.

[0033] In some embodiments, the target component or target blood component is a component capable of simultaneously binding two BC2LCNs. In some embodiments, the target component or target blood component is BC2LCN-binding SerpinA3. In some embodiments, the target component or target blood component is BC2LCN-binding Gal3BP. In these embodiments, the cancer may be as described above, but may be, for example, one or more cancers selected from the group consisting of pancreatic cancer, colon cancer, rectal adenocarcinoma, bladder cancer, breast cancer, uterine cancer, hepatocellular carcinoma, thyroid cancer, melanoma, esophageal cancer, bile duct cancer, ovarian cancer, non-Hodgkin's lymphoma, and chronic myeloid leukemia. In some embodiments, the cancer may be pancreatic cancer. In some embodiments, the pancreatic cancer may be at a stage selected from the group consisting of stages I and II. In some embodiments, the pancreatic cancer may be at stage I. In some embodiments, the pancreatic cancer may be at stage II.

[0034] In one embodiment, the cutoff value can be, for example, 0, the first quartile, average, third quartile, or maximum value of the concentration of a target component (e.g., a target blood component) in a healthy subject group, or a value between any two of these (e.g., the third quartile or maximum value, or a value between them). In one embodiment, the cutoff value can be, for example, 0, the first quartile, average, third quartile, or maximum value of the concentration of a target component (e.g., a target blood component) in a cancer patient group, or a value between any two of these. In some embodiments, the cutoff value may be the cutoff value at the point on the ROC curve that is closest to the point of sensitivity 1 and specificity 1, a value greater than the cutoff value, or a value less than the cutoff value. In some embodiments, the cutoff value may also be the cutoff value at the point where the Youden Index is at its maximum, a value greater than the cutoff value, or a value less than the cutoff value.

[0035] The cutoff value can be determined by balancing the sensitivity and false positive rate. The cutoff value can be set low when it is desired to detect as many cancer patients as possible even if there are many false positives (when it is desired to increase the sensitivity), and can be set high when it is desired to reduce false positives (when it is acceptable to reduce the sensitivity instead). Those skilled in the art can set the cutoff value appropriately according to the purpose of the test.

[0036] In the analytical 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 can be obtained that the subject may have cancer. Thus, the analytical 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.

[0037] 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 can be obtained that the subject may have pancreatic cancer. 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.

[0038] In the analysis method of the present invention, a target component (e.g., a target blood component) can be detected even in a biological sample (e.g., a blood sample) of a patient having early cancer (stages I and II). Thus, the analysis method of the present invention can include providing information indicating that a subject may have early 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 the subject is greater than a cutoff value. The early cancer can be early pancreatic cancer. The stage classification of pancreatic cancer can be determined by a physician based on the UICC TNM Classification of Malignant Tumours, 8th Edn. Wiley-Blackwell; 2017.94-95). Stage I includes stages 1A and 1B, and stage II includes stages 2A and 2B. Stages III and IV are synonymous with stages 3 and 4, respectively.

[0039] [Table 1]

[0040] The provision of information can be achieved by transmitting or outputting information. The output of information can be on a display or in print.

[0041] The analytical method of the present invention may further comprise determining that the subject has a likelihood of having cancer.

[0042] According to the present invention, there is provided a method for treating cancer, comprising subjecting at least a portion of a subject determined to possibly have cancer in the analysis method of the present invention to a cancer therapy. The cancer therapy may be one or more selected from the group consisting of radiation therapy, chemotherapy, and surgical therapy. The cancer therapy may be a standard therapy. The radiation therapy may include irradiating the cancer with radiation. The chemotherapy may include administering an anticancer drug to the subject. The surgical therapy may include resecting the cancer. Examples of chemotherapy for cancer (e.g., pancreatic cancer) include, for example, forfirinox therapy, gemcitabine-nab-paclitaxel therapy (GnP therapy), gemcitabine-S1 therapy (GS therapy), liposomal irinotecan-5FU / LV therapy (Nal-IRI / FL therapy), gemcitabine therapy, and S1 therapy, and the subject may be treated with any one or more of these therapies. Examples of anticancer agents include one or more selected from the group consisting of platinum preparations such as oxaliplatin, irinotecan, liposomal irinotecan, levofolinate, gemcitabine, nab-paclitaxel, 5-fluorouracil (5-FU), and S-1. According to the present invention, a pharmaceutical composition can be provided that contains one or more therapeutically effective amounts of anticancer agents selected from the group consisting of the above-mentioned anticancer agents, and is used to treat cancer in a subject determined to have a possibility of having cancer by the analysis method of the present invention. The pharmaceutical composition may further contain a pharmacologic active ingredient and a pharmacologic acceptable excipient. The pharmacologic acceptable excipient can be appropriately selected by a person skilled in the art.

[0043] In one embodiment of the analytical method of the present invention, 1. A method for analyzing a biological sample (e.g., a blood sample) obtained from a subject, comprising: A method is provided, comprising analyzing a component (e.g., a blood component) contained in a biological sample (e.g., a blood sample) by a sandwich assay or immunochromatographic assay performed 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.

[0044] In some embodiments, the first molecule can be immobilized on a support (e.g., a substrate) and the second molecule can be labeled. In some embodiments, the first molecule can be labeled and the second molecule can be immobilized on a support.

[0045] For the immobilization, known immobilization methods such as a direct method, a polystyrene tag method (WO2018 / 190357), a biotin-streptavidin method, a HaloTag method, a method using chemical bonds such as an amine coupling method, and a V5 tag method can be used. In the direct method, the capture molecule to be immobilized is directly contacted with the support, and thus immobilized on the support. In the polystyrene tag method, the capture molecule is immobilized on a polystyrene plate via a polystyrene tag fused to the capture molecule. In the biotin-streptavidin method, the capture molecule is immobilized on the support by binding a capture molecule modified with biotin to a support coated with streptavidin. In the HaloTag method, the capture molecule is immobilized on a support coated with HaloTag via a HaloTag protein fused to the capture molecule. In the amine coupling method, the carboxyl group formed on the solid surface is activated and proteins and the like can be immobilized by chemical bonds via the amino group. In the V5 tag method, a capturing molecule linked to a V5 tag is immobilized on a support coated with an anti-V5 tag antibody by binding the capturing molecule to the support.

[0046] As the label, substrates (chromogenic substrates, fluorescent substrates, and luminescent substrates) and enzymes used in enzyme antibody techniques (e.g., peroxidase, glucose oxidase, and alkaline phosphatase) can be used in sandwich assays. Labeling can be performed by a conventional method by those skilled in the art. Labeling can be performed, for example, by covalent bonding. Labeling can be performed, for example, by reacting a biotin-linked antibody with an avidin-linked label. As the substrate, for example, a fluorescent substrate can be used. As the peroxidase, for example, horseradish peroxidase can be used. Horseradish peroxidase generates color, fluorescence, or chemiluminescence by adding a chromogenic substrate, fluorescent substrate, or luminescent substrate. Therefore, the presence of the labeled capture molecule can be detected using color, fluorescence, or chemiluminescence as an indicator. Colorimetric substrates for horseradish peroxidase include, for example, tetramethylbenzidine (TMB), o-phenylenediamine (OPD), 2,2-azinobis[3-ethylbenzo-thiazoline-6-sulfonic acid (ABTS), and Amplex™ Red, which can be used to detect labeled molecules in the presence of hydrogen peroxide. Luminescent substrates for alkaline phosphatase include p-nitrophenyl phosphate (pNPP), 4-methylumbelliferyl phosphate (4-MUP), and AttoPhos™, which can be used to detect captured molecules. Glucose oxidase oxidizes glucose to generate gluconic acid and hydrogen peroxide. Hydrogen peroxide can be easily detected, for example, using a colorimetric probe for detecting hydrogen peroxide (e.g., peroxidase). Hydrogen peroxide can be colored, for example, in the presence of peroxidase and its colorimetric substrate.

[0047] In immunochromatographic assays, dyes (eg, colorants such as gold colloid) can be used as labels.

[0048] In one embodiment, the first molecule is BC2LCN. In this embodiment, recombinant BC2LCN can be used as BC2LCN. BC2LCN may be directly immobilized, or may be, for example, biotin-labeled and immobilized on a support coated with streptavidin (or neutravidin). Alternatively, BC2LCN may be fused to a HaloTag protein and immobilized on a support coated with a HaloTag ligand. BC2LCN may also be fused to a polystyrene tag and immobilized directly on a polystyrene plate. In both sandwich assays and immunochromatographic assays, when the first molecule is BC2LCN, the target component is one that simultaneously binds to both the first molecule BC2LCN and the second molecule BC2LCN. Therefore, the target component detected by sandwich assays and immunochromatographic assays in which the first molecule is BC2LCN is a component that can simultaneously bind to multiple BC2LCNs. In the immunochromatographic assay, an antibody against BC2LCN or a carbohydrate antigen that binds to BC2LCN, such as an H-type 1 carbohydrate antigen or an H-type 3 carbohydrate antigen, can be immobilized on the control line.

[0049] In one embodiment, the first molecule is an antibody that binds to SerpinA3. In this embodiment, it is considered that SerpinA3 having a BC2LCN-binding glycan is detected. The antibody that binds to SerpinA3 can be prepared by a conventional method using its binding ability to SerpinA3 as an index.

[0050] The antibody that binds to SerpinA3 may be further confirmed by its ability to bind to SerpinA3 having a BC2LCN-binding glycan. As used in the Examples, SerpinA3 having a BC2LCN-binding glycan can be purified as a component that reacts with immobilized BC2LCN and an antibody that binds to SerpinA3. The antibody that binds to SerpinA3 may be selected based on whether or not it binds to the purified component.

[0051] In embodiments in which the first molecule is an antibody that binds to SerpinA3, the first molecule can be immobilized to a support (e.g., a substrate or bead) and the second molecule can be labeled; or, the first molecule can be labeled and the second molecule can be immobilized to a support.

[0052] In one embodiment, the first molecule is an antibody that binds to Gal3BP. In this embodiment, Gal3BP having a BC2LCN-binding glycan is considered to be detected. The antibody that binds to Gal3BP can be prepared by a standard method using its binding ability to Gal3BP as an index.

[0053] The antibody that binds to Gal3BP may be further confirmed by its ability to bind to Gal3BP having a BC2LCN-binding glycan. As used in the Examples, Gal3BP having a BC2LCN-binding glycan can be purified as a component that reacts with immobilized BC2LCN and an antibody that binds to Gal3BP. The antibody that binds to Gal3BP may be selected based on whether or not it binds to the purified component.

[0054] In embodiments in which the first molecule is an antibody that binds to Gal3BP, the first molecule can be immobilized to a support (e.g., a substrate or bead) and the second molecule can be labeled; or, the first molecule can be labeled and the second molecule can be immobilized to a support.

[0055] According to the present invention, there is provided a method for predicting whether a subject has cancer, a method for obtaining preliminary information 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 selected from the group consisting of a component capable of simultaneously binding two BC2LCNs, BC2LCN-binding SerpinA3, and BC2LCN-binding Gal3BP), or a method for analyzing whether a subject has cancer, comprising: The method comprises: analyzing the presence or concentration of a component (e.g., a blood component) in a biological sample (e.g., a blood sample), wherein the component (e.g., the blood component) is a component selected from the group consisting of a component capable of simultaneously binding two BC2LCNs, a BC2LCN-binding SerpinA3, and a BC2LCN-binding Gal3BP; or Analyzing a component (e.g., a blood component) contained in a biological sample (e.g., a blood sample) by a sandwich assay or immunochromatographic assay carried out 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 which is BC2LCN; A method is provided.

[0056] According to the present invention, there is provided a method 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) being selected from the group consisting of a component capable of simultaneously binding two BC2LCNs, SerpinA3 binding to BC2LCN, and Gal3BP binding to BC2LCN. The method may include contacting 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 that is BC2LCN with a 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) by a sandwich assay or immunochromatographic assay performed with 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 that is BC2LCN.

[0057] According to the present invention, a kit for sandwich assay or immunochromatographic assay is provided, which comprises 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 can measure the amount of glycoprotein in a subject's biological sample (e.g., a blood sample) using the first molecule and the second molecule. In the kit of the present invention, in one embodiment, the first molecule is immobilized on a substrate or strip, and the second molecule is labeled. In the kit of the present invention, in one embodiment, the first molecule is labeled and the second molecule is immobilized on a substrate or strip. In one embodiment, the first molecule is labeled and the second molecule is contained in a gel or immobilized on a gel. The kit of the present invention may further comprise a chromogenic substrate when the label is an enzyme used in enzyme antibody techniques. When the biological sample is a blood sample, the component is a blood component.

[0058] 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.

[0059] The kit of the present invention can be used for detecting a molecule of interest, or for detecting, determining, predicting, or diagnosing cancer.

[0060] 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. The combination of the present invention can be used to detect a component selected from the group consisting of a component that can simultaneously bind two BC2LCNs, BC2LCN-binding SerpinA3, and BC2LCN-binding Gal3BP in a sample (e.g., a biological sample or a biological sample (e.g., a blood sample)). In the above combination, one of the first molecule and the second molecule may be immobilized on a support, and the other may be labeled for detection. The above 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 the above combination is provided. The above combination can be a kit for sandwich assay, a kit for immunochromatography assay, a kit for lectin electrophoresis, a kit for mass spectrometry, a kit for lectin affinity chromatography, or a kit for μTAS. The kit for lectin electrophoresis may include a gel containing lectin. The kit for lectin affinity chromatography may include a column containing a support on which lectin is immobilized. These kits may further include purified SerpinA3, purified BC2LCN-binding SerpinA3, purified Gal3BP, or purified BC2LCN-binding Gal3BP as a standard substance. The kit for μTAS may include a microfluidic chip. The microfluidic chip has a detection unit, and the detection unit may have immobilized thereon a molecule that binds one or more selected from the group consisting of SerpinA3, BC2LCN-binding SerpinA3, Gal3BP, and BC2LCN-binding Gal3BP, and a protein complex containing these.

[0061] In the present invention 1. A method for analyzing a biological sample obtained from a subject, comprising: A method comprising: analyzing a component (sometimes referred to as a "target component") contained in a biological sample by an assay carried out using a first molecule selected from the group consisting of a molecule that binds to a BC2LCN-binding glycan, a molecule that binds to SerpinA3, and a molecule that binds to Gal3BP, and a second molecule that is BC2LCN. In the methods of the present invention, the signal obtained by the assay may decrease due to a decrease in the amount of cancer in the body (e.g., the cancer is removed). Thus, in the methods of the present invention, the assay signal may indicate the amount of cancer in the body. Also, in the methods of the present invention, the signal obtained by the assay may increase due to recurrence. Thus, in the methods of the present invention, an increase in the signal may indicate recurrence of the cancer.

[0062] According to the present invention, there is provided a method comprising isolating one or more components selected from the group consisting of components capable of simultaneously binding to two BC2LCNs, SerpinA3 and Gal3BP, from a biological sample obtained from a subject. According to the present invention, there is also provided a method comprising detecting one or more components selected from the group consisting of components capable of simultaneously binding to two BC2LCNs, SerpinA3 and Gal3BP, from a biological sample obtained from a subject. According to the present invention, there is also provided a method comprising: 1. A method for analyzing a biological sample obtained from a subject, comprising: and isolating one or more components selected from the group consisting of components capable of simultaneously binding to two BC2LCNs, SerpinA3 and Gal3BP, in a biological sample obtained from a subject; method is provided. Separation means separating a component of interest from at least one or more other components. Separation may include concentrating, enriching, and purifying the component of interest. Concentration means increasing the concentration. Enrichment and purification mean selectively increasing the abundance ratio of the component of interest relative to other components. Enrichment and purification may involve concentrating the component of interest. Separation may also include binding to immobilized molecules. In this case, the component of interest is separated from other components that do not bind by binding to the immobilized molecules. Separation may include, in addition to adsorption of the component of interest to the immobilized surface, removing the components that have not been adsorbed. Removal of the components that have not been adsorbed may be performed by washing the surface. Removal of the components that have not been adsorbed may also be performed by removing the liquid phase. Removal of the components that have not been adsorbed may also be performed by recovering the beads if the solid phase is present on the surface of the beads. In this embodiment, the invention may further include measuring the amount of the separated component. Measurement may be performed by various assays well known to those skilled in the art, thereby obtaining an assay signal. The measured amount can be compared to other indicative amounts, such as cut-off values. The cut-off values ​​can be as described above.

[0063] In the method of the present invention, if the measured amount is high compared to other indicative amounts, it may indicate the possibility that the subject from which the biological sample is derived has cancer. Thus, the method of the present invention may 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 may be reduced by reducing the amount of cancer in the body (e.g., removing the cancer). Thus, in the method of the present invention, the assay signal may indicate the amount of cancer in the body. Also, in the above method, the signal obtained by the assay may be increased by recurrence. Thus, in the method of the present invention, an increase in the signal may indicate recurrence of cancer. The method of the present invention may be performed on two or more biological samples collected at different times. Thus, in one embodiment, the biological sample may be two or more biological samples collected at different times. In one embodiment, the method of the present invention may further include comparing the signal obtained by the assay (corresponding to the measured amount) with the signal obtained in another sample that may be collected at the different time. In this way, the present invention can be used to monitor the course of cancer treatment and / or recurrence in a patient.

[0064] The method of the present invention may be carried out in combination with an existing method. For example, a method including measuring the amount of CA19-9 in a body fluid sample is known as an existing method. CA19-9 has an increased concentration in a body fluid sample having cancer. In addition, its concentration increases when the amount of cancer increases, and its concentration decreases when the amount of cancer decreases. Thus, by combining CA19-9 with the method of the present invention, the accuracy and / or specificity of cancer detection by analyzing a body fluid sample can be increased. CA19-9 can be an indicator of pancreatic cancer, particularly pancreatic cancer such as stage III and IV. Therefore, the present invention may further include measuring the concentration of CA19-9 in a biological sample.

[0065] One or more components selected from the group consisting of SerpinA3 and Gal3BP to be measured may be further analyzed using the presence of BC2LCN-binding glycan modification as an index. Whether or not one or more components selected from the group consisting of SerpinA3 and Gal3BP have BC2LCN-binding glycan modification can be determined using a factor that binds to the glycan modification (glycan) as described above. Alternatively, one or more components selected from the group consisting of SerpinA3 and Gal3BP may be detected after separation using a factor that binds to the glycan modification (glycan). One or more components selected from the group consisting of SerpinA3 and Gal3BP may be detected after separation using a molecule that binds to SerpinA3 and a molecule that binds to Gal3BP. EXAMPLES

[0066] Example 1: Detection of glycoproteins in serum of cancer patients using rBC2LCN Glycoproteins in the sera of patients with various cancers were detected by a sandwich assay using rBC2LCN.

[0067] First, biotinylated rBC2LCN was diluted to 0.3 μg / mL with PBS and added to an avidin plate (blocking-less type) (Sumitomo Bakelite, BS-X7603) at 50 μL / well and incubated at room temperature for 1 hour. The resulting rBC2LCN-immobilized plate was washed five times with PBS / 0.1% Tween 20 and 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 applied at 50 μL / well and reacted at room temperature for 1 hour. After washing five times with PBS / 0.1% Tween 20, 50μL / well of TMB solution (Fujifilm Wako Pure Chemical Industries, Ltd.) was applied, and color development was allowed to occur at room temperature for 30 minutes. 50μL / well of 1N HCl was added to stop the color reaction, and OD450 / 620 was measured. The average of the values ​​obtained is shown. In the sandwich assay using this rBC2LCN, a higher OD450 / 620 value indicates the presence of a large amount of glycoprotein that binds to rBC2LCN.

[0068] The results are shown in Figure 1. As shown in Figure 1, it was revealed that the glycoproteins detected by the sandwich assay using rBC2LCN had higher concentrations in pancreatic cancer patients, colon cancer patients, bladder cancer patients, breast cancer patients, non-Hodgkin's lymphoma patients, uterine cancer patients, ovarian cancer patients, and chronic myeloid leukemia patients than the maximum values ​​observed in the healthy subject group.

[0069] The detected glycoprotein must be able to simultaneously bind to two molecules of immobilized rBC2LCN and detection rBC2LCN. The results of this example revealed that components (e.g., blood components) that can simultaneously bind to two molecules of rBC2LCN are widely present in cancer patients and have higher concentrations than healthy subjects, and are therefore useful for analyzing components (e.g., blood components).

[0070] Example 2: Identification of glycoproteins recognized by rBC2LCN In this example, glycoproteins in the serum of pancreatic cancer patients and healthy individuals were precipitated using rBC2LCN-conjugated beads, the precipitated glycoproteins were eluted with rBC2LCN, and the resulting eluate was subjected to electrophoresis, and glycoproteins specific to pancreatic cancer were identified by mass spectrometry.

[0071] Biotinylated rBC2LCN was bound to DynabeadsM280 Streptavidin (Invitrogen). The mixture was incubated with pancreatic cancer and healthy donor serum overnight at 4°C, washed, and then 0.2M fucose was added and incubated at room temperature for 1 hour to elute the glycoproteins bound to rBC2LCN. The resulting eluate was electrophoresed, transferred to a PVDF membrane, and blotted with HRP-labeled rBC2LCN. As a result, no reactivity was observed in the rBC2LCN blot with healthy donor serum, but reactivity was observed in the vicinity of 75-150 kDa with pancreatic cancer serum. Therefore, the 100-150 kDa area circled in red was excised from the electrophoresed and silver-stained gel. A peptide mixture was obtained from each excised gel piece through trypsin hydrolysis. The peptide mixture was subjected to LC-MS / MS to obtain MS / MS data. The results of the amino acid sequence database search 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.

[0072] Therefore, glycoproteins that showed binding to rBC2LCN-immobilized beads from the sera of pancreatic cancer patients and healthy controls were electrophoresed and Western blotted using antibodies against SerpinA3 and Gal3BP and HRP-labeled secondary antibodies. As a result, as shown in Figure 3, SerpinA3 and Gal3BP were significantly more strongly detected in the sera of pancreatic cancer patients than in healthy controls. SerpinA3 and Gal3BP were obtained as proteins that adsorb to beads with rBC2LCN, and therefore these glycoproteins were found to be reactive to rBC2LCN.

[0073] Normal pancreatic tissue and pancreatic cancer tissue were obtained from patients and subjected to immunohistochemical staining. Immunohistochemical staining was performed according to a conventional method. As the primary antibody, anti-SerpinA3 antibody (R&D Systems, product number: MAB12945, dilution ratio: 200 times) was used for staining SerpinA3, and anti-Gal3BP antibody (R&D Systems, product number: AF2226, 1 μg / mL) was used for staining Gal3BP. In addition, biotinylated anti-mouse IgG (Nichirei Biosciences, product number: 424021, dilution ratio: undiluted) and biotinylated anti-goat IgG (Nichirei Biosciences, product number: 414011, dilution ratio: undiluted) were used as secondary antibodies. The results were as shown in FIG. 9. As shown in FIG. 9, almost no staining was observed in the normal pancreatic tissue, whereas in the pancreatic cancer tissue, strong expression of SerpinA3 and Gal3BP was evident in the pancreatic duct.

[0074] It is suggested that SerpinA3 and Gal3BP detected in the blood originate from the pancreatic ducts of pancreatic cancer tissue.

[0075] Example 3: Analysis of sera from cancer patients in a sandwich assay using rBC2LCN and SerpinA3 or Gal3BP antibodies Therefore, a sandwich assay of rBC2LCN and SerpinA3 antibody was constructed to analyze the serum of healthy subjects and patients with various cancers, including pancreatic cancer. Biotinylated rBC2LCN was diluted to 0.3 μg / mL with PBS, added to an avidin plate (blocking-less type) (Sumitomo Bakelite, BS-X7603) at 50 μL / well, and incubated at room temperature for 1 hour. The obtained rBC2LCN-immobilized plate was washed five times with PBS / 0.1% Tween20, and 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% Tween20, and 50 μL / well of HRP-labeled SerpinA3 antibody (1 μg / mL; R&D Systems, Cat#: AF1295) was applied 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 Industries, Ltd.) was applied and allowed to develop at room temperature for 30 minutes. The color reaction was stopped by adding 50 μL / well of 1N HCL, and OD450 / 620 was measured. The average values ​​obtained are shown.

[0076] The results were as shown in Figure 4. As shown in Figure 4, the glycoprotein detected by the sandwich assay of rBC2LCN and SerpinA3 antibody (i.e., SerpinA3 having a specific glycan that binds to BC2LCN lectin) was barely detectable in healthy subjects, whereas it was strongly detected in all of the cancers examined, including colon cancer, pancreatic cancer, bladder cancer, gastric cancer, small cell lung cancer, breast cancer, non-Hodgkin's lymphoma, cervical cancer, rectal adenocarcinoma, stem cell cancer, thyroid cancer, prostate cancer, uterine cancer, melanoma, esophageal cancer, ovarian cancer, bile duct cancer, and chronic myeloid leukemia.

[0077] Next, a sandwich assay of rBC2LCN and Gal3BP antibody was constructed to analyze the serum of healthy subjects and patients with various cancers, including pancreatic cancer. Biotinylated rBC2LCN was diluted to 0.3 μg / mL in PBS, added to an avidin plate (blocking-less type) (Sumitomo Bakelite, BS-X7603) at 50 μL / well, and incubated at room temperature for 1 hour. The resulting rBC2LCN-immobilized plate was washed five times with PBS / 0.1% Tween20, 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% Tween20, and 50 μL / well of HRP-labeled Gal3BP antibody (1 μg / mL; R&D Systems, Cat#: AF2226) was applied and reacted at room temperature for 1 hour. After washing five times with PBS / 0.1% Tween 20, 50μL / well of TMB solution (Fujifilm Wako Pure Chemical Industries) was applied, coloring was allowed to occur at room temperature for 30 minutes, and 50μL / well of 1N HCL was added to stop the coloring reaction, and OD450 / 620 was measured. A graph was created using the average of the values ​​obtained. The average of the values ​​obtained is shown.

[0078] The results were as shown in Figure 5. As shown in Figure 5, the glycoprotein detected by the sandwich assay of rBC2LCN and Gal3BP antibody (i.e., Gal3BP having a specific glycan that binds to BC2LCN lectin) was barely detectable in healthy subjects, whereas it was strongly detected in all of the cancers examined, including colon cancer, pancreatic cancer, bladder cancer, gastric cancer, small cell lung cancer, breast cancer, non-Hodgkin's lymphoma, cervical cancer, rectal adenocarcinoma, stem cell cancer, thyroid cancer, prostate cancer, uterine cancer, melanoma, esophageal cancer, ovarian cancer, bile duct cancer, and chronic myeloid leukemia.

[0079] In addition, in Figure 10, healthy subjects, pancreatitis patients, and pancreatic cancer patients were compared separately. As a result, in the sandwich assay of rBC2LCN and rBC2LCN, the sandwich assay of rBC2LCN and anti-SerpinA3 antibody, the sandwich assay of rBC2LCN and anti-Gal3BP antibody, and the sandwich assay of anti-SerpinA3 antibody and anti-SerpinA3 antibody, stronger signals were detected in pancreatic cancer patients compared to healthy subjects and pancreatitis patients. This suggests that these assays can distinguish pancreatic cancer patients from healthy subjects and pancreatitis patients.

[0080] These results demonstrated that SerpinA3, which has a specific glycan that binds to BC2LCN lectin, and Gal3BP, which has a specific glycan that binds to BC2LCN lectin, are widely present in the sera of cancer patients.

[0081] In addition, using serum samples from other healthy individuals and pancreatic cancer patients, sandwich assays using rBC2LCN and rBC2LCN, sandwich assays using rBC2LCN and anti-SerpinA3 antibody, sandwich assays using rBC2LCN and anti-Gal3BP antibody, and sandwich assays using anti-SerpinA3 antibody (R&D Systems, Cat#: AF1295) and anti-SerpinA3 antibody (R&D Systems, Cat#: AF1295) were performed using the methods described above.

[0082] The results are shown in FIG. 6. As shown in FIG. 6, in the sandwich assay using rBC2LCN and rBC2LCN, the sandwich assay using rBC2LCN and anti-SerpinA3 antibody, and the sandwich assay using rBC2LCN and anti-Gal3BP antibody, the measurement intensity was higher in the sandwich assay for the serum of pancreatic cancer patients than in the serum of healthy subjects. In contrast, in the sandwich assay using anti-SerpinA3 antibody and anti-SerpinA3 antibody, the measurement intensity was equivalent between the serum of healthy subjects and the serum of pancreatic cancer patients. This result suggests that SerpinA3 is present in both the serum of healthy subjects and the serum of pancreatic cancer patients, but that SerpinA3 is specifically modified with a sugar that can be recognized by rBC2LCN in pancreatic cancer patients.

[0083] The results obtained above were analyzed, and an ROC curve was drawn with specificity on the horizontal axis and sensitivity on the vertical axis. The results are shown in FIG. As shown in the upper left panel of Figure 7, the sandwich assay using rBC2LCN and rBC2LCN (rBC2-rBC2) had an area under the ROC curve (AUC) of 0.679, the cutoff in the ROC curve (obtained from the point on the ROC curve that is the shortest distance from sensitivity 1 and specificity 1) was 0.329, the specificity at that time was 0.820, and the sensitivity was 0.575. As shown in the upper right panel of Figure 7, the sandwich assay using rBC2LCN and anti-SerpinA3 antibody (rBC2-SerpinA3) had an area under the ROC curve (AUC) of 0.892, and the cutoff in the ROC curve (obtained from the point on the ROC curve that is the shortest distance from sensitivity 1 and specificity 1) was 0.014, with the specificity at that time being 0.840 and the sensitivity being 0.825. As shown in the lower left panel of Figure 7, the sandwich assay using rBC2LCN and anti-Gal3BP antibody (rBC2-Gal3BP) had an area under the ROC curve (AUC) of 0.743, the cutoff in the ROC curve (obtained from the point on the ROC curve that is the shortest distance from sensitivity 1 and specificity 1) was 0.01, and the specificity at that time was 0.900 and the sensitivity was 0.575. Furthermore, as shown in the lower right panel of Figure 7, in a sandwich assay using anti-SerpinA3 antibody and anti-SerpinA3 antibody (SerpinA3-SerpinA3), the area under the ROC curve (AUC) was 0.718, the cutoff in the ROC curve (obtained from the point on the ROC curve that is the shortest distance from sensitivity 1 and specificity 1) was 1.056, the specificity at that time was 0.8, and the sensitivity was 0.65.

[0084] From the above results, it was revealed that the sandwich assay using rBC2LCN and anti-SerpinA3 antibody (rBC2-SerpinA3) had the highest accuracy, and the sandwich assay using rBC2LCN and anti-Gal3BP antibody (rBC2-Gal3BP) showed the highest specificity. It was also revealed that the sandwich assay using rBC2LCN and rBC2LCN (rBC2-rBC2) and the sandwich assay using anti-SerpinA3 antibody and anti-SerpinA3 antibody (SerpinA3-SerpinA3) also showed a certain level of usefulness.

[0085] Furthermore, ROC curves were created to examine whether the assay could distinguish between healthy subjects (n=49), pancreatitis patients (n=9), and pancreatic cancer patients (n=85). As a result, as shown in Figure 11, it was revealed that the sandwich assay using rBC2LCN and anti-SerpinA3 antibody (rBC2-SerpinA3) and the sandwich assay using rBC2LCN and anti-Gal3BP antibody (rBC2-Gal3BP) showed the best AUC. It was also revealed that the sandwich assay using rBC2LCN and rBC2LCN (rBC2-rBC2) and the sandwich assay using anti-SerpinA3 antibody and anti-SerpinA3 antibody (SerpinA3-SerpinA3) had a certain degree of usefulness.

[0086] Example 5: Analysis of serum from stage-specific pancreatic cancer patients In this example, serum samples from patients with various pancreatic cancers, from stage 1A to stage 4, were evaluated by sandwich assay.

[0087] The stage of cancer was determined by a physician based on the UICC TNM classification (UICC: TNM Classification of Malignant Tumours, 8th Edn. Wiley-Blackwell; 2017.94-95). The sera of the cancer patients were subjected to a sandwich assay using rBC2LCN and rBC2LCN, a sandwich assay using rBC2LCN and an anti-SerpinA3 antibody, a sandwich assay using rBC2LCN and an anti-Gal3BP antibody, and a sandwich assay using an anti-SerpinA3 antibody and an anti-SerpinA3 antibody, as described in Examples 1 and 3. The results were as shown in Figures 8A to 8D.

[0088] As shown in Figures 8A to 8C, samples showing high measurement intensity were observed at all stages from stages 1 to 4 in the sandwich assay using rBC2LCN and rBC2LCN, the sandwich assay using rBC2LCN and anti-SerpinA3 antibody, and the sandwich assay using rBC2LCN and anti-Gal3BP antibody. As shown in FIG. 8D, the sandwich assay using anti-Serpin A3 antibody and anti-Serpin A3 antibody showed high measurement intensity in all samples, similar to that shown in FIG.

[0089] An ROC curve was created between stage I pancreatic cancer patients (n=9) and healthy subjects (n=49). The results are shown in FIG. 12. As shown in FIG. 12, stage I pancreatic cancer patients could be detected well in all sandwich assays. An ROC curve was created between stage II pancreatic cancer patients (n=51) and healthy subjects (n=49). The results are shown in FIG. 13. As shown in FIG. 13, stage II pancreatic cancer patients could be detected well in all sandwich assays. An ROC curve was created between stage III pancreatic cancer patients (n=8) and healthy subjects (n=49). The results are shown in FIG. 14. As shown in FIG. 14, stage III pancreatic cancer patients could be detected well in all sandwich assays. An ROC curve was created between stage IV pancreatic cancer patients (n=17) and healthy subjects (n=49). The results are shown in FIG. 15. As shown in FIG. 15, all of the sandwich assays shown were able to successfully detect stage IV pancreatic cancer patients.

[0090] Pancreatic cancer patients underwent surgery to remove pancreatic cancer. Pre- and post-operative blood samples (n=38) were analyzed by sandwich assay. The results of the sandwich assay with rBC2LCN and anti-SerpinA3 antibody are shown in FIG. 16, the results of the sandwich assay with rBC2LCN and anti-Gal3BP antibody are shown in FIG. 17, and the results of the CA19-9 assay are shown in FIG. 18. In each figure, 30POD means 30 days after surgery, and 3m means 3 months after surgery. As shown in FIGS. 16 to 18, the signal of each sandwich assay was reduced by the removal of the cancer. Therefore, it can be understood that in these assays, the results of the removal of the cancer affect the component content in the blood.

[0091] Furthermore, blood samples from patients with recurrence were assayed over time. The assay was a sandwich assay using BC2LCN and anti-SerpinA3 antibody. The results are shown in FIG. 19. As shown in FIG. 19, in patient A, the assay signal decreased over time after surgery. However, when blood from patient A with recurrence was analyzed, the signal significantly increased. This suggests that the assay system of the present invention can be used to detect recurrence.

[0092] Therefore, by setting appropriate cutoff values, the sandwich assay using rBC2LCN and rBC2LCN, the sandwich assay using rBC2LCN and anti-SerpinA3 antibody, and the sandwich assay using rBC2LCN and anti-Gal3BP antibody can detect a wide range of pancreatic cancer, from early stages such as stages 1 and 2 to stage 4.

Claims

1. 1. A method for analyzing a biological sample obtained from a subject, comprising: A method comprising: analyzing a component contained in a biological sample by an assay performed using 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.

2. The method of claim 1 , wherein the subject has or is at risk of having cancer.

3. The method of claim 2 , wherein the subject has or is at risk of having pancreatic cancer.

4. The method of claim 3, wherein the subject has or is at risk of having stage 1 or 2 pancreatic cancer.

5. 1. A method for analyzing a biological sample obtained from a subject, comprising: The method includes analyzing a component contained in a biological sample by an assay carried out using a first molecule selected from the group consisting of a molecule that binds to a BC2LCN-binding glycan, a molecule that binds to SerpinA3, and a molecule that binds to Gal3BP, and a second molecule which is BC2LCN; The subject has or is at risk of having stage 1 or 2 pancreatic cancer. method.

6. A kit for a sandwich assay, a kit for immunochromatography assay, a kit for lectin electrophoresis, a kit for mass spectrometry, 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 which is BC2LCN, and capable of measuring the amount of glycoprotein in a subject's biological sample using the first molecule and the second molecule.

7. The kit according to claim 6, which is used to detect cancer.

8. The kit according to claim 7 , wherein the cancer is pancreatic cancer.

9. The kit of claim 8 , wherein the cancer is stage 1 or 2 pancreatic cancer.

10. A kit for sandwich assay, kit for immunochromatography assay, kit for lectin electrophoresis, kit for mass spectrometry, kit for lectin affinity chromatography or kit for μTAS, which comprises a first molecule selected from the group consisting of a molecule that binds to BC2LCN, SerpinA3, and Gal3BP, and a second molecule that is a molecule that binds to a BC2LCN-binding glycan, and which can measure the amount of glycoprotein in a subject's biological sample using the first molecule and the second molecule, and which is used to detect stage 1 or 2 pancreatic cancer.

11. A method for detecting a specific component in a biological sample obtained from a subject, comprising: The specific component is a component selected from the group consisting of a component capable of simultaneously binding two BC2LCNs, BC2LCN-binding SerpinA3, and BC2LCN-binding Gal3BP; isolating from a biological sample obtained from a subject one or more components selected from the group consisting of components capable of simultaneously binding to two BC2LCNs, SerpinA3 and Gal3BP; The subject has stage I or II pancreatic cancer or is suspected of having stage I or II pancreatic cancer; method.

12. The method of claim 11 further comprising measuring the amount of the separated components.

13. The method of claim 12, further comprising comparing the amount of the measured component to a cutoff value.

14. The method according to claim 13, wherein the cutoff value is a value equal to or greater than the average amount of the component corresponding to the measured component in healthy subjects and equal to or less than the average amount of the component corresponding to the measured component in pancreatic cancer patients.

15. A method for detecting a specific component in a biological sample obtained from a subject, comprising: The specific component is a component selected from the group consisting of a component capable of simultaneously binding two BC2LCNs, BC2LCN-binding SerpinA3, and BC2LCN-binding Gal3BP; isolating from a biological sample obtained from a subject one or more components selected from the group consisting of components capable of simultaneously binding to two BC2LCNs, SerpinA3 and Gal3BP; The component is selected from the group consisting of BC2LCN-binding SerpinA3 and BC2LCN-binding Gal3BP. method.

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