JP2024066398A - Cancer testing method, reagent, kit and device - Google Patents

Abstract

To provide a method which enables highly efficient cancer testing with high sensitivity.SOLUTION: A cancer testing method is provided, comprising (1) binding at least one lectin selected from the group consisting of TJA II, ADA, LFA, WGA, PVL, PltB, BCoV, SubB2M, MAL, HSA, MAH, ACG, rACG, rGal8N, SNA, SSA, TJAI, rPSL1a, and rGC2 to a biological sample, and (2) analyzing the presence/absence or abundance of a biological component binding to the lectin in the biological sample.SELECTED DRAWING: None

Description

The present invention relates to a method, a reagent, a kit, and an apparatus for testing for cancer. More specifically, the present invention relates to a method for testing for cancer using a lectin that binds to a biological component associated with cancer.

Currently, it is said that one in two Japanese people will develop some form of cancer in their lifetime. There are many different types of cancer, and each type of cancer has different pathological tissue/cell morphology, gene expression, and cell surface protein/glycan expression. Therefore, diagnostic methods and types of cancer markers also differ depending on the cancer cell type.

For example, breast cancer is screened using non-invasive diagnostic methods such as mammography, and if malignant lesions or cancer are suspected, fine needle aspiration cytology or needle biopsy (tissue biopsy) is performed to collect a small amount of cells or tissue. Prostate cancer is screened using a blood test (PSA test), and then diagnosed with a rectal examination and transrectal prostate ultrasound. If cancer is suspected, evaluation is performed using a needle biopsy for histopathological diagnosis. Lung cancer is screened using a chest X-ray, and if a lesion is suspected, a sputum test is performed to check for the presence of cancer cells in the sputum. In addition, evaluation is performed using an endoscopy or needle biopsy for histopathological diagnosis.

In any case, a biopsy is necessary to make a definitive diagnosis of cancer, but if a diagnosis cannot be made with a small number of cells, additional tissue sampling is required, which places a heavy burden on the patient. Therefore, there is a demand for the development of a cancer testing method that can make a definitive diagnosis with high efficiency even with a small number of cells obtained by fine needle aspiration, etc. In particular, when deciding on a patient's treatment plan, determining the malignancy of the cancer is the most important diagnostic requirement.

Traditionally, there has been active research into cancer testing and the development of anticancer drugs by recognizing proteins present on the surface of various cancer cells. However, because the same proteins are also expressed in normal cells, most of these methods have been ineffective in terms of specificity.

In recent years, attention has been focused on changes in the glycans of glycoproteins and glycolipids in the serum of cancer patients, and testing techniques using antibodies and lectins that can recognize and detect glycans that increase specifically in various cancers as glycan epitopes, even if the amount of proteins with glycans is the same. For example, a method for diagnosing pancreatic cancer by measuring the amount of multiple specific glycans present in serum (Patent Documents 1 and 2), a method for diagnosing pancreatic cancer by detecting diseased human haptoglobin using a basidiomycete-derived lectin specific for fucose α1→6 (Patent Document 3), and a method for detecting cancer cells by recognizing glycoproteins present on the cell surface of breast cancer cells and prostate cancer cells using BC2LCN lectin that specifically recognizes Fucα1-2Galβ1-3GlcNAc/GalNAc (Patent Document 4) have been proposed.

JP 2012-63139 A JP 2013-83490 A WO2011/089988 WO2017/061449

The objective of the present invention is to provide a method, reagent, kit, and device that can perform cancer testing with high efficiency and high sensitivity using a small amount of biological sample, determine whether a test subject has cancer, determine the state of the cancer (e.g., malignancy, progression, stage, etc.), determine the effectiveness of treatment in a test subject with cancer that has been treated, and collect data for these purposes.

As a result of intensive research to achieve the above object, the present inventors have found that cancer can be tested by binding at least one lectin selected from the group consisting of TJA II, ADA, LFA, WGA, PVL, PltB, BCoV, SubB2M, MAL, HSA, MAH, ACG, rACG, rGal8N, SNA, SSA, TJAI, rPSL1a, and rGC2 to a biological sample and analyzing the presence or absence or amount of a biological component that binds to the lectin in the biological sample, thereby completing the present invention.
That is, the present invention includes the following aspects.
[1] A cancer testing method comprising:
(1) binding at least one lectin selected from the group consisting of TJA II, ADA, LFA, WGA, PVL, PltB, BCoV, SubB2M, MAL, HSA, MAH, ACG, rACG, rGal8N, SNA, SSA, TJAI, rPSL1a, and rGC2 to a biological sample;
(2) Analyzing the presence or absence or amount of a biological component that binds to the lectin in a biological sample;
A method comprising:
[2] Further, (3) contacting the biological component with a second component;
The method according to claim 1, comprising:
[3] A method for collecting data for determining whether a subject is afflicted with cancer, comprising:
(1) binding at least one lectin selected from the group consisting of TJA II, ADA, LFA, WGA, PVL, PltB, BCoV, SubB2M, MAL, HSA, MAH, ACG, rACG, rGal8N, SNA, SSA, TJAI, rPSL1a, and rGC2 to a biological sample from a test individual;
(2) Measuring the presence or absence or amount of a biological component that binds to the lectin in a biological sample;
(3) comparing the presence or absence or the amount of the biological component with the presence or absence or the amount of a biological component that binds to the lectin in a biological sample from a healthy subject;
(4) collecting data on the presence or absence or the difference in the amount of the biological component between the test individual and a healthy individual in order to determine whether the test individual is affected by cancer;
A method comprising:
[4] further, (5) collecting data on the presence or absence or the difference in the amount of the biological component between the test individual and a healthy subject in order to determine the cancer state of the test individual;
The method according to [3], comprising:
[5] A method for collecting data to determine the efficacy of a treatment in a subject suffering from a cancer to which the treatment has been administered, comprising:
(1) binding at least one lectin selected from the group consisting of TJA II, ADA, LFA, WGA, PVL, PltB, BCoV, SubB2M, MAL, HSA, MAH, ACG, rACG, rGal8N, SNA, SSA, TJAI, rPSL1a, and rGC2 to a biological sample from a test individual;
(2) Measuring the presence or absence or amount of a biological component that binds to the lectin in a biological sample;
(3) comparing the presence or absence or the amount of the biological component with the presence or absence or the amount of the biological component that binds to the lectin in a biological sample of the test individual before treatment, which has been previously measured;
(4) collecting data on the presence or absence or difference in the amount of the biological component before and after the treatment to determine the effectiveness of the treatment;
A method comprising:
[6] The method according to any one of [1] to [5], wherein the lectin is at least one lectin selected from the group consisting of TJA II, ADA, and rGC2.
[7] The method according to any one of [1] to [6], wherein the biological sample is a body fluid sample from a test individual.
[8] The method according to [7], wherein the body fluid sample is a blood-derived sample.
[9] The method according to any one of [1] to [6], wherein the biological sample is a tumor tissue or a surrounding tissue thereof excised from an organ, organ, or tissue of a test subject, or a tissue sample or cell sample derived from a biopsy material.
[10] The method according to any one of [1] to [9], wherein the biological component that binds to the lectin is a protein.
[11] The method according to [10], wherein the protein is at least one selected from the group consisting of von Willebrand factor, α ₂ -macroglobulin, fibronectin, and fibrinogen β chain.
[12] The method according to any one of [1] to [11], wherein the cancer is pancreatic cancer.
[13] A reagent, kit, or device for testing for cancer, comprising:
(1) A reagent, kit, or device comprising at least one lectin selected from the group consisting of TJA II, ADA, LFA, WGA, PVL, PltB, BCoV, SubB2M, MAL, HSA, MAH, ACG, rACG, rGal8N, SNA, SSA, TJAI, rPSL1a, and rGC2.
[14] The reagent, kit, or device described in [13], wherein the lectin is at least one lectin selected from the group consisting of TJA II, ADA, and rGC2.
[15] The reagent, kit, or device according to [13] or [14], wherein the cancer is pancreatic cancer.

According to the present invention, it is possible to test for cancer with high efficiency and high sensitivity using a small amount of biological sample, to determine whether a test subject is suffering from cancer, to determine the state of the cancer (e.g., malignancy, progression, stage, etc.), to determine the effectiveness of treatment in a test subject suffering from cancer that has been treated, and to collect data for these purposes.
In particular, according to the present invention, pancreatic cancer, which is difficult to detect early, can be detected at an early stage (for example, stages IA and IB, which will be described later).
Therefore, the present invention can be used in the fields of cancer research, cancer diagnosis, and drug discovery and medicine through cancer treatment.

FIG. 1 shows the results of staining the tumor site of a clinical pancreatic cancer xenograft mouse model with TJA II. FIG. 2 shows the results of staining the tumor site of a clinical pancreatic cancer xenograft mouse model with ADA. FIG. 3 shows the results of electrophoresis of eluates of glycoproteins bound to ADA in the serum of pancreatic cancer patients and healthy subjects, which were then transferred to a PVDF membrane and blotted with ADA lectin. FIG. 4 shows the results of electrophoresis of eluates of glycoproteins bound to rGC2 in the serum of pancreatic cancer patients and healthy subjects, which were then transferred to a PVDF membrane and blotted with rGC2 lectin. FIG. 5 shows the results of electrophoresis of glycoproteins from the sera of pancreatic cancer and healthy subjects that exhibited binding to ADA-immobilized beads, followed by Western blotting using an antibody against von Willebrand factor and an HRP-labeled secondary antibody. FIG. 6 shows the results of electrophoresis of glycoproteins from the sera of pancreatic cancer and healthy subjects that exhibited binding to rGC2-immobilized beads, followed by Western blotting using an antibody against fibronectin and an HRP-labeled secondary antibody. FIG. 7 shows the results of electrophoresis of glycoproteins from the sera of pancreatic cancer and healthy subjects that exhibited binding to MAL-immobilized beads, followed by Western blotting using an antibody against von Willebrand factor and an HRP-labeled secondary antibody. FIG. 8 shows the results of electrophoresis of glycoproteins from the sera of pancreatic cancer and healthy subjects that exhibited binding to SNA-immobilized beads, followed by Western blotting using an antibody against von Willebrand factor and an HRP-labeled secondary antibody. FIG. 9 shows the results of measuring fibronectin in the serum of pancreatic cancer patients and healthy subjects using the HRP-labeled anti-fibronectin antibody-rGC2 solid-phase ELISA for detecting fibronectin prepared in (Example 5-1). FIG. 10 shows the results of measuring von Willebrand factor in the serum of pancreatic cancer patients and healthy subjects using the HRP-labeled anti-von Willebrand factor antibody-MAL solid-phase ELISA for detecting von Willebrand factor prepared in (Example 5-2). FIG. 11 shows the results of measuring von Willebrand factor in the serum of pancreatic cancer patients and healthy subjects using the HRP-labeled anti-von Willebrand factor antibody-SNA solid-phase ELISA for detecting von Willebrand factor prepared in (Example 5-3). FIG. 12 shows the results of measuring von Willebrand factor in the serum of pancreatic cancer patients and healthy subjects using the HRP-labeled anti-von Willebrand factor antibody-HSA solid-phase ELISA for detecting von Willebrand factor prepared in (Example 5-4). FIG. 13 shows the results of measuring von Willebrand factor in the serum of pancreatic cancer patients and healthy subjects using the HRP-labeled SNA-anti-von Willebrand factor antibody solid-phase ELISA for detecting von Willebrand factor prepared in (Example 5-5). FIG. 14 shows the results of measuring α ₂ -macroglobulin in the serum of pancreatic cancer patients and healthy subjects using the HRP-labeled anti-α ₂ -macroglobulin antibody-TJA II solid-phase ELISA for detecting α ₂ -macroglobulin prepared in (Example 5-6). FIG. 15 shows the results of measuring α ₂ -macroglobulin in the serum of pancreatic cancer patients and healthy subjects using the HRP-labeled TJA II-anti-α ₂ -macroglobulin antibody solid-phase ELISA for detecting α ₂ -macroglobulin prepared in (Example 5-7).

As used herein, "cancer" includes malignant tumors and malignant sarcomas, such as epithelial cancers such as tongue cancer, laryngeal cancer, pharyngeal cancer, esophageal cancer, lung cancer, gastric cancer, duodenal cancer, liver cancer, bile duct cancer, gallbladder cancer, pancreatic cancer, colorectal cancer, kidney cancer, bladder cancer, urothelial cancer, prostate cancer, uterine cancer, ovarian cancer, testicular cancer, breast cancer, and thyroid cancer, leukemia, malignant lymphoma, plasma cell tumor, myeloma, melanoma, and brain tumor.
As used herein, "epithelial cancer" includes tongue cancer, laryngeal cancer, pharyngeal cancer, esophageal cancer, lung cancer, gastric cancer, duodenal cancer, liver cancer, bile duct cancer, gallbladder cancer, pancreatic cancer, colorectal cancer, kidney cancer, bladder cancer, urothelial cancer, prostate cancer, uterine cancer, ovarian cancer, testicular cancer, breast cancer, thyroid cancer, and the like.
As used herein, "digestive system epithelial cancer" includes tongue cancer, pharyngeal cancer, esophageal cancer, gastric cancer, duodenal cancer, liver cancer, bile duct cancer, gallbladder cancer, pancreatic cancer, colon cancer, and the like.
In this specification, "pancreatic cancer" generally refers to cancer that originates in the pancreas. The pancreas has exocrine glands that secrete digestive enzymes (amylase, trypsin, lipase, etc.) and endocrine glands that secrete hormones (insulin, etc.). Pancreatic cancer is broadly divided into two types: exocrine system (digestive enzyme secretion system) cancer and endocrine system (hormone secretion system) cancer, with exocrine system cancer accounting for 95% of cases, and invasive pancreatic ductal cancer arising from the epithelium of the pancreatic duct is the most common, accounting for 85% of the total. Therefore, pancreatic cancer generally refers to this invasive pancreatic ductal cancer. In this specification, "epithelial cancer" or "digestive system epithelial cancer" includes invasive pancreatic ductal cancer.

In this specification, the "state of cancer" includes, for example, malignancy, progression (stage), histological type, degree of differentiation, recurrence and metastasis.
Various indices, from clinical to histopathological, are used to judge the malignancy of cancer. In general, the indices of the site of cancer occurrence, the histological type of cancer, and the degree of differentiation of cancer are widely used. In terms of the site of occurrence, the gallbladder, biliary tract, and pancreas are classified as the most malignant cancers because they are highly progressive, invasive, and metastatic, and have a poor prognosis (5-year relative survival rate). On the other hand, the prostate, breast, and thyroid are classified as the least malignant cancers because they progress slowly, have a low metastatic rate, and have a good prognosis. The uterine body, large intestine, cervix, stomach, ovary, lung, esophagus, and liver are considered to have the lowest 5-year relative survival rate and the highest malignancy, in that order.
Histological types are cancers derived from the same tissue that are further classified according to the type of cell from which they originate. For example, lung cancer can be broadly classified into squamous cell carcinoma, adenocarcinoma, large cell carcinoma, and small cell carcinoma.
The degree of differentiation of cancer is the degree of abnormality from normal tissue or cells, and the more the structure and shape of cancer tissue or cancer cells deviate from the structure and shape of normal tissue or cells, the higher the malignancy is judged to be. In terms of structure, the more unclear the tissue boundary is, or the more irregular the cell arrangement is, the more malignant it is judged to be. In terms of shape, the more irregular the nucleus or cytoplasm is, the larger the cytoplasm, nucleus, or nucleolus is, the more staining each is, or the more nucleoli there are, the more malignant it is judged to be. The progression of cancer is usually classified as a "disease stage." The disease stage is generally written using Roman numerals, and in pancreatic cancer, there are stages 0 to IV as it progresses from early stages. The disease stage is determined by the size of the cancer, its spread to the surrounding area (infiltration), and whether it has metastasized to lymph nodes or other organs. It is very important to examine the condition of the whole body and perform tests to understand the disease stage in order to decide on a treatment policy.
For example, one of the methods for classifying the stages of pancreatic cancer used in Japan is the classification according to the "Pancreatic Cancer Treatment Guidelines, 7th Edition, July 2016 (edited by the Japan Pancreas Society)" (Kanbara Publishing), which is determined by combining the following three TNM classifications (TNM classification) (see table below).

The stages of pancreatic cancer in this specification are based on the above classification.

In this specification, an "individual" refers to a mammal, such as a human, monkey, cow, horse, goat, dog, cat, mouse, rat, or rabbit, and in particular a human.

In this specification, the term "subject" refers to an individual who is not known to have cancer or an individual who is known to have cancer, and also includes individuals who have undergone treatment such as surgery or administration of anticancer drugs. In the former case (individuals who are not known to have cancer and individuals who are known to have cancer), it is possible to determine whether the individual has cancer as well as its malignancy or the degree of acquired drug resistance, and in the latter case (individuals who have undergone treatment such as surgery or administration of anticancer drugs), it is possible to determine the prognosis or the effectiveness of treatment.

In this specification, the term "healthy individual" refers to an individual who is at least not suspected of having cancer.

As used herein, the term "biological sample" includes tumor tissue or its surrounding tissue excised from an organ, organ or tissue of a subject, or a tissue or cell sample derived from a biopsy material, a body fluid sample of a subject, and the like.
As used herein, a "body fluid sample" includes whole blood, serum, plasma, and blood-derived samples including synovial fluid, interstitial fluid, lymph, saliva, urine, cerebrospinal fluid, tissue extracts, and the like of a subject.
The above biological samples include those collected from test individuals, those that have been subjected to a prescribed treatment after collection, those that have been cultured after collection, and those that have been subjected to a prescribed treatment therefor.

The "biological component" as used herein is not particularly limited as long as it shows different affinities (abundance, detection amount) for the lectin as used herein between healthy subjects and cancer patients (for example, a biological component that is detected in a characteristically higher amount, or in a characteristically lower amount or not detected at all in cancer patients by the lectin), but examples of the biological component include proteins, glycoproteins, lipids, glycolipids, and in particular glycoproteins.
Examples of the glycoprotein include Von Willebrand factor, α ₂ -macroglobulin, fibronectin, and fibrinogen β chain.
Among glycoproteins, those that show different affinities (abundance, detection amount) for the lectins herein between healthy subjects and patients with early stage cancer are preferred, as they can contribute to early detection of cancer. Examples of such glycoproteins include von Willebrand factor and _α2 -macroglobulin. Von Willebrand factor is detected by the lectins herein in a characteristically higher amount in stage I pancreatic cancer patients than in healthy subjects. Moreover, _α2 -macroglobulin is detected in a characteristically lower amount in stage I pancreatic cancer patients than in healthy subjects.

As used herein, the term "lectin" refers to a protein that specifically binds to a sugar chain. Lectins may be natural lectins isolated from plants, fungi, animals, etc., recombinant lectins, lectins chemically synthesized based on the amino acid sequence of natural lectins, and lectins with a structure that does not exist in nature and whose sequence (structure) is artificially designed. Preferably, recombinant lectins are used.
Examples of the "lectin" include TJA II, ADA, LFA, WGA, PVL, PltB, BCoV, SubB2M, MAL, HSA, MAH, ACG, rACG, rGal8N, SNA, SSA, TJAI, rPSL1a, rGC2, etc., and preferably TJA II, ADA, rGC2, etc.
Among the above lectins, "TJA II" is a lectin derived from Trichosanthes japonica, and is known to have specificity for the structure of α1-2Fuc.
Of the above lectins, "ADA" is a lectin derived from Allomyrina dichtoma, and is known to have specificity for the structures of α2-6Sia, Forssman antigen, type A antigen, and type B antigen.
Among the above lectins, "rGC2" stands for "recombinant GC2." GC2 is a lectin derived from Geodia cydonium, and is known to have specificity for the structures α1-2Fuc (H), αGalNAc (A), and αGal (B).
Among the above lectins, "MAL" is a lectin derived from Maackia amurensis, and is known to have specificity for the structure of α2-3Sia.
Of the above lectins, "SNA" is a lectin derived from Sambucus nigra, and is known to have specificity for the structure of α1-6Sia.
Lectins can be commercially available or produced according to any method known to those skilled in the art, for example, natural lectins can be obtained using known protein isolation methods, and recombinant lectins can be obtained by expression using recombinant DNA molecules prepared by recombining nucleic acids, for example, by cloning, DNA shuffling, or other well-known molecular biology techniques.

The term "antibody" as used herein includes monoclonal and polyclonal antibodies. Preferably, the antibody is a monoclonal antibody. The antibody may be derived from a mammal (e.g., human, mouse, rat, rabbit, sheep, camel, etc.) or may be a recombinant antibody. Furthermore, the antibody may be an antibody having a structure that does not exist in nature, the sequence (structure) of which has been artificially designed, such as a humanized antibody or a chimeric antibody.
In addition, the term "antibody" as used herein also includes "functional antibody fragments". The term "functional antibody fragments" refers to partial fragments of antibodies that have antigen-binding activity, and includes Fab, F(ab')2, scFv, and the like. In addition, Fab', which is a monovalent fragment of the variable region of an antibody obtained by treating F(ab')2 under reducing conditions, is also included in the functional antibody fragments. However, the functional fragments are not limited to these molecules as long as they have the ability to bind to antigens. The functional fragments include not only those obtained by treating the full-length antibody protein molecule with an appropriate enzyme, but also proteins produced in appropriate host cells using genetically engineered antibody genes.
The antibody may be commercially available or may be produced according to any method known to those skilled in the art. For example, the antibody may be produced using a specific antigen according to cell fusion technology, gene recombination technology, phage display, etc.

As used herein, "specificity" and "specifically bind" mean binding with high affinity to a target.

1. Cancer Testing Method The present invention includes a cancer testing method.
(1) binding at least one lectin selected from the group consisting of TJA II, ADA, LFA, WGA, PVL, PltB, BCoV, SubB2M, MAL, HSA, MAH, ACG, rACG, rGal8N, SNA, SSA, TJAI, rPSL1a, and rGC2 to a biological sample;
(2) Analyzing the presence or absence or amount of a biological component that binds to the lectin in a biological sample;
including.

Among the above lectins, "ADA" can specifically bind to sugar chains bound to von Willebrand factor present in biological samples derived from cancer patients.
Among the above lectins, "MAL" can specifically bind to sugar chains bound to von Willebrand factor present in biological samples derived from cancer patients.
Among the above lectins, "SNA" can specifically bind to sugar chains bound to von Willebrand factor present in biological samples derived from cancer patients.
Among the above lectins, "HSA" can specifically bind to sugar chains bound to von Willebrand factor present in biological samples derived from cancer patients.
Among the above lectins, "rGC2" can specifically bind to glycan bound to fibronectin, glycan bound to fibrinogen β-chain, and glycan bound to _α2 -macroglobulin present in biological samples from cancer patients.
Among the above lectins, "TJA II" can specifically bind to sugar chains bound to α ₂ -macroglobulin present in biological samples from healthy subjects, as opposed to biological samples from cancer patients.

The above-mentioned lectins can specifically bind to "biological components" present on the cell surface of cancer cells in cancer tissue or cultured cancer cells, and can also specifically bind to "biological components" that have been released from the cell surface of cancer cells in cancer tissue or cultured cancer cells and are present in body fluids or culture supernatants.
Alternatively, the above-mentioned lectin can specifically bind to "biological components" present on the cell surface of normal cells in normal tissues or cultured normal cells, and can also specifically bind to "biological components" that have been released from the cell surface of normal cells in normal tissues or cultured normal cells and are present in body fluids or culture supernatants.

The above lectins are known substances and can be produced by known methods. They are also available from Vector Laboratories, Cosmo Bio Co., Ltd., Fujifilm Wako Pure Chemical Industries, Seikagaku Corporation, Tokyo Chemical Industry Co., Ltd., etc.

The lectin may be labeled. Lectin can be labeled by a standard method using fluorescence, an enzyme, a nucleic acid chain, biotin, magnetic beads, or the like.
For example, lectins can be fluorescently labeled using fluorescent dyes such as "Cy3", "Cy5", "FITC", "Hilyte Fluor ^™ 647", "phycoerythrin", and "allophycocyanin".
By using the publicly known method of Hosaka et al. (Iijima et al. (2009) ChemBioChem, 10, 999-1006), it is possible to prepare a mutant in which a fluorescently labeled amino acid is introduced into a specific site of a lectin.
In addition to fluorescent dyes, magnetic beads can also be used for labeling. For example, lectins labeled with magnetic beads can be prepared using protocols provided by reagent manufacturers such as ThermoFisher.
Labeling can also be performed using enzymes such as "horseradish peroxidase" and "alkaline phosphatase" or "detection systems using biotin-avidin reaction". In this case, the primary amino group ( _NH2 group) or thiol group (SH group, sulfhydryl group) of the lectin can be labeled using enzymes or biotin activated with NHS group or maleimide group by using protocols provided by reagent manufacturers such as Dojindo.

The lectin may be immobilized on a carrier. There are no particular limitations on the method for immobilizing the lectin on an insoluble carrier, and known methods such as chemical binding (immobilization by covalent bonding) and physical adsorption can be used. It is also possible to immobilize the lectin on an insoluble carrier by utilizing a very strong binding reaction such as the avidin-biotin reaction. In this case, a biotinylated lectin in which biotin is bound to the lectin may be immobilized on a streptavidin plate coated with streptavidin. The lectin may also be immobilized on an insoluble carrier via various linkers commonly used in this field.

The "biological sample" in the above cancer testing method includes those mentioned above, but among them, a body fluid sample is preferred from the viewpoint of the burden on the subject, and in particular a sample derived from blood.

The "biological components" in the above cancer testing method include those mentioned above, among which glycoproteins are mentioned, and in particular von Willebrand factor, α ₂ -macroglobulin, fibronectin and fibrinogen β chain.

In the above cancer testing method, the "cancer" includes those mentioned above, particularly pancreatic cancer.

The above step (1) is not particularly limited as long as it is a step that can bind the above lectin to the above biological sample, but can be carried out, for example, by the following procedure.

(1-1) When the biological sample is a body fluid sample When a body fluid sample such as blood is used as the biological sample, the sample is contacted with a solution containing the lectin without going through a purification step, or after dilution, or after being concentrated in advance to only a protein fraction, whereby the lectin binds to the biological components in the body fluid sample.
Alternatively, a body fluid sample containing a biological component, optionally labeled with an enzyme, fluorescence, biotin, or the like, can be contacted with a support on which the above-mentioned lectin has been immobilized, such as an ELISA plate, magnetic beads, a filter, a slide glass, etc. This causes the biological component in the body fluid sample to bind to the lectin immobilized on the support.
Alternatively, a biological sample can be reacted with a support on which a known antibody or lectin has been immobilized, and then reacted with the above-mentioned lectin that has been labeled with an enzyme, fluorescence, biotin, or the like, if necessary.

(1-2) When the biological sample is a cell sample When test cells are cultured in a culture vessel while attached to a substrate (e.g., a bead-shaped, hollow fiber-shaped, or flat substrate), the lectin is added to a solution in which the substrate is present. The "solution" referred to here may be a culture solution, or a buffer solution or physiological saline solution after removing medium components. This causes the lectin to bind to biological components in the cell sample.
Even if the test cells are cultured in a suspended state, the lectin will bind to the biological component if added to the solution. The "solution" referred to here may be a culture solution, or a buffer solution or physiological saline solution after removing the medium components.

(1-3) When the biological sample is a tissue sample, the above-mentioned lectin solution is brought into contact with a tissue slice, either as is or after chemical fixation, so that the lectin binds to biological components in the tissue slice.
Alternatively, a tissue sample, either raw or chemically fixed, may be sliced in a standard manner and attached to a slide glass to form a pathological section, which may then be contacted with the lectin solution, causing the lectin to bind to biological components in the tissue section.

The above step (2) is not particularly limited as long as it is a step that can analyze the presence or absence or amount of a biological component that binds to the above lectin in a biological sample, and can be analyzed by known methods such as fluorescent staining, flow cytometry, ELISA, and lectin blotting. For example, it can be performed according to the following procedure depending on the type of sample.

(2-1) When the biological sample is a body fluid sample When the biological sample is a body fluid sample and is directly contacted with a solution containing the lectin, it is necessary to separate the biological components bound to the lectin from the free lectin for analysis. Examples of the separation method include known methods such as chromatography, high performance liquid chromatography, electrophoresis, capillary electrophoresis, capillary chip electrophoresis, and methods using an automated immunoanalyzer such as LiBASys (manufactured by Shimadzu Corporation). Specific conditions may be set so that the biological components bound to the lectin can be separated, and other conditions may be in accordance with known methods. For example, when separation is performed using HPLC, the method may be performed in accordance with the method described in Anal. Chem. 65, 5, 613-616 (1993) or JP-A-9-301995, and when capillary electrophoresis is used, the method may be performed in accordance with the method described in J. Chromatogr. 593 253-258 (1992), Anal. Chem. 64 1926-1932 (1992), WO2007/027495, etc. In addition, when using, for example, LiBASys as the automated immunoanalyzer, the method may be similar to that described in Biological Sample Analysis, Vol. 22, No. 4, 303-308 (1999). After separation, the presence or absence or the amount of the biological component can be analyzed by analyzing the amount of the lectin bound to the biological component.

(2-2) When the biological sample is a cell sample Whether the test cells are cultured by attaching them to a substrate or in a suspension state, the presence or absence or the amount of the biological component can be analyzed by adding the above-mentioned lectin to the cell sample and then analyzing the amount of the biological component bound to the lectin on the surface of the test cells.
For example, when test cells are attached to a substrate and cultured, a fluorescently labeled lectin can be added to a solution containing the substrate as described above, and then the reactivity of the labeled lectin with a biological component specifically expressed on the surface of cancer cells or normal cells can be analyzed by a fluorescence microscope, ELISA, etc., to examine cancer cells or normal cells. According to such an analysis method, a sample in which a label such as fluorescence is not examined (it is at the same level as the background value) during biopsy can be evaluated as a sample in which no cancer cells are present.
For example, when test cells are cultured in a suspension state or when tissue collected by biopsy or the like is enzymatically treated to dissociate the cells, a fluorescently labeled lectin is added to a solution containing the test cells, as described above, and then the solution is analyzed using flow cytometry analysis. The labeled lectin directly labels only cancer cells or normal cells with a fluorescent label or the like, so that flow cytometry analysis can be applied. Specifically, by performing flow cytometry analysis using a FACS device, it is possible to reliably test whether cancer cells are present even in a small amount of sample.
Alternatively, the lectin may be immobilized on a transparent substrate such as a slide glass, and a test sample containing cancer cells suspended in a solution may be directly or after dilution, or may be concentrated in advance to a protein fraction only, and then labeled with "Cy3-NHS ester" or the like, and reacted with the immobilized lectin, and the binding may be analyzed using a plate reader, a fluorescent scanner, an evanescent wave excitation fluorescent inspection system, or the like.

In addition, for quality control of cancer cells maintained for research purposes, etc., cell samples can be collected periodically or as needed, and the labeling intensity, such as the fluorescence intensity, of the labeled lectin can be analyzed.

(2-3) When the biological sample is a tissue sample, the tissue sample is contacted with the above-mentioned lectin solution as a tissue slice or as a tissue section, and the presence or absence or the amount of the biological component can be analyzed by analyzing the amount of the biological component bound to the lectin on the surface of the tissue slice or in the tissue section.

In addition, in the above step (2), in order to facilitate analysis of the amount of the biological component bound to the lectin, the lectin may be labeled as described above. For example, fluorescent labeling can enable analysis with higher sensitivity.

The cancer testing method may further include (3) contacting the biological component with a second component. The order of step (3) is not particularly limited, and may be, for example, before or after step (1).
The second component is not particularly limited, but examples thereof include a labeled antibody or labeled lectin that binds to a biological component to which the lectin is bound (lectin-biological component complex), a labeled antibody or labeled lectin that binds to a biological component, and a labeled antibody or labeled lectin that binds to the lectin.
When the biological sample is a body fluid sample, the step (3) can be carried out, for example, by the following procedure.
(a) A body fluid sample is reacted with the lectin in a solution (step (1)), and then a labeled antibody or a labeled lectin that binds to the lectin-biological component complex is added to the solution for contact (step (3)), after which the amount of label is measured to analyze the presence or absence or amount of the biological component in the body fluid sample (step (2), "sandwich assay method"). In particular, the use of the "lectin-lectin sandwich method" or the "lectin-antibody sandwich method" enables a more sensitive analysis. Of these, the "lectin-antibody sandwich method" is preferred.
In this procedure, the lectin to be reacted with the body fluid sample may be immobilized on a support.
(b) Alternatively, after contacting a body fluid sample with a solution of a labeled antibody or labeled lectin that binds to a biological component (step (3)), the body fluid sample is contacted with a support on which the lectin is immobilized (step (1)), and the amount of the biological component present can be analyzed by measuring the amount of label on the support surface (step (2)).

The cancer testing method may further include (4) separating the lectin-bound biological component or cancer cells containing the biological component from normal cells. When the method includes step (4), step (3) may not be included. The order of step (4) is not particularly limited, and may be, for example, before or after step (2), or before or after step (3).
When the test sample is a cell sample, step (4) can be carried out, for example, by the following procedure.
In the case of cells in a suspension, after step (1), cancer cells containing the biological components bound to the lectin can be separated from normal cells using a cell sorter or a magnetic cell separator.
Specifically, the above-mentioned fluorescently labeled lectin is added to a solution containing a cell sample (culture medium, or a buffer solution or physiological saline solution after removing the medium components, etc.), and the fluorescently labeled lectin is allowed to bind to the biological components in the cell sample. Then, by using a cell sorter in combination with flow cytometry analysis, for example, by using a flow cytometer equipped with a cell sorter, it is possible to isolate only live cancer cells.
In addition, when using lectins labeled with magnetic beads, the biological components in a cell sample can be bound to the magnetic bead-labeled lectins, and then the sample can be supplied to a magnetic cell separator to isolate only cancer cells.

The "cancer" in the above cancer testing method includes those mentioned above, particularly pancreatic cancer. The present inventors have found that α ₂ -macroglobulin, von Willebrand factor, fibronectin, and fibrinogen β-chain derived from pancreatic cancer patients show different affinities for the above lectins compared to those derived from healthy subjects. This is thought to be because the types and/or amounts of sugar chains bound to these proteins are different between pancreatic cancer patients and healthy subjects, and therefore the affinities for lectins are also different.

2. Method for collecting data for determining whether a subject has cancer The present invention also includes a method for collecting data for determining whether a subject has cancer, the method comprising:
(1) binding at least one lectin selected from the group consisting of TJA II, ADA, LFA, WGA, PVL, PltB, BCoV, SubB2M, MAL, HSA, MAH, ACG, rACG, rGal8N, SNA, SSA, TJAI, rPSL1a, and rGC2 to a biological sample;
(2) Measuring the presence or absence or amount of a biological component that binds to the lectin in a biological sample;
(3) comparing the presence or absence or the amount of the biological component with the presence or absence or the amount of a biological component that binds to the lectin in a biological sample from a healthy subject;
(4) collecting data on the presence or absence or the difference in the amount of the biological component between the test individual and a healthy individual in order to determine whether the test individual is affected by cancer;
including.

Examples of "subject individuals" in the above data collection method include those mentioned above.

The "cancer," "biological sample," "lectin," and steps (1) and (2) in the above data collection method are the same as those in the above cancer testing method. In addition, the above data collection method can also include steps (3) and (4) in the above cancer testing method.

Step (3) in the above data collection method is a step of comparing the presence or absence or amount of a biological component that binds to the lectin in the biological sample of the test individual with the presence or absence or amount of a biological component that binds to the lectin in the biological sample of a healthy subject, i.e., a step of performing similar steps (1) and (2) using a biological sample of a healthy subject instead of the biological sample of the test individual, and comparing the presence or absence or amount of a biological component that binds to the lectin obtained with the result of the test individual.

Step (4) in the data collection method is a step of collecting data on the presence or absence or difference in the amount of a biological component that binds to the lectin in a test individual and a healthy individual in order to determine whether the test individual is affected by cancer. When the biological component is present only in the test individual or a healthy individual, or when the amount of the biological component present in the test individual is at a significantly different level compared to a healthy individual, the test individual is determined to be affected by cancer or highly likely to be affected by cancer.

The data collection method may further include (5) collecting data on the presence or absence or difference in the amount of the biological component between the test individual and a healthy subject in order to determine the cancer state of the test individual.

The "cancer state" in step (5) above is the same as that described above, but particularly includes the degree of malignancy.
The malignancy of cancer can be evaluated, for example, from the obtained fluorescence intensity. In this case, the tissue sample or cell sample may be fixed before use, but a more quantitative evaluation of the malignancy of cancer can be achieved by fluorescently labeling the biological components in an unfixed cell sample (e.g., a body fluid sample) with the above-mentioned labeled lectin or the like and then applying a flow cytometry analysis method. In addition, by using a cell sorter in combination, the proportion of fluorescently stained cancer cells in a biopsy sample can be accurately analyzed.
After separating a membrane protein fraction from a tissue or cell sample by a known method, the fraction can be suspended in a buffer or physiological saline and subjected to an analysis step. In this case, the fraction can be analyzed in the same manner as a body fluid sample.
A specific determination can be made as follows.
When the amount of biological components that bind to the above-mentioned lectin analyzed in the biological sample of the test individual is at a different level compared to that of healthy individuals or patients with low-grade cancer, the cancer of the test individual can be determined to be highly malignant.
Furthermore, when analyzing the amount of biological components that bind to the above-mentioned lectin in a test individual, the amount of biological components that bind to the above-mentioned lectin in healthy individuals or patients with low-grade cancer may be analyzed as a control and compared.
Furthermore, the amount of the biocomponent that binds to the lectin in a biological sample from a healthy person or a patient with a low-grade cancer is analyzed in advance, a cutoff value for the amount is determined, and if the amount of the biocomponent that binds to the lectin in the biological sample of the test individual exceeds the cutoff value (when the lectin specifically binds to a biocomponent derived from cancer cells) or falls below the cutoff value (when the lectin specifically binds to a biocomponent derived from normal cells), the cancer of the test individual can be judged to be highly malignant. In this case, when the biological sample is a cell sample, the amount of the biocomponent that binds to the lectin may be expressed as the proportion of cells expressing the biocomponent that binds to the lectin.
The above lectin can specifically test cancer cells, and since its binding activity with highly malignant cancer cells is at a different level from its binding activity with normal cells, it is possible to test cancer at an early stage and evaluate the malignancy. The above data collection method is a useful method for collecting data for determining whether or not a test individual is affected by cancer, particularly highly malignant cancer.

3. Method for collecting data to determine the efficacy of a treatment in a treated individual suffering from cancer The present invention also includes a method for collecting data to determine the efficacy of a treatment in a treated individual suffering from cancer, the method comprising:
(1) binding at least one lectin selected from the group consisting of TJA II, ADA, LFA, WGA, PVL, PltB, BCoV, SubB2M, MAL, HSA, MAH, ACG, rACG, rGal8N, SNA, SSA, TJAI, rPSL1a, and rGC2 to a biological sample from a test individual;
(2) Measuring the presence or absence or amount of a biological component that binds to the lectin in a biological sample;
(3) comparing the presence or absence or the amount of the biological component with the presence or absence or the amount of the biological component that binds to the lectin in a biological sample of the test individual before treatment, which has been previously measured;
(4) collecting data on the presence or absence or difference in the amount of the biological component before and after the treatment to determine the effectiveness of the treatment;
including.

The "subject," "cancer," "biological sample," "lectin," and steps (1) and (2) in the above data collection method are the same as those in the above data collection method for determining whether a subject has cancer. In addition, the above data collection method can also include steps (3) and (4) in the above cancer testing method.

Step (3) in the data collection method is a step of comparing the presence or absence or amount of a biological component that binds to the lectin in the biological sample of the test individual with the presence or absence or amount of a biological component that binds to the lectin in the biological sample of the test individual before treatment that has been previously measured. In other words, steps (1) and (2) of analyzing the biological component that binds to the lectin in the test individual before and after treatment are the same.

Step (4) in the above data collection method is a step of collecting data on the presence or absence or difference in the amount of the biological component before and after the treatment in order to determine the effectiveness of the treatment.
For example, treatment methods such as chemotherapy and surgical therapy can be determined depending on the amount of biological components that bind to the above-mentioned lectin in a test individual.
Specifically, when the lectin among the above-mentioned lectins specifically binds to a biological component derived from a cancer cell, if the amount of the biological component that binds to the lectin is low, chemotherapy can be selected in the hope that an anticancer drug will be effective. On the other hand, if the amount is high, it can be determined that the malignancy is high and chemotherapy will not have a significant effect on extending life, and surgical therapy or pain relief care can be selected.
Furthermore, when the lectins among the above-mentioned lectins specifically bind to biological components derived from normal cells, if the amount of the biological component that binds to the lectin is high, chemotherapy can be selected in anticipation of the efficacy of anticancer drugs, whereas if the amount is low, it can be determined that the malignancy is high and chemotherapy will not have a significant effect on extending life.
Furthermore, by periodically analyzing the amount of biological components that bind to the above-mentioned lectins in biological samples from cancer patients, it is possible to determine an appropriate treatment method for each case.

Furthermore, the prognosis of a patient suffering from cancer such as pancreatic cancer can be determined based on the amount of a biological component that binds to the lectin analyzed in a biological sample from the patient. For example, when the amount of a biological component that binds to the lectin differs, the prognosis can be evaluated as poor.
Furthermore, when a subject suffering from cancer is subjected to a surgical treatment such as surgery, a chemical or immunological treatment such as anticancer drug treatment, or a radiation therapy, the effectiveness of the therapeutic effect of the treatment can be assessed by observing the difference in the amount of biological components that bind to the above-mentioned lectin between a biological sample taken before the treatment and a biological sample taken after the treatment.

In this way, the above-mentioned data collection method is a useful method for collecting data for determining the effectiveness of cancer treatment. Therefore, the method is a useful method for collecting data for selecting a treatment method for a test subject suffering from cancer, and further for predicting the prognosis of the test subject.

4. Reagent, kit, or device for testing for cancer The present invention further includes a reagent, kit, or device for testing for cancer. The reagent, kit, or device includes:
(1) Contains at least one lectin selected from the group consisting of TJA II, ADA, LFA, WGA, PVL, PltB, BCoV, SubB2M, MAL, HSA, MAH, ACG, rACG, rGal8N, SNA, SSA, TJAI, rPSL1a and rGC2.

The above "cancer" and "lectin" are the same as those in the cancer testing method described above.

The reagent, kit, or device further comprises:
(2) a labeling agent; and (3) a reagent, means, or device for detecting the label.

The labeling agent (2) above is an agent for labeling a lectin or antibody with a fluorescent dye, an enzyme, biotin, etc., or magnetic beads, and a lectin or antibody labeled with a fluorescent dye, an enzyme, biotin, etc., or magnetic beads can be obtained from the above (1) and (2).
Furthermore, instead of the above (1) and (2), a lectin or antibody bound in advance with a labeling agent can be used together with the above (3) as a reagent, kit, or device.

The above-mentioned (3) reagent, means or device may be, for example, a fluorescent microscope or plate reader in the case of fluorescent labeling, an image analyzer in the case of enzyme labeling or biotin labeling, or reagents, tools, equipment, etc. used therefor. These may also have a means (e.g., an automatic dispenser) for contacting the lectin with a cell surface or tissue surface or adding the lectin to a body fluid sample.

The above-mentioned reagent, kit, or device may further include (4) a second component. The second component is the same as that in the above-mentioned cancer testing method.

The above-mentioned reagent, kit, or device may further include (5) a means or device for analyzing the label and separating the labeled cells. The means or device is, for example, a means or device for detecting and separating labels such as fluorescent labels or magnetic bead labels, and specifically, a flow cytometer equipped with a cell sorter, a magnetic cell separation device, etc.

The above-mentioned reagents, kits, or devices can be used in the above-mentioned cancer testing methods, data collection methods for determining whether a subject has cancer, and data collection methods for determining the effectiveness of a treatment in a subject with cancer that has been treated.

The terms and concepts used in the present invention are based on the meanings of terms commonly used in the relevant field, and the various techniques used to implement the present invention can be easily and reliably implemented by a person skilled in the art based on known literature, etc., except for those techniques whose sources are specifically indicated.

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these.
Example 1: Evaluation of binding activity of lectin to cancer cells and cancer tissues (Example 1-1) TJA II staining of tumor sites in clinical pancreatic cancer xenograft mouse model In this example, the binding activity of TJA II to tumors in clinical pancreatic cancer xenograft mouse model was evaluated immunohistochemically.
Tumors excised from clinical pancreatic cancer xenograft mouse models were fixed in formalin, embedded in paraffin, and then tissue sections were prepared. Each tissue section obtained was stained with horseradish peroxidase (HRP)-labeled TJA II and stained with hematoxylin/eosin, and the tissue images were observed under a microscope.
As a result, it was found that in clinical pancreatic cancer xenograft mouse model tumors, cancer cells were strongly stained with HRP-labeled TJA II, particularly in the ductal structure area, and that the surrounding normal pancreatic cell areas were not stained at all (Figure 1).

(Example 1-2) ADA staining of tumor sites in clinical pancreatic cancer xenograft mouse models ADA staining of tumor sites in clinical pancreatic cancer xenograft mouse models was performed using the same procedure as in (Example 1-1). As a result, it was found that cancer cells in the clinical pancreatic cancer xenograft mouse model tumors were strongly stained with HRP-labeled ADA, and that the glandular structure sites were particularly strongly stained, while the surrounding normal pancreatic cell sites were not stained at all (Figure 2).

Example 2: Identification of glycoproteins recognized by lectins (Example 2-1) Identification of glycoproteins recognized by ADA In this example, glycoproteins in the serum of pancreatic cancer patients and healthy subjects were precipitated using ADA-bound beads, the precipitated glycoproteins were eluted, and the resulting eluate was subjected to electrophoresis, and glycoproteins specific to pancreatic cancer were identified by mass spectrometry.
Biotinylated ADA was bound to DynabeadsM280 Streptavidin (Invitrogen). Then, the mixture was incubated with pancreatic cancer and healthy serum overnight at 4°C, washed, and heated at 95°C for 5 minutes with Tris buffer containing 0.2% SDS to elute the glycoprotein bound to ADA. The resulting eluate was electrophoresed, transferred to a PVDF membrane, and blotted with HRP-labeled ADA. As a result, no reactivity was observed in the ADA blot in the healthy serum, but reactivity was observed in the vicinity of 250 kDa in the pancreatic cancer serum (Figure 3). Therefore, the glycoprotein solution precipitated with ADA-immobilized beads from the same serum used in the lectin blot was electrophoresed and a silver-stained gel was prepared using a silver staining MS kit (Fujifilm Wako Pure Chemical, 299-58901), and the band near 160 kDa was excised. A peptide mixture was obtained from each excised gel piece through trypsin hydrolysis. The peptide mixture was subjected to LC-MS/MS to acquire MS/MS data. The results of an amino acid sequence database search of the MS/MS data were output as a list of peptide identifications. As a result, Von Willebrand factor was acquired as a candidate protein detected by ADA in cancer patients.

(Example 2-2) Identification of glycoproteins recognized by rGC2 In this example, glycoproteins in the serum of pancreatic cancer patients and healthy subjects were precipitated using rGC2-bound beads, the precipitated glycoproteins were eluted, and the resulting eluate was subjected to electrophoresis, and glycoproteins specific to pancreatic cancer were identified by mass spectrometry.
The same method as in (Example 2-1) was carried out. As a result, no reactivity was observed in the rGC2 blot in the serum of healthy subjects, but reactivity was observed in the vicinity of 250 kDa, 125 kDa, and 60 kDa in the serum of pancreatic cancer patients (FIG. 4). Therefore, a glycoprotein solution precipitated with rGC2-immobilized beads from the same serum used in the lectin blot was electrophoresed to prepare a silver-stained gel using a silver staining MS kit (FUJIFILM Wako Pure Chemical Industries, 299-58901), and bands near 250 kDa, 125 kDa, and 60 kDa were excised. Identification was carried out from each excised gel piece using the same method as in (Example 2-1). As a result, fibronectin, α ₂ -macroglobulin, and fibrinogen β chain were obtained as candidate proteins detected by rGC2 in cancer patients in order of molecular weight.

Example 3: Validation of glycoprotein identification results (Example 3-1) Western blotting using anti-von Willebrand factor antibody for glycoproteins that exhibited binding to ADA-immobilized beads In this example, glycoproteins that exhibited binding to ADA-immobilized beads from the serum of pancreatic cancer patients and healthy subjects were electrophoresed and Western blotted using an antibody against von Willebrand factor and an HRP-labeled secondary antibody.
[Sample preparation]
Three lots each of healthy subjects and pancreatic cancer patients (early stage pancreatic cancer, late stage pancreatic cancer) identical to those used in Example 2 were used to precipitate glycoproteins in the serum using ADA-immobilized beads in a manner similar to that used in Example 2-1, and then the glycoproteins were eluted to obtain glycoprotein solutions.
2-Mercaptoethanol was diluted to 2% (v/v) with 2x Laemmli Sample Buffer (BIORAD, 1610737) to prepare a sample buffer. 1 μL of the glycoprotein solution was diluted to 10 μL with the sample buffer to prepare a sample solution. This sample solution was heated at 95° C. for 10 minutes.
[Electrophoresis]
A running buffer and an electrophoresis gel U-PAGEL H (3-10%) (ATTO, UH-R310) were placed in the electrophoresis tank, and the entire amount of the sample solution prepared in the previous step was added to each well, followed by electrophoresis at a constant voltage of 180 V for 75 minutes.
[Transcription]
The gel removed from the glass was immersed in transfer buffer ((48 mM Tris, 39 mM glycine)/20% methanol) and shaken at room temperature for 10 minutes. At the same time, two sheets of transfer filter paper (BIO-RAD, 1703967) and a PVDF membrane activated with methanol (ATTO, WSE-4051) were immersed in the transfer buffer and shaken at room temperature for 10 minutes. The filter paper, PVDF membrane, and gel were placed in a Transblot SD cell and operated at a constant voltage of 25 V for 30 minutes to transfer the glycoprotein to the PVDF membrane.
[Western blot]
A blocking solution was prepared by diluting Block Ace (KAC, UKB80) to 1% (w/v) with PBS/0.05% Tween 20. The PVDF membrane after transfer was placed in the blocking solution and shaken at room temperature for 1 hour for blocking.
A sheep anti-human von Willebrand factor antibody (R&D SYSTEMS, AF2764) was diluted with the blocking solution to 0.5 μg/mL to prepare a primary antibody solution. The primary antibody solution was added to the PVDF membrane that had been shaken for 1 hour, and the membrane was then shaken at room temperature for 1 hour.
The PVDF membrane was shaken for 1 hour and then washed three times with PBS/0.05% Tween 20. A secondary antibody solution containing rabbit anti-sheep IgG antibody (HRP-labeled) (abcam, ab6747) diluted 5,000-fold with blocking solution was then added, and the membrane was shaken at room temperature for 30 minutes.
The PVDF membrane was again washed three times with PBS/0.05% Tween 20, after which ECL Prime (cytiva, RPN2232) was applied and reacted at room temperature for 5 minutes, and the Western blot image was photographed with a CCD imager (GE Healthcare, LAS4000).
As a result, as shown in Figure 5, a band was observed near the molecular weight of von Willebrand factor, 250 kDa, confirming that the candidate protein was von Willebrand factor and that the identification results were valid. It was also revealed that von Willebrand factor, which exhibits binding affinity to ADA-immobilized beads, was detected significantly more strongly in the sera of pancreatic cancer patients than in the sera of healthy subjects.

(Example 3-2) Western blotting using anti-fibronectin antibody for glycoproteins that exhibited binding to rGC2-immobilized beads In this example, glycoproteins that exhibited binding to rGC2-immobilized beads from the sera of pancreatic cancer and healthy subjects were electrophoresed and Western blotted using an antibody against fibronectin and an HRP-labeled secondary antibody.
The experiment was carried out in the same manner as in Example 3-1. A sheep anti-human von Willebrand factor antibody (R&D SYSTEMS, AF2764) was used as the primary antibody, and a rabbit anti-sheep IgG antibody (HRP-labeled) (abcam, ab6747) was used as the secondary antibody.
As a result, as shown in Figure 6, a band was observed near the molecular weight of 250 kDa of fibronectin, confirming that the candidate protein was fibronectin and the identification results were valid. It was also revealed that fibronectin, which exhibits binding to rGC2-immobilized beads, was detected significantly more strongly in the sera of pancreatic cancer patients than in the sera of healthy subjects.

Example 4: Detection of von Willebrand factor exhibiting binding affinity to sialic acid-binding lectin (Example 4-1) Western blotting using anti-von Willebrand factor antibody for glycoproteins exhibiting binding affinity to MAL-immobilized beads In this example, glycoproteins exhibiting binding affinity to MAL-immobilized beads, which is the same sialic acid-binding lectin as ADA, from the sera of pancreatic cancer and healthy subjects were electrophoresed and Western blotted using an antibody against von Willebrand factor and an HRP-labeled secondary antibody.
The experiment was carried out in the same manner as in Example 3-1. A sheep anti-human von Willebrand factor antibody (R&D SYSTEMS, AF2764) was used as the primary antibody, and a rabbit anti-sheep IgG antibody (HRP-labeled) (abcam, ab6747) was used as the secondary antibody.
As a result, as shown in Figure 7, a band was observed near the molecular weight of 250 kDa of von Willebrand factor, and it was revealed that von Willebrand factor, which exhibits binding affinity to MAL-immobilized beads like ADA, was detected significantly more strongly in the sera of pancreatic cancer patients than in the sera of healthy subjects.

(Example 4-2) Western blotting using anti-von Willebrand factor antibody for glycoproteins that exhibited binding to SNA-immobilized beads In this Example, glycoproteins from the sera of pancreatic cancer and healthy subjects that exhibited binding to SNA-immobilized beads, which is the same sialic acid-binding lectin as ADA, were electrophoresed and Western blotted using an antibody against von Willebrand factor and an HRP-labeled secondary antibody.
The experiment was carried out in the same manner as in Example 3-1. A sheep anti-human von Willebrand factor antibody (R&D SYSTEMS, AF2764) was used as the primary antibody, and a rabbit anti-sheep IgG antibody (HRP-labeled) (abcam, ab6747) was used as the secondary antibody.
As a result, as shown in Figure 8, a band was observed near the molecular weight of 250 kDa of von Willebrand factor, and it was revealed that von Willebrand factor, which exhibits binding affinity to SNA-immobilized beads like ADA, was detected significantly more strongly in the sera of pancreatic cancer patients than in the sera of healthy subjects.
Combined with the results of Example 4-1, it was suggested that it is possible to find differences between the sera of healthy subjects and those of pancreatic cancer patients by detecting von Willebrand factor, which exhibits binding affinity to sialic acid-binding lectin.

Example 5: Sample measurement using lectin ELISA The list of serum samples used for measurement is as follows.

Example 5-1: Sample measurement using HRP-labeled anti-fibronectin antibody-rGC2 solid-phase ELISA for detecting fibronectin In this example, a lectin ELISA for detecting fibronectin was prepared using an avidin plate solid-phased with rGC2 and an HRP-labeled anti-fibronectin antibody, and fibronectin was measured in serum samples from pancreatic cancer and healthy subjects.
[Lectin ELISA Preparation]
A solution of biotinylated rGC2 dissolved in PBS was added to an avidin plate (blocking-less type) (manufactured by Sumitomo Bakelite, product number: BS-X7603), and incubated at room temperature.
After washing the plate with 0.1% Tween/PBS, each lot of serum from healthy subjects and pancreatic cancer patients was diluted 100-fold with PBS, and 50 μL of each was added to each well.
After incubation at room temperature and washing the plate with 0.1% Tween/PBS, HRP-labeled anti-fibronectin antibody was added to each well and incubated at room temperature.
After washing the plate with 0.1% Tween/PBS, 50 μL of TMB Solution (manufactured by Fujifilm Wako Pure Chemical Industries, product number: 208-17371) was added to each well and incubated at room temperature, after which 50 μL of 1N HCl was added to each well.
[Sample Measurement]
The values obtained by subtracting the OD 620 nm (second wavelength) measurement value from the OD 450 nm (main wavelength) measurement value were compared for each sample. The measured value corresponds to the amount of fibronectin having a sugar chain structure recognized by rGC2.
As a result, the measured values tended to be low in healthy subjects and high in pancreatic cancer patients (Figure 9). In other words, it was revealed that fibronectin with the glycan structure recognized by rGC2 tended to be low in the serum of healthy subjects and high in the serum of pancreatic cancer patients.
From the above, it was demonstrated that it is possible to distinguish between healthy subjects and pancreatic cancer patients by measurement using HRP-labeled anti-fibronectin antibody-rGC2 solid-phase ELISA for detecting fibronectin.

Example 5-2: Sample measurement using HRP-labeled anti-von Willebrand factor antibody-MAL solid-phase ELISA for detecting von Willebrand factor In this example, a lectin ELISA for detecting von Willebrand factor was prepared using an avidin plate solid-phased with MAL and an HRP-labeled anti-von Willebrand factor antibody, and von Willebrand factor was measured in serum samples from pancreatic cancer and healthy subjects.
[Lectin ELISA Preparation]
The same procedure as in Example 5-1 was followed.
[Sample Measurement]
The values obtained by subtracting the OD 620 nm (second wavelength) from the OD 450 nm (main wavelength) were compared for each sample. The measured values correspond to the amount of von Willebrand factor having a sugar chain structure recognized by MAL.
As a result, the measured values tended to be low in healthy subjects and high in pancreatic cancer patients (Figure 10). In other words, it was revealed that von Willebrand factor having a glycan structure recognized by MAL tends to be low in the serum of healthy subjects and high in the serum of pancreatic cancer patients.
From the above, it was shown that it is possible to distinguish between healthy subjects and pancreatic cancer patients by measuring with HRP-labeled anti-von Willebrand factor antibody-MAL solid-phase ELISA for detecting von Willebrand factor. In addition, since the von Willebrand factor in stage IB pancreatic cancer patient samples (357) was greater than that in healthy subject samples, it was shown that it is possible to distinguish early stage I pancreatic cancer.

Example 5-3: Sample measurement using HRP-labeled anti-von Willebrand factor antibody-SNA solid-phase ELISA for detecting von Willebrand factor In this example, a lectin ELISA for detecting von Willebrand factor was prepared using an avidin plate with solid-phase SNA and an HRP-labeled anti-von Willebrand factor antibody, and von Willebrand factor was measured in serum samples from pancreatic cancer and healthy subjects.
[Lectin ELISA Preparation]
The same procedure as in Example 5-1 was followed.
[Sample Measurement]
The values obtained by subtracting the OD 620 nm (second wavelength) from the OD 450 nm (main wavelength) were compared for each sample. The measured values correspond to the amount of von Willebrand factor having a sugar chain structure recognized by SNA.
As a result, the measured values tended to be low in healthy subjects and high in pancreatic cancer patients (Figure 11). In other words, it was revealed that von Willebrand factor having a glycan structure recognized by SNA tended to be low in the serum of healthy subjects and high in the serum of pancreatic cancer patients.
From the above, it was shown that it is possible to distinguish between healthy subjects and pancreatic cancer patients by measuring with HRP-labeled anti-von Willebrand factor antibody-SNA solid-phase ELISA for detecting von Willebrand factor. In addition, since the von Willebrand factor in stage IB pancreatic cancer patient samples (357) was greater than that in healthy subject samples, it was shown that it is possible to distinguish early stage I pancreatic cancer.

Example 5-4: Sample measurement using HRP-labeled anti-von Willebrand factor antibody-HSA solid-phase ELISA for detecting von Willebrand factor In this example, a lectin ELISA for detecting von Willebrand factor was prepared using an avidin plate solid-phased with Streptococcus gordonii-derived lectin (HSA) and an HRP-labeled anti-von Willebrand factor antibody, and von Willebrand factor was measured in serum samples from pancreatic cancer and healthy subjects.
[Lectin ELISA Preparation]
The same procedure as in Example 5-1 was followed.
[Sample Measurement]
The values obtained by subtracting the OD 620 nm (second wavelength) measurement value from the OD 450 nm (main wavelength) measurement value were compared for each sample. The measured value corresponds to the amount of von Willebrand factor having a sugar chain structure recognized by HSA.
As a result, the measured values tended to be low in healthy subjects and high in pancreatic cancer patients (Figure 12). In other words, it was revealed that von Willebrand factor having a glycan structure recognized by HSA tends to be low in the serum of healthy subjects and high in the serum of pancreatic cancer patients.
From the above, it was demonstrated that it is possible to distinguish between healthy subjects and pancreatic cancer patients by measurement using HRP-labeled anti-von Willebrand factor antibody-HSA solid-phase ELISA for detecting von Willebrand factor.

(Example 5-5) Sample measurement using HRP-labeled SNA-anti-von Willebrand factor antibody solid-phase ELISA for detecting von Willebrand factor In this example, a lectin ELISA for detecting von Willebrand factor was prepared using an avidin plate with immobilized anti-von Willebrand factor antibody and HRP-labeled SNA, and von Willebrand factor was measured in serum samples from pancreatic cancer and healthy subjects.
[Lectin ELISA Preparation]
A solution of biotinylated anti-von Willebrand factor antibody dissolved in PBS was added to an avidin plate (blocking-less type) (manufactured by Sumitomo Bakelite, product number: BS-X7603), and incubated at room temperature.
After washing the plate with 0.1% Tween/PBS, each lot of serum from healthy subjects and pancreatic cancer patients was diluted 100-fold with PBS, and 50 μL of each was added to each well.
After incubation at room temperature and washing the plate with 0.1% Tween/PBS, HRP-labeled SNA was added to each well and incubated at room temperature.
After washing the plate with 0.1% Tween/PBS, 50 μL of TMB Solution (manufactured by Fujifilm Wako Pure Chemical Industries, product number: 208-17371) was added to each well and incubated at room temperature, after which 50 μL of 1N HCl was added to each well.
[Sample Measurement]
The values obtained by subtracting the OD 620 nm (minor wavelength) measurement value from the OD 450 nm (main wavelength) measurement value were compared for each sample. The measurement value corresponds to the amount of von Willebrand factor having a sugar chain structure recognized by SNA among the von Willebrand factor in serum.
As a result, the measured values tended to be low in healthy subjects and high in pancreatic cancer patients (Figure 13). In other words, it was revealed that von Willebrand factor having a glycan structure recognized by SNA tends to be low in the serum of healthy subjects and high in the serum of pancreatic cancer patients.
From the above, it was shown that it is possible to distinguish between healthy subjects and pancreatic cancer patients by measuring using HRP-labeled SNA-anti-von Willebrand factor antibody solid-phase ELISA for detecting von Willebrand factor. In addition, since the von Willebrand factor in stage IB pancreatic cancer patient samples (357) was greater than that in healthy subject samples, it was shown that it is possible to distinguish early stage I pancreatic cancer.

(Examples 5-6) Sample measurement using HRP-labeled anti-α2-macroglobulin antibody-TJA II solid-phase ELISA for detecting _α2 -macroglobulin In this example, a lectin ELISA for detecting _α2 -macroglobulin was prepared using an avidin plate on which TJA II was solidified and an HRP-labeled anti- _α2 -macroglobulin antibody, _{and α2} -macroglobulin in serum samples from pancreatic cancer and healthy individuals was measured.
[Lectin ELISA Preparation]
The same procedure as in Example 5-1 was followed.
[Sample Measurement]
The values obtained by subtracting the OD 620 nm (minor wavelength) measurement value from the OD 450 nm (main wavelength) measurement value were compared for each sample. The measured value corresponds to the amount of α ₂ -macroglobulin having a sugar chain structure recognized by TJA II.
As a result, the measured values tended to be high in healthy subjects and low in pancreatic cancer patients (Figure 14). In other words, it was revealed that _α2 -macroglobulin, which has a glycan structure recognized by TJA II, tends to be abundant in the sera of healthy subjects and to be abundant in the sera of pancreatic cancer patients.
From the above, it was demonstrated that it is possible to distinguish between healthy subjects and pancreatic cancer patients by performing measurement using HRP-labeled anti-α ₂ -macroglobulin antibody-TJA II solid-phase ELISA for detecting α ₂ -macroglobulin.

(Examples 5-7) Sample measurement using HRP-labeled TJA II-anti- _α2 -macroglobulin antibody solid-phase ELISA for detecting _α2 -macroglobulin In this example, a lectin ELISA for detecting _α2 -macroglobulin was prepared using an avidin plate on which anti- _α2 -macroglobulin antibody was solidified and HRP-labeled TJA II, and _α2 -macroglobulin in serum samples from pancreatic cancer and healthy individuals was measured.
[Lectin ELISA Preparation]
The same procedure as in Example 5-5 was followed.
[Sample Measurement]
The values obtained by subtracting the OD 620 nm (minor wavelength) values from the OD 450 nm (main wavelength) values were compared for each sample. The measured values correspond to the amount of _α2 -macroglobulin in serum that has _a glycan structure recognized by TJA II.
As a result, the measured values tended to be high in healthy subjects and low in pancreatic cancer patients (Figure 15). In other words, it was revealed that _α2 -macroglobulin, which has a glycan structure recognized by TJA II, tends to be abundant in the sera of healthy subjects and to be abundant in the sera of pancreatic cancer patients.
These results suggest that the use of HRP-labeled TJA II-anti- _α2 -macroglobulin antibody solid-phase ELISA for detecting _α2 -macroglobulin makes it possible to distinguish between healthy subjects and pancreatic cancer patients. In addition, the amount of _α2 -macroglobulin in stage IB pancreatic cancer patient specimens (357) was less than _that in healthy subject specimens, suggesting that early stage I pancreatic cancer can be distinguished.

Claims (15)

A method for testing for cancer, comprising:
(1) binding at least one lectin selected from the group consisting of TJA II, ADA, LFA, WGA, PVL, PltB, BCoV, SubB2M, MAL, HSA, MAH, ACG, rACG, rGal8N, SNA, SSA, TJAI, rPSL1a, and rGC2 to a biological sample;
(2) Analyzing the presence or absence or amount of a biological component that binds to the lectin in a biological sample;
A method comprising: Furthermore, (3) contacting the biological component with a second component;
The method of claim 1 , comprising: 1. A method for collecting data to determine whether a subject is afflicted with cancer, comprising:
(1) binding at least one lectin selected from the group consisting of TJA II, ADA, LFA, WGA, PVL, PltB, BCoV, SubB2M, MAL, HSA, MAH, ACG, rACG, rGal8N, SNA, SSA, TJAI, rPSL1a, and rGC2 to a biological sample from a test individual;
(2) Measuring the presence or absence or amount of a biological component that binds to the lectin in a biological sample;
(3) comparing the presence or absence or the amount of the biological component with the presence or absence or the amount of a biological component that binds to the lectin in a biological sample from a healthy subject;
(4) collecting data on the presence or absence or the difference in the amount of the biological component between the test individual and a healthy individual in order to determine whether the test individual is affected by cancer;
A method comprising: Furthermore, (5) collecting data on the presence or absence or the difference in the amount of the biological component between the test individual and a healthy subject in order to determine the cancer state of the test individual;
The method of claim 3, comprising: 1. A method for collecting data to determine the efficacy of a treatment in a subject suffering from cancer to which the treatment has been administered, comprising:
(1) binding at least one lectin selected from the group consisting of TJA II, ADA, LFA, WGA, PVL, PltB, BCoV, SubB2M, MAL, HSA, MAH, ACG, rACG, rGal8N, SNA, SSA, TJAI, rPSL1a, and rGC2 to a biological sample from a test individual;
(2) Measuring the presence or absence or amount of a biological component that binds to the lectin in a biological sample;
(3) comparing the presence or absence or the amount of the biological component with the presence or absence or the amount of the biological component that binds to the lectin in a biological sample of the test individual before treatment, which has been previously measured;
(4) collecting data on the presence or absence or difference in the amount of the biological component before and after the treatment to determine the effectiveness of the treatment;
A method comprising: The method according to any one of claims 1 to 5, wherein the lectin is at least one lectin selected from the group consisting of TJA II, ADA, and rGC2. The method according to any one of claims 1 to 6, wherein the biological sample is a body fluid sample from a test individual. The method of claim 7, wherein the body fluid sample is a blood-derived sample. The method according to any one of claims 1 to 6, wherein the biological sample is a tumor tissue or its surrounding tissue excised from an organ, organ or tissue of a test subject, or a tissue sample or cell sample derived from a biopsy material. The method according to any one of claims 1 to 9, wherein the biological component that binds to the lectin is a protein. The method according to claim 10, wherein the protein is at least one selected from the group consisting of Von Willebrand factor, α ₂ -macroglobulin, fibronectin and fibrinogen β chain. The method according to any one of claims 1 to 11, wherein the cancer is pancreatic cancer. A reagent, kit, or device for testing for cancer, comprising:
(1) A reagent, kit, or device comprising at least one lectin selected from the group consisting of TJA II, ADA, LFA, WGA, PVL, PltB, BCoV, SubB2M, MAL, HSA, MAH, ACG, rACG, rGal8N, SNA, SSA, TJAI, rPSL1a, and rGC2. The reagent, kit, or device according to claim 13, wherein the lectin is at least one lectin selected from the group consisting of TJA II, ADA, and rGC2. The reagent, kit, or device according to claim 13 or 14, wherein the cancer is pancreatic cancer.