WO2022163798A1

WO2022163798A1 - Method for testing possibility, severity, or progression of inflammatory disease

Info

Publication number: WO2022163798A1
Application number: PCT/JP2022/003246
Authority: WO
Inventors: 清史東; 幸一斉藤; 佳太山田
Original assignee: 住友化学株式会社; 学校法人大谷学園
Priority date: 2021-02-01
Filing date: 2022-01-28
Publication date: 2022-08-04
Also published as: JPWO2022163798A1; TW202246772A

Abstract

The present invention addresses the problem of providing a feature of testing the possibility, severity, or progression of an inflammatory disease, in particular, a chronic inflammatory disease. The problem is solved by measuring the amount or concentration of a specific N-linked sugar chain in a biological sample collected from a subject.

Description

A method for examining the possibility of having an inflammatory disease, its severity, or the degree of progression

The present invention relates to a method for examining the possibility of affliction of an inflammatory disease, its severity or progression, and the like.

There are various types of diseases involving inflammation. For example, neurodegenerative diseases such as Alzheimer's disease; autoimmune diseases such as rheumatoid arthritis; cancer; skin inflammatory diseases such as psoriasis; ing.

In clinical settings, C-reactive protein (CRP) is widely used as a blood biomarker for testing inflammation. However, since the CRP level also increases in acute inflammation caused by a mild cold or the like, the CRP level may also increase in healthy subjects who are not suffering from the target inflammatory disease.

The object of the present disclosure is to provide techniques for examining the possibility of affliction of inflammatory diseases, especially chronic inflammatory diseases, and their severity or progression.

As a result of intensive research in view of the above problems, the present inventors have found that a method comprising the step of measuring the amount or concentration of a specific N-linked sugar chain in a biological sample collected from a subject has the potential to cause inflammatory diseases. It has been found that the sex, its severity or progression can be tested. Based on this knowledge, the inventor of the present invention has completed the invention of the present disclosure as a result of further research. That is, the present invention includes the following aspects.

Section 1. A method for examining the possibility of contracting an inflammatory disease, its severity, or the degree of progression, comprising the step of (1) measuring the amount or concentration of N-linked sugar chains in a biological sample collected from a subject. , wherein the N-linked sugar chain is (i) a sialo-glycan and (ii) an asialo-glycan.

Section 2. Item 1, wherein the sialic acid contained in the sialoglycan is N-acetylneuraminic acid.

Section 3. Item 1 or 2, wherein the asialo-glycan comprises at least a high-mannose-type sugar chain.

Item 4. The method according to any one of Items 1 to 3, wherein the asialo-glycan comprises at least a complex-type sugar chain.

Item 5. Item 4, wherein the complex-type sugar chain is a biantennary-type sugar chain.

Item 6. Item 5, wherein the biantennary-type sugar chain is a sugar chain containing core fucose.

Item 7. The sialo-glycan is a disialo-diantennary sugar chain 1, and the asialo-glycan comprises a high-mannose-type sugar chain 2, an asialo-diantennary sugar chain digalactose 3, and an asialo-diantennary sugar chain monogalactose 4. At least one asialo-glycan selected from the group:

, the method according to any one of items 1 to 6.

Item 8. In step (1), the ratio of the amount or concentration of the sialo-glycan to the asialo-glycan (ratio A: sialo-glycan/asialo-glycan), and/or the amount or concentration of the asialo-glycan to the sialo-glycan Item 8. The method according to any one of Items 1 to 7, comprising the step of calculating at least one value selected from the group consisting of concentration ratios (ratio B: asialo-glycan/sialo-glycan).

Item 9. Furthermore, (2a) based on the comparison result between the ratio A and a preset reference value (reference value A), and/or the comparison result between the ratio B and a preset reference value (reference value B) 9. The method of paragraph 8, comprising determining the likelihood that the subject is suffering from an inflammatory disease, or the severity or progression of the inflammatory disease of the subject, based on:

Item 10. The reference value A is the maximum value, mean value, or percentile of the ratio A in a biological sample collected from a subject not suffering from an inflammatory disease or a subject with an inflammatory disease of any severity or any degree of progression. or the minimum value and/or the reference value B in a biological sample taken from a subject not suffering from an inflammatory disease or a subject with any severity or any degree of progression of an inflammatory disease 10. Method according to clause 9, wherein the ratio B is the maximum, mean, percentile or minimum value.

Item 11. Items 1 to 10, wherein the biological sample is a body fluid or a sample derived from a body fluid.

Item 12. Item 12. The method according to Item 11, wherein the bodily fluid is at least one selected from the group consisting of whole blood, serum, and plasma.

Item 13. Item 13. The method according to any one of items 1 to 12, wherein the sample to be measured in the step (1) is a sample obtained without subjecting the biological sample to sialidase treatment.

Item 14. The method according to any one of items 1 to 13, wherein the sample to be measured in the step (1) is a sample obtained by subjecting the biological sample to N-linked sugar chain release treatment.

Item 15. Item 15. The method according to Item 14, wherein the method for measuring the amount or concentration of N-linked sugar chains in step (1) includes a chromatography method.

Item 16. Item 16. The method according to any one of Items 1 to 15, wherein the inflammatory disease is a chronic inflammatory disease.

Item 17. Item 16, wherein the chronic inflammatory disease is neurodegenerative disease, autoimmune disease, cancer, inflammatory skin disease, or collagen disease.

Item 18. The chronic inflammatory disease is mild cognitive impairment, Alzheimer's disease, multiple sclerosis, Parkinson's disease, rheumatoid arthritis, autoimmune bullous disease, pancreatic cancer, stomach cancer, ovarian cancer, uterine cancer, cholangiocarcinoma, atopic dermatitis, psoriasis, Item 18. The method of Item 17, which is systemic scleroderma or systemic lupus erythematosus.

Item 19. A method for examining the possibility of having a disease selected from the group consisting of neurodegenerative disease, autoimmune disease, cancer, skin inflammatory disease, and collagen disease, and its severity or progression,
(1) comprising a step of measuring the amount or concentration of an N-linked sugar chain in a biological sample collected from a subject, wherein the N-linked sugar chain is (i) a sialo-glycan and (ii) an asialo-glycan ,Method.

Item 20. the possibility of affliction with an inflammatory disease comprising at least one selected from the group consisting of a sugar chain and an antibody against the sugar chain, wherein the sugar chain is (i) a sialo-glycan and (ii) an asialo-glycan; A test that tests severity or progress.

Item 21. The sialo-glycan is a disialo-diantennary sugar chain 1, and the asialo-glycan comprises a high-mannose-type sugar chain 2, an asialo-diantennary sugar chain digalactose 3, and an asialo-diantennary sugar chain monogalactose 4. At least one asialo-glycan selected from the group:

21. The test agent according to item 20.

Item 22. Item 20 or 21, wherein the inflammatory disease is a chronic inflammatory disease.

Item 23. Item 22, wherein the chronic inflammatory disease is neurodegenerative disease, autoimmune disease, cancer, inflammatory skin disease, or collagen disease.

Item 24. The chronic inflammatory disease is mild cognitive impairment, Alzheimer's disease, multiple sclerosis, Parkinson's disease, rheumatoid arthritis, autoimmune bullous disease, pancreatic cancer, stomach cancer, ovarian cancer, uterine cancer, cholangiocarcinoma, atopic dermatitis, psoriasis, Item 24. The test agent according to item 23, which is systemic scleroderma or systemic lupus erythematosus.

Item 25. Neurodegenerative disease, autoimmune disease, cancer, comprising at least one selected from the group consisting of a sugar chain and an antibody against the sugar chain, wherein the sugar chain is (i) a sialo-glycan and (ii) an asialo-glycan , skin inflammatory disease, and collagen disease, a test agent for testing the possibility of contracting a disease, its severity, or the degree of progress thereof.

The present disclosure also includes the following aspect as another aspect:
Item 20. A method for assisting in determining the likelihood of having an inflammatory disease, its severity or progression, comprising:
(1) comprising a step of measuring the amount or concentration of an N-linked sugar chain in a biological sample collected from a subject, wherein the N-linked sugar chain is (i) a sialo-glycan and (ii) an asialo-glycan ,Method.
Item 21. A method for measuring sugar chains in a subject, comprising:
(1) comprising a step of measuring the amount or concentration of an N-linked sugar chain in a biological sample collected from a subject, wherein the N-linked sugar chain is (i) a sialo-glycan and (ii) an asialo-glycan ,Method.
Item 22. A method of diagnosing an inflammatory disease in a subject, comprising:
(1) comprising a step of measuring the amount or concentration of an N-linked sugar chain in a biological sample collected from a subject, wherein the N-linked sugar chain is (i) a sialo-glycan and (ii) an asialo-glycan and the ratio of the amount or concentration of the sialo-glycan to the asialo-glycan (ratio A: sialo-glycan/asialo-glycan), and/or the ratio of the amount or concentration of the asialo-glycan to the sialo-glycan (ratio B: a step of calculating at least one value selected from the group consisting of asialo-glycan/sialo-glycan),
(2a) when the ratio A is a preset reference value (reference value A) or more and/or when the ratio B is a preset reference value (reference value B) or less, the subject and (3a) determining that the subject is likely to be suffering from an inflammatory disease in step (2a). A method comprising applying a diagnostic method.
Item 23. A method of diagnosing and treating an inflammatory disease in a subject, comprising:
(1) comprising a step of measuring the amount or concentration of an N-linked sugar chain in a biological sample collected from a subject, wherein the N-linked sugar chain is (i) a sialo-glycan and (ii) an asialo-glycan and the ratio of the amount or concentration of the sialo-glycan to the asialo-glycan (ratio A: sialo-glycan/asialo-glycan), and/or the ratio of the amount or concentration of the asialo-glycan to the sialo-glycan (ratio B: calculating at least one value selected from the group consisting of asialo-glycan/sialo-glycan);
(2a) when the ratio A is a preset reference value (reference value A) or more and/or when the ratio B is a preset reference value (reference value B) or less, the subject is likely to be suffering from an inflammatory disease, or (3b) if the ratio A is a preset reference value (reference value A) or more, and / or the ratio B is preset determining that the subject has a high possibility of suffering from an inflammatory disease when it is equal to or less than the determined reference value (reference value B), and determining that the subject has a high possibility of suffering from an inflammatory disease and (4) a subject determined to have a high possibility of suffering from an inflammatory disease in said step (2a), or said administering an inflammatory disease treatment to the subject diagnosed as having the inflammatory disease in step (3b).
Item 24. At least one agent selected from the group consisting of a sugar chain and an antibody against the sugar chain for use as an agent for testing the possibility of having an inflammatory disease, its severity, or the degree of progression, wherein the sugar is A drug, wherein the chain is (i) a sialo-glycan and (ii) an asialo-glycan.
Item 25. Use of at least one drug selected from the group consisting of a sugar chain and an antibody against the sugar chain for examining the possibility of contracting an inflammatory disease, its severity, or the degree of progression, wherein the sugar chain is Use of (i) sialo-glycan and (ii) asialo-glycan.
Item 26. The use of at least one agent selected from the group consisting of sugar chains and antibodies against the sugar chains for the manufacture of test agents for testing the possibility of having an inflammatory disease, its severity, or the degree of progression, Use wherein the sugar chain is (i) a sialo-glycan and (ii) an asialo-glycan.

According to the present disclosure, it is possible to provide techniques for examining the possibility of having inflammatory diseases, particularly chronic inflammatory diseases, and their severity or progress.

Elution pattern of 2-aminobenzoic acid-labeled N-linked sugar chains isolated from serum of healthy subjects and HPLC analysis performed in Example 1(c), and N-bonds identified by mass spectral analysis structure of the type sugar chain. In the structure, ◇ represents N-acetylneuraminic acid (NeuAc), ○ represents galactose (Gal), ● represents mannose (Man), ■ represents N-acetylglucosamine (GlcNAc), and △ represents fucose (Fuc). The vertical axis indicates fluorescence intensity (arbitrary unit), and the horizontal axis indicates elution time (minutes). Fig. 2 shows asialo-glycan levels in sera of healthy subjects and patients with Alzheimer's disease, as measured in Example 1(d). The vertical axis indicates the high mannose type (M6) sugar chain value (No.1/No.2). The middle bar represents the mean and the bars above and below it represent the standard deviation. Fig. 2 shows asialo-glycan levels in sera of healthy subjects and patients with Alzheimer's disease, as measured in Example 1(d). The vertical axis indicates the value of the asialo di-chain sugar chain digalactose sugar chain (No.1/No.3). The middle bar represents the mean and the bars above and below it represent the standard deviation. Fig. 2 shows asialo-glycan levels in sera of healthy subjects and patients with Alzheimer's disease, as measured in Example 1(d). The vertical axis indicates the value of monogalactose sugar chain of asialo diantyl sugar chain (No.1/No.4). The middle bar represents the mean and the bars above and below it represent the standard deviation. Fig. 2 shows asialo-glycan levels in sera of healthy subjects and patients with Alzheimer's disease, as measured in Example 1(d). The vertical axis indicates the value of (asialo double-chain sugar chain digalactose + asialo double-chain sugar chain monogalactose) sugar chain (No.1/(No.3+No.4)). The middle bar represents the mean and the bars above and below it represent the standard deviation. FIG. 1 shows asialo-glycan levels in sera of healthy subjects, patients with mild cognitive impairment, and patients with Alzheimer's disease (moderate and severe) in Example 3. FIG. The vertical axis indicates the value of the asialo di-chain sugar chain digalactose sugar chain (No.1/No.3). The middle bar represents the mean and the bars above and below it represent the standard deviation. FIG. 1 shows asialo-glycan levels in plasma of healthy subjects (70s), mild cognitive impairment patients (70s), and Parkinson's disease patients (70s), which were performed in Example 4. FIG. The vertical axis indicates the value of the asialo di-chain sugar chain digalactose sugar chain (No.1/No.3). The middle bar represents the mean and the bars above and below it represent the standard deviation. FIG. 1 shows the plasma CRP concentrations of healthy subjects (70s), mild cognitive impairment patients (70s), and Parkinson's disease patients (70s) in Comparative Example 1. FIG. The vertical axis indicates CRP concentration. The middle bar represents the mean and the bars above and below it represent the standard deviation. 1 shows the asialo-glycan levels of each plasma of healthy subjects (age 50s) and multiple sclerosis patients (ages 50s) performed in Example 5. FIG. The vertical axis indicates the value of the asialo di-chain sugar chain digalactose sugar chain (No.1/No.3). The middle bar represents the mean and the bars above and below it represent the standard deviation. FIG. 1 shows the plasma CRP concentrations of healthy subjects (50's) and multiple sclerosis patients (50's) in Comparative Example 2. FIG. The vertical axis indicates CRP concentration. The middle bar represents the mean and the bars above and below it represent the standard deviation. 2 shows the asialo-glycan levels of each plasma of healthy subjects and patients with rheumatoid arthritis performed in Example 6. FIG. The vertical axis indicates the value of the asialo di-chain sugar chain digalactose sugar chain (No.1/No.3). The middle bar represents the mean and the bars above and below it represent the standard deviation. 3 shows CRP concentrations in plasma of healthy subjects and patients with rheumatoid arthritis in Comparative Example 3. FIG. The vertical axis indicates CRP concentration. The middle bar represents the mean and the bars above and below it represent the standard deviation. FIG. 4 shows anti-galactose-deficient IgG antibody concentrations (CA/RF) in each plasma of healthy subjects and rheumatoid arthritis patients performed in Comparative Example 4. FIG. The vertical axis indicates the CA/RF concentration. The middle bar represents the mean and the bars above and below it represent the standard deviation. Fig. 2 shows asialo-glycan levels in sera of healthy subjects, pancreatic cancer stage I and stage III patients, which were carried out in Example 7; The vertical axis indicates the value of the asialo di-chain sugar chain digalactose sugar chain (No.1/No.3). The middle bar represents the mean and the bars above and below it represent the standard deviation. FIG. 10 shows asialo-glycan levels in sera of healthy subjects, gastric cancer patients, and cholangiocarcinoma patients in Example 8. FIG. The vertical axis indicates the value of the asialo di-chain sugar chain digalactose sugar chain (No.1/No.3). The middle bar represents the mean and the bars above and below it represent the standard deviation. 2 shows CRP concentrations in sera of healthy subjects, gastric cancer patients, and cholangiocarcinoma patients in Comparative Example 5. FIG. The vertical axis indicates CRP concentration. The middle bar represents the mean and the bars above and below it represent the standard deviation. FIG. 10 shows asialo-glycan levels in sera of healthy subjects and psoriasis patients, which were carried out in Example 9. FIG. The vertical axis indicates the value of the asialo di-chain sugar chain digalactose sugar chain (No.1/No.3). The middle bar represents the mean and the bars above and below it represent the standard deviation. 10 shows asialo-glycan levels in sera of healthy subjects, systemic scleroderma, and systemic lupus erythematosus patients, as measured in Example 10. FIG. The vertical axis indicates the value of the asialo di-chain sugar chain digalactose sugar chain (No.1/No.3). The middle bar represents the mean and the bars above and below it represent the standard deviation.

In this specification, the expressions "contain" and "include" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

In this specification, the expression "and/or" includes the meaning when either "and" or "or" is selected. That is, the expression "A and/or B" includes both the meanings of "A or B" and "A and B."

1. Method for Examining Possibility of Having a Target Disease, Its Severity or Progression In one aspect, the present disclosure is a method for examining the possibility of having an inflammatory disease, its severity or its progress, comprising: (1) comprising a step of measuring the amount or concentration of an N-linked sugar chain in a biological sample collected from a subject, wherein the N-linked sugar chain is (i) a sialo-glycan and (ii) an asialo-glycan , method, related. Further, the present disclosure is a method for examining the possibility of having a disease selected from the group consisting of neurodegenerative diseases, autoimmune diseases, cancers, skin inflammatory diseases, and collagen diseases, and the severity or progress thereof. (1) measuring the amount or concentration of an N-linked sugar chain in a biological sample collected from a subject, wherein the N-linked sugar chain is (i) a sialo-glycan and (ii) an asialo-glycan; is about a method. In this specification, these two methods may be collectively referred to as "the inspection method of the present disclosure". In the present specification, "inflammatory diseases", "diseases selected from the group consisting of neurodegenerative diseases, autoimmune diseases, cancers, skin inflammatory diseases, and collagen diseases" are sometimes referred to as "target diseases". . These methods are described below.

1-1. Process (1)
Inflammatory diseases are not particularly limited as long as they are accompanied by inflammation and/or inflammation is involved in the onset process. Inflammatory diseases include acute inflammatory diseases and chronic inflammatory diseases. Examples of acute inflammatory diseases include acute gastritis, acute interstitial pneumonia, acute hepatitis, fulminant hepatitis, acute pancreatitis, acute gastroenteritis, and acute nephritis. Chronic inflammatory diseases include, for example, neurodegenerative diseases, autoimmune diseases, cancers, skin inflammatory diseases, collagen diseases and the like. In addition to these, chronic inflammatory diseases include, for example, aging, lifestyle-related diseases such as obesity/diabetes and hypertension, arteriosclerosis, heart failure, chronic periodontitis, idiopathic interstitial pneumonia, chronic gastritis, viral Chronic hepatitis, alcoholic liver disease, non-alcoholic steatohepatitis, primary biliary cholangitis, chronic pancreatitis, chronic kidney disease, ulcerative colitis, Crohn's disease, inflammatory bowel disease, lymphedema, and the like. Inflammatory diseases are preferably chronic inflammatory diseases, more preferably neurodegenerative diseases, autoimmune diseases, cancers, skin inflammatory diseases, collagen diseases and the like, and still more preferably neurodegenerative diseases, autoimmune diseases, cancers and the like.

The neurodegenerative disease is not particularly limited as long as it is a disease that accompanies neurodegeneration. Neurodegenerative diseases include cognitive impairment diseases. Examples of neurodegenerative diseases include mild cognitive impairment, Alzheimer's disease, multiple sclerosis, Parkinson's disease, preclinical Alzheimer's disease, cerebrovascular dementia, dementia with Lewy bodies, etc., preferably mild cognitive impairment, Alzheimer's disease, multiple sclerosis, Parkinson's disease and the like. Alzheimer's disease is an irreversible progressive central disease that usually presents with symptoms such as dementia, behavioral disorders, and personality changes. Cognitive impairment diseases collectively called dementia and Alzheimer's Transitional Mild Cognitive Impairment are also included in neurodegenerative diseases. Mild cognitive impairment is an impairment in one of the cognitive functions (memory, decision making, reasoning, execution, etc.). As a result, slight deterioration in cognitive function is observed, but it does not interfere with daily life. state. Preclinical Alzheimer's disease refers to a state in which no clinical symptoms appear prior to mild cognitive impairment. Since preclinical Alzheimer's disease may eventually progress to Alzheimer's disease, preclinical Alzheimer's disease is also included in the classification of neurodegenerative diseases herein. It is known that neuroinflammation is deeply involved in cognitive dysfunction observed in Alzheimer's disease. In addition, multiple sclerosis, in which immune cells are activated and inflammation occurs in the central nerves (brain, spinal cord) and optic nerves, and activated microglia secrete inflammatory cytokines, selectively causing cell death in dopaminergic neurons. The Parkinson's disease it causes is also a neurodegenerative disease.

Autoimmune diseases are not particularly limited as long as they involve immune reactions against self-tissues. Specifically, autoimmune diseases are, for example, autoimmune diseases in which, for example, self tissues are recognized as foreign substances and become antigens, and after autoantibodies are produced against the antigens, immune cells are activated and inflammation occurs in specific tissues. It is a disease that occurs and destroys tissues. Examples of autoimmune diseases include rheumatoid arthritis, autoimmune bullous disease, and autoimmune hepatitis, preferably rheumatoid arthritis, autoimmune bullous disease, and more preferably rheumatoid arthritis. In rheumatoid arthritis, which is a typical autoimmune disease, autoantibodies such as anti-cyclic citrullinated peptide antibodies (anti-CCP antibodies) and anti-galactose-deficient IgG antibodies (CA/RF) are produced as autoantibodies, and inflammatory reactions are induced in joint tissues. is destroyed. Also, in autoimmune bullous disease, skin tissue is destroyed by autoantibodies.

Cancer is not particularly limited, and examples thereof include pancreatic cancer, stomach cancer, ovarian cancer, uterine cancer, bile duct cancer, renal cancer, liver cancer, esophageal cancer, colon cancer, leukemia, lung cancer, prostate cancer, skin cancer, and breast cancer. . Among these, pancreatic cancer, gastric cancer, ovarian cancer, uterine cancer, bile duct cancer, etc. are preferred, and pancreatic cancer, gastric cancer, bile duct cancer, etc. are more preferred. Chronic inflammation is also one of the causes of cancer. For example, chronic pancreatitis progresses to pancreatic cancer, chronic gastritis progresses to stomach cancer, chronic cholangitis progresses to cholangiocarcinoma, and chronic hepatitis progresses to liver cancer. As the mechanism of carcinogenesis, inflammatory cytokines, reactive oxygen species, prostaglandins secreted from inflammatory cells induce cell proliferation, cell death, DNA mutation and methylation, and angiogenesis, and tumors are formed. It is

Inflammatory skin diseases are not particularly limited, and include, for example, atopic dermatitis and psoriasis. In atopic dermatitis, IL-4 and IL-13 produced by Th2 cells at the inflammatory site, and in psoriasis, IL-17 produced by Th17 cells are deeply involved in their onset.

Collagen disease is a general term for diseases that cause inflammation and degeneration in the connective tissue and blood vessels of the skin and internal organs, and inflammation in various organs, and is not particularly limited in this respect. Collagen diseases commonly exhibit, for example, systemic symptoms such as fever, arthritis, and general malaise, and visceral symptoms such as skin symptoms and muscle symptoms. One of the causes of connective tissue diseases is an abnormality of the immune system caused by autoantibodies such as antinuclear antibodies and anti-double-stranded DNA antibodies. Collagen diseases include, for example, systemic lupus erythematosus, systemic scleroderma, dermatomyositis, polyarteritis nodosa, mixed connective tissue disease, Sjögren's syndrome, etc., preferably systemic lupus erythematosus, systemic scleroderma, etc. is mentioned.

There are no particular restrictions on the severity and progression of the target disease. Severity is based on grade values such as mild, moderate, and severe, and evaluation values such as MMSE value, cancer stage, and psoriasis PASI score. The degree of progression is a concept defined from the viewpoint of the pathology of the target disease and the progression of symptoms, and can be based on the above values.

The subject is the target organism of the test method of the present disclosure, and its species is not particularly limited. Species of the subject include, for example, various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits, preferably humans.

The subject's condition related to the target disease is not particularly limited. Subjects include, for example, specimens that are unknown whether or not they have the target disease, specimens that have no history of the target disease, specimens that have a history of the target disease and have received treatment for the target disease, and those who have the target disease by other testing methods. Specimens that have already been determined to have (or not have) disease, interview method, questionnaire test method, score method, pathological diagnosis method, diagnostic imaging method, blood biochemical test method, etc. Examples include specimens for which the degree and progress have been determined. When the subject is a human, any person, including those who are considered to be healthy, can be the test subject regardless of their past medical history. In the case of a person who is considered to be healthy, it is effective for early detection and early diagnosis of target diseases in general medical examinations, comprehensive medical examinations, and the like. In addition, subjects who are suspected of having the target disease and diagnosed with its severity or progress by the target disease test by interview method, questionnaire test method, score method, pathological diagnosis method, diagnostic imaging method, blood biochemical test method, etc. In the case of, the test method of the present disclosure can assist in diagnosing the target disease.

The biological sample is not particularly limited as long as it can contain N-linked sugar chains. Examples of biological samples include body fluids such as blood, cerebrospinal fluid, urine, body secretions, saliva, and sputum; feces, and samples derived from these body fluids or feces. Of these, body fluids or samples derived from body fluids are preferred, and blood samples are more preferred, for examinations of target diseases in general medical examinations, complete medical checkups, and the like. Blood samples include whole blood, plasma, serum, and the like. A blood sample can be prepared by appropriately processing whole blood drawn from a subject. It is desirable that the blood sampling be performed by medical personnel such as doctors and nurses. There is no particular limitation on the treatment performed when preparing a blood sample from collected whole blood, and any clinically acceptable treatment may be performed. Serum is a portion of whole blood from which blood cells and specific blood clotting factors have been removed, and can be obtained, for example, as the supernatant after blood coagulation. Plasma is a portion of whole blood from which blood cells have been removed, and can be obtained, for example, as a supernatant when whole blood is subjected to centrifugation under non-clotting conditions. In addition, the biological sample to be subjected to step (1) may be one that has been appropriately stored at a low temperature such as freezing in an intermediate stage of the preparation process or a later stage of the preparation process.

Among the sugar chains in glycoproteins, N-linked sugar chains are sugar chains that are attached to the nitrogen atom of the amide group of the side chain of the asparagine residue of the protein. Also called sugar chain. N-linked sugar chains are classified into high mannose sugar chains, hybrid sugar chains and complex sugar chains. In these three types of sugar chains, the five sugars (core five sugars) on the reducing end side near the asparagine residue of the protein to which the sugar chain binds are common to two N-acetylglucosamine residues and three mannose residues. Each sugar located on the non-reducing end side (hereinafter referred to as non-reducing end sugar) other than the core pentasaccharide has its own characteristics. Typical examples of core pentasaccharides are shown below.

In the high mannose-type sugar chain, all the sugars on the non-reducing end side are mannose sugar chains. A complex-type sugar chain is a sugar chain that does not contain mannose in the sugar on the non-reducing end side, and examples of the sugar on the non-reducing end side include sialic acid, galactose, and N-acetylglucosamine. A hybrid sugar chain is a sugar chain other than high-mannose type and complex type sugar chains, and specifically, a sugar chain containing mannose in a part of the sugar chain on the non-reducing end side. Among the three types of sugar chains, complex-type sugar chains are particularly rich in structural diversity, with different degrees of branching such as biantennary, triantennary, and tetraantennary, and sialic acid Examples include the presence or absence of residues, a core fucose structure in which a fucose residue is linked to an N-acetylglucosamine residue at the reducing end, and a polylactosamine structure in which galactose and glucosamine at the non-reducing end are repeated. Core fucose means fucose bound to the reducing terminal N-acetylglucosamine residue in the core pentasaccharide.

Sialic acid is a general term for modifications of neuraminic acid containing 9 carbon atoms and having an amino group and a carboxylic acid, and includes N-acetylneuraminic acid, N-glycolylneuraminic acid, and deaminoneuraminic acid. . In particular, N-acetylneuraminic acid exists in various species and is the most predominant sialic acid. The N-linked sugar chains to be measured in step (1) are sialo-glycans that are sugar chains having sialic acid and asialo-glycans that are sugar chains not having sialic acid, and the test method of the present disclosure comprises: It measures the amount or concentration of sialo-glycans and asialo-glycans in a biological sample collected from a subject. As one aspect thereof, the N-linked sugar chains to be measured in step (1) are (i) disialo diantennary sugar chain 1 and (ii) high mannose type sugar chain 2 and asialo diantennary sugar chain digalactose. 3, and at least one asialo-glycan selected from the group consisting of asialo-2-chain sugar chain monogalactose 4:

is.

In the above sugar chain structure, each monosaccharide residue is indicated by an abbreviation. Thus, NeuAc indicates an N-acetylneuraminic acid residue, Gal indicates a galactose residue, Man indicates a mannose residue, GlcNAc indicates an N-acetylglucosamine residue, and Fuc indicates a fucose residue. The term "residue" as used herein is not particularly limited as long as it is a residue of a sugar that constitutes a sugar chain, and is usually a group obtained by removing an atom that detaches from a sugar via a glycosidic bond. In the above sugar chain structure, - indicates a glycosidic bond that forms a sugar chain. In addition, in the asialo double-chain sugar chain monogalactose 4, Gal is linked by a glycosidic bond to only one of the two GlcNAc shown next to each other with the vertical line interposed therebetween. For this reason, there are two types of asialo dichain monogalactose 4, depending on the Gal linkage position. In the above sugar chain structure, the binding mode and binding positions between sugar residues are not particularly limited, and all combinations are included.

An example of the structural formula of the disialo-2-chain sugar chain 1 is the formula (A).

An example of the structural formula of the high mannose-type sugar chain 2 is formula (B).

An example of the structural formula of the asialo di-chain sugar chain digalactose 3 is the formula (C).

Examples of structural formulas of the asialo dichain monogalactose 4 include formula (D1) and formula (D2).

In the examples of the structural formulas above, the notation of the binding mode and binding position follows the usual notation for sugar chains. That is, the notation sugar residue Aα (or β) XY sugar residue B means that the hydroxy group at the X position of sugar residue A and the hydroxy group at the Y position of sugar residue B are α (or β) glycosides. Indicates that they are connected by a bond. Specifically, for example, the notation NeuAα2-6Galβ1-4GlcNAc means that the hydroxy group at the 2-position of N-acetylneuraminic acid and the hydroxy group at the 6-position of galactose are linked by an α-glycosidic bond, and the hydroxy group at the 1-position of the galactose is A sugar chain structure in which the group and the hydroxy group at the 4-position of N-acetylglucosamine are linked by a β-glycosidic bond.

In step (1), the amount or concentration of N-linked sugar chains in the biological sample is measured.

From the viewpoint of test efficiency, N-linked sugar chains to be measured include (i) sialo-glycans and (ii) asialo-glycans. and (ii) at least one asialo-glycan selected from the group consisting of high-mannose-type sugar chain 2, asialo di-chain sugar chain digalactose 3, and asialo di-chain sugar chain monogalactose 4, N-linked sugar chains of fewer types (eg, 30 or less, 20 or less, 10 or less, 5 or less) are preferred. From the viewpoint of test efficiency, it is particularly preferable that the N-linked sugar chains to be measured are (i) sialo-glycans and (ii) only asialo-glycans. and (ii) only at least one asialo-glycan selected from the group consisting of high mannose-type sugar chain 2, asialo di-chain sugar chain digalactose 3, and asialo di-chain sugar chain monogalactose 4 be.

The sample to be measured in step (1) is not particularly limited as long as it is a sample capable of measuring the amount or concentration of N-linked sugar chains in the biological sample, and may be the biological sample itself, or the biological sample may be subjected to some treatment. It may be a sample obtained by

The sample to be measured may be a sample obtained by subjecting a biological sample to sialidase treatment as long as the sialo-glycan to be measured, or the disialo diantennary sugar chain 1, remains in one embodiment, but is preferably a sample. is a sample obtained without subjecting the biological sample to sialidase treatment.

The sample to be measured is preferably a sample obtained by subjecting a biological sample to N-linked sugar chain release treatment. The release treatment is a treatment for releasing N-linked sugar chains from glycoproteins in the sample. Examples of the treatment method include enzymatic methods using N-glycosidase F (also called glycopeptidase, PNGase, glycanase, glycoamidase, etc.), glycopeptidase A, etc., and non-enzymatic methods such as hydrazinolysis. be done. In particular, an enzymatic method using N-glycosidase F can be mentioned as a good example. At that time, a protease such as trypsin can be used together. After the biological sample undergoes N-linked sugar chain release treatment, it is preferable to undergo purification treatment of the free N-linked sugar chain. The purification method is not particularly limited, for example, as long as it is a method of selectively capturing and purifying sugar chains from a mixture in a sample. After precipitating proteins with an alcohol such as ethanol, solid-liquid separation such as centrifugation is performed. method of purification using a sugar chain-binding carrier (for example, BlotGlyco (manufactured by Sumitomo Bakelite Co., Ltd.), which is a commercially available sugar chain-capturing bead).

The sample to be measured is preferably a sample obtained through N-linked sugar chain labeling treatment. The labeling method preferably includes a fluorescent labeling method. Pyridyl amination and 2-aminobenzamidation are the most widely used fluorescent labeling methods, but both labeling methods require harsh labeling conditions, such as the need to heat under acidic conditions. Therefore, from the viewpoint of more stable retention of sialic acid residues, the fluorescent labeling method is preferably a method that allows sugar chain labeling under relatively mild conditions, particularly the 2-aminobenzoic acid method.

The sample to be measured is particularly preferably a sample that has not undergone sialidase treatment and has undergone N-linked sugar chain release treatment and N-linked sugar chain labeling treatment.

The method for measuring the amount or concentration of N-linked sugar chains is not particularly limited as long as the method allows the measurement. From the viewpoint of detection sensitivity, stability of sialic acid residues, and the like, the method is preferably a chromatographic method. Chromatographic methods include normal phase chromatography (eg, hydrophilic interaction chromatography, etc.), ion exchange chromatography, reversed phase chromatography, adsorption chromatography, size exclusion chromatography, and the like. The sugar chain to be measured in the resulting chromatogram can be identified by mass spectrometry or by using a standard sample. Sugar chains used as standard samples (sialo-glycans (in one embodiment, disialo-2-chain sugar chain 1), asialo-glycans (in one embodiment, high-mannose-type sugar chain 2, asialo-2-chain sugar chain digalactose 3, Asialo dichain monogalactose 4)) can be synthesized according to a known method, or can be used as various commercial products. As the disialo diantennary sugar chain 1, for example, a sialyl sugar chain (manufactured by Theraproteins, catalog number: GTP 2N(2,6)-2A) can be used, and as the high mannose type sugar chain 2, for example, Oligomannosidic (Man6GlcNAc2) Theraproteins, catalog number: GTP Man6) can be used, and Asialo diantennary plus proximal α1→6Fucose (Theraproteins, catalog number: GTP 0N-2A+F) can be used as the asialo double-chain sugar chain digalactose 3. As the asialo double-chain sugar chain monogalactose 4, for example, Asialo diantennary minus 1 Gal plus proximal α1→6Fucose (manufactured by Theraproteins, catalog number: GTP 0N-2A-1G+F) can be used.

Measurement methods other than chromatography include mass spectrometry, immunoassay, and electrophoresis (for example, fluorescence-labeled sugar (FACE) analysis by polyacrylamide gel electrophoresis). Immunoassays can be widely employed regardless of whether they are direct, indirect, homogeneous, heterogeneous, competitive, or non-competitive methods. As immunoassays, more specifically, for example, ELISA (e.g., direct method, indirect method, sandwich method, competitive method, etc.), radioimmunoassay (RIA), immunoradiometric assay (IRMA), enzyme immunoassay (EIA), sandwich EIA, immunochromatography, western blot, immunoprecipitation, slot or dot blot assay, immunohistochemical staining, fluorescence immunoassay, immunoassay using avidin-biotin or streptavidin-biotin system, immunoassay using surface plasmon resonance (SPR) method, and the like. Substances that are used in immunoassays and have binding properties to sugar chains to be measured include, for example, antibodies, enzymes, and lectins. As these substances, those synthesized according to known methods can be used, and various commercially available products can also be used.

As a method for measuring the amount or concentration of N-linked sugar chains, one type can be used alone, or two or more types can be used in combination.

After measuring the amount or concentration of the N-linked sugar chain, the ratio of the amount or concentration of the sialo-glycan to the amount or concentration of the asialo-glycan is further calculated to obtain By calculating the ratio between the amount or concentration of 1 and the amount or concentration of the asialo-glycan, it is possible to obtain an index for determining the possibility of contracting the target disease, its severity, or the degree of progression. Therefore, in step (1), the ratio of the amount or concentration of the sialo-glycan to the asialo-glycan (ratio A: sialo-glycan/asialo-glycan) and/or the amount of the asialo-glycan to the sialo-glycan is preferably Alternatively, a step of calculating at least one ratio selected from the group consisting of concentration ratios (ratio B: asialo-glycan/sialo-glycan). In addition, as one embodiment, the ratio of the amount or concentration of disialoditennary sugar chain 1 to asialo-glycan (ratio A: disialoditennary sugar chain 1/asialoglycan), and/or the disialo-diantennary sugar chain A step of calculating at least one ratio selected from the group consisting of the ratio of the amount or concentration of the asialo-glycan to 1 (ratio B: asialo-glycan/disialo diantennary 1). In addition, sialo-glycans, as one aspect, disialoglycans 1, have little difference in amount or concentration between serum samples. The amount or concentration of protein or major serum proteins such as albumin or immunoglobulins may be measured and ratio A and/or ratio B calculated. In that case, the amount or concentration of total serum protein can be measured by the BCA method, the Bradford method, the Lowry method, or the like. In addition, the amount or concentration of major serum proteins such as albumin and immunoglobulin can be measured by ELISA or the like using antibodies specific to each.

According to the test method of the present disclosure including step (1), it is possible to provide an index for determining the possibility of contracting the target disease, its severity, or the degree of progression. This can assist in determining the possibility of contracting the target disease, its severity, or the degree of progression.

1-2. Step (2a)
As an aspect, the inspection method of the present disclosure further includes (2a) based on the comparison result between the ratio A and a preset reference value (reference value A), and / or the ratio B and the preset reference value The step (step (2a)) of determining the possibility that the subject is afflicted with the target disease, or the severity or progression of the target disease in the subject, based on the result of comparison with (reference value B) preferably included. Here, "possibility of suffering from the target disease" means "possibility of suffering from the target disease at the time of collection of the biological sample", and "severity or progress of the target disease" means "biological sample It means the degree of the state of the target disease at the time of collection and the change in the degree over time.

According to the test method of the present disclosure including the step (2a), it is possible to determine the possibility of suffering from the target disease and its severity or progression. In addition, since the test method of the present disclosure can determine the possibility of suffering from the target disease with higher accuracy, its severity or progress, the test method of the present disclosure including this step (2a) can be used to determine the target disease A subject suffering from can be more reliably determined as "having the target disease" (that is, the possibility of misdetermining "not having the target disease" can be further reduced) Also, the severity or progression of the target disease can be determined more accurately.

The preset reference values (reference value A and reference value B, respectively) can be appropriately set by those skilled in the art from the viewpoint of sensitivity, specificity, positive predictive value, negative predictive value, etc.

For example, when determining the possibility of having a target disease (embodiment 1), the reference value is the maximum value of ratio A or ratio B in a biological sample collected from a subject not suffering from the target disease, It can be an average value, a percentile value, or a minimum value. More specifically, for example, by measuring the amount or concentration of N-linked sugar chains in biological samples collected from subjects suffering from the target disease and subjects not suffering from the target disease, Calculate the ratio A and / or ratio B, using the calculated value, statistical analysis based on the analysis of the Receiver Operating Characteristic (ROC) curve (more specifically, using the Youden index method is exemplified.). The reference value is, for example, the 10th to 90th percentile value, 30th to 70th percentile value, 40th to 60th percentile value, 45th to 55th percentile value of ratio A or ratio B in a biological sample collected from a subject not suffering from the target disease It can be a percentile value. In aspect 1, when the ratio A is greater than or equal to the reference value A and/or when the ratio B is less than or equal to the reference value B, it is determined that the subject is highly likely to have the target disease. When the ratio A is equal to or lower than the reference value A and/or when the ratio B is equal to or higher than the reference value B, it can be determined that the subject has a low possibility of suffering from the target disease.

For example, when determining the severity or progression of the target disease (embodiment 2), the reference value is the ratio A Or it can be the maximum, average, percentile, or minimum value of the ratio B. More specifically, for example, the amount or concentration of N-linked sugar chains is measured in a subject with any severity or any degree of progression of the target disease, and the ratio A and/or ratio B is calculated. , using the calculated value, statistical analysis based on analysis of the Receiver Operating Characteristic (ROC) curve, etc. (more specifically, a method using the Youden index is exemplified) can be set by The reference value is, for example, the 10th to 90th percentile value, 30th to 70th percentile value, 40 to It can be the 60th percentile value, the 45th-55th percentile value. In aspect 2, when the ratio A is greater than or equal to the reference value A and/or the ratio B is less than or equal to the reference value B, the severity or progression of the target disease in the subject is less than or equal to the reference value If the ratio A is about the same as the reference value A (e.g., within the range of the reference value A), and/or the ratio B is about the same as the reference value B ( For example, within the range of the reference value B), the severity or progression of the target disease in the subject can be determined to be comparable to the severity or progression of the reference value, and the ratio When A is the reference value A or less and/or when the ratio B is the reference value B or more, the severity or progression of the target disease in the subject is higher than the severity or progression of the reference value can be determined to be low.

The standard value is usually a numerical value of 1 point, but in the case of mode 2, it can be a numerical range with a certain width.

The reference value can also be the ratio A or ratio B in a biological sample collected from the same subject a certain period of time ago. The “fixed period” is not particularly limited as long as it is a period during which the ratio A and/or the ratio B can change within the same subject. Examples include periods of 1 month to 10 years, 2 months to 5 years, 3 months to 2 years, and 4 months to 1 year.

1-3. Step (2b)
As a modification of the test method of the present disclosure, instead of the above step (2a), (2b) the ratio A calculated in step (1) is the ratio in a biological sample collected from the same subject a certain period of time ago A, and/or if the ratio B calculated in step (1) is lower than the ratio B in a biological sample collected from the same subject a certain period of time ago, the subject has a future target disease (step (2b)). The future risk of contracting the target disease is determined by the test method including the step (2b).

"A certain period of time" is not particularly limited as long as it is a period during which the ratio A and/or the ratio B can change within the same subject. Examples include periods of 1 month to 10 years, 2 months to 5 years, 3 months to 2 years, and 4 months to 1 year.

The degree of "high" is not particularly limited, but the ratio A calculated in step (1) is 1.1 times or more, 1.5 times or more, 2 More than twice, more than three times is exemplified.

The degree of "low" is not particularly limited, but the ratio B calculated in step (1) is 90% or less, 70% or less, 50% of the ratio B in the biological sample collected from the same subject a certain period of time ago. % or less and 30% or less.

2. If it is determined that the subject is likely to be suffering from the target disease by the test method of the present disclosure including the diagnostic step (2a) for the target disease with higher accuracy , or the severity of the target disease or When the degree of progression is determined, the test method of the present disclosure further includes (3a) a subject determined to have a high possibility of suffering from the target disease in step (2a), or the severity of the target disease or By combining the step of applying another diagnostic method for the target disease to the subject whose progression has been determined (step (3a)), the possibility of contracting the target disease, its severity, or Progression can be diagnosed. In addition, since the test method of the present disclosure can determine the possibility of suffering from the target disease with higher accuracy, its severity or progress, by combining the step (3a) with the test method of the present disclosure, true A subject suffering from the target disease can be more reliably diagnosed as "suffering from the target disease" (that is, the possibility of misdiagnosing "not suffering from the target disease", and the step (3a ) can be more reduced), and the severity or progression of the target disease can be more accurately diagnosed.

The method of diagnosing the possibility of morbidity, severity, or progression of the target disease applied in step (3a) is not particularly limited, and various known diagnostic methods can be adopted. Examples of the diagnostic method include an interview method, a questionnaire test method, a score method, a pathological diagnosis method, an image diagnosis method, and a blood biochemistry test method. The inquiry method is not particularly limited as long as it is an inquiry by a doctor, and includes both direct inquiry by a doctor and indirect inquiry by a doctor (through another person). The questionnaire test method is not particularly limited. For example, as a test method to measure the cognitive function of neurodegenerative diseases, the revised Hasegawa Intelligence Rating Scale (HDS-R), the Mini-Mental State Examination (MMSE: Mini-Mental State Examination), etc. mentioned. In particular, MMSE is the most widely used test method in the world for the purpose of screening for cognitive impairment diseases, mainly dementia. The scoring method is not particularly limited, and for rheumatoid arthritis, which is one of the autoimmune diseases, there are ARA classification, 2010 ACR/EULAR Classification criteria, severity classification, and the like. SCORD (Severity Scoring of Atopic Dermatitis) and EASI (Eczema Area and Severity Index) are used for atopic dermatitis, which is one of the chronic inflammatory diseases of the skin, and BSA (Body Surface Area) is used for psoriasis. ) and PASI (Psoriasis Area and Severity Index) are used as methods for diagnosing the severity and progression of disease. The pathological diagnosis method is also not particularly limited, and in the case of cancer, stage classification is performed by HE staining, and grade classification is performed based on tissue structure, nuclear heteromorphism, and nuclear fission image, and the severity and progression of cancer can be examined. In addition, the Ki-67 value is calculated by immunohistochemical staining using an antibody against Ki-67, which is a cell proliferation marker, and the severity and progress can be determined from the value. In the case of neurodegenerative diseases, diagnostic imaging methods include CT and MRI to examine morphological abnormalities in the brain such as cerebral atrophy and sulciventricular enlargement, cerebral perfusion scintigraphy (SPECT) to see cerebral blood flow, oxygen Examples include positron emission tomography (PET), which examines consumption and glucose consumption. In the case of rheumatoid arthritis, simple X-ray examination, joint ultrasound, MRI, etc., and in the case of cancer, PET, CT, MRI, ultrasound, etc. are mentioned. Blood biochemical test methods vary depending on the target disease, for example, Alzheimer's disease is amyloid β peptide, rheumatoid arthritis is anti-CCP antibody and CA RF, pancreatic cancer is CA19-9, gastric cancer is CEA, atopic dermatitis is TARC. Anti-double-stranded DNA antibody for systemic lupus erythematosus, and anti-nuclear antibody for scleroderma.

3. In one aspect of the present disclosure, a test agent for a target disease contains at least one selected from the group consisting of a sugar chain and an antibody against the sugar chain, and the sugar chain is (i) a sialo sugar chain and (ii) an asialo sugar chain. and (ii) high mannose-type sugar chain 2, asialo diantennary sugar chain digalactose 3, and asialo diantennary sugar chain monogalactose. It relates to a test agent for a target disease, which is at least one asialo-glycan selected from the group consisting of 4 (herein, also referred to as the “test agent of the present disclosure”). This will be explained below.

The test agent of the present disclosure is used to test the possibility of suffering from the target disease, its severity or progress.

When the test agent of the present disclosure contains a sugar chain, it includes (i) a sialo-glycan and (ii) an asialo-glycan as a sugar chain, and in one aspect, (i) a disialo-2-chain sugar chain 1, and ( ii) contains at least one asialo-glycan selected from the group consisting of 2 high-mannose-type sugar chains, 3 asialo-2-chain sugar chains digalactose, and 4 asialo-2-chain sugar chains monogalactose; These sugar chains can be used as standard samples.

When the test agent of the present disclosure contains an antibody, the antibody includes (xi) an antibody against a sialo-glycan and (xii) an antibody against an asialo-glycan; and (xii) at least one asialo-sugar selected from the group consisting of an antibody against high mannose-type sugar chain 2, an antibody against asialo double-chain sugar chain digalactose 3, and an antibody against asialo double-chain sugar chain monogalactose 4 Contains antibodies against chains. These antibodies can be used to detect target sugar chains in immunoassays and the like.

For example, using hybridoma technology, the antibody can be prepared from sialo-glycans (in one embodiment, disialo-2-chain sugar chain 1) or asialo-glycans (in one embodiment, high-mannose-type sugar chain 2, asialo-2-chain sugar chain, digalactose 3, or after obtaining a hybridoma that produces an antibody that binds to the asialo double-chain sugar chain monogalactose 4), the culture supernatant can be purified. More specifically, antibodies can be obtained, for example, as follows. First, lymphocytes are prepared from lymph nodes or spleen, fused with myeloma cells such as SPM4-0, and cultured. A hybridoma culture supernatant is collected after a certain period of time (for example, 4 weeks) after the fusion, and hybridomas in the culture supernatant that react with sugar chains are selected. Antibodies can be obtained from the supernatant obtained by culturing a large amount of selected hybridomas by a salting-out method and an affinity purification method using a Protein G column or the like.

The test agent of the present disclosure may be in the form of a composition containing at least one selected from the group consisting of sugar chains and antibodies against the sugar chains. The composition may contain other ingredients as needed. Other components include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, and perfumes. , chelating agents and the like.

The test agent of the present disclosure may be used in a kit containing at least one selected from the group consisting of sugar chains and antibodies against the sugar chains. The kit may contain instruments, reagents, and the like that can be used to carry out the testing method of the present disclosure.

Instruments include, for example, test tubes, ultrafiltration units, chromatography columns (eg Sephadex columns, etc.). In addition, reagents include SDS, 2-mercaptoethanol, enzymes for releasing N-linked sugar chains from glycoproteins (e.g., glycosidase F, etc.), sugar labeling agents (e.g., 2-aminobenzoic acid, etc.), sodium acetate, boric acid, sodium cyanoborohydride and the like.

The invention of the present disclosure will be described in detail below based on examples, but the invention of the present disclosure is not limited by these examples.

Example 1. Measurement of asialo-glycan levels (a) Preparation of serum-derived N-linked sugar chains Add 485 μl of purified water to 15 μl of serum from healthy subjects, mix, and then add to AMICON ULTRA-0.5 (manufactured by Merck, catalog number: C82301). and centrifuged at 14000 x g for 25 minutes. 420 μl of purified water was added to the concentrate, the mixture was concentrated again, and the concentrate was dried under reduced pressure. To this dried sample, 70 μl of purified water, 8 μl of 10% SDS, and 0.6 μl of 2-mercaptoethanol were added and mixed, followed by heating at 100° C. for 10 minutes. Next, 8 μl of 10% nonidet P-40 aqueous solution and 10 μl of 1 M phosphate buffer (pH 7.5) were added to the reaction solution and mixed, followed by N-Glycosidase F (manufactured by Poche, catalog number: 06538355103). was added with 3.5 U and allowed to stand at 37°C for 12 hours. After heating this reaction solution at 100° C. for 10 minutes, 232 μl of ethanol was added and mixed, followed by centrifugation at 18000×g for 10 minutes to recover the supernatant. The recovered supernatant was dried under reduced pressure to obtain a serum-derived N-linked sugar chain.

(b) Fluorescence labeling of serum-derived N-linked sugar chains 2% boric acid, 3% sodium cyanoborohydride) (100 μl) was added and mixed, and allowed to stand at 80° C. for 90 minutes. After adding 100 μl of purified water to this reaction solution and mixing, it was applied to a Sephadex LH-20 column (0.75 cm in diameter, 30 cm in length), and the fluorescent fraction was recovered and dried under reduced pressure. This fraction was used as serum-derived 2AA-labeled N-linked sugar chains.

(c) HPLC analysis of serum-derived 2AA-labeled N-linked sugar chains Serum-derived 2AA-labeled N-linked sugar chains were analyzed using an Asahi Shodex NH2P-50 4E column (4.6 mm diameter, 250 mm length). The analysis conditions were as follows: column temperature 50° C.; mobile phase A: acetonitrile solution containing 2% acetic acid; mobile phase B: aqueous solution containing 5% acetic acid and 3% triethylamine. The flow rate was 1 ml/min, and linear gradient elution was performed so that the mobile phase B was 30% for 2 minutes after sample injection and 95% after 80 minutes. The amount of 2AA-labeled N-linked sugar chain subjected to analysis was 0.3 μl in terms of serum.

As shown in Figure 1, mass spectrometry analysis identified the sugar chain with an elution time of around 48 minutes as the disialodichain sugar chain (No. 1). Similarly, asialo-glycans were also identified as follows. That is, the sugar chain around 25 minutes of elution time is high mannose type (M6) sugar chain (No.2), the sugar chain around 24 minutes of elution time is digalactose (No.3), and the sugar chain around 23 minutes of elution time is The sugar chain was identified as an asialo-2-chain sugar chain monogalactose (No.4).

(d) Calculation of serum asialo-glycan levels of healthy subjects and patients with Alzheimer's disease Serum from 19 healthy subjects and 10 Alzheimer's disease patients was subjected to sugar chains No. 1 to No. 4 according to the above (a) to (c). to calculate the asialo-glycan value shown below: (asialo-glycan value) = (disialo-2-chain sugar chain peak area value) / (asialo-glycan peak area value), A comparison was made between sera from healthy subjects and those from Alzheimer's patients.

As a result, as shown in FIGS. 2 to 5, serum asialo-glycan levels were significantly higher in Alzheimer's disease patients than in healthy subjects, and there was a correlation between the incidence of Alzheimer's disease and these asialo-glycan levels. It was shown that

Example 2. Testing for neurodegenerative diseases using asialo-glycan levels as an index 1
Based on the results of Example 1, the vertical axis is the sensitivity (positive rate) (%), and the horizontal axis is the value obtained by subtracting the specificity (%) from 100% (100% - specificity (%)) (false positive rate ) was created using the medical statistics software GraphPad Prism. Based on the ROC curve, a reference value was calculated using Area Under the Curve (AUC) and Youden index as indices. In addition, the sensitivity, specificity and accuracy of the test method of the present disclosure determined based on the calculated reference values were calculated.

As a result, as shown in Table 1, it was shown that the test method of the present disclosure has high accuracy (sensitivity, specificity, accuracy).

Example 3. Testing for neurodegenerative diseases using asialo-glycan levels as an index 2
Healthy subjects, patients with mild cognitive impairment (MMSE = 23-27), patients with moderate Alzheimer's disease (MMSE = 18 or 19), and patients with severe Alzheimer's disease (MMSE = 2 or 4). The asialo-glycan values (No.1/No.3) were calculated according to (a) to (d).

As a result, asialo-glycan levels (No. 1/No. 3) increased according to the degree of neurodegenerative disease, as shown in FIG. It was shown to be inversely correlated. From this, it was shown that the test method of the present disclosure can test the possibility of having a neurodegenerative disease, its severity, or progress.

Example 4. Testing for neurodegenerative diseases using asialo-glycan levels as an index 3
The asialo-glycan levels ( No.1/No.3) was calculated.

As a result, as shown in Figure 7, compared to healthy subjects, patients with mild cognitive impairment and Parkinson's disease had higher plasma asialo-glycan levels and an AUC value of 1 for both. From this, it was shown that the examination method of the present disclosure has a high ability to examine the possibility of having mild cognitive impairment and Parkinson's disease, which are neurodegenerative diseases.

Comparative example 1. Examination of neurodegenerative diseases using CRP level as an index1
The CRP concentration of the same sample used in Example 4 was measured. The CRP concentration was measured by SRL Co., Ltd. using an in vitro diagnostic N-latex CRPII (manufactured by Siemens Healthcare Diagnostics Co., Ltd.).

As a result, as shown in Figure 8, the CRP concentration in the plasma was higher in patients with mild cognitive impairment than in healthy subjects, but the CRP concentration in Parkinson's disease was lower than in healthy subjects. In addition, regarding the ability to test the possibility of having mild cognitive impairment and Parkinson's disease, the AUC values were 0.56 and 0.90, respectively, and the test method of the present disclosure is more likely to have mild cognitive impairment and Parkinson's disease than the CRP test method. It was shown to be highly capable of testing possibilities. In addition, individual differences in asialo-glycan levels in healthy subjects were relatively small, whereas individual differences in CRP concentrations in healthy subjects were relatively large (see standard deviation bars in FIGS. 7 and 8).

Example 5. Testing for neurodegenerative diseases using asialo-glycan levels as an index 4
Asialo-glycan levels (No.1/No.3) according to (a) to (d) of Example 1 for plasma from 5 specimens each from healthy subjects (50s) and multiple sclerosis patients (50s) was calculated.

As a result, as shown in Figure 9, multiple sclerosis patients had higher plasma asialo-glycan levels and an AUC value of 0.92 compared to healthy subjects. From this, it was shown that the testing method of the present disclosure is highly capable of testing for the possibility of having multiple sclerosis, which is a neurodegenerative disease.

Comparative example 2. Examination of neurodegenerative diseases using CRP level as an index 2
Using the same sample as the sample used in Example 5, the CRP concentration was measured in the same manner as in Comparative Example 1.

As a result, as shown in Figure 10, CRP concentrations were lower in multiple sclerosis patients than in healthy subjects. In addition, the AUC value for the ability to test for the possibility of multiple sclerosis is 0.80, and the test method of the present disclosure has a higher ability to test for the possibility of multiple sclerosis than the CRP test method. It was shown to be In addition, individual differences in asialo-glycan levels in healthy subjects were relatively small, whereas individual differences in CRP concentrations in healthy subjects were relatively large (see standard deviation bars in FIGS. 9 and 10).

Example 6. Examination of autoimmune diseases using asialo-glycan levels as an index . Plasma samples from 5 healthy subjects (in their 70s) and 5 samples from rheumatoid arthritis patients (in their 70s) were analyzed according to (a) to (d) of Example 1. The chain value (No.1/No.3) was calculated.

As a result, as shown in FIG. 11, rheumatoid arthritis patients had a higher plasma asialo-glycan level and an AUC value of 1 compared to healthy subjects. It was shown that the ability to test for the possibility of morbidity is high.

Comparative example 3. Examination of Autoimmune Diseases Using CRP Levels as Indicators Using the same specimens as those used in Example 6, the CRP concentration was measured in the same manner as in Comparative Example 1.

As a result, as shown in FIG. 12, the AUC value for the ability to diagnose rheumatoid arthritis was 0.84, and the test method of the present disclosure was more likely to have rheumatoid arthritis, an autoimmune disease, than the CRP test method. It was shown that the ability to inspect the In addition, individual differences in asialo-glycan levels in healthy subjects were relatively small, whereas individual differences in CRP concentrations in healthy subjects were relatively large (see standard deviation bars in FIGS. 11 and 12).

Comparative example 4. Autoimmune disease test using anti-galactose-deficient IgG antibody (CA/RF) level as an index The CA/RF concentration, which is one of the test methods for rheumatoid arthritis, was measured for the same sample as used in Example 6. . Plasma CA/RF concentrations were measured by SRL Co., Ltd. using an in-vitro diagnostic agent, Picolumi CA/RF (manufactured by Sekisui Medical Co., Ltd.).

As a result, as shown in FIG. 13, the ability to diagnose rheumatoid arthritis by the CA/RF test method has an AUC value of 0.92, and the test method of the present disclosure is more autoimmune than the CA/RF test method. It has been shown to be highly capable of testing for the possibility of having a rheumatoid arthritis.

Example 7. Cancer test using asialo-glycan levels as an indicator 1
The asialo-glycan levels (No. 1/No. 3) were calculated according to Example 1 (a) to (d) for sera from 5 samples each from a healthy subject, stage I pancreatic cancer, and stage III patient.

As a result, as shown in FIG. 14, the average value of asialo-glycan levels was higher than that of healthy subjects according to pancreatic cancer stage I, stage III, and the severity or progression of pancreatic cancer. It was shown that the examination method can examine the possibility, severity and progression of pancreatic cancer.

Example 8. Cancer test using asialo-glycan levels as an indicator 2
The asialo-glycan levels (No. 1/No. 3) were calculated according to Example 1 (a) to (d) for serum samples from 5 healthy subjects, 5 gastric cancer stage II patients, and 5 cholangiocarcinoma stage III patients.

As a result, as shown in FIG. 15, gastric cancer patients and cholangiocarcinoma patients had higher serum asialo-glycan levels and AUC values of 0.80 and 1, respectively, compared to healthy subjects. From this, it was shown that the testing method of the present disclosure is highly capable of testing the possibility of cancer.

Comparative example 5. Cancer test using CRP value as an index The CRP concentration of the same specimen as used in Example 8 was measured in the same manner as in Comparative Example 1.

As a result, as shown in FIG. 16, the AUC values for the ability to diagnose gastric cancer and cholangiocarcinoma were 0.56 and 0.72, respectively, and the test method of the present disclosure reduced the possibility of affliction with cancer more than the CRP test method. It was shown that the ability to inspect is high. Moreover, individual differences in asialo-glycan levels in healthy subjects were relatively small, whereas individual differences in CRP concentrations in healthy subjects were relatively large (see standard deviation bars in FIGS. 15 and 16).

Example 9. Asialo-glycan levels (No. 1/No.3) was calculated.

As a result, as shown in FIG. 17, psoriasis patients had higher plasma asialo-glycan levels and an AUC value of 0.88 compared to healthy subjects. It was shown that the ability to inspect the possibility of

Example 10. Examination of connective tissue disease using asialo-glycan levels as an index . Serum from healthy subjects (female, 5 samples), systemic sclerosis (female, 4 samples), and systemic lupus erythematosus patients (female, 5 samples), Example 1 The asialo-glycan values (No.1/No.3) were calculated according to (a) to (d) of .

As a result, as shown in Fig. 18, systemic scleroderma patients and systemic lupus erythematosus patients had higher serum asialo-glycan levels than healthy subjects, with AUC values of 0.95 and 0.80, respectively. From this, it was shown that the testing method of the present disclosure is highly capable of testing for the possibility of contracting collagen disease.

Claims

A method for examining the possibility of having an inflammatory disease, its severity or progression, comprising:
(1) comprising a step of measuring the amount or concentration of an N-linked sugar chain in a biological sample collected from a subject, wherein the N-linked sugar chain is (i) a sialo-glycan and (ii) an asialo-glycan ,Method.
2. The method according to claim 1, wherein the sialic acid contained in the sialo-glycan is N-acetylneuraminic acid.
3. The method according to claim 1 or 2, wherein the asialo-glycan comprises at least a high-mannose-type sugar chain.
The method according to any one of claims 1 to 3, wherein the asialo-glycan comprises at least a complex-type sugar chain.
5. The method according to claim 4, wherein the complex-type sugar chain is a biantennary-type sugar chain.
6. The method according to claim 5, wherein the biantennary-type sugar chain is a sugar chain containing core fucose.
The sialo-glycan is a disialo-diantennary sugar chain 1, and the asialo-glycan comprises a high-mannose-type sugar chain 2, an asialo-diantennary sugar chain digalactose 3, and an asialo-diantennary sugar chain monogalactose 4. At least one asialo-glycan selected from the group:

, the method according to any one of claims 1 to 6.
In step (1), the ratio of the amount or concentration of the sialo-glycan to the asialo-glycan (ratio A: sialo-glycan/asialo-glycan), and/or the amount or concentration of the asialo-glycan to the sialo-glycan 8. The method according to any one of claims 1 to 7, comprising a step of calculating at least one value selected from the group consisting of a concentration ratio (ratio B: asialo-glycan/sialo-glycan).
Furthermore, (2a) based on the comparison result between the ratio A and a preset reference value (reference value A), and/or the comparison result between the ratio B and a preset reference value (reference value B) 9. The method of claim 8, comprising determining the likelihood that the subject is suffering from an inflammatory disease, or the severity or progression of the inflammatory disease of the subject, based on.
The reference value A is the maximum value, mean value, or percentile of the ratio A in a biological sample collected from a subject not suffering from an inflammatory disease or a subject with an inflammatory disease of any severity or any degree of progression. or the minimum value and/or the reference value B in a biological sample taken from a subject not suffering from an inflammatory disease or a subject with any severity or any degree of progression of an inflammatory disease 10. The method of claim 9, wherein the ratio B is the maximum, mean, percentile or minimum value.
The method according to any one of claims 1 to 10, wherein said biological sample is a body fluid or a sample derived from a body fluid.
12. The method of claim 11, wherein said bodily fluid is at least one selected from the group consisting of whole blood, serum, and plasma.
The method according to any one of claims 1 to 12, wherein the sample to be measured in step (1) is a sample obtained without subjecting the biological sample to sialidase treatment.
The method according to any one of claims 1 to 13, wherein the sample to be measured in step (1) is a sample obtained by subjecting the biological sample to N-linked sugar chain release treatment.
15. The method according to claim 14, wherein the method for measuring the amount or concentration of N-linked sugar chains in step (1) comprises chromatography.
16. The method of any of claims 1-15, wherein the inflammatory disease is a chronic inflammatory disease.
17. The method of claim 16, wherein the chronic inflammatory disease is neurodegenerative disease, autoimmune disease, cancer, inflammatory skin disease, or collagen disease.
The chronic inflammatory disease is mild cognitive impairment, Alzheimer's disease, multiple sclerosis, Parkinson's disease, rheumatoid arthritis, autoimmune bullous disease, pancreatic cancer, stomach cancer, ovarian cancer, uterine cancer, cholangiocarcinoma, atopic dermatitis, psoriasis, 18. The method of claim 17, which is systemic scleroderma or systemic lupus erythematosus.
A method for examining the possibility of having a disease selected from the group consisting of neurodegenerative disease, autoimmune disease, cancer, skin inflammatory disease, and collagen disease, and its severity or progression,
(1) comprising a step of measuring the amount or concentration of an N-linked sugar chain in a biological sample collected from a subject, wherein the N-linked sugar chain is (i) a sialo-glycan and (ii) an asialo-glycan ,Method.
the possibility of affliction with an inflammatory disease comprising at least one selected from the group consisting of a sugar chain and an antibody against the sugar chain, wherein the sugar chain is (i) a sialo-glycan and (ii) an asialo-glycan; A test that tests severity or progress.
The sialo-glycan is a disialo-diantennary sugar chain 1, and the asialo-glycan comprises a high-mannose-type sugar chain 2, an asialo-diantennary sugar chain digalactose 3, and an asialo-diantennary sugar chain monogalactose 4. At least one asialo-glycan selected from the group:

21. The test agent according to claim 20.
The test agent according to claim 20 or 21, wherein the inflammatory disease is a chronic inflammatory disease.
The test agent according to claim 22, wherein the chronic inflammatory disease is neurodegenerative disease, autoimmune disease, cancer, inflammatory skin disease, or collagen disease.
The chronic inflammatory disease is mild cognitive impairment, Alzheimer's disease, multiple sclerosis, Parkinson's disease, rheumatoid arthritis, autoimmune bullous disease, pancreatic cancer, stomach cancer, ovarian cancer, uterine cancer, cholangiocarcinoma, atopic dermatitis, psoriasis, The test agent according to claim 23, which is systemic scleroderma or systemic lupus erythematosus.
Neurodegenerative disease, autoimmune disease, cancer, comprising at least one selected from the group consisting of a sugar chain and an antibody against the sugar chain, wherein the sugar chain is (i) a sialo-glycan and (ii) an asialo-glycan , skin inflammatory disease, and collagen disease, a test agent for testing the possibility of contracting a disease, its severity, or the degree of progress thereof.