Method for detecting or quantifying glycoprotein

Abstract

To provide a method for detecting or quantifying glycoprotein that can be used for detection or determination of a disease, such as an arteriosclerotic disease, and an agent that can be used for the method.SOLUTION: There is provided a method for detecting or quantifying glycoprotein including: a peptide consisting of an amino acid sequence represented by the sequence number 1 in a biological sample or an amino acid sequence in which 1-5 amino acid residues are substituted, deleted, added and / or inserted, of amino acid sequences described in the sequence number 1; and a sugar chain coupled to the peptide and represented by the following formula (I): (Hex)a(HexNAc)b(Deoxyhex)c(NeuAc)d+(Man)3(GlcNAc)2 (I) (respective signs in the formula are as defined in the description), the method including (1) detecting or quantifying the sugar chain in the glycoprotein to detect or quantify the glycoprotein.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for detecting or quantifying glycoproteins associated with diseases such as arteriosclerosis. [Background technology]

[0002] Lipoprotein lipase (LPL, hereafter simply referred to as "LPL") is synthesized and secreted from adipose tissue and is thought to be present on the surface of endothelial cells in capillaries. LPL, with apoprotein C-II as an activator, breaks down TG in lipoprotein particles in circulating blood to generate free fatty acids.

[0003] Human LPL is made from the translation region of exons 1 to 10 of the LPL gene, and its structure is known. For example, the sequence registered in GenBank Accession No. NP_000228 is a precursor LPL protein sequence consisting of 475 amino acid residues. This consists of a signal peptide consisting of 27 amino acid residues and a mature LPL consisting of 448 amino acid residues. Furthermore, it has been reported that the glycosylation sites of human LPL are asparagine residue 43 at the N-terminus and asparagine residue 359 at the C-terminus (Non-Patent Document 1). In this specification, the numbers are given with the N-terminal amino acid residue of mature LPL being numbered 1.

[0004] The present inventors have found that 20 kDa (LPL-20) and 21 kDa (LPL-21) molecules, which are the C-terminal portion of the LPL protein, exist in human blood, and have clarified that the difference between these two types of molecules is mainly due to the sugar chains attached to the LPL protein portion. They also found that the ratio of LPL-20 and LPL-21 differs between people with a history of arteriosclerosis and healthy people, and that the ratio of LPL-20 is higher in people with a history of arteriosclerosis. They have proposed that arteriosclerosis can be examined by detecting or quantifying these two types of molecules as biomarkers (Patent Document 1).

[0005] However, no structural analysis of the glycan of the LPL protein has been performed, and the specific glycan structure is unknown. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2010-17107 A [Non-patent literature]

[0007] [Non-Patent Document 1] J Lipid Research; 35; 1511-1523 (1994) Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a method for detecting or quantifying glycoproteins which can be used for detecting or determining diseases such as arteriosclerotic diseases, and a reagent which can be used for said method. [Means for solving the problem]

[0009] As a result of intensive research to solve the above-mentioned problems, the inventors have discovered that by detecting or quantifying a glycoprotein having a specific glycan, it is possible to detect or determine diseases such as arteriosclerosis diseases and arteriosclerosis-related diseases, i.e., that the glycoprotein can be used as a biomarker, and have discovered a method for detecting or quantifying the glycoprotein and a reagent that can be used for the method, thereby completing the present invention. That is, the present invention includes the following aspects. [1] A peptide having an amino acid sequence as shown in SEQ ID NO: 1 in a biological sample, or an amino acid sequence in which 1 to 5 amino acid residues have been substituted, deleted, added and / or inserted from the amino acid sequence as shown in SEQ ID NO: 1, and a compound of the following formula (I) bound to the peptide: (Hex)a (HexNAc) b (Deoxyhex) c (NeuAc) d +(Man)3(GlcNAc)2(I) [In the formula, Man is mannose, GlcNAc is N-acetylglucosamine, Hex is a hexose, HexNAc is N-acetylhexosamine, Deoxyhex is deoxyhexose, NeuAc is N-acetylneuraminic acid, a is 0 to 4; b is 0 to 3; c is 0 or 1 d is 0 to 3; however, If a is 2 and b is 1, then d is not 1, If a is 3 and b is 1, then d is not O or 1.] A method for detecting or quantifying a glycoprotein containing a sugar chain represented by the formula: (1) A method comprising detecting or quantifying the glycoprotein by detecting or quantifying the sugar chain in the glycoprotein. [2] The method according to [1], wherein the detection or quantification of the glycan is carried out using at least one method selected from the group consisting of liquid chromatography, capillary electrophoresis, mass spectrometry, NMR, and a method using a binder that specifically binds to the glycan. [3] The method according to [2], wherein the binder that specifically binds to the glycan includes at least one selected from the group consisting of a lectin, an antibody, and an aptamer. [4] The method according to [2] or [3], wherein the method using a conjugate that specifically binds to the glycan is at least one selected from the group consisting of lectin blotting, lectin array, lectin affinity chromatography, enzyme immunoassay, fluorescent enzyme immunoassay, chemiluminescent enzyme immunoassay, chemiluminescent immunoassay, electrochemiluminescence immunoassay, fluorescent antibody method, radioimmunoassay, Western blotting, immunoblotting, latex agglutination, and immunochromatography. [5] The method according to either [3] or [4], wherein the lectin comprises a mannose-recognition lectin. [6] The method according to [5], wherein the mannose-recognizing lectin comprises at least one selected from the group consisting of GNA (Galanthus nivalis) lectin, NPA (Narcissus pseudonarcissus) lectin, HHL (Hippeastrum Hybrid) lectin, Artocarpin (Artocarpus integrifolia) lectin, CCA (Castanea crenata, Japanese chestnut) lectin, and Calsepa (Calystegia sepium) lectin. [7] The glycan is In the above formula (I), a sugar chain (Ia) is one in which a is 0, b is 1, c is 0 or 1, and d is 0. A sugar chain (Ib) in which a is 2, b is 0, c is 0 or 1, and d is 0 in the formula (I), and In the above formula (I), a is 2, b is 2, c is 0 or 1, and d is 2. The method according to any one of [1] to [6], comprising at least one selected from the group consisting of: [8] Furthermore, (2) When the glycoprotein is detected or quantified in a larger amount than in healthy subjects, the patient is judged to have an arteriosclerosis disease or an arteriosclerosis-related disease. The method according to any one of [1] to [7], comprising: [9] The method according to [8], wherein the arteriosclerotic disease includes at least one selected from the group consisting of acute coronary syndrome, cerebral infarction and aortic dissection.

[10] A peptide having an amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence in which 1 to 5 amino acid residues have been substituted, deleted, added and / or inserted from the amino acid sequence represented by SEQ ID NO: 1, and a compound represented by the following formula (I) bound to the peptide: (Hex) a (HexNAc) b (Deoxyhex) c (NeuAc) d +(Man)3(GlcNAc)2(I) [In the formula, Man is mannose, GlcNAc is N-acetylglucosamine, Hex is a hexose, HexNAc is N-acetylhexosamine, Deoxyhex is deoxyhexose, NeuAc is N-acetylneuraminic acid, a is 0 to 4; b is 0 to 3; c is 0 or 1 d is 0 to 3; however, If a is 2 and b is 1, then d is not 1, If a is 3 and b is 1, then d is not O or 1.] A reagent for detecting or quantifying a glycoprotein having a sugar chain represented by the formula:

[11] The reagent described in

[10] , wherein the binder comprises at least one selected from the group consisting of a lectin, an antibody, and an aptamer.

[12] The reagent described in

[11] , wherein the binder comprises a mannose-recognizing lectin.

[13] The reagent described in

[12] , wherein the mannose-recognizing lectin includes at least one selected from the group consisting of GNA (Galanthus nivalis) lectin, NPA (Narcissus pseudonarcissus) lectin, HHL (Hippeastrum Hybrid) lectin, Artocarpin (Artocarpus integrifolia) lectin, CCA (Castanea crenata, Japanese chestnut) lectin, and Calsepa (Calystegia sepium) lectin.

[14] The glycan is In the above formula (I), a sugar chain (Ia) is one in which a is 0, b is 1, c is 0 or 1, and d is 0. A sugar chain (Ib) in which a is 2, b is 0, c is 0 or 1, and d is 0 in the formula (I), and In the above formula (I), a is 2, b is 2, c is 0 or 1, and d is 2. The reagent according to any one of

[10] to

[13] , comprising at least one selected from the group consisting of:

[15] The reagent according to any one of

[10] to

[14] , for use in detecting or determining an arteriosclerosis disease or an arteriosclerosis-related disease.

[16] The reagent described in

[15] , wherein the arteriosclerotic disease includes at least one selected from the group consisting of acute coronary syndrome, cerebral infarction, and aortic dissection.

[17] A peptide having an amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence in which 1 to 5 amino acid residues have been substituted, deleted, added and / or inserted from the amino acid sequence represented by SEQ ID NO: 1, and a compound represented by the following formula (I) bound to the peptide: (Hex) a (HexNAc) b (Deoxyhex) c (NeuAc) d +(Man)3(GlcNAc)2(I) [In the formula, Man is mannose, GlcNAc is N-acetylglucosamine, Hex is a hexose, HexNAc is N-acetylhexosamine, Deoxyhex is deoxyhexose, NeuAc is N-acetylneuraminic acid, a is 0 to 4; b is 0 to 3; c is 0 or 1 d is 0 to 3; however, If a is 2 and b is 1, then d is not 1, If a is 3 and b is 1, then d is not O or 1.] A biomarker for detecting or determining an arteriosclerosis disease or an arteriosclerosis-related disease, comprising a glycoprotein having a glycan represented by the formula:

[18] The biomarker described in

[17] , wherein the arteriosclerotic disease includes at least one selected from the group consisting of acute coronary syndrome, cerebral infarction, and aortic dissection.

[19] A method for detecting or determining an arteriosclerosis disease or an arteriosclerosis-related disease, comprising: (1) A peptide having an amino acid sequence shown in SEQ ID NO: 1 in a biological sample, or an amino acid sequence in which 1 to 5 amino acid residues have been substituted, deleted, added, and / or inserted from the amino acid sequence shown in SEQ ID NO: 1, and a peptide having the following formula (I) bound to the peptide: (Hex) a (HexNAc) b (Deoxyhex) c (NeuAc) d +(Man)3(GlcNAc)2(I) [In the formula, Man is mannose, GlcNAc is N-acetylglucosamine, Hex is a hexose, HexNAc is N-acetylhexosamine, Deoxyhex is deoxyhexose, NeuAc is N-acetylneuraminic acid, a is 0 to 4; b is 0 to 3; c is 0 or 1 d is 0 to 3; however, If a is 2 and b is 1, then d is not 1, If a is 3 and b is 1, then d is not O or 1.] detecting or quantifying a glycoprotein (A) containing a sugar chain represented by the formula: (2) A peptide having an amino acid sequence shown in SEQ ID NO: 1 in a biological sample, or an amino acid sequence in which 1 to 5 amino acid residues have been substituted, deleted, added, and / or inserted from the amino acid sequence shown in SEQ ID NO: 1, and a peptide having the following formula (II) bound to the peptide: (Hex) a (HexNAc) b (Deoxyhex) c (NeuAc) d +(Man)3(GlcNAc)2(II) [Wherein, Man is mannose, GlcNAc is N-acetylglucosamine, Hex is a hexose, HexNAc is N-acetylhexosamine, Deoxyhex is deoxyhexose, NeuAc is N-acetylneuraminic acid, a is 2 or 3; b is 1, c is 0 or 1 d is 0 or 1; however, If a is 2, then d is 1.] and detecting or quantifying a glycoprotein (B) containing a sugar chain represented by the formula: (3) (3-1) determining that the patient has an arteriosclerosis disease or an arteriosclerosis-related disease when a ratio A / (A+B) of the amount of the detected or quantified glycoprotein (A) to the total amount of the detected or quantified glycoprotein (A) and the glycoprotein (B) is higher than that of a healthy subject; (3-2) determining that the patient has an arteriosclerosis disease or an arteriosclerosis-related disease when the ratio B / (A+B) of the detected or quantified amount of the glycoprotein (B) to the total detected or quantified amounts of the glycoprotein (A) and the glycoprotein (B) is lower than that of a healthy subject; The method includes carrying out at least one of (3-1) or (3-2).

[20] The method according to

[19] , wherein the detection or quantification of the glycan is carried out using at least one method selected from the group consisting of liquid chromatography, capillary electrophoresis, mass spectrometry, NMR, and a method using a binder that specifically binds to the glycan.

[21] The method according to

[20] , wherein the binder that specifically binds to the glycan includes at least one selected from the group consisting of a lectin, an antibody, and an aptamer.

[22] The method according to

[20] or

[21] , wherein the method using a binder that specifically binds to the glycan is at least one selected from the group consisting of lectin blotting, lectin array, lectin affinity chromatography, enzyme immunoassay, fluorescent enzyme immunoassay, chemiluminescent enzyme immunoassay, chemiluminescent immunoassay, electrochemiluminescence immunoassay, fluorescent antibody method, radioimmunoassay, Western blotting, immunoblotting, latex agglutination, and immunochromatography.

[23] The method according to either

[21] or

[22] , wherein the lectin used in the step (1) for detecting or quantifying the glycan comprises a mannose-recognizing lectin.

[24] The method described in

[23] , wherein the mannose-recognizing lectin includes at least one selected from the group consisting of GNA (Galanthus nivalis) lectin, NPA (Narcissus pseudonarcissus) lectin, HHL (Hippeastrum Hybrid) lectin, Artocarpin (Artocarpus integrifolia) lectin, CCA (Castanea crenata, Japanese chestnut) lectin, and Calsepa (Calystegia sepium) lectin.

[25] The glycan in the glycoprotein (A) In the above formula (I), a sugar chain (Ia) is one in which a is 0, b is 1, c is 0 or 1, and d is 0. A sugar chain (Ib) in which a is 2, b is 0, c is 0 or 1, and d is 0 in the formula (I), and In the above formula (I), a is 2, b is 2, c is 0 or 1, and d is 2. The method according to any one of

[19] to

[24] , comprising at least one selected from the group consisting of:

[26] The method according to any one of

[21] to

[25] , wherein the lectin used in the step (2) for detecting or quantifying the glycan comprises a terminal galactose-recognizing lectin.

[27] The method described in

[26] , wherein the terminal galactose-recognizing lectin comprises at least one selected from the group consisting of CA (Colchicum autumnale) lectin, CAA (Caragana arborescens) lectin, ECL (Erythrina cristagalli) lectin, MAL (Maackia amrensis Lectin I) lectin, RCA120 (Ricinus communis) lectin, and galectin.

[28] The method according to any one of

[19] to

[27] , wherein the arteriosclerotic disease includes at least one selected from the group consisting of acute coronary syndrome, cerebral infarction, and aortic dissection. Effect of the Invention

[0010] The glycoprotein of the present invention is present in human blood and has sugar chains that differ in patients suffering from diseases such as arteriosclerosis compared to healthy individuals. The glycoprotein can be detected or quantified based on its sugar chain, and therefore there is no need to detect or quantitate by measuring the molecular weight of the protein. Therefore, according to the present invention, the glycoprotein can be used as a biomarker for diseases such as arteriosclerotic diseases. Furthermore, according to the present invention, by detecting or quantifying the glycoprotein, the onset of diseases such as arteriosclerosis can be easily detected, and the early stage, progression, and aggravation of the disease can be judged. Furthermore, the therapeutic effect against the disease and the efficacy of preventive and therapeutic drugs can be evaluated. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 shows the results of separating LPL-20 and LPL-21 by SDS-polyacrylamide gel electrophoresis (PAGE), followed by staining of proteins in the gel with Coomassie brilliant blue. [Diagram 2] FIG. 2 shows representative examples of HPLC charts of the glycans of glycoprotein (A) and glycoprotein (B). [Diagram 3] FIG. 3 shows the results of measuring LPL-20 and LPL-21 based on absorbance (450 nm) using the ELISA method using F8F9 antibody / biotinylated-GNA lectin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The "biological sample" in the present invention is not particularly limited, but is preferably one that is minimally invasive to the living body, and examples thereof include those secreted from humans, such as blood, serum, plasma, saliva, semen, mucous membrane, tears, and urine, and those collected by biopsy. Preferred examples include blood, serum, and plasma.

[0013] The "glycoprotein" of the present invention (also referred to herein as "glycoprotein (A)" or "LPL-20") comprises a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence in which 1 to 5 amino acid residues have been substituted, deleted, added, and / or inserted within the amino acid sequence shown in SEQ ID NO: 1, and a glycoprotein represented by the following formula (I): (Hex) a (HexNAc) b (Deoxyhex) c (NeuAc) d +(Man)3(GlcNAc)2(I) [In the formula, Man is mannose, GlcNAc is N-acetylglucosamine, Hex is a hexose, HexNAc is N-acetylhexosamine, Deoxyhex is deoxyhexose, NeuAc is N-acetylneuraminic acid, a is 0 to 4; b is 0 to 3; c is 0 or 1 d is 0 to 3; however, If a is 2 and b is 1, then d is not 1, If a is 3 and b is 1, then d is not O or 1.] It contains a glycan represented by the formula:

[0014] The "peptide" of the glycoprotein has the amino acid sequence shown in SEQ ID NO:1: [ka] or the amino acid sequence of SEQ ID NO:1 in which 1 to 5 amino acid residues have been substituted, deleted, added and / or inserted. Here, "the amino acid sequence shown in SEQ ID NO: 1" refers to the C-terminal portion of the LPL protein, i.e., the portion of amino acids up to the 142nd amino acid from the C-terminus of the amino acid sequence of mature LPL (SEQ ID NO: 2), which consists of a total of 448 amino acid residues, and more specifically, the portion of amino acids from the 307th to 448th amino acids from the N-terminus of mature LPL. [ka]

[0015] The above-mentioned "amino acid sequence in which 1 to 5 amino acid residues have been substituted, deleted, added and / or inserted from the amino acid sequence set forth in SEQ ID NO: 1" is not particularly limited as long as it is substantially the same as the peptide shown in SEQ ID NO: 1 or has an activity equivalent thereto. The above amino acid substitution is preferably a conservative substitution. Here, "conservative substitution" means replacing an amino acid residue with another chemically similar amino acid residue so as not to substantially alter the activity of the peptide. For example, a hydrophobic residue may be replaced with another hydrophobic residue, or a polar residue may be replaced with another polar residue having the same charge. Examples of functionally similar amino acids that can be substituted in this way include non-polar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Positively charged (basic) amino acids include arginine, histidine, and lysine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Preferably, the amino acid residue at the 5th and / or 10th position counting from the N-terminus of the amino acid sequence shown in SEQ ID NO:1 is a tyrosine residue, which may be modified or unmodified.

[0016] In the present invention, examples of the "N-acetylhexosamine" in the above formula (I) include N-acetylglucosamine, N-acetylgalactosamine, and the like. The "hexose" in the above formula (I) includes, for example, mannose, galactose, glucose, etc. The "deoxyhexose" in the above formula (I) includes, for example, fucose.

[0017] Preferably, the sugar chain is In the above formula (I), a sugar chain (Ia) is one in which a is 0, b is 1, c is 0 or 1, and d is 0. A sugar chain (Ib) in which a is 2, b is 0, c is 0 or 1, and d is 0 in the formula (I), and In the above formula (I), a is 2, b is 2, c is 0 or 1, and d is 2. The composition includes at least one selected from the group consisting of:

[0018] More preferably, the sugar chain includes at least one type selected from the group consisting of sugar chains having the structural formulas in Table 1 below. [Table 1]

[0019] The "disease" in the present invention is not particularly limited as long as the sugar chain of the "glycoprotein" in the present invention differs between patients suffering from the disease and healthy individuals, and examples of the disease include arteriosclerosis and arteriosclerosis-related diseases. The term "arteriosclerotic disease" in the present invention includes any disease caused by arteriosclerosis. Arteriosclerosis is classified into types such as atherosclerosis, arteriolar sclerosis, and Menkelberg type (medial) sclerosis depending on the manner and site of occurrence of arteriosclerosis. Although the arteriosclerosis in the present invention is not particularly limited, examples thereof include those caused by atherosclerosis. Examples of arteriosclerotic diseases include arteriosclerosis itself, and further include ischemic heart disease such as myocardial infarction and angina pectoris caused by vascular stenosis in the heart or brain due to arteriosclerosis, cerebral infarction, cerebral hemorrhage, aortic aneurysm, aortic dissection, nephrosclerosis in the renal artery and renal failure caused by it, and obliterative arteriosclerosis in the peripheral artery. Preferably, at least one selected from the group consisting of acute coronary syndrome, cerebral infarction, and aortic dissection is included. In the present invention, "arteriosclerosis-related disease" means a disease that originates from chronic inflammation in arteriosclerosis, and examples thereof include non-alcoholic hepatitis (NASH), diabetes, rheumatoid arthritis, psoriasis, dementia, Parkinson's disease, and cancer, and preferably includes non-alcoholic hepatitis (NASH), rheumatoid arthritis, and dementia.

[0020] The present invention includes a method for detecting or quantifying the above-mentioned glycoprotein in a biological sample, which comprises: (1) detecting or quantifying the glycoprotein by detecting or quantifying the glycan in the glycoprotein.

[0021] In the above method, the detection or quantification of the sugar chain is preferably carried out using at least one method selected from the group consisting of liquid chromatography, capillary electrophoresis, mass spectrometry, NMR, and a method using a binder that specifically binds to the sugar chain. These methods can be carried out according to known methods and procedures. Examples of the above-mentioned liquid chromatography method or capillary electrophoresis method include a method in which a glycan is released from a glycoprotein, purified, and then labeled with fluorescence or the like, and the labeled glycan is analyzed by liquid chromatography or capillary electrophoresis to estimate, identify, detect, or quantify the glycan structure. Examples of the above-mentioned mass spectrometry or NMR method include a method in which glycans are released and purified from glycoproteins, and then analyzed by mass spectrometry such as MALDI-TOF MS or ESI-MS or NMR to estimate, identify or detect the glycan structure. Furthermore, examples of methods using a binder that specifically binds to the above-mentioned glycan include methods in which a lectin or antibody that recognizes the glycan is used as the binder to estimate, identify or detect the partial structure of the glycan by the sandwich method or the like.

[0022] The above-mentioned "binding substance that specifically binds to a glycan" is not particularly limited as long as it specifically binds to the above-mentioned glycan, and examples thereof include at least one selected from the group consisting of lectins, antibodies, and aptamers. Here, the term "specifically binds to a sugar chain" means that the sugar chain essentially binds only to a specific sugar chain structure or partial structure.

[0023] The above-mentioned "lectin" is not particularly limited as long as it specifically binds to the above-mentioned sugar chain, and examples thereof include mannose-recognizing lectin. The "mannose-recognizing lectin" may be, for example, at least one selected from the group consisting of GNA (Galanthus nivalis) lectin, NPA (Narcissus pseudonarcissus) lectin, HHL (Hippeastrum Hybrid) lectin, Artocarpin (Artocarpus integrifolia) lectin, CCA (Castanea crenata, Japanese chestnut) lectin, and Calsepa (Calystegia sepium) lectin. Among these, GNA lectin is preferred.

[0024] The above-mentioned "method using a conjugate that specifically binds to a sugar chain" is not particularly limited, but may include, for example, at least one selected from the group consisting of lectin blotting, lectin array, lectin affinity chromatography, enzyme immunoassay, fluorescent enzyme immunoassay, chemiluminescent enzyme immunoassay, chemiluminescent immunoassay, electrochemiluminescence immunoassay, fluorescent antibody method, radioimmunoassay, Western blotting, immunoblotting, latex agglutination, and immunochromatography. These methods can be carried out according to known methods and procedures. For example, the lectin blotting method includes a method in which a protein is subjected to electrophoresis, transferred to a membrane, and then a protein having a target sugar chain is detected with a labeled lectin. Furthermore, the lectin array method includes, for example, a method in which a labeled target protein is reacted with an array on which various lectins are immobilized, and the sugar chain structure of the target protein is estimated, identified or detected.

[0025] The method for detecting or quantifying a glycoprotein further comprises: (2) When the glycoprotein is detected or quantified to be greater than that in healthy subjects, it is determined that the patient has an arteriosclerosis disease or an arteriosclerosis-related disease.

[0026] The determination of arteriosclerosis or arteriosclerosis-related disease in step (2) above may involve, for example, statistical processing of data obtained from multiple patients to determine standard values ​​(cutoff values) for onset or aggravation, thereby detecting the onset of the disease, determining early lesions, progression, and aggravation of the disease, and determining the presence or absence or level of risk thereof.

[0027] [Reagents for detecting or quantifying glycoproteins] The present invention includes a reagent for detecting or quantifying the above-mentioned glycoprotein. The reagent includes a binder that specifically binds to the sugar chain. Examples of the "binder" in the reagent include those described in the method for detecting or quantifying the above-mentioned glycoprotein. The reagent can be used in a method using a conjugate that specifically binds to the above-mentioned sugar chain, and can be used for detecting or determining arteriosclerosis or arteriosclerosis-related diseases.

[0028] [Biomarkers for detecting or determining arteriosclerosis or arteriosclerosis-related diseases] The present invention includes a biomarker for detecting or determining an arteriosclerosis disease or an arteriosclerosis-related disease. The biomarker includes the above-mentioned glycoprotein. The biomarker can be detected or quantified by a method using a conjugate that specifically binds to the sugar chain, and can be used to detect or determine arteriosclerosis or arteriosclerosis-related diseases.

[0029] [Method for detecting or assessing arteriosclerosis or arteriosclerosis-related disease] The present invention includes a method for detecting or determining an arteriosclerosis disease or an arteriosclerosis-related disease, the method comprising the steps of: (1) detecting or quantifying the above-mentioned glycoprotein (hereinafter referred to as "glycoprotein (A)" in this method) by detecting or quantifying the sugar chain; (2) A peptide having an amino acid sequence shown in SEQ ID NO: 1 in a biological sample, or an amino acid sequence in which 1 to 5 amino acid residues have been substituted, deleted, added, and / or inserted from the amino acid sequence shown in SEQ ID NO: 1, and a peptide having the following formula (II) bound to the peptide: (Hex) a (HexNAc) b (Deoxyhex) c (NeuAc) d +(Man)3(GlcNAc)2(II) [Wherein, Man is mannose, GlcNAc is N-acetylglucosamine, Hex is a hexose, HexNAc is N-acetylhexosamine, Deoxyhex is deoxyhexose, NeuAc is N-acetylneuraminic acid, a is 2 or 3; b is 1, c is 0 or 1 d is 0 or 1; however, If a is 2, then d is 1.] (Herein, also referred to as "glycoprotein (B)" or "LPL-21") is detected or quantified by detecting or quantifying the sugar chain. (3) (3-1) determining that the patient has an arteriosclerosis disease or an arteriosclerosis-related disease when a ratio A / (A+B) of the amount of the detected or quantified glycoprotein (A) to the total amount of the detected or quantified glycoprotein (A) and the glycoprotein (B) is higher than that of a healthy subject; (3-2) determining that the patient has an arteriosclerosis disease or an arteriosclerosis-related disease when the ratio B / (A+B) of the detected or quantified amount of the glycoprotein (B) to the total detected or quantified amounts of the glycoprotein (A) and the glycoprotein (B) is lower than that of a healthy subject; Among the above, at least one of (3-1) or (3-2) shall be carried out. Includes.

[0030] "N-acetylhexosamine", "hexose" and "deoxyhexose" in the above formula (II) have the same meanings as in the above formula (I).

[0031] In the above-mentioned method for detecting or determining arteriosclerosis or arteriosclerosis-related diseases, the detection or quantification of sugar chains in steps (1) and (2) can be performed using methods similar to those used for detecting or quantitating glycoproteins. However, because glycoprotein (B) has a different glycan structure from glycoprotein (A), the "conjugate that specifically binds to the glycan" used in step (1) and the "conjugate that specifically binds to the glycan" used in step (2) are different. For example, a mannose-recognizing lectin is used in step (1), and a terminal galactose-recognizing lectin is used in step (2).

[0032] The above-mentioned "terminal galactose-recognizing lectin" includes at least one selected from the group consisting of CA (Colchicum autumnale) lectin, CAA (Caragana arborescens) lectin, ECL (Erythrina cristagalli) lectin, MAL (Maackia amrensis Lectin I) lectin, RCA120 (Ricinus communis) lectin, and galectin.

[0033] The determination of an arteriosclerosis disease or an arteriosclerosis-related disease in the above step (3) includes, for example, statistically processing data obtained from multiple patients to determine a standard value (cutoff value) for onset, and determining that the patient has the disease when the standard value is exceeded or fallen below the standard value. EXAMPLES

[0034] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these. [Example 1] Analysis of LPL glycan structure by LC-MS (liquid chromatography mass spectrometry) (1) Purification of LPL protein from plasma After the operation using a cardiopulmonary bypass, 1000 mL of post-heparin plasma (PHP, LPL protein concentration 536 ng / mL) was added to a heparin column (100 mL), and then 1000 mL of buffer A (10 mM KPB, pH 7.0 / 10% glycerol / 0.1% Chaps / 3 mM Benzamidine / 1 mM EDTA / 0.02% NaN3) was poured through the column to remove components that were not adsorbed to the heparin column. Then, 210 mL (LPL protein concentration 4.5 μg / mL) was collected using 0.8 M NaCl-containing buffer A (Biochim. Biophys. Acta 1003; 254-269 (1989)). To further purify the LPL protein, affinity chromatography was performed in the next step using agarose resin (1.9 mg of monoclonal antibody bound per 1 mL of resin) bound to mouse anti-human LPL monoclonal antibody (B4D4), which specifically recognizes the C-terminal region of LPL. 0.5 mL of the mouse anti-human LPL monoclonal antibody gel was packed into a column. First, the column was washed with 10 mL of an acidic solution for elution of samples (100 mM Glycine / 10% glycerol / 0.15 M NaCl / 0.1% Chaps / 0.02% NaN3, pH 2.7) to elute nonspecifically bound antibodies. The column was immediately neutralized with 10 mL of Tris buffer (20 mM Tris / 0.1% Chaps / 0.02% NaN3, pH 7.4) containing 0.2 M NaCl. 10 mL of the 0.8 M NaCl elution fraction from the heparin column was added to the column containing 0.5 mL of the gel after washing and neutralization, and the mixture was mixed by inversion at room temperature for 1 hour. The column was left to stand to allow proteins that did not bind to the gel to flow out, and the column was washed with Tris buffer containing 10% glycerol and 1 M NaCl. The proteins bound to the column (specifically LPL protein bound) were eluted with the above-mentioned acidic solution for elution of samples, and after neutralization, the LPL protein was recovered (0.5 mL, LPL protein concentration 41 μg / mL). The recovered LPL sample was concentrated from 500 μL to 60 μL using Amicon-YM10 (Merck) to prepare a sample containing LPL (60 μL, LPL protein concentration 0.4 μg / μL) in order to analyze the recovered LPL sample by SDS-polyacrylamide gel electrophoresis. The mouse anti-human LPL monoclonal antibody (B4D4) used was that described in JP-B-6-2075 (Patent No. 1879346).

[0035] (2) Separation of glycoprotein (A) (LPL-20) and glycoprotein (B) (LPL-21) (electrophoresis) The above prepared samples (4 μg protein per lane) were subjected to 12% SDS-polyacrylamide gel electrophoresis (PAGE) to separate LPL-20 and LPL-21, and the proteins in the gel were stained with Coomassie Brilliant Blue. The results are shown in Figure 1.

[0036] (3) Release of N-glycans The gel bands obtained were each cut into pieces approximately 2 mm square and transferred to a 1.5 mL tube. 100 μL of 25 mM ammonium bicarbonate aqueous solution / 50% acetonitrile solution was added to the tube containing the gel bands, shaken for 10 minutes, and then the solution was removed. 100 μL of 25 mM ammonium bicarbonate aqueous solution / 50% acetonitrile solution was added again, shaken for 30 minutes, and then the solution was removed. 100 μL of acetonitrile was added and the solution was removed. The gel pieces were degassed and dried at room temperature for 5 minutes using a centrifugal dryer (SpeedVac). 50 μL of 10 mM DTT (dithiothreitol) was added to the dried gel pieces, and reacted at room temperature for 30 minutes. 10 μL of 100 mM IAA (iodoacetamide) was added and reacted at room temperature for 45 minutes in the dark. 100 μL of 25 mM ammonium bicarbonate aqueous solution / 50% acetonitrile solution was added, shaken for 10 minutes, and then the solution was removed. 100 μL of acetonitrile was added, and the solution was removed. The mixture was degassed and dried at room temperature for 5 minutes using a centrifugal dryer (SpeedVac). 100 μL of 4 units / μL trypsin solution was added to the dried gel pieces and reacted at 37°C for 2 hours. Trypsin was inactivated by heating at 90°C for 10 minutes. 5 μL of N-glycosidase solution was added and reacted at 37°C overnight. The tube was centrifuged to precipitate the gel pieces, and the supernatant was collected in a new tube. 50 μL of pure water was added to the gel pieces to wash the gel, and the solution was collected. The same procedure was repeated. 50 μL of acetonitrile was added to wash the gel, and the solution was collected. The same procedure was repeated two more times. The resulting solution was concentrated and dried, and redissolved in 20 μL of pure water.

[0037] (4) Purification of N-glycans using glycan capture carriers 5 mg of particles with hydrazide groups, which are carriers for capturing sugar chains (BlotGlyco (R)), Sumitomo Bakelite Co., Ltd., BS-45414) was added to a disposable column, and 20 μL of the above sugar chain solution and 180 μL of 2% acetic acid / acetonitrile solution were added and reacted at 80 °C for 1 hour. The reaction was carried out in an open system, and it was visually confirmed that the solvent had completely evaporated and the particles had dried up. After washing the particles with a guanidine solution, water, methanol, and triethylamino solution, 10% acetic anhydride / methanol was added and reacted at room temperature for 30 minutes to cap the unreacted hydrazide groups. After capping, the particles were washed with pure water. Next, 20 μL of ultrapure water and 180 μL of 2% acetic acid / acetonitrile solution were added to the disposable column containing the particles, and the reaction was carried out at 70 °C for 1.5 hours. The reaction was carried out in an open system, and it was visually confirmed that the solvent had completely evaporated and the particles had dried up.

[0038] (5) 2AB labeling of N-glycans Labeling with 2-aminobenzamide (AB) was performed. 50 μL of a solution prepared by dissolving 2-AB and sodium cyanoborohydride in a 30% acetic acid / dimethyl sulfoxide (DMSO) mixed solvent to final concentrations of 0.35 M and 1 M, respectively, was added to a disposable column containing the particles, and the mixture was allowed to react at 60°C for 2 hours.

[0039] (6) Removal of excess 2AB 50 μL of the reaction solution was collected and diluted 10-fold with acetonitrile, then added to a silica column (BlotGlyco kit accessory) to adsorb the labeled glycan onto the silica gel. After washing the column with acetonitrile, the labeled glycan was collected with 50 μL of ultrapure water.

[0040] (7) Detection of labeled glycans The resulting labeled glycans were analyzed by LC-MS under the following conditions. LC equipment: Nexera (Shimadzu Corporation) ·LC analysis conditions Color :ACQUITY UPLC (R) Glycan BEH Amide,1.7μm(2.1mm ID x 150mm L.) Color temperature: 40℃ Mobile phase A: 40% acetonitrile aqueous solution containing 0.1% formic acid Mobile phase B: 90% acetonitrile aqueous solution containing 0.1% formic acid Concentration gradient conditions: shown in the table below. [Table 2] Flow rate: 0.2mL / min Injection volume: 1μL Fluorescence detector: RF-20Axs (excitation wavelength 330 nm, fluorescence wavelength 420 nm) ·Mass spectrometer: LCMS-IT-TOF (Shimadzu Corporation) ·Mass spectrometry conditions Ionization mode: ESI negative ion mode

[0041] (8) A representative example of the HPLC chart obtained in the above study is shown in Figure 2. As a result of mass spectrometry, the glycans shown in Figure 2 were detected from each band. A glycan characteristic of glycoprotein (A) was detected at the position enclosed by the dotted line. In addition, a glycan characteristic of glycoprotein (B) was also detected from glycoprotein (B). From this, it is believed that the presence or absence of the target glycoprotein can be confirmed by directly measuring the glycans bound to the glycoprotein.

[0042] (9) Detection of LPL-20 by lectin Here, we will describe in detail the detection using the plant-derived lectin Galanthus nivalis GNA (hereafter, GNA lectin). GNA lectin is known to have a strong affinity for mannose-containing glycans, and as a result of (7), it is believed to strongly recognize the glycan with the following structure, which was only present in LPL-20. [ka] LPL-20 was detected by immobilization of mouse anti-human LPL monoclonal antibody (F8F9) obtained from mouse ascites and lectin ELISA (Enzyme-Linked Immunosorbent Assay). First, F8F9-F(ab')2, in which the antibody site (Fc) that may react with lectin was removed using Ficin protease, was used. First, 100 μL of 10.0 μg / mL F8F9-F(ab')2 antibody solution was dispensed into a 96-well plate (NUNC Maxisorp) and left to stand overnight at 4°C to immobilize the antibody. After removing the antibody solution, the plate was washed three times with a washing solution (0.05% Tween 20, 20 mM PBS, pH 7.2). Then, 100 μL of 0.5% PVA (polyvinyl alcohol) aqueous solution was dispensed and left at 4 °C for 24 hours to perform blocking. After removing the blocking solution, the plate was washed three times with the above washing solution before use. As specimens to be added, LPL samples purified from plasma by the method (1) were gel-filtered with Sephadex G-200 or Sephadex G-75, and LPL-20, a mixture of LPL-20 and LPL-21, and three fractions containing LPL-21 were used. Each of the three fractions was added to each well with the antibody immobilized at a concentration of 0 ng, 80 ng, and 160 ng / 100 μL, and the antigen-antibody reaction was carried out at room temperature for 30 minutes. After removing the unreacted LPL sample, the plate was washed three times with a washing solution. Next, 100 μL of 40 μg / mL biotinylated-GNA lectin was dispensed into each well and reacted at room temperature for 1 hour. The lectin solution was removed, and the plate was washed three times with washing solution, after which 100 μL of a 1000-fold diluted solution of VECTASTAIN ABC reagent (Vector, PK-6100), a complex of avidin, biotin, and HRP, was dispensed and reacted at room temperature for 30 minutes. After removing the solution, the plate was washed three times. Next, 100 μL of TMB solution (Nacalai Tesque, 05299) was added and reacted at room temperature for 30 minutes, and then 100 μL of 1N sulfuric acid was added to stop the reaction. After stopping the reaction, the absorbance (450 nm) was measured, and the results obtained are shown in Figure 3. As expected, strong absorbance was obtained with LPL-20, while it was confirmed that there was no reaction with LPL-21.

Claims

1. A peptide consisting of the amino acid sequence shown in Sequence ID No. 1 in a biological sample, or an amino acid sequence in which 1 to 5 amino acid residues are substituted, deleted, added, and / or inserted from the amino acid sequence described in Sequence ID No. 1, and the following formula (I) bound to the peptide: (Hex) a (HexNAc) b (Deoxyhex) c (NeuAc) d +(Man) 3 (GlcNAc) 2 (I) [In the formula, Man is mannose, GlcNAc is N-acetylglucosamine, Hex is a hexose, HexNAc is N-acetylhexosamine, Deoxyhex is deoxyhexose, NeuAc is N-acetylneuraminic acid, a is between 0 and 4. b is between 0 and 3. c is either 0 or 1 d is between 0 and 3. however, If a is 2 and b is 1, then d is not 1. If a is 3 and b is 1, then d is neither 0 nor 1. A method for detecting or quantifying glycoproteins containing sugar chains represented by, (1) A method comprising detecting or quantifying a glycoprotein by detecting or quantifying the sugar chain in the glycoprotein.

2. The method according to claim 1, wherein the detection or quantification of the glycan is performed by at least one method selected from the group consisting of liquid chromatography, capillary electrophoresis, mass spectrometry, NMR, and a method using a conjugate that specifically binds to the glycan.

3. The method according to claim 2, wherein the conjugate that specifically binds to the sugar chain comprises at least one selected from the group consisting of lectins, antibodies, and aptamers.

4. The method according to claim 2, wherein the method using a conjugate that specifically binds to the sugar chain is at least one selected from the group consisting of lectin blotting, lectin arraying, lectin affinity chromatography, enzyme immunoassay, fluorescent enzyme immunoassay, chemiluminescent enzyme immunoassay, chemiluminescent immunoassay, electrochemiluminescent immunoassay, fluorescent antibody assay, radioimmunoassay, Western blotting, immunoblotting, latex agglutination, and immunochromatography.

5. The method according to claim 3, wherein the lectin includes a mannose-recognizing lectin.

6. The method according to claim 5, wherein the mannose-recognizing lectin comprises at least one selected from the group consisting of GNA (Galanthus nivalis) lectin, NPA (Narcissus pseudonarcissus) lectin, HHL (Hippeastrum Hybrid) lectin, Artocarpin (Artocarpus integrifolia) lectin, CCA (Castanea crenata, Japanese chestnut) lectin, and Calsepa (Calystegia sepium) lectin.

7. The aforementioned sugar chain, In the above formula (I), a sugar chain (Ia) is such that a is 0, b is 1, c is 0 or 1, and d is 0. In formula (I), a sugar chain (Ib) is such that a is 2, b is 0, c is 0 or 1, and d is 0, and In the above formula (I), a is 2, b is 2, c is 0 or 1, and d is 2 (Ic) The method according to claim 1, comprising at least one selected from the group consisting of the following.

8. moreover, (2) If the glycoprotein is detected or quantified in a higher amount compared to healthy individuals, it shall be determined to be an arteriosclerotic disease or an arteriosclerosis-related disease. The method according to any one of claims 1 to 7, including the method described in any one of claims 1 to 7.

9. The method according to claim 8, wherein the arteriosclerotic disease includes at least one selected from the group consisting of acute coronary syndrome, cerebral infarction, and aortic dissection.

10. A peptide consisting of the amino acid sequence shown in Sequence ID No. 1, or an amino acid sequence in which 1 to 5 amino acid residues are substituted, deleted, added, and / or inserted from the amino acid sequence described in Sequence ID No. 1, and the following formula (I) bound to the peptide: (Hex) a (HexNAc) b (Deoxyhex) c (NeuAc) d +(Man) 3 (GlcNAc) 2 (I) [In the formula, Man is mannose, GlcNAc is N-acetylglucosamine, Hex is a hexose, HexNAc is N-acetylhexosamine, Deoxyhex is deoxyhexose, NeuAc is N-acetylneuraminic acid, a is between 0 and 4. b is between 0 and 3. c is either 0 or 1 d is between 0 and 3. however, If a is 2 and b is 1, then d is not 1. If a is 3 and b is 1, then d is neither 0 nor 1. A reagent for detecting or quantifying glycoproteins containing a glycan represented by [a specific symbol], comprising a conjugate that specifically binds to the glycan.

11. The reagent according to claim 10, wherein the conjugate comprises at least one selected from the group consisting of lectins, antibodies, and aptamers.

12. The reagent according to claim 11, wherein the conjugate comprises a mannose-recognizing lectin.

13. The reagent according to claim 12, wherein the mannose-recognizing lectin comprises at least one selected from the group consisting of GNA (Galanthus nivalis) lectin, NPA (Narcissus pseudonarcissus) lectin, HHL (Hippeastrum Hybrid) lectin, Artocarpin (Artocarpus integrifolia) lectin, CCA (Castanea crenata, Japanese chestnut) lectin, and Calsepa (Calystegia sepium) lectin.

14. The aforementioned sugar chain, In the above formula (I), a sugar chain (Ia) is such that a is 0, b is 1, c is 0 or 1, and d is 0. In formula (I), a sugar chain (Ib) is such that a is 2, b is 0, c is 0 or 1, and d is 0, and In the above formula (I), a is 2, b is 2, c is 0 or 1, and d is 2 (Ic) The reagent according to claim 10, comprising at least one selected from the group consisting of the following.

15. A reagent according to any one of claims 10 to 14, for use in detecting or determining arteriosclerotic diseases or arteriosclerosis-related diseases.

16. The reagent according to claim 15, wherein the arteriosclerotic disease comprises at least one selected from the group consisting of acute coronary syndrome, cerebral infarction, and aortic dissection.

17. A peptide consisting of the amino acid sequence shown in Sequence ID No. 1, or an amino acid sequence in which 1 to 5 amino acid residues are substituted, deleted, added, and / or inserted from the amino acid sequence described in Sequence ID No. 1, and the following formula (I) bound to the peptide: (Hex) a (HexNAc) b (Deoxyhex) c (NeuAc) d +(Man) 3 (GlcNAc) 2 (I) [In the formula, Man is mannose, GlcNAc is N-acetylglucosamine, Hex is a hexose, HexNAc is N-acetylhexosamine, Deoxyhex is deoxyhexose, NeuAc is N-acetylneuraminic acid, a is between 0 and 4. b is between 0 and 3. c is either 0 or 1 d is between 0 and 3. however, If a is 2 and b is 1, then d is not 1. If a is 3 and b is 1, then d is neither 0 nor 1. A biomarker for the detection or determination of atherosclerotic disease or atherosclerosis-related disease, comprising a glycoprotein containing a sugar chain as shown.

18. The biomarker according to claim 17, wherein the arteriosclerotic disease comprises at least one selected from the group consisting of acute coronary syndrome, cerebral infarction, and aortic dissection.

19. A method for detecting or determining arteriosclerotic disease or arteriosclerosis-related disease, (1) A peptide consisting of an amino acid sequence in a biological sample that is represented by the amino acid sequence shown in Sequence ID No. 1, or an amino acid sequence in which 1 to 5 amino acid residues are substituted, deleted, added and / or inserted from the amino acid sequence described in Sequence ID No. 1, and the following formula (I) bound to the peptide: (Hex) a (HexNAc) b (Deoxyhex) c (NeuAc) d +(Man) 3 (GlcNAc) 2 (I) [In the formula, Man is mannose, GlcNAc is N-acetylglucosamine, Hex is a hexose, HexNAc is N-acetylhexosamine, Deoxyhex is deoxyhexose, NeuAc is N-acetylneuraminic acid, a is between 0 and 4. b is between 0 and 3. c is either 0 or 1 d is between 0 and 3. however, If a is 2 and b is 1, then d is not 1. If a is 3 and b is 1, then d is neither 0 nor 1. To detect or quantify a glycoprotein (A) containing a glycan shown by, by detecting or quantifying the glycan, (2) A peptide consisting of an amino acid sequence in a biological sample that is represented by the amino acid sequence shown in Sequence ID No. 1, or an amino acid sequence in which 1 to 5 amino acid residues are substituted, deleted, added and / or inserted from the amino acid sequence described in Sequence ID No. 1, and the following formula (II) bound to the peptide: (Hex) a (HexNAc) b (Deoxyhex) c (NeuAc) d +(Man) 3 (GlcNAc) 2 (II) [In the formula, Man is mannose, GlcNAc is N-acetylglucosamine, Hex is a hexose, HexNAc is N-acetylhexosamine, Deoxyhex is deoxyhexose, NeuAc is N-acetylneuraminic acid, a is either 2 or 3, b is 1, c is either 0 or 1 d is either 0 or 1, however, If a is 2, then d is 1. To detect or quantify a glycoprotein (B) containing a glycan shown by, by detecting or quantifying the glycan, (3) (3-1) If the ratio A / (A+B) of the detected or quantified amount of glycoprotein (A) to the total amount of detected or quantified amounts of glycoprotein (A) and glycoprotein (B) is higher than that of a healthy person, it shall be determined that the person has an arteriosclerotic disease or an arteriosclerosis-related disease. (3-2) If the ratio B / (A+B) of the detected or quantified amount of glycoprotein (B) to the total detected or quantified amounts of glycoprotein (A) and glycoprotein (B) is lower than that of a healthy person, it shall be determined that the person has an arteriosclerotic disease or an arteriosclerosis-related disease. A method comprising performing at least one of (3-1) or (3-2).

20. The method according to claim 19, wherein the detection or quantification of the glycan is performed by at least one method selected from the group consisting of liquid chromatography, capillary electrophoresis, mass spectrometry, NMR, and a method using a conjugate that specifically binds to the glycan.

21. The method according to claim 20, wherein the conjugate that specifically binds to the sugar chain comprises at least one selected from the group consisting of lectins, antibodies, and aptamers.

22. The method according to claim 20, wherein the method using a conjugate that specifically binds to the sugar chain is at least one selected from the group consisting of lectin blotting, lectin arraying, lectin affinity chromatography, enzyme immunoassay, fluorescence enzyme immunoassay, chemiluminescence enzyme immunoassay, chemiluminescence immunoassay, electrochemiluminescence immunoassay, fluorescence antibody assay, radioimmunoassay, Western blotting, immunoblotting, latex agglutination, and immunochromatography.

23. The method according to claim 21, wherein the lectin used for detecting or quantifying the sugar chain in step (1) includes a mannose-recognizing lectin.

24. The method according to claim 23, wherein the mannose-recognizing lectin comprises at least one selected from the group consisting of GNA (Galanthus nivalis) lectin, NPA (Narcissus pseudonarcissus) lectin, HHL (Hippeastrum Hybrid) lectin, Artocarpin (Artocarpus integrifolia) lectin, CCA (Castanea crenata, Japanese chestnut) lectin, and Calsepa (Calystegia sepium) lectin.

25. The sugar chain in the glycoprotein (A) is In the above formula (I), a sugar chain (Ia) is such that a is 0, b is 1, c is 0 or 1, and d is 0. In formula (I), a sugar chain (Ib) is such that a is 2, b is 0, c is 0 or 1, and d is 0, and In the above formula (I), a is 2, b is 2, c is 0 or 1, and d is 2 (Ic) The method according to claim 19, comprising at least one selected from the group consisting of the following.

26. The method according to claim 21, wherein the lectin used for detecting or quantifying the sugar chain in step (2) comprises a terminal galactose-recognizing lectin.

27. The method according to claim 26, wherein the terminal galactose-recognizing lectin comprises at least one selected from the group consisting of CA (Colchicum autumnale) lectin, CAA (Caragana arborescens) lectin, ECL (Erythrina cristagalli) lectin, MAL (Maackia amrensis Lectin I) lectin, RCA120 (Ricinus communis) lectin, and galectin.

28. The method according to any one of claims 19 to 27, wherein the arteriosclerotic disease includes at least one selected from the group consisting of acute coronary syndrome, cerebral infarction, and aortic dissection.

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