Development of blood fibrosis markers for nonalcoholic steatohepatitis

Abstract

A method for evaluating the progression of hepatic fibrosis in non-alcoholic steatohepatitis, said method comprising measuring the amount of a sugar chain having a structure represented by formula (I) and / or a precursor sugar chain of the biosynthesis of a sugar chain having a structure represented by formula (I) in a sample.

Description

[Technical Field]

[0001] The present invention relates to a marker for liver disease. In particular, the present invention relates to a blood fibrosis marker for nonalcoholic steatohepatitis. [Background technology]

[0002] Because the progression of liver fibrosis in nonalcoholic steatohepatitis (NASH) leads to the development of liver cirrhosis and liver cancer, early detection of fibrosis is crucial. While histological assessment by liver biopsy is the most accurate method for assessing liver fibrosis, it places a significant burden on patients and carries the risk of infection. Therefore, blood hyaluronic acid (Non-Patent Document 1), type IV collagen, and M2BPGi (Non-Patent Document 2) have been used to non-invasively assess liver fibrosis. However, these levels are elevated in other diseases, so highly disease-specific diagnostic markers are needed. For example, M2BPGi was discovered as an indicator of liver fibrosis in patients with hepatitis C (Non-Patent Document 2), but it has not been optimized for assessing liver fibrosis in NASH. Previously, the inventors have found that serum expression of the N-linked glycan on α1-antitrypsin (A3F) significantly increases during the fibrosis process in NASH disease (Patent Document 1). However, A3F correlates better with inflammation in liver tissue than fibrosis indicators such as factor F. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. WO2017-126514 [Non-patent literature]

[0004] [Non-Patent Document 1] Suzuki A, Angulo P, Lymp J, Li D, Satomura S, Lindor K. Hyaluronic acid, an accurate serum marker for severe hepatic fibrosis in patients with non-alcoholic fatty liver disease. Liver Int 2005; 25:779-786. [Non-patent document 2] Tianhui L, Xiaoming W, Morten A, Diana JL,and Federica G. Molecular Serum Markers of Liver Fibrosis. Biomark Insights. 2012; 7: 105-117. [Non-patent document 3] Kuno A, Ikehara Y, Tanaka Y, et al. A serum "sweet-doughnut" protein facilitates fibrosis evaluation and therapy assessment in patients with viral hepatitis. Sci Rep. 2013; 3:1065. Summary of the Invention [Problem to be solved by the invention]

[0005] It was necessary to find a disease-specific, non-invasive marker that could evaluate the progression of liver fibrosis in NASH. [Means for solving the problem]

[0006] The present inventors conducted extensive research to solve the above-mentioned problems and found that the expression of blood glycans (A2F bisect) and their biosynthetic precursor glycans increases with the progression of liver fibrosis in NASH. Furthermore, the present inventors found that the A2F bisect glycan bound to IgA2 and the biosynthetic precursor of the A2F bisect glycan bound to IgA2 are preferred markers. Based on these findings, the present inventors have completed the present invention.

[0007] Thus, the present invention provides the following: (1) Formula (I) in a sample: [ka] A method for evaluating the progression of liver fibrosis in NASH, comprising measuring the amount of a glycan having a structure represented by formula (I) and / or a biosynthetic precursor glycan of a glycan having a structure represented by formula (I). (2) The method according to (1), which comprises measuring the total amount of biosynthetic precursor glycans of glycans having the structure represented by formula (I). (3) The biosynthetic precursor glycans of the glycans having the structure represented by formula (I) are glycans 1, 2A, 2B, 3A, 3B, 4, 5A, and 5B: [ka] The method according to (1) or (2), wherein the compound has one or more structures selected from the group consisting of: (4) A method according to any one of (1) to (3), wherein a glycan having a structure represented by formula (I) and / or a biosynthetic precursor glycan of a glycan having a structure represented by formula (I) is bound to IgA2. (5) The method according to any one of (1) to (4), wherein the sample is a blood sample. (6) A marker for evaluating the progression of liver fibrosis in NASH, comprising the formula (I): [ka] and / or a biosynthetic precursor sugar chain of a sugar chain having a structure represented by formula (I). (7) The biosynthetic precursor glycans of the glycans having the structure represented by formula (I) are glycans 1, 2A, 2B, 3A, 3B, 4, 5A, and 5B: [ka] The marker according to (6), which has one or more structures selected from the group consisting of: (8) A marker according to (6) or (7), in which a glycan having a structure represented by formula (I) and / or a biosynthetic precursor glycan of a glycan having a structure represented by formula (I) is bound to IgA2. (9) A kit for evaluating the progression of liver fibrosis in NASH, comprising: [ka] and / or a kit comprising a sugar chain having a structure represented by formula (I) and / or a biosynthetic precursor sugar chain of a sugar chain having a structure represented by formula (I). (10) The biosynthetic precursor glycans of the glycans having the structure represented by formula (I) are glycans 1, 2A, 2B, 3A, 3B, 4, 5A, and 5B: [ka] The kit according to (9), which has one or more structures selected from the group consisting of: (11) The kit according to (9) or (10), wherein the means is an antibody, antibody fragment, interacting protein, or peptide specific to a protein to which a glycan having a structure represented by formula (I) and / or a biosynthetic precursor glycan of a glycan having a structure represented by formula (I) is bound. (12) The kit according to (11), wherein the antibody, antibody fragment, interacting protein, or peptide is specific to IgA2. [Effects of the Invention]

[0008] According to the present invention, a disease-specific, non-invasive marker capable of determining hepatic fibrosis in NASH is provided. Therefore, by using the marker of the present invention, hepatic fibrosis in NASH can be accurately determined and the burden on patients can be reduced. The marker of the present invention can be used to accurately examine the progression of NASH and the therapeutic effect of NASH. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 is a graph showing the expression level of A2F bisect associated with fibrosis. [Figure 2] Figure 2 is a graph showing the changes in expression of the biosynthetic precursor glycans of A2F bisect during fibrillation. The arrows in the figure indicate the A2F biosynthetic pathway. The structures of glycans 1, 2 (2A, 2B), 3 (3A, 3B), 4, and 5 (5A, 5B) are shown herein. [Figure 3] FIG. 3 is a graph showing the results of analyzing the correlation between pathological fibrosis evaluation in liver biopsies and fibrosis evaluation factors in the blood and the expression of A2F bisect. [Figure 4] FIG. 4 is a graph comparing the expression levels of A2F bisect between a NASH fibrosis group and a hepatitis C (HCV) patient group. [Figure 5] Figure 5 shows the results of ROC analysis using specimens with defined fibrosis stages (F3 or higher). From left to right, the ROC curves are for type IV collagen 7s, Fib4, A2F bisect, and the sum of the biosynthetic precursor glycans 1, 2A, 2B, 3A, 3B, 4, 5A, and 5B of A2F bisect. [Figure 6]Figure 6 shows the results of N-linked glycan analysis in the flow-through fraction and protein G-bound fraction of NASH F3 / 4 pooled serum. The left panel shows SDS-PAGE (CBB staining) of serum and each fraction, the middle panel shows an analysis chart of N-linked glycans by MALDI-TOF MS, and the right panel shows an enlarged view of the area around m / z 3100 (highlighted area) on the chart. [Figure 7] FIG. 7 shows the results of N-linked glycan analysis and carrier analysis of protein bands detected by CBB staining after separating the protein G-binding fraction of NASH F3 / 4 pooled serum by SDS-PAGE. [Figure 8] FIG. 8 shows the results of examining serum IgA2 concentrations with progression of fibrosis using an IgA2 ELISA kit. DETAILED DESCRIPTION OF THE INVENTION

[0010] In one aspect, the present invention provides a method for detecting a compound of formula (I) in a sample: [ka] and / or the amount of a biosynthetic precursor sugar chain of a sugar chain having a structure represented by formula (I).

[0011] The glycan having the structure shown in formula (I) is called A2F bisect. A2F bisect is an N-linked glycan that is bound to proteins via an asparagine residue. The biosynthetic precursor glycan of A2F bisect may be any glycan located upstream in the biosynthetic pathway of A2F bisect. Examples of the biosynthetic precursor glycans of A2F bisect include glycans 1, 2A, 2B, 3A, 3B, 4, 5A, and 5B: [ka] Examples of such sugar chains include, but are not limited to, sugar chains having the structure shown in the following formula (for convenience in this specification, these sugar chains may be referred to as sugar chains 1, 2A, 2B, 3A, 3B, 4, 5A, and 5B).

[0012] In formula (I) and sugar chains 1, 2A, 2B, 3A, 3B, 4, 5A, and 5B, GlcNAc represents N-acetylglucosamine, Man represents mannose, Fuc represents fucose, Gal represents galactose, and Neu5Ac represents N-acetylneuraminic acid. In formula (I) and sugar chains 1, 2A, 2B, 3A, 3B, 4, 5A, and 5B, a1-3, a1-6, and a2-6 represent α1-3, α1-6, and α2-6 glycosidic bonds, respectively, and b1-2 and b1-4 represent β1-2 and β1-4 glycosidic bonds, respectively. In formula (I) and sugar chains 1, 2A, 2B, 3A, 3B, 4, 5A, and 5B, GlcNAcb1- at the reducing end represents a bond to asparagine in the protein.

[0013] In the above-described methods of the present invention, A2F bisect and / or its biosynthetic precursor glycans are used as markers for liver fibrosis in NASH. A2F bisect alone may be used as a marker, or one or more of its biosynthetic precursor glycans may be used as markers. Alternatively, A2F bisect and one or more of its biosynthetic precursor glycans may be used as markers. Using two or more glycans as markers and calculating their sum can improve the accuracy of assessing the progression of liver fibrosis. For example, two or more of A2F bisect's biosynthetic precursor glycans may be used as markers and calculating their sum to assess the progression of liver fibrosis. In one specific example of the method of the present invention, the progression of liver fibrosis is assessed by calculating the amount of A2F bisect alone. In a further specific example of the method of the present invention, the progression of liver fibrosis is evaluated by calculating the sum of the amounts of seven types of sugar chains, sugar chains 1, 2A, 2B, 3A, 3B, 4, 5A, and 5B.

[0014] The present inventors have found that A2F bisect glycosylation of IgA2 is promoted with the progression of liver fibrosis, while serum IgA2 does not significantly increase. Therefore, in the present invention, preferred markers of liver fibrosis in NASH are A2F bisect bound to IgA2 (immunoglobulin IgA2 protein) and the biosynthetic precursor glycosylation of A2F bisect bound to IgA2. It was previously unknown that A2F bisect and its biosynthetic precursor glycosylation can be used as markers of liver fibrosis progression in NASH, and that A2F bisect bound to IgA2 and the biosynthetic precursor glycosylation of A2F bisect bound to IgA2 can be used as preferred markers of liver fibrosis progression in NASH.

[0015] The IgA2 protein may be a wild-type or mutant. The IgA2 mutant may be a mutant due to, for example, an in vivo mutation or polymorphism. The IgA2 mutant may have an amino acid sequence in which one to several amino acids are deleted, substituted, added, or inserted in the amino acid sequence of wild-type IgA2. Here, several means two, three, four, five, six, seven, eight, or nine. In the mutant IgA2 protein, it is preferable that the asparagine residue to which a sugar chain can be bound is conserved.

[0016] The sample used in the method of the present invention may be obtained from any subject, but is preferably obtained from a subject suffering from or suspected of having NASH. The sample does not need to be obtained by an invasive method such as liver biopsy. Examples of samples include blood, urine, cerebrospinal fluid, lymphatic fluid, saliva, sweat, etc., but blood samples are preferred, and serum is more preferred.

[0017] Means and methods for measuring the expression level (sometimes simply referred to as "quantity") of A2F bisect and / or its biosynthetic precursor glycans in a sample are known to those skilled in the art. For example, as described in Example 1, a protein fraction in serum is precipitated and purified by a technique such as ethanol precipitation, and labeled glycans are prepared by glycoblotting and sialic acid linkage-specific amide labeling (SALSA). The amount of the resulting labeled glycans may be measured by analyzing them by MALDI-TOF mass spectrometry. During measurement, analysis is performed using the absolute quantitative value of each glycan detected by adding an internal standard glycan of known concentration, or the relative quantitative value calculated from the total amount of N-linked glycans in serum. Alternatively, for example, the protein to which A2F bisect and / or its biosynthetic precursor glycans are bound may be isolated using antibodies, antibody fragments, interacting proteins, or peptides such as peptide M specific for the protein, and the amount of A2F bisect and / or its biosynthetic precursor glycans bound to the protein may be measured using, for example, known methods such as chromatography or mass spectrometry. The antibody may be either a monoclonal or polyclonal antibody, but is preferably a monoclonal antibody. In the chromatography, a lectin that binds to A2F bisect and / or its biosynthetic precursor sugar chain may be used.

[0018] Means and methods for measuring the amount of A2F bisect bound to IgA2 in a sample and the amount of its biosynthetic precursor glycans bound to IgA2 are also known. First, IgA2 in the sample is isolated. For example, IgA2 may be isolated using an anti-IgA2 antibody, a fragment of the antibody, or another protein or peptide that interacts with IgA2. Isolation may also be performed using immunoprecipitation or affinity chromatography. Next, the amount of A2F bisect and / or its biosynthetic precursor bound to the isolated IgA2 is measured. The method for measuring the amount of these glycans is as described above.

[0019] In the methods of the present invention, the greater the amount of A2F bisect and / or its biosynthetic precursor sugar chains in a sample, the more advanced the patient's liver fibrosis can be assessed. Conversely, the smaller the amount of A2F bisect and / or its biosynthetic precursor sugar chains in a sample, the less advanced the patient's liver fibrosis can be assessed. For example, the progression of liver fibrosis can be assessed by comparing the amount of A2F bisect and / or its biosynthetic precursor sugar chains in a sample from a subject without liver fibrosis with the amount of these sugar chains in a NASH patient sample. Furthermore, the progression of liver fibrosis can be assessed by determining a cutoff value using an ROC curve, as described in Example 4, for example.

[0020] Assessment of the progression of liver fibrosis involves determining the stage of liver fibrosis. An example of a classification of liver fibrosis stages is the Shin-Inuyama Classification: F0: no fibrosis, F1: fibrous expansion in the portal venous area, F2: bridging fibrosis, F3: bridging fibrosis with distortion of the lobular structure, F4: cirrhosis.

[0021] In another aspect, the present invention provides a marker for assessing the progression of hepatic fibrosis in NASH, the marker comprising A2F bisect and / or a biosynthetic precursor glycan of A2F bisect. A2F bisect and its biosynthetic precursor glycan are as described above.

[0022] The markers of the present invention may contain only A2F bisect, or may contain one or more biosynthetic precursor glycans of A2F bisect, or may contain A2F bisect and one or more biosynthetic precursor glycans of A2F bisect. One specific example of a marker of the present invention is a marker containing only A2F bisect. Another specific example of a marker of the present invention is a marker containing seven glycans: glycans 1, 2A, 2B, 3A, 3B, 4, 5A, and 5B.

[0023] Preferred markers of the present invention are A2F bisect bound to IgA2 and biosynthetic precursor glycans of A2F bisect bound to IgA2.

[0024] By measuring the amount of the marker of the present invention in a sample, the progression of liver fibrosis in a patient can be evaluated. The evaluation of liver fibrosis has been described above, but will be described in more detail below. The method of evaluating the progression of liver fibrosis in NASH of the present invention includes comparing the amounts of A2F bisect and its biosynthetic precursor sugar chains on glycoproteins contained in blood, as well as A2F bisect bound to IgA2 and the biosynthetic precursor sugar chain of A2F bisect bound to IgA2, with reference values. For example, if the amount of any of A2F bisect and its biosynthetic precursor sugar chains, A2F bisect bound to IgA2 and the biosynthetic precursor sugar chain of A2F bisect bound to IgA2 in a subject is equal to or greater than the threshold (reference value) for determining whether the subject is a healthy individual, liver fibrosis in NASH can be determined.

[0025] As mentioned above, the progression of liver fibrosis can be classified as follows: F0: no fibrosis, F1: fibrotic expansion in the portal vein area, F2: fibrotic bridging, F3: fibrotic bridging accompanied by distortion of the lobule structure, and F4: cirrhosis. The method of the present invention for evaluating the progression of liver fibrosis in a subject with NASH can determine which stage of the F0-F4 stage the subject's liver is at. In this case, the range of reference values ​​for the subject at various stages is measured in advance, and if the amount of A2F bisect and its biosynthetic precursor sugar chain, A2F bisect bound to IgA2, or the biosynthetic precursor sugar chain of A2F bisect bound to IgA2 in the subject falls within a specific range, the subject is likely to be at the corresponding stage. The classification is not limited to the above F0-F4, and different classifications may also be used.

[0026] The marker of the present invention can be used in combination with other liver fibrosis markers to improve the accuracy of evaluation of the progression of liver fibrosis in NASH. Examples of other liver fibrosis markers include, but are not limited to, type IV collagen 7S, hyaluronic acid, M2BPGi, etc.

[0027] In yet another aspect, the present invention provides a kit for assessing the progression of liver fibrosis in NASH. The kit of the present invention is used to carry out the above-described method for assessing the progression of liver fibrosis in NASH. The kit of the present invention comprises a means for measuring the amount of A2F bisect and / or its biosynthetic precursor glycan, and / or A2F bisect and / or its biosynthetic precursor glycan. A2F bisect and its biosynthetic precursor glycan are as described above.

[0028] The means for measuring the amount of A2F bisect and / or its biosynthetic precursor glycans in the kits of the present invention is not particularly limited, and may be a means for separating proteins in a sample to which A2F bisect and / or its biosynthetic precursor glycans are bound. Examples of such means include antibodies, antibody fragments, interacting proteins, or peptides such as peptide M that are specific for blood-secreted proteins to which A2F bisect and / or its biosynthetic precursor glycans are bound. A specific example of blood-secreted proteins to which A2F bisect and / or its biosynthetic precursor glycans are bound is IgA2. The antibodies may be monoclonal or polyclonal, with monoclonal antibodies being preferred. Further examples of means for measuring the amount of A2F bisect and / or its biosynthetic precursor glycans include lectins that bind to A2F bisect and / or its biosynthetic precursor glycans, and chromatographic supports containing such lectins. Further examples of the above means include reagents used in glycoblotting and sialic acid linkage-specific amide labeling (SALSA).

[0029] Next, we will describe the method for measuring the markers. The amount of markers in each blood sample can be measured by MS. For example, N-linked glycans are released from serum by digestion with PNGase F, and labeled glycans are prepared by glycoblotting and sialic acid linkage-specific amide labeling (SALSA). This allows the labeled glycans to be analyzed by MALDI-TOF MS. Alternatively, quantification can be achieved without MS, for example, by isolating glycan components from plasma and reacting them with a lectin (E4-PHA) that specifically recognizes A2F bisect and its biosynthetic precursor glycans. It is preferable to use a lectin labeled with an isotope or fluorescent reagent.

[0030] Furthermore, A2F bisect and its biosynthetic precursor glycans bound to specific proteins or peptides can be used as markers of the invention. In this case, this fraction can be isolated using an antibody that selectively binds to the glycan-bound protein or peptide, and quantified by reacting it with a lectin that specifically recognizes A2F bisect and its biosynthetic precursor glycans present on the protein. It is preferable to use a lectin labeled with an isotope or fluorescent reagent. It is preferable to use IgA2 or IgM as the glycan-bound protein. Even without separating into the above fractions, lectins or antibodies that recognize A2F bisect and its biosynthetic precursor sugar chains bound to specific proteins or peptides can be used.

[0031] In yet another aspect, the present invention provides a method for evaluating the progression of liver fibrosis in NASH, comprising the following steps: (a) separating IgA2 or IgM in a sample, and then (b) Measure the amount of A2F bisect and / or its biosynthetic precursor glycans bound to IgA2 or IgM. The present invention provides a method comprising: Examples of means for separating IgA2 or IgM contained in a sample include, but are not limited to, anti-IgA2 antibodies or anti-IgM antibodies, fragments of these antibodies, proteins or peptides that interact with IgA2 or IgM, etc., and these means can be appropriately selected and used. The present inventors have found that Protein G, which is originally used to separate IgG, can be suitably used for this purpose.

[0032] The method, marker, and kit of the present invention are specific to NASH and can evaluate the progression of liver fibrosis in NASH. Furthermore, the method, marker, and kit of the present invention can also be used to perform the evaluation non-invasively.

[0033] The method, marker and kit of the present invention can be used to evaluate the therapeutic effect of NASH.The method, marker and kit of the present invention can also be used to evaluate the prognosis of NASH.Furthermore, the method, marker and kit of the present invention can also be used to evaluate the effectiveness of therapeutic drugs for NASH.These evaluations can also be carried out non-invasively.

[0034] The present invention will be described in more detail and specifically below with reference to examples, but the examples are not intended to limit the scope of the present invention. This application claims priority to Japanese Patent Application No. 2019-129798, filed on July 12, 2019, the entire contents of which are incorporated herein by reference. [Example]

[0035] To verify the utility of A2F bisect and / or its biosynthetic precursor glycans, comprehensive N-linked glycan analysis was performed using serum samples from NASH patients F0, F1 or F2 (F1 / F2), and F3 or F4 (F3 / F4) (20, 22, and 28 samples, respectively). Serum protein fractions were prepared by ethanol precipitation, and labeled glycans were prepared by glycoblotting and sialic acid linkage-specific amide labeling (SALSA). The labeled glycans were analyzed by MALDI-TOF MS.

[0036] The results of MALDI-TOF MS analysis of A2F bisect are shown in Figure 1. It was confirmed that the expression level of A2F bisect increases with the progression of fibrosis. Similarly, increased expression of glycans located upstream in the A2F bisect biosynthetic pathway with fibrosis was also confirmed (Figure 2). In Figure 2, glycans 1 to 5 represent the glycans 1, 2 (2A, 2B), 3 (3A, 3B), 4, and 5 (5A, 5B) described above. These results confirmed that the expression of not only A2F bisect but also its biosynthetic precursors increases with liver fibrosis. [Example]

[0037] Next, we performed a correlation analysis between A2F bisect expression levels and pathological fibrosis assessment in liver biopsies and fibrosis assessment factors in the blood (Figure 3). The results showed that A2F bisect expression levels were highly correlated with F factor and fib4 index, which are indicators of fibrosis, but not with CRP, which assesses inflammation, and showed an inverse correlation with steatosis. These results confirmed that changes in A2F bisect expression are highly correlated with fibrosis factors in NASH. [Example]

[0038] To investigate the disease specificity of A2F bisect glycans, we measured A2F bisect glycans in serum samples from hepatitis C patients (HCV: 25 samples) using the same method and compared their expression levels with those of NASH fibrosis groups (F0, F1 / F2, F3 / F4) (Fig. 4). Although hepatitis C patients included cases with advanced liver fibrosis, the expression levels of A2F bisect glycans were not as high as those of the NASH F3 / 4 group. In NASH, the expression level increased as fibrosis progressed, and the expression of A2F bisect glycans in the F3 / 4 group was more than twice as high as that of hepatitis C patients. Therefore, A2F bisect glycans are suggested to be a disease-specific marker that can evaluate the progression of liver fibrosis in NASH disease. [Example]

[0039] To evaluate the sensitivity of A2F bisect glycans for fibrosis discrimination, ROC analysis was performed using specimens with fibrosis stages defined by liver biopsy and pathological diagnosis (F0 (20 specimens), F1 / 2 (22 specimens), F3 / 4 (28 specimens)). The results are shown in Figure 5. The AUC score increased when the sum (SUM2) of the expression levels of the precursor glycans shown in Figure 2 (glycan 1, glycan 2 (2A, 2B), glycan 3 (3A, 3B), glycan 4, and glycan 5 (5A, 5B)) was used, compared with the expression level of A2F bisect glycans alone. The AUC score of this sum was found to be comparable to the AUC scores of type IV collagen 7s (labeled 4-7s in Figure 5) and Fib-4. Differences were observed between the curve shapes of the sum of precursor glycans and type IV collagen 7s. The combined analysis of the sum of precursor glycans and type IV collagen 7s showed significantly higher area under curvature (AUC) scores than either the sum of precursor glycans or type IV collagen 7s alone. The cutoff value for severe fibrosis diagnosis in the ROC curve for type IV collagen 7s was 6 ng / mL, and false positives exceeding this value were 5 of 42 samples (5 F1 / 2 samples). However, by using a cutoff value of 139.7 pmol / 2.5 uL serum for the sum of A2F bisect and biosynthetic precursor glycans 1, 2A, 2B, 3A, 3B, and 4, false positives due to type IV collagen 7s could be reduced to 40% (3 of 42 samples). [Example]

[0040] To identify the protein to which the A2F bisect glycan binds, the following experiment was performed. NASH F3 / 4 pooled serum was fractionated using protein G into a flow-through fraction and a protein G-bound fraction (elution fraction), and these were confirmed by SDS-PAGE. N-linked glycan analysis of the flow-through fraction and elution fraction was performed using glycoblotting. The results are shown in Figure 6. It was revealed that the elution fraction contained a large amount of A2F bisect glycans, whose expression was found to be elevated in NASH fibrosis.

[0041] Protein G elution fractions from NASH F3 / 4 pooled serum were separated by SDS-PAGE, and N-linked glycan analysis was performed on the protein bands detected by Coomassie Brilliant Blue (CBB) staining. The results are shown in Figure 7. A2F bisect glycans were detected in bands 8, 9, and 13, and quantification using internal standard glycans revealed that band 9 contained the most A2F bisect glycans, accounting for 75%. Identification of the proteins contained in each band by peptide mass fingerprinting (PMF) revealed that band 8 contained complement C3 and IgM, and band 9 contained IgA.

[0042] The elution fraction prepared from standard serum using protein G was digested with trypsin, and peptides bound to the A2F bisect glycan were comprehensively analyzed by LC-MS. As a result, IgA2 was identified among the IgM and IgA subclasses.

[0043] To evaluate serum IgA2 concentrations during fibrosis progression, we measured serum IgA2 concentrations in F0, F1 / 2, and F3 / 4 (20, 32, and 35 specimens, respectively) using an IgA2 ELISA (Enzyme-Linked ImmunoSorbent Assay) kit. The results are shown in Figure 8. Although serum IgA2 concentrations increased with the progression of fibrosis, no significant increase in IgA2 protein expression was observed. Therefore, it was demonstrated that A2F bisect glycosylation of IgA2 is promoted with the progression of fibrosis. These results suggest that the A2F bisect glycan bound to IgA2 is a potent biomarker for liver fibrosis in NASH. [Industrial Applicability]

[0044] The present invention is useful for diagnosing liver diseases, etc. In particular, the present invention is useful for evaluating the progression of liver fibrosis in NASH, etc. Therefore, the present invention is useful in the fields of diagnostic agents for liver diseases, research on liver diseases, etc.

Claims

1. The compound of formula (I) in the sample: 【Chemical 1】 and / or a biosynthetic precursor sugar chain of a sugar chain having a structure represented by formula (I), The biosynthetic precursor sugar chains of the sugar chains having the structure represented by formula (I) are sugar chains 1, 2A, 2B, 3A, 3B, 4, 5A, and 5B: 【Chemistry 2】 and having one or more structures selected from the group consisting of: a sugar chain having a structure represented by formula (I) and / or a biosynthetic precursor sugar chain of a sugar chain having a structure represented by formula (I) is bound to IgA, the sample is a blood sample; method.

2. The method according to claim 1, comprising measuring the total amount of biosynthetic precursor sugar chains of a sugar chain having a structure represented by formula (I).

3. A marker for assessing the progression of liver fibrosis in NASH, comprising a compound represented by formula (I): 【Chemistry 3】 and / or a biosynthetic precursor sugar chain of a sugar chain having a structure represented by formula (I), The biosynthetic precursor sugar chains of the sugar chains having the structure represented by formula (I) are sugar chains 1, 2A, 2B, 3A, 3B, 4, 5A, and 5B: 【Chemistry 4】 and having one or more structures selected from the group consisting of: a sugar chain having a structure represented by formula (I) and / or a biosynthetic precursor sugar chain of a sugar chain having a structure represented by formula (I) is bound to IgA; marker.

4. A kit for assessing the progression of liver fibrosis in NASH, comprising: 【Chemical 1】 and / or a means for measuring the amount of a sugar chain having a structure represented by formula (I) and / or a biosynthetic precursor sugar chain of a sugar chain having a structure represented by formula (I), The biosynthetic precursor sugar chains of the sugar chains having the structure represented by formula (I) are sugar chains 1, 2A, 2B, 3A, 3B, 4, 5A, and 5B: 【Chemistry 4】 and having one or more structures selected from the group consisting of: the means is an antibody, antibody fragment, interacting protein, or peptide specific to a protein to which a sugar chain having a structure represented by formula (I) and / or a biosynthetic precursor sugar chain of a sugar chain having a structure represented by formula (I) is bound, the protein to which the sugar chain having the structure represented by formula (I) and / or the biosynthetic precursor sugar chain of the sugar chain having the structure represented by formula (I) is bound is IgA; kit.

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