JPWO2017126514A1 - Non-alcoholic steatohepatitis detection method - Google Patents

Abstract

  The present invention provides a novel marker that serves as an index for NASH detection (particularly, an index for distinguishing NASH from NAFL), a NASH detection method using the marker, and a kit for detecting NASH using the marker. . Specifically, the marker is sugar chain A3F bound to α1-antitrypsin.

Description

  The present invention relates to a marker serving as an index for detection of nonalcoholic steatohepatitis (NASH), a method for detecting NASH using the marker, and a NASH detection kit including the marker.

  Non-alcoholic fatty liver disease (hereinafter referred to as NAFLD) is a group of diseases that occur in people who do not have a clear drinking history, excluding chronic liver diseases such as viral and autoimmunity. It is defined as NAFLD is further classified into non-alcoholic fatty liver (hereinafter referred to as NAFL) and non-alcoholic steatohepatitis (hereinafter referred to as NASH). NAFL rarely progresses in pathology and has little pathological significance. On the other hand, NASH against the background of lifestyle-related diseases such as obesity and diabetes is a disease with a poor prognosis that causes a risk of liver cancer or liver-related death through cirrhosis, and requires early diagnosis and treatment.

  Whether or not you have NAFLD can be diagnosed by confirming fatty liver by imaging, non-viral by blood, and confirming that it is not caused by a large amount of alcohol drinking through a history of drinking. A definitive diagnosis that a patient diagnosed with NAFLD suffers from NASH, not NAFL, is currently performed by liver biopsy, inflammation in the lobule, balloon-like degeneration of hepatocytes, Mallory Denk body Alternatively, pathological determination using Matteoni classification based on the presence or absence of fibrosis (Non-Patent Document 1), NAFLD Activity Score (NAS) calculated from the scores of fat accumulation, inflammation, and hepatocyte balloon-like hypertrophy, or the pathology of liver fibrosis Judgment (Non-Patent Document 2) is a diagnostic criterion. However, liver biopsy has a large burden on patients, and there are many problems such as sampling errors and variations between evaluators. Therefore, development of a noninvasive diagnostic method replacing liver biopsy is required.

In recent years, studies have been conducted to use protein-linked sugar chains as diagnostic markers for diseases such as cancer. For example, a Mac-2-binding protein sugar chain-modified isomer (M2BPGi) is known as a sugar chain marker reflecting the progress of the fibrosis stage of the liver based on data of hepatitis C patients (Non-patent Document 3). ), A measurement kit is marketed in Japan (HISCL (registered trademark) M2BPGi reagent, Sysmex Corporation). Regarding the relationship between protein-binding sugar chain markers and NASH, for example, Non-Patent Document 4 describes that M2BPGi reflects the progression of the fibrosis stage in NASH patients, but regarding the selection of NASH patients and NAFL patients, Neither listed nor suggested. In Non-Patent Document 5, since the fucosylated Mac-2 binding protein (Mac2bp) is related to the total amount of Mac2bp, the total amount of Mac2bp is measured instead of the sugar chain, and compared with non-NASH (NAFL) patients. It is described that Mac2bp is significantly increased in NASH patients.
Non-Patent Document 6 describes that serum fucosylated haptoglobin (Fuc-Hpt) is significantly increased in NASH patients compared to non-NASH (NAFL) patients. Non-Patent Document 7 describes that NASH patients can be more efficiently selected from NAFLD patients by the combined use of Fuc-Hpt and Mac2bp.

  α1-antitrypsin (AAT) is the most major protease inhibitor in the blood and inhibits various serine proteases. It is one of the acute phase reactants that are mainly produced in hepatocytes and increase in the blood during various inflammations, and is known to be an indicator of inflammatory diseases and malignant tumors. Non-Patent Document 8 discloses that the amount of sugar chain A3F (3) 1G3S3 (outer arm fucosylation) bound to AAT in blood is higher than that in healthy subjects, cirrhosis patients caused by hepatitis C, C It is described that it rises in patients with hepatitis caused by hepatitis B. However, the relationship between AAT and NASH, or the relationship between sugar chains bound to AAT and NASH is not known.

Gastroenterology, Volume 116, Issue 6, 1413-1419 (1999) Hepatology, Volume 41, Issue 6, 1313-1321 (2005) Scientific Reports, Volume 3, 1065 (2013) Journal of Gastroenterology, Volume 50, 776-784 (2015) Proteomics Clinical Applications, Volume 7, 648-656 (2013) PLos ONE, Volume 8, Issue 6, e66328 (2013) Hepatology, Volume 62, Issue 5, 1433-1443 (2015) PLos ONE, Volume 5, Issue 8, e12419 (2010)

  An object of the present invention is to provide a novel marker that serves as an index for NASH detection (particularly, an index for distinguishing NASH from NAFL), a NASH detection method using the marker, and a kit for detecting NASH using the marker. It is to provide. Further, the present invention relates to a NASH diagnosis method using the marker, a NASH progress or mitigation monitoring method, a NASH therapeutic effect evaluation method, and a method for evaluating the effectiveness of a substance for treating NASH.

（式中、Ｎｅｕ５ＡｃはＮ−アセチルノイラミン酸、Ｇａｌはガラクトース、ＧｌｃＮＡｃはＮ−アセチルグルコサミン、Ｍａｎはマンノースを意味する。）
で示される構造を含むフコシル化糖鎖のみ、ＮＡＦＬ患者と比較してＮＡＳＨ患者において有意に増加することを見出し、新規のＮＡＳＨ検出用マーカーとして同定した。本明細書において式（Ｉ）の構造を含むフコシル化糖鎖を「Ａ３Ｆ」ともいう。本明細書において、上記本発明のマーカーを「ＡＡＴ結合糖鎖Ａ３Ｆ」とも言う。As a result of intensive studies, the present inventors have found that among about 20 sugar chains bonded to α1-antitrypsin (AAT), the compound of formula (I)

(In the formula, Neu5Ac means N-acetylneuraminic acid, Gal means galactose, GlcNAc means N-acetylglucosamine, and Man means mannose.)
It was found that only fucosylated glycans containing the structure represented by (1) were significantly increased in NASH patients compared to NAFL patients, and were identified as novel NASH detection markers. In the present specification, the fucosylated sugar chain containing the structure of formula (I) is also referred to as “A3F”. In the present specification, the marker of the present invention is also referred to as “AAT-linked sugar chain A3F”.

で示される構造を含むフコシル化糖鎖の量を測定することを特徴とする、非アルコール性脂肪肝炎（ＮＡＳＨ）の検出方法。
（２）非アルコール性脂肪肝炎（ＮＡＳＨ）に罹患していない被検体由来の対照試料における量と比較して、被検試料における量が多いことを、該被検試料が非アルコール性脂肪肝炎（ＮＡＳＨ）に罹患している被検体由来の試料であることの指標とする、（１）記載の方法。
（３）該非アルコール性脂肪肝炎（ＮＡＳＨ）に罹患していない被検体が、非アルコール性脂肪肝（ＮＡＦＬ）に罹患している被検体である、（２）記載の方法。
（４）被検試料が、非アルコール性脂肪性肝疾患（ＮＡＦＬＤ）に罹患している被検体由来である、（１）〜（３）いずれかに記載の方法。
（５）該被検試料が血液又は血清である、（１）〜（４）いずれかに記載の方法。
（６）該測定が、免疫測定法、質量分析法又は液体クロマトグラフィーを用いて行うものである、（１）〜（５）いずれかに記載の方法。
（７）該免疫測定法がＥＬＩＳＡである（６）記載の方法。
（８）該質量分析法がＭＡＬＤＩ法、ＬＣ／ＭＳ法又はＬＣ／ＭＳ／ＭＳ法である（６）記載の方法。
（９）α１‐アンチトリプシンに結合している式（Ｉ）：

で示される構造を含むフコシル化糖鎖からなる、非アルコール性脂肪肝炎（ＮＡＳＨ）検出用マーカー。
（１０）式（Ｉ）：

で示される構造を含むフコシル化糖鎖及び／又は
α１‐アンチトリプシンに対する抗体若しくはその断片
を含む、非アルコール性脂肪肝炎（ＮＡＳＨ）検出用キット。
（１１）α１‐アンチトリプシンに対する抗体を用いて、被検試料における、α１‐アンチトリプシンに結合している式（Ｉ）で示される構造を含むフコシル化糖鎖の量を測定することを特徴とする、非アルコール性脂肪肝炎（ＮＡＳＨ）の検出方法。
（１２）（２）〜（８）いずれかに記載の方法で非アルコール性脂肪肝炎（ＮＡＳＨ）を検出する工程、及び
薬物療法、運動療法及び食事療法からなる群より選ばれる１以上の治療により非アルコール性脂肪肝炎（ＮＡＳＨ）が検出された被検体を治療する工程を含む、非アルコール性脂肪肝炎（ＮＡＳＨ）治療方法。
（１３）被検試料における、ＡＡＴ結合糖鎖Ａ３Ｆの量が、０．８μＭ以上である場合に、該被検試料が非アルコール性脂肪肝炎（ＮＡＳＨ）に罹患している被検体由来の試料であるとする、（２）〜（８）いずれかに記載の方法。（実施例１）
（１４）被検試料における、ＡＡＴ結合糖鎖Ａ３Ｆの量が、０．３μＭ以上である場合に、該被検試料が非アルコール性脂肪肝炎（ＮＡＳＨ）に罹患している被検体由来の試料であるとする、（２）〜（８）いずれかに記載の方法。（実施例１）
（１５）被検試料における、ＡＡＴ結合糖鎖Ａ３Ｆの量が、９μＭ以上である場合に、該被検試料が非アルコール性脂肪肝炎（ＮＡＳＨ）に罹患している被検体由来の試料であるとする、（２）〜（８）いずれかに記載の方法。（実施例２）That is, the present invention relates to the following.
(1) Formula (I) bound to α1-antitrypsin in a test sample:

A method for detecting non-alcoholic steatohepatitis (NASH), comprising measuring the amount of a fucosylated sugar chain comprising the structure represented by:
(2) Compared to the amount in a control sample derived from a subject not suffering from non-alcoholic steatohepatitis (NASH), the amount of the test sample is higher than the amount in the test sample. (1) The method according to (1), wherein the sample is derived from a subject suffering from NASH.
(3) The method according to (2), wherein the subject not suffering from non-alcoholic steatohepatitis (NASH) is a subject suffering from non-alcoholic fatty liver (NAFL).
(4) The method according to any one of (1) to (3), wherein the test sample is derived from a subject suffering from nonalcoholic fatty liver disease (NAFLD).
(5) The method according to any one of (1) to (4), wherein the test sample is blood or serum.
(6) The method according to any one of (1) to (5), wherein the measurement is performed using an immunoassay, mass spectrometry, or liquid chromatography.
(7) The method according to (6), wherein the immunoassay is ELISA.
(8) The method according to (6), wherein the mass spectrometry method is a MALDI method, an LC / MS method, or an LC / MS / MS method.
(9) Formula (I) bound to α1-antitrypsin:

A marker for detecting non-alcoholic steatohepatitis (NASH), comprising a fucosylated sugar chain comprising the structure represented by
(10) Formula (I):

A kit for detecting non-alcoholic steatohepatitis (NASH), comprising a fucosylated sugar chain comprising the structure represented by the above and / or an antibody against α1-antitrypsin or a fragment thereof.
(11) Using the antibody against α1-antitrypsin, measuring the amount of fucosylated sugar chain containing the structure represented by formula (I) bound to α1-antitrypsin in a test sample A method for detecting nonalcoholic steatohepatitis (NASH).
(12) By the process of detecting nonalcoholic steatohepatitis (NASH) by the method according to any one of (2) to (8), and one or more treatments selected from the group consisting of drug therapy, exercise therapy and diet therapy A method for treating nonalcoholic steatohepatitis (NASH), comprising a step of treating a subject in which nonalcoholic steatohepatitis (NASH) is detected.
(13) When the amount of AAT-linked sugar chain A3F in the test sample is 0.8 μM or more, the test sample is a sample derived from a subject suffering from nonalcoholic steatohepatitis (NASH) The method according to any one of (2) to (8). Example 1
(14) When the amount of AAT-linked sugar chain A3F in the test sample is 0.3 μM or more, the test sample is a sample derived from a subject suffering from nonalcoholic steatohepatitis (NASH) The method according to any one of (2) to (8). Example 1
(15) When the amount of AAT-linked sugar chain A3F in the test sample is 9 μM or more, the test sample is derived from a subject suffering from nonalcoholic steatohepatitis (NASH) The method according to any one of (2) to (8). (Example 2)

The present invention also includes the following.
(1-1) (A) a step of measuring the amount of AAT-linked sugar chain A3F in a test sample;
(B) Compared with the amount in a control sample derived from a subject not suffering from non-alcoholic steatohepatitis (NASH), the amount of the test sample measured in step (A) is large. A method for screening non-alcoholic steatohepatitis (NASH) patients, comprising a step of indicating that the sample is a sample derived from a non-alcoholic steatohepatitis (NASH) patient.
(1-2) A method for monitoring progression or alleviation of nonalcoholic steatohepatitis (NASH), which comprises measuring the amount of AAT-linked sugar chain A3F in a test sample.
(1-3) A method for evaluating the therapeutic effect of nonalcoholic steatohepatitis (NASH), which comprises measuring the amount of AAT-linked sugar chain A3F in a test sample.
(1-4) A method for evaluating the effectiveness of a substance for treating nonalcoholic steatohepatitis (NASH), which comprises measuring the amount of AAT-linked sugar chain A3F in a test sample.

  The marker of the present invention can detect NASH by distinguishing NASH from NAFL, NASH diagnosis method, NASH progression or mitigation monitoring method, NASH therapeutic effect evaluation method, NASH therapeutic substance Useful for evaluating efficacy. Furthermore, measurement can be performed using blood or serum, and the burden on the patient and medical staff is lighter than that of liver biopsy, and the above diagnosis and evaluation can be performed more quickly and easily.

(A) Serum concentration of AAT-linked sugar chain A3F for each specimen, and (B) Box-and-whisker diagram prepared from (A) (Example 1) ROC curve of AAT-linked sugar chain A3F in diagnosis of NASH (Example 1) (A) Serum concentration of AAT-linked sugar chain A3F for each specimen, and (B) Box-and-whisker diagram prepared from (A) (Example 2) ROC curve of AAT-linked sugar chain A3F in diagnosis of NASH (Example 2)

  The terms used in the present specification are used in the meaning normally used in the art unless otherwise specified.

  “Test sample” means a biological material isolated from a subject. For example, blood, lymph, cerebrospinal fluid, urine and processed products thereof are used, preferably blood, and more preferably serum and plasma obtained by separating the blood.

  “Subject” means any animal. For example, mammals such as humans, monkeys, mice, rats, rabbits and the like can be mentioned. Preferably, it is human or mouse.

  Specific embodiments of the present invention are described below, but the present invention is not limited thereto.

I. NASH Detection Marker As shown in the examples of the present specification, AAT-linked sugar chain A3F was found as a novel NASH detection marker. That is, the present invention includes a non-alcoholic steatohepatitis (NASH) detection marker comprising AAT-linked sugar chain A3F.

  AAT-linked sugar chain A3F, which is a marker of the present invention, is a known substance.

α1-antitrypsin (AAT, alpha-1-antitrypsin) is a protein consisting of 418 amino acids, and the sequence is registered and published in GenBank under the accession number AAB59375. In addition, it is registered with Uniprot as accession number P01009, among which isoforms and many natural mutants are published.
Asparagine at position 70, 107 or 271 is known as an N-glycosylation position on the AAT sequence (J Proteome Res. 2014; 13 (7): 3131-43). The reason why the marker of the present invention can be used as a NASH detection marker is that the ratio of A3F in the total amount of sugar chains binding to the three glycosylation positions is significantly higher in NASH patients than in NAFL patients. There can be many.

  AAT includes not only the proteins and natural mutants registered above, but also mutants that can occur in vivo, and 1 to several, 1-5, 1-3, 1, or 2 in the protein. Proteins in which the amino acids are deleted, substituted, and / or added, and are caused by mutations based on polymorphisms or mutations. However, it is preferable that the asparagine at position 70, 107 or 271 which is the N-glycosylation position is conserved.

（式（ＩＩ）又は式（ＩＩＩ）中のα２―３／６はα２―３グリコシド結合又はα２―６グリコシド結合を、β１−３／４はβ１−３グリコシド結合又はβ１−４グリコシド結合を意味する。）Specifically, the NASH marker of the present invention is a sugar chain containing the structure represented by formula (I), wherein any sugar residue of the sugar chain is fucosylated (Fuc). . More specifically, the NASH marker of the present invention includes a sugar chain containing the structure represented by the formula (I), wherein any one N-acetylglucosamine (GlcNAc) is fucosylated. It is done. More specifically, it is a sugar chain containing the structure represented by formula (I), and any one N— in the three branched sugar chains present on the non-reducing end side (sugar chain head) is present. A sugar chain in which acetylglucosamine (GlcNAc) is fucosylated (outer arm fucosylation) or a sugar chain containing the structure represented by formula (I), and present at the reducing end closest to the asparagine residue Examples include those in which acetylglucosamine (GlcNAc) is fucosylated (core fucosylation).
The structure represented by the formula (I) includes, for example, the structure represented by the following formula (II) or formula (III).

(In formula (II) or formula (III), α2-3 / 6 means α2-3 glycoside bond or α2-6 glycoside bond, β1-3 / 4 means β1-3 glycoside bond or β1-4 glycoside bond To do.)

II. NASH Detection Method NASH can be detected using the “I. NASH detection marker”. That is, the present invention includes a method for detecting nonalcoholic steatohepatitis (NASH), which comprises measuring the amount of AAT-linked sugar chain A3F in a test sample.

で示される構造を含むフコシル化糖鎖（Ａ３Ｆ）の量
を測定することにより、ＮＡＳＨを検出することができる。The marker of the present invention can be used to detect NASH. That is, in the test sample,
Formula (I) bound to AAT

NASH can be detected by measuring the amount of fucosylated sugar chain (A3F) containing the structure represented by

  Examples of the method for measuring the amount of AAT-linked sugar chain A3F include an immunoassay method and a method using mass spectrometry. It can also be measured with reference to the following examples.

  As an immunoassay, a known immunological method can be used. Examples of known immunological methods include ELISA, RIA, and Western blot.

As mass spectrometry, specifically, for example,
(A) separating AAT from the test sample; and
(B) The method etc. which include the process of measuring the quantity of A3F couple | bonded with isolate | separated AAT are mentioned.

  Step (a) may be any method as long as it can specifically recognize AAT and separate AAT from a test sample containing other proteins. At this time, the AAT may be a wild type or a mutant type, and may be a full length or a fragment as long as it contains a site to which A3F is bound. More specifically, it may be a protein or fragment containing asparagine at position 70, 107 or 271 on the AAT sequence.

Specific examples include an immunoprecipitation method using an anti-AAT antibody and a method of separating AAT from a test sample by an antibody affinity column.
First, an antibody against AAT is bound to agarose beads or magnetic beads. Here, the binding mode may be a covalent bond or a biotin-avidin bond. Next, the test sample is brought into contact with the antibody-bound beads, and after the AAT in the test sample is bound to the antibody on the beads, the beads are sufficiently washed, and further, acid, alkali, or high salt concentration AAT is separated by releasing AAT from the antibody on the beads using a solution.

  Step (b) may be any method as long as it can measure the amount of A3F bound to AAT separated in step (a). For example, a method of measuring by known mass spectrometry or chromatography is exemplified. Examples include MS method, MS / MS method, LC / MS method, LC / MS / MS method, HPLC, gas chromatography, liquid chromatography and the like. Moreover, a well-known immunoassay method can also be utilized similarly to a process (a).

  In addition, as the amount of AAT-linked sugar chain A3F that is the marker of the present invention, it is not always necessary to determine the absolute amount, and the peak specific to each AAT-linked sugar chain A3F detected by the above-described measurement method or the like can be quantified, It can also be determined by determining the ratio to the reference peak. Specific methods include a method for quantifying the height of each detected peak, a method for quantifying the peak area, etc., and since it is a measurement method having quantitativeness in liquid chromatography, it is limited to one of them. However, the MS method is preferable because the method of quantifying the peak area has good accuracy.

  Specifically, the AAT having the A3F which is the marker of the present invention is obtained from the mixture of AAT fragments obtained by fragmenting the AAT separated in the step (a) with a protease or the like. A method for detecting fragments with a mass spectrometer is mentioned. The AAT separated in step (a) is denatured, reductively alkylated, and peptide fragmented using a protease. Any protease may be used as long as it decomposes the protein into peptide fragments, and examples include trypsin and lysyl endopeptidase. The AAT fragment can be directly measured with a mass spectrometer, but it is preferable to concentrate it in advance using an antibody, a lectin or the like. Specifically, for example, AAL (Aurelia aurantia lectin), AOL (Apergillus oryzae L-fucose-specific lectin), LTL (Lotus tetragonolus lectin lectin) It is desirable to concentrate AAT using a lectin that binds to fucose, such as. The measurement by the mass spectrometer is not a problem even with the mass spectrometer alone, but it is desirable to combine it with chromatography such as liquid chromatography or capillary electrophoresis. A method for measuring the amount of A3F includes a method for measuring a peak area.

  In addition, after the sugar chain part of AAT separated in step (a) is decomposed and released using glycanase or hydrazine, the sugar chain part (A3F) to be measured is derivatized as necessary. Alternatively, the amount can be measured by chemical modification. Alternatively, A3F can be separated by liquid chromatography or the like and the amount thereof can be measured.

  Examples of a method for decomposing a sugar chain part from AAT include a hydrazine decomposition method and an enzyme (N-glycanase) digestion method. Among these, the enzymatic (N-glycanase) digestion method is preferable for quantitatively cleaving the sugar chain.

The method for labeling and derivatizing A3F to be detected in the sugar chain prepared as described above is not particularly limited, but when using a mass spectrometry apparatus, a labeling method for increasing ionization efficiency, more specifically, Specifically, a labeling method using N ^α -((aminooxy) acetyl) tryptophanylargineline methyl ester (aoWR) is particularly preferable. When a fluorescence detector is used, it is preferable to label the sugar chain part with 2-aminopyridine. Further, as a method for derivatizing the sugar chain moiety labeled as described above, when aoWR is used for labeling, for example, J. Org. Frukawa et al. , Anal. Chem. 80 (2008) 1094-1101 and the like, and when 2-aminopyridine is used for the label, Y.C. Otake et al. , J Biochem (Tokyo) 129 (2001) 537-42, and the like.

  As described above, as a method for separating labeled and derivatized A3F, electrophoresis or the like can be used in addition to liquid chromatography, preferably liquid chromatography can be used. The conditions for liquid chromatography are not particularly limited, but a reverse phase or normal phase column is desirable, and any specification is possible as long as the eluent can be sent stably. Absent.

  Next, the amount of A3F is measured. This method is not particularly limited as long as A3F can be selectively detected. Specific measurement methods include an ultraviolet-visible light absorption method, a fluorescence detection method, a mass spectrometry method, a nuclear magnetic resonance method, a method using an antibody specific for the sugar chain of the present invention, and the like. Method or fluorescence detection method is desirable.

  When detecting using a fluorescence detection method, it is necessary to label a sugar chain in advance with a fluorescent substance such as 2-aminopyridine. The detection conditions of the fluorescence detection method are not particularly limited as long as the sugar chain (A3F) to be detected can be detected. When 2-aminopyridine is used as the labeling compound, it is preferable to select a wavelength of 280 nm to 330 nm for excitation light and a wavelength of 350 nm to 420 nm for fluorescence detection.

  In addition, when mass spectrometry is used, it is desirable to add an ionic compound to the sugar chain in advance by the aoWR described above. When detection is performed using mass spectrometry, the detection range of the mass spectrometer is not particularly limited as long as the sugar chain (A3F) to be detected can be detected. The ionization method may be MALDI, ESI (electrospray ionization), APCI (atmospheric pressure chemical ionization), or the like, but MALDI is most preferable. The mass spectrometer may be any of a quadrupole type, a TOF type, an ion trap type, a magnetic field type, and a Fourier transform type, but a quadrupole type with high quantitativeness, a TOF type with high sensitivity, and an ion trap type are particularly preferable. In addition, ions to be detected are not limited to parent ions, but may be related ions such as fragment ions, additional ions, and dimer ions. Examples of the method for measuring the amount of the NASH marker of the present invention thus obtained include a method for measuring the peak area of the peak corresponding to the sugar chain (A3F) to be detected. In addition, when detecting using mass spectrometry, LS / MS which also has the function of a liquid chromatography can also be used.

In addition to the method described above, a step of separating a glycoprotein having A3F which is a marker of the present invention from a test sample, and a step of measuring the amount of AAT contained in the separated glycoprotein having A3F Including methods can also be used. That is, the step of measuring the marker in the method of the present invention comprises:
(C) separating the glycoprotein having A3F from the total glycoprotein in the body fluid, and (d) measuring the amount of AAT in the separated glycoprotein,
Is a NASH detection method.

  When comparing two or more measurements, using one or more statistical analyzes (eg, t-test, Welch's T test, Wilcoxon rank sum test, ANOVA, recursive partitioning, random forest, etc.) Can be compared.

  The marker of the present invention may be used alone, but may be used in combination with other markers known to be capable of detecting other NASH (for example, Fuc-Hpt, Mac2bp, etc.).

III. NASH Diagnosis Method NASH can be diagnosed using the above-mentioned “I. NASH detection marker”. The amount of the marker of the present invention is small in the test sample in healthy subjects and NAFL patients, and the amount in the test sample is significantly increased in NASH patients. Therefore, in “II. NASH detection method”, the amount in the test sample is larger than the amount in the control sample derived from a subject not suffering from NASH (a healthy person and / or a NAFL patient). NASH can be detected by using the test sample as an indicator that the sample is derived from a subject suffering from NASH.
In other words, the present invention
(A) a step of measuring AAT-linked sugar chain A3F in a test sample;
(B) Compared with the amount in the control sample derived from the subject not suffering from NASH, the amount in the test sample measured in the step (A) is large. It includes a method for screening a NASH patient, which comprises a step of indicating that there is an index.

  In clinical practice, NAFLD has established diagnostic criteria and confirmed that it is not a viral disease by known blood tests using HBs antigen, HCV antibody, various autoantibodies, etc. Diagnosis can be made by confirming that a large amount of drinking is not caused by an inquiry of drinking history (for example, the amount of drinking is less than 20 g / day or less than 140 g / week in terms of ethanol). Therefore, it is important to make a definitive diagnosis that a patient diagnosed with NAFLD is suffering from NASH with a poor prognosis, not NAFL, whose pathological condition rarely progresses. For a subject diagnosed as having NAFLD by the method, it is very useful in that it can be diagnosed whether or not it is suffering from NASH. That is, in the “II. NASH detection method”, the measured amount in a test sample derived from a NAFLD patient is the same as a subject who is not affected by NASH and suffers from NAFLD (ie, affected by NAFL. By using a large amount in the test sample as an indicator that the test sample is derived from a subject suffering from NASH as compared to the amount in the control sample derived from NASH can be detected (diagnosed) and NASH patients can be screened.

  The amount in the control sample is preferably an average value of a plurality of samples. The amount in the control sample may be measured simultaneously with the test sample, or may be measured in advance to set a cutoff value.

  The “amount of NASH marker of the present invention” is small in a test sample derived from a subject not suffering from NASH, but significantly increased in a test sample derived from a NASH patient. Therefore, when “the amount of NASH marker of the present invention” in the test sample or the calculated value based on the amount of NASH marker in the test sample is significantly larger than the “amount in the control sample” in “II. When it is larger than the set cutoff value, it can be said that the test sample is likely to be a sample derived from a NASH patient.

The “cut-off value” in the present invention refers to the amount of NAS marker obtained by “II. NASH detection method” or a calculated value based on the amount of NASH marker and is affected by NASH patients and NASH. Refers to a value that distinguishes non-subject subjects (healthy and / or NAFL patients). Although the determination method of a cutoff value is not specifically limited, You may determine a cutoff value using a ROC curve like the following Example.
When NASH is detected using a cut-off value, the cut-off value varies depending on the NASH definition and the marker measurement method. Therefore, it is preferable to confirm in advance the measurement values of the subject and NASH patients who are not affected by NASH, determine the cutoff value, and detect the target marker accordingly. According to the embodiment of the present invention, when the marker is measured using the serum of a subject, NASH can be detected with reference to the numerical value obtained in the embodiment of the present invention.

As described above, diagnosis can be performed using the marker of the present invention to treat a NASH patient. Specifically, in “II. Method for detecting NASH”, the amount of the test sample is larger than that of the control sample derived from the subject not suffering from NASH. NASH is detected by the step of detecting NASH and one or more treatments selected from the group consisting of drug therapy, exercise therapy and diet therapy by using as an index that the sample is derived from a subject suffering from A NASH treatment method comprising a step of treating a subject.
In other words, the present invention
(A) a step of measuring the amount of AAT-linked sugar chain A3F in the test sample;
(B) Compared with the amount in the control sample derived from the subject not suffering from NASH, the amount of the test sample measured in the step (A) is larger, indicating that the test sample is a sample derived from the NASH patient. Detecting NASH as an index of
(C) A NASH treatment method including the step of treating a subject in which NASH is detected by one or more treatments selected from the group consisting of drug therapy, exercise therapy and diet therapy.
Examples of the drug therapy include insulin resistance improving drugs (for example, pioglitazone), vitamin E (for example, α-tocophenol) and the like.

IV. NASH Progression / Reduction Monitoring Method Using the “I. NASH detection marker”, NASH progression / reduction in a subject can be monitored. Specifically, the progress or reduction of NASH can be monitored by a method characterized by measuring the amount of AAT-linked sugar chain A3F in a test sample.
In other words, the present invention
(D) measuring the amount of AAT-linked sugar chain A3F in the first test sample;
(E) a step of measuring AAT-linked sugar chain A3F in a second test sample derived from the same subject,
(F) A method of monitoring the progress or reduction of NASH including the step of comparing the measured value of the first test sample with the measured value of the second test sample is included. The results of the method indicate the course of NASH in the subject (ie progression or reduction if there is a change).

  The marker of the present invention is measured at least twice over a certain period and the measured values are compared. It may be performed a plurality of times at intervals of a certain period or more, and the changes over time of the measured values may be compared. If there is a change in the measured value of the marker of the present invention over time, the change may indicate progression or reduction of NASH in the subject. When the measurement value of the marker of the present invention increases over time by comparing the measurement value of the first test sample and the measurement value of the second test sample, the result is It means the progression (deterioration) of NASH. When the measurement value of the marker of the present invention decreases with time by comparing the measurement value of the first test sample with the measurement value of the second test sample, the result is It means reduction of NASH.

  The method can be performed to monitor the progress of NASH in a subject suffering from NASH. Moreover, in order to monitor the level of predisposition to NASH, it can be used with a subject who does not have NASH (for example, a subject suspected of being likely to develop NASH). It is suggested that NASH is not so severe that the marker measurement value of a test subject approaches the measurement value of a person (a healthy person or a NAFL patient) who does not suffer from NASH. On the contrary, as the marker measurement value of the subject becomes farther from the measurement value of the person who is not affected with NASH (a healthy person or a NAFL patient), the NASH is serious or the deterioration of the disease state is suggested.

V. Method for evaluating the therapeutic effect of NASH The therapeutic effect of NASH in a subject can be evaluated using the “I. NASH detection marker”. Specifically, the therapeutic effect of NASH can be evaluated by a method characterized by measuring the amount of AAT-linked sugar chain A3F in a test sample.
That is, the present invention includes a method for evaluating the therapeutic effect of NASH by performing the steps (D) to (F) over time, such as before, during, and after NASH treatment. The result of the method indicates the therapeutic effect of NASH in the subject.

  The marker of the present invention is measured at least twice over a certain period and the measured values are compared. It may be performed a plurality of times at intervals of a certain period or more, and the changes over time of the measured values may be compared. When there is a change over time in the measurement value of the marker of the present invention, the change can indicate the presence or absence of a therapeutic effect of NASH in the subject. When the measurement value of the marker of the present invention is decreased by comparing the measurement value of the first test sample and the measurement value of the second test sample, the result is the treatment of NASH in the test sample. It means that there is an effect. By comparing the measured value of the first test sample with the measured value of the second test sample, if the measured value of the marker of the present invention is increased or unchanged, the result is It means that there is no effect on the treatment of NASH in the specimen.

The NASH treatment includes drug therapy, exercise therapy, diet therapy, and other treatment methods such as surgical treatment performed for the purpose of NASH treatment. In the present invention, the period during which NASH treatment is performed is referred to as NASH treatment period.
In the method for evaluating the therapeutic effect of the present invention, it is desirable that the NASH treatment is performed for a certain period or more, and the measurement of the marker of the present invention is performed after a certain period or more in the NASH treatment period. In the present invention, the fixed period is not particularly limited, but is preferably 1 week, more preferably 1 month, 3 months, 6 months, 1 year, and may be a period of 1 year or more. For example, a mode in which a marker in a test sample of a subject is measured periodically every three months is desirable.

VI. Method for Evaluating Effectiveness of Substance for Treating NASH The effectiveness of a substance for treating NASH can be evaluated using the “I. NASH detection marker”. Specifically, the effectiveness of a substance for treating NASH can be evaluated by a method characterized by measuring the amount of AAT-linked sugar chain A3F in a test sample.
That is, it includes a method for evaluating the effectiveness of the substance by performing the steps (D) to (F) over time, such as before, during, and after administration of the substance for treating NASH. . The results of the method indicate the effectiveness of the material.

  The marker of the present invention is measured at least twice over a certain period and the measured values are compared. It may be performed a plurality of times at intervals of a certain period or more, and the changes over time of the measured values may be compared. Where there is a change in the measured value of the marker of the present invention over time, the change can indicate the effectiveness of the substance. When the measurement value of the marker of the present invention is decreased by comparing the measurement value of the first test sample and the measurement value of the second test sample, the result is that the substance is used for the treatment of NASH. Means effective. When the measured value of the marker of the present invention is increased or unchanged by comparing the measured value of the first test sample with the measured value of the second test sample, the result is the substance Means ineffective in the treatment of NASH.

VII. The kit for detecting NASH The present invention includes a kit for detecting NASH used in the methods II to VI described above. Specifically, a non-alcoholic steatohepatitis (NASH) detection kit containing an AAT-linked sugar chain A3F can be mentioned.

In addition, a non-alcoholic steatohepatitis (NASH) detection kit containing an antibody against α1-antitrypsin (AAT) or a fragment thereof is also included. The kit can be used for isolation of AAT to which A3F is bound from a test sample and measurement of the amount of AAT to which A3F is bound in the test sample.
The type and origin of the antibody are not particularly limited as long as it has a specific binding property to AAT. Monoclonal antibodies and polyclonal antibodies can be used, but monoclonal antibodies are preferred. The antibody may be labeled.
The antibody production method is not particularly limited, and a known method can be used. Specifically, a monoclonal antibody or a polyclonal antibody can be produced by immunizing an animal with AAT or a partial sequence peptide thereof.

  The present invention will be described in more detail below with reference to examples of the present invention, but the present invention is not limited thereto.

Example 1
Specimens Analysis was performed using sera of patients diagnosed with NAFL by liver biopsy (17 specimens) and sera of patients diagnosed with NASH by liver biopsy (28 specimens). Diagnosis of NAFL and NASH was performed by Matteoni classification based on pathological findings. Matteoni classification is a classification method for nonalcoholic fatty liver disease based on the presence or absence of inflammation in the leaflets, balloon-like degeneration of hepatocytes, Mallory-Denck body or fibrosis. In cases where inflammation is accompanied by intralobular inflammation, type 2 is classified as type 1, when type 1 is accompanied by balloon-like degeneration of hepatocytes, type 3 is classified as type 3, and type 3 is accompanied by Mallory-Denk or fibrosis. Type 1 and type 2 are diagnosed as NAFL, type 3 and type 4 as NASH.

Separation of AAT 50 μL of NAFL and NASH patient serum was filtered through a 0.22 μm filter (Cellulose Acetate Spin Filters, Agilent, Santa Clara, Calif.) And then diluted with 150 μL of Buffer A (Agilent, Santa Clara, Calif.). The diluted serum was applied to a MARS HSA / IgG spin cartridge (Agilent, Santa Clara, Calif.) Equilibrated with Buffer A. Centrifugation (100 × g, 2 minutes, 4 ° C.) was performed to recover the eluate 1. The MARS HSA / IgG spin cartridge was washed three times with 267 μL of Buffer A, and all the eluates were combined with eluate 1 to make eluate 2. Of about 1000 μL of eluate 2, 200 μL was applied to a MARS Human-14 spin cartridge (Agilent, Santa Clara, Calif.) Equilibrated with Buffer A. Centrifuge (100 × g, 2 minutes, 4 ° C.), allow to stand at room temperature for 5 minutes, wash 3 times with 267 μL of Buffer A, and add 2.5 mL of Buffer B (Agilent, Santa Clara, Calif.) The minute was eluted. The solvent of the fraction containing AAT was replaced with phosphate buffered saline using Spin Concentrators, 5K MWCO (Agilent, Santa Clara, Calif.). NuPAGE LDS Sample Buffer (4X) (Invitrogen, Waltham, MA) was added to this solution and heated at 70 ° C. for 15 minutes to denature the protein. Ten μg of denatured protein was separated with NuPAGE Novex 4-12% Bis-Tris Gel 1.0 mm, 10 Well (Invitrogen, Waltham, MA), and the protein was stained with SimplyBlue SafeStain (Invitrogen, Waltham, MA). A band corresponding to AAT was cut out, and protein extraction and trypsin digestion were performed from the gel using In-Gel Tryptic Digest Kit (Pierce Biotechnology, Waltham, MA). 200 μL of purified water was added to the gel after trypsin digestion, shaken at room temperature for 30 minutes, and the supernatant was transferred to a new sample tube (sample solution A). Next, 400 μL of 12.5 mM ammonium bicarbonate buffer / 50% acetonitrile was added to the gel, shaken at room temperature for 30 minutes, and the supernatant was combined with sample solution A (sample solution B). Further, 200 μL of acetonitrile was added to the gel, shaken at room temperature for 30 minutes, and the supernatant was combined with the sample solution B. This solution was dried under reduced pressure, dissolved in 100 μL of 25 mM ammonium bicarbonate buffer, heated at 90 ° C. for 10 minutes, and then cooled on ice (sample solution C).

Sugar chain analysis of AAT 1 U PNGaseF (Basel, Switzerland) was added to sample solution C, and allowed to stand at 37 ° C. overnight. In this solution, the sugar chain contained in 50 μL was labeled with aoWR by a glycoblotting method using BlotGlyco (Sumitomo Bakelite Co. Ltd., Tokyo, Japan). At this time, the COOH group possessed by the sialic acid in the sample was methyl esterified with 1-methyl-3-p-tolyltriazene (Tokyo Chemical Industry Co., Ltd, Tokyo, Japan). When performing the above-mentioned glycoblotting, A3 glycan (Ludger, Oxfordshire, UK) was also labeled with aoWR. The COOH group of sialic acid in A3 glycan was ethyl esterified with 1-ethyl-3-p-tolyltriazene (Tokyo Chemical Industry Co., Ltd, Tokyo, Japan). After the aoWR labeling reaction, the labeled sugar chain was eluted with 100 μL of purified water. To this amount, 2.5 pmol of A3 glycan labeled with aoWR was added to 50 μL, and the sugar chain was purified by GlycoWorks HILIC uElution Plate (Waters, Milford, Mass.). MALDI-TOF (/ TOF) analysis was performed with UltraFlex II (Bruker Daltonics GmbH, Bremen, Germany) using 2,5-dihydroxybenzoic acid (10 mg / mL) as a matrix. A3F was quantified from the ratio of the A3F peak to the A3 glycan peak in the sample.

  Based on the amount of serum subjected to sugar chain analysis, the serum concentration of AAT-linked sugar chain A3F was calculated and compared between NAFL patients and NASH patients. FIG. 1 (A) shows the serum concentration of each AAT-linked sugar chain A3F for each specimen, and FIG. 1 (B) shows a boxplot created from FIG. 1 (A). In addition, Table 1 shows the average value (Mean) and standard deviation (SD) of NAFL and NASH. The t test was used for the test of significant difference.

The concentration of AAT-linked sugar chain A3F in the serum of NASH patients was significantly increased as compared with NAFL patients. Therefore, it was shown that AAT-linked sugar chain A3F is useful as a NASH marker in distinguishing NASH patients from NAFL patients.
The ROC (receiver operating characteristics) curve of the AAT-linked sugar chain A3F in the diagnosis of NASH is shown in FIG. The ROC curve was created using R (Ver 3.3.2). The area under the ROC curve obtained from the ROC curve was 0.775. In the ROC curve, when the point at which the sum of specificity and sensitivity is the maximum is taken as the cutoff value, the value was 0.8 μM. When the cutoff value was 0.8 μM, the sensitivity in diagnosis of NASH was 64% and the specificity was 82%.
Since the AAT-linked sugar chain A3F concentration in the NASH patient serum was 0.3 μM or higher, false negatives can be eliminated by setting the cut-off value to 0.3 μM. When the cutoff value was 0.3 μM, the sensitivity in NASH diagnosis was 100% and the specificity was 29.4%.

Example 2
Specimens Analysis was performed using sera of patients diagnosed with NAFL by liver biopsy (31 specimens) and sera of patients diagnosed with NASH by liver biopsy (38 specimens). The diagnosis of NAFL and NASH was performed by Matteoni classification based on pathological findings as in Example 1.

Purification of AAT by immunoprecipitation A 0.45 μm centrifugal filter unit (Ultrafree-MC HV Centrifugal Filter, Millipore, Billerica, Massachusetts) and 300 μL Affi-Gel Blue Gel (Bio-Rad, Alfred Nobel Drive Hercules, Calif.) 70 μL of Protein G Sepharose 4 Fast Flow (GE Healhcare, Chicago, Illinois) was added and equilibrated with 20 mM sodium phosphate buffer (pH 7.0) to prepare column A. 50 μL of serum from NAFL and NASH patients was diluted with 150 μL of 20 mM sodium phosphate buffer (pH 7.0), and filtered through a 0.45 μm centrifugal filter unit (Ultrafree-MC HV Centrifugal Filter). The filtered serum was applied to column A and mixed by shaking for 10 minutes at room temperature. The mixture was centrifuged (2500 × g, 1 minute, room temperature), and the eluate was applied again to column A, and mixed by shaking for 10 minutes at room temperature. Centrifugation (2500 × g, 1 minute, room temperature) was performed to collect the eluate (eluate 3). Column A was washed with 250 μL of 20 mM sodium phosphate buffer (pH 7.0), and the washing solution was combined with eluent 3 to prepare eluent 4. Into a new 0.45 μm centrifugal filter unit (Ultrafree-MC HV Centrifugal Filter), 20 μL of Alpha-1 Antitrypsin Select (GE Healhcare) was added and equilibrated with 150 mM sodium chloride / 20 mM sodium phosphate buffer (pH 7.0). To be column B. The solvent was changed to 150 mM sodium chloride / 20 mM sodium phosphate buffer (pH 7.0) by adding a 5 M sodium chloride aqueous solution to the eluate 4, which was applied to the column B and mixed by shaking at room temperature for 30 minutes. The mixture was centrifuged (100 × g, 5 minutes, room temperature), and the eluate was again applied to column B, and mixed by shaking at room temperature for 30 minutes. Centrifugation (100 × g, 5 minutes, room temperature) and column B was washed 3 times with 200 μL of 150 mM sodium chloride / 20 mM sodium phosphate buffer (pH 7.0). To column B, 200 μL of 2M magnesium chloride / 20 mM Tris-HCl buffer (pH 7.0) was added and mixed by shaking for 1 minute at room temperature. Centrifugation (1000 × g, 1 minute, room temperature) was carried out to recover the eluate 5 containing AAT. 200 μL of 2M magnesium chloride / 20 mM Tris-HCl buffer (pH 7.0) was again added to column B, and the mixture was mixed by shaking at room temperature for 1 minute. The mixture was centrifuged (1000 × g, 1 minute, room temperature), and the eluate was combined with eluate 5. The solvent of this solution was replaced with 10 mM ammonium bicarbonate solution using Amicon (registered trademark) Ultra 0.5 mL Centrifugal Filters (MWCO = 3 kDa) (Millipore) to obtain a purified AAT solution.

Measurement of AAT concentration by ELISA AAT concentrations of serum and purified AAT solution were measured by Human Serpin A1 DuoSet ELISA (R & D Systems, Inc., McKinley Place NE, MN). Capture antibody made up to 400 ng / mL with phosphate buffered saline was added to Nunc (registered trademark) MaxiSorp ^™ 384 well plate (Nunc, Roskilde, Denmark) at 15 μL / well and allowed to stand overnight at 4 ° C. The plate was washed 3 times with 90 μL of washing solution (0.05% Tween20 / PBS) and blocked with 70 μL of 5% Tween20 / PBS. The sample was washed twice with 90 μL of washing solution and suspended in 5% Tween 20 / PBS at 15 μL / well, and allowed to stand at room temperature for 2 hours. The antibody was washed 4 times with 90 μL of the washing solution, and a detection antibody adjusted to 150 ng / mL with 5% Tween20 / PBS was added at 15 μL / well, and left at room temperature for 2 hours. The plate was washed 4 times with 90 μL of the washing solution, HRP-labeled streptavidin solution diluted 1000-fold with 5% Tween20 / PBS was added at 15 μL / well, and the mixture was allowed to stand at room temperature for 30 minutes. The plate was washed 4 times with 90 μL of washing solution, TMB solution (Dako, Santa Clara, California) was added at 15 μL / well, and the mixture was allowed to stand at room temperature for 30 minutes. The reaction was stopped with 15 μL of 1N sulfuric acid, and the absorbance at 450 nm was measured using Benchmark Plus (Bio-Rad). Using AAT derived from human plasma purchased from Sigma (St. Louis, Missouri) as a standard in AAT quantification, the AAT concentration in the sample was quantified.

Sugar chain analysis of AAT Reduction / alkylation treatment was performed on 10 μg of AAT, 0.1 μg of trypsin was added, and the mixture was allowed to stand at 37 ° C. overnight. After trypsin was inactivated by heating at 90 ° C. for 10 minutes, 1 U PNGase F (Roche, Basel, Switzerland) was added, and the mixture was allowed to stand at 37 ° C. overnight. The enzyme was inactivated by heating at 90 ° C. for 10 minutes. The sugar chain released from AAT was quantified in the same manner as in “AAT sugar chain analysis” in Example 1. For the purpose of accurate quantification of A3F derived from AAT, it is reported to contain 15 pmol A3F per μg (Anal Chim Acta. (2015) 25; 866: 59-68.) Sugar derived from α1-acid glycoprotein A calibration curve was created with the chain, and the quantitative value of A3F was calculated.

  From the A3F quantitative value (pmol / μg AAT) by MALDI-TOF MS and the serum AAT concentration (mg / mL) measured by ELISA, the serum concentration (μM) of AAT-A3F was calculated, and the value was calculated for NAFL patients. And NASH patients. FIG. 3 (A) shows the serum concentration of each AAT-linked sugar chain A3F for each specimen, and FIG. 3 (B) shows a boxplot created from FIG. 3 (A). Table 2 shows the mean value (Mean) and standard deviation (SD) of NAFL and NASH. The t test was used for the test of significant difference.

The concentration of AAT-linked sugar chain A3F in the serum of NASH patients was significantly increased as compared with NAFL patients. Therefore, it was shown that AAT-linked sugar chain A3F is useful as a NASH marker in distinguishing NASH patients from NAFL patients.
The ROC curve of AAT-linked sugar chain A3F in the diagnosis of NASH is shown in FIG. The ROC curve was created using R (Ver 3.3.2). The area under the ROC curve obtained from the ROC curve was 0.655. In the ROC curve, when the point at which the sum of specificity and sensitivity is maximized is taken as the cutoff value, the value was 9 μM. When the cutoff value was 9 μM, the sensitivity in diagnosis of NASH was 58% and the specificity was 74%.

  The marker of the present invention can distinguish NASH from NAFL, and is a method for diagnosing NASH, a method for monitoring NASH progression or mitigation, a method for evaluating the therapeutic effect of NASH, and evaluating the effectiveness of a substance for treating NASH. Useful for.

Claims (10)

Formula (I) bound to α1-antitrypsin in the test sample:

A method for detecting non-alcoholic steatohepatitis, comprising measuring the amount of a fucosylated sugar chain having a structure represented by: A subject whose test sample suffers from non-alcoholic steatohepatitis, that the amount in the test sample is greater than the amount in a control sample derived from a subject who does not suffer from non-alcoholic steatohepatitis The method according to claim 1, wherein the method is an index indicating that the sample is derived from the source. The method according to claim 2, wherein the subject not suffering from non-alcoholic steatohepatitis is a subject suffering from non-alcoholic steatohepatitis. The method according to any one of claims 1 to 3, wherein the test sample is derived from a subject suffering from non-alcoholic fatty liver disease. The method according to any one of claims 1 to 4, wherein the test sample is blood or serum. The method according to any one of claims 1 to 5, wherein the measurement is performed using an immunoassay, mass spectrometry, or liquid chromatography. The method according to claim 6, wherein the immunoassay is ELISA. The method according to claim 6, wherein the mass spectrometry is a MALDI method, an LC / MS method or an LC / MS / MS method. Formula (I) bound to α1-antitrypsin:

A marker for detecting non-alcoholic steatohepatitis, comprising a fucosylated sugar chain comprising the structure represented by Formula (I):

A kit for detecting non-alcoholic steatohepatitis, comprising a fucosylated sugar chain comprising the structure represented by the above and / or an antibody against α1-antitrypsin or a fragment thereof.

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