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US20130281309A1 - Novel biomarker for non-alcoholic fatty liver disease, and method for detecting non-alcoholic fatty liver disease by using the biomarker - Google Patents

Novel biomarker for non-alcoholic fatty liver disease, and method for detecting non-alcoholic fatty liver disease by using the biomarker Download PDF

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Publication number
US20130281309A1
US20130281309A1 US13/119,724 US200913119724A US2013281309A1 US 20130281309 A1 US20130281309 A1 US 20130281309A1 US 200913119724 A US200913119724 A US 200913119724A US 2013281309 A1 US2013281309 A1 US 2013281309A1
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fatty liver
amino acid
liver disease
biomarker
detection
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Kohji Meno
Hideaki Suzuki
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MCBI Inc
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MCBI Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8114—Kunitz type inhibitors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/81—Protease inhibitors
    • G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • G01N2333/811—Serine protease (E.C. 3.4.21) inhibitors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/08—Hepato-biliairy disorders other than hepatitis
    • G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin

Definitions

  • the present invention relates to novel biomarkers for nonalcoholic fatty liver disease and methods for detecting nonalcoholic fatty liver disease using such biomarkers.
  • the commonly used means to differentiate between normal and non-normal states of a human subject using his or her biological materials are mainly those which have been used in the field of diagnostics. Most frequently used are those methods which target biomarkers in blood. It has been practiced in this field to comparatively measure the amount of a specific protein or a peptide that is less than 10,000 in molecular weight or, in the case of enzyme protein, enzyme activities in samples from normal (healthy) subjects and those from diseased individuals to help diagnosis.
  • measurements are done on a fixed number each of samples from healthy controls and patients with certain disease with respect to the amount(s) or activity (activities) of single or multiple specific proteins or peptides and the ranges of abnormal and normal values are respectively determined.
  • the sample to be evaluated is then analyzed by the same method and the resultant value is judged with respect to whether it is in normal or abnormal range.
  • the amount(s) of specified protein(s) or peptide(s) in test samples are determined by the use of enzyme-linked immmunosorbent assay (ELISA) which uses a primary, or secondary, antibody labeled with an enzyme reacting with a substrate that yields a color upon reaction, chemiluminescent immunoassay (CLIA), radioimmunoassay (RIA) which uses a primary, or secondary, antibody labeled with a radioisotope, and, if the protein is an enzyme, the measurement of the activity of the enzyme by adding its substrate and determining the intensity of produced color, etc.
  • ELISA enzyme-linked immmunosorbent assay
  • CLIA chemiluminescent immunoassay
  • RIA radioimmunoassay
  • target protein or peptide is captured by beads (including magnetic ones) with linked antibody to the protein or peptide, eluted from the beads, and determined by mass spectrometry.
  • intact proteins have been reported to be analyzed by mass spectrometry using above-mentioned methods after digestion with trypsin etc. (Patent Document 1).
  • intact target proteins are selected either by fractionation or by adsorption to an adsorbant specific to them and then determined by mass spectrometry.
  • Nonalcoholic fatty liver disease is abbreviated as NAFLD and patients with this disease, despite the fact that they have no drinking habit (less than 20 g daily), give histological findings characterized by hepatic fatty deposition reminiscent of those found in alcoholic hepatic damage.
  • the disease caused viruses such as HCV or HBV or of autoimmune origin is excluded.
  • the disease is regarded as a phenotype in the liver of metabolic syndrome accompanying obesity.
  • NAFLD is divided into simple fatty liver and nonalcoholic steatohepatitis. The latter, abbreviated as NASH, is a progressive disease. NASH frequently accompanies fibrosis and has been known to often progress to hepatic cirrhosis and further to hepatic cancer. These features have attracted attention to this disease (Non-patent Document 1).
  • simple fatty liver is called fatty liver.
  • Fatty liver is suspected in regular checkups from high levels of triglyceride in blood and diagnosed by abdominal ultrasonography and CT. Attention is warranted to the fact that 40% of fatty liver in non-drinker is accompanied by hepatic damage. Despite the observation that nonalcoholic fatty liver progresses to NASH, no convenient blood test is available for NASH.
  • NASH is regarded as a severe type of NAFLD
  • routine blood chemistry specifically the values of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) increase only slightly and the disease is often overlooked.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • Patent Document 1 JP-A-2004-333274
  • Patent Document 2 JP-A-2006-308533
  • Non-Patent Document 1 “A Guide to Diagnosis and Treatment of NASH and NAFLD,” edited by Japanese Hepatology Association, 2006 (in Japanese)
  • Non-Patent Document 2 Benkirane, N. et al, J. Biol. Chem. Vol. 268, 26279-26285, 1993
  • the present invention aims to present methods to detect nonalcoholic fatty liver disease including nonalcoholic steatohepatitis by using a protein or its partial peptide that differs in presence or absence, or in quantity between healthy human subjects and patients with nonalcoholic fatty liver disease or nonalcoholic steatohepatitis and further aims to present biomarkers comprising said protein and said partial peptide to be used to detect nonalcoholic fatty liver disease including nonalcoholic steatohepatitis.
  • a biomarker for detection of nonalcoholic fatty liver disease comprising at least one protein or peptide selected from the group consisting of inter-alpha-trypsin inhibitor heavy chain H4 precursor consisting of amino acid sequence expressed by Sequence No. 1, 35 kDa protein fragment consisting of amino acid sequence expressed by Sequence No. 2 of inter-alpha-trypsin inhibitor heavy chain H4 precursor, and partial peptide consisting of amino acid sequence expressed by Sequence No. 3 of inter-alpha-trypsin inhibitor heavy chain H4 precursor.
  • a biomarker for detection of nonalcoholic fatty liver disease comprising protein fragment or peptide of not less than 5 amino acid residues arising from at least one protein or peptide selected from the group consisting of inter-alpha-trypsin inhibitor heavy chain H4 precursor consisting of amino acid sequence expressed by Sequence No. 1, 35 kDa protein fragment consisting of amino acid sequence expressed by Sequence No. 2 of inter-alpha-trypsin inhibitor heavy chain H4 precursor, and partial peptide consisting of amino acid sequence expressed by Sequence No. 3 of inter-alpha-trypsin inhibitor heavy chain H4 precursor.
  • a biomarker for detection of nonalcoholic steatohepatitis comprising at least one protein or peptide selected from the group consisting of inter-alpha-trypsin inhibitor heavy chain H4 precursor consisting of amino acid sequence expressed by Sequence No. 1, 35 kDa protein fragment consisting of amino acid sequence expressed by Sequence No. 2 of inter-alpha-trypsin inhibitor heavy chain H4 precursor, and partial peptide consisting of amino acid sequence expressed by Sequence No. 3 of inter-alpha-trypsin inhibitor heavy chain H4 precursor.
  • a biomarker for detection of nonalcoholic steatohepatitis comprising protein fragment or peptide of not less than 5 amino acid residues arising from at least one protein or peptide selected from the group consisting of inter-alpha-trypsin inhibitor heavy chain H4 precursor consisting of amino acid sequence expressed by Sequence No. 1, 35 kDa protein fragment consisting of amino acid sequence expressed by Sequence No. 2 of inter-alpha-trypsin inhibitor heavy chain H4 precursor, and partial peptide consisting of amino acid sequence expressed by Sequence No. 3 of inter-alpha-trypsin inhibitor heavy chain H4 precursor.
  • the present invention it is possible to diagnose a subject as to whether said subject has suffered from nonalcoholic fatty liver disease or nonalcoholic steatohepatitis by determining in biological material obtained from said subject the kind and amount of at least one protein or at least one partial peptide derived by digestion, etc. of said protein selected from the group consisting of inter-alpha-trypsin inhibitor heavy chain H4 precursor consisting of amino acid sequence expressed by Sequence No. 1, 35 kDa protein fragment consisting of amino acid sequence expressed by Sequence No. 2 of inter-alpha-trypsin inhibitor heavy chain H4 precursor, and partial peptide consisting of amino acid sequence expressed by Sequence No. 3 of inter-alpha-trypsin inhibitor heavy chain H4 precursor.
  • the present invention presents a diagnostic system that is high in both accuracy and specificity.
  • the present invention enables highly accurate diagnosis of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis in which there have been no specific test methods for such biological materials as blood. It is also possible with the present invention to diagnose the degree to which the liver disease has progressed to hepatic cancer. Further, the biomarkers disclosed in the present invention are highly useful in judgment of drug efficacy.
  • FIG. 1 illustrates the results of applying immunoblot method using BMPEP1117R as primary antibody in patients with fatty liver (FL), nonalcoholic steatohepatitis (NS) and chronic hepatitis (CH).
  • FL fatty liver
  • NS nonalcoholic steatohepatitis
  • CH chronic hepatitis
  • FIG. 2 proves that the immunoblot shown in FIG. 1 of Example 2 determined the 35 kDa protein fragment of inter-alpha-trypsin inhibitor heavy chain H4 precursor.
  • FIG. 3 illustrates the results of determination by immunoMS method of serum concentration of glycated partial peptide consisting of amino acid sequence expressed by Sequence No. 3 of inter-alpha-trypsin inhibitor heavy chain H4 precursor wherein healthy controls are shown as NR.
  • FIG. 4 illustrates the scatter plots for serum concentrations as determined by immunoMS method of glycated partial peptide consisting of amino acid sequence expressed by Sequence No. 3 of inter-alpha-trypsin inhibitor heavy chain H4 precursor wherein fatty liver (FL) and NASH are compared.
  • FIG. 5 shows the ROC curve and the value for AUC of serum concentrations determined by immunoMS method of glycated partial peptide consisting of amino acid sequence expressed by Sequence No. 3 of inter-alpha-trypsin inhibitor heavy chain H4 precursor and demonstrates that said partial peptide is capable of differentially diagnose between fatty liver (FL) and NASH.
  • FIG. 6 illustrates the fact that BMPEP1117R actually captured the 35 kDa protein fragment of inter-alpha-trypsin inhibitor heavy chain H4 precursor and that the captured fragment reacted with antibody specific to its C-terminus (BMPEP1117C).
  • the present invention is a method for determining the kind and the amount of intact protein and/or its partial peptide when test subject is suffering from nonalcoholic fatty liver disease or nonalcoholic steatohepatitis as well as for diagnosing whether test subject is suffering from nonalcoholic fatty liver disease or nonalcoholic steatohepatitis and, if test subject is diagnosed to be suffering from nonalcoholic fatty liver disease or nonalcoholic steatohepatitis, for elucidating the degree to which the liver disease has progressed.
  • a peptide is generally said to be a chemical entity, made by polymerizing a number of amino acids, of less than 10,000 in molecular weight or by polymerizing several to less than about 50 amino acid residues.
  • a partial peptide of an intact protein can be used as a biomarker for detection of nonalcoholic fatty liver disease or nonalcoholic steatohepatitis
  • such partial peptide is defined as a peptide of less than 10,000 in molecular weight consisting of a part of the amino acid sequence of the intact protein.
  • Such peptide may arise as a partial peptide during the expression by transcription followed by synthesis by translation before maturing into an intact protein or as a peptide produced by enzyme digestion in the body after the intact protein has been synthesized. It is possible that, when the body is in abnormal state suffering from such disease as nonalcoholic fatty liver disease or nonalcoholic steatohepatitis, the mechanism for protein synthesis and regulation is de-regulated.
  • the present invention is also a method for determining if test subject is in normal state or is suffering from either nonalcoholic fatty liver disease or nonalcoholic steatohepatitis by using the degree of protein synthesis and/or protein digestion as an indicator.
  • the detection of nonalcoholic fatty liver disease or nonalcoholic steatohepatitis in the present invention means evaluation and differentiation, i.e., diagnosis of test subject as to whether the subject is suffering from nonalcoholic fatty liver disease or nonalcoholic steatohepatitis.
  • the present invention can also include the evaluation of patient's risk of suffering from more serious liver disease.
  • the examples of intact protein that can be used as a biomarker for nonalcoholic fatty liver disease or nonalcoholic steatohepatitis include inter-alpha-trypsin inhibitor heavy chain H4 precursor consisting of amino acid sequence expressed by Sequence No. 1 and the 35 kDa protein fragment consisting of amino acid sequence expressed by Sequence No. 2.
  • Further biomarkers for nonalcoholic fatty liver disease or nonalcoholic steatohepatitis of the present invention include any protein fragment of greater than 10,000 in molecular weight arising from inter-alpha-trypsin inhibitor heavy chain H4 precursor consisting of amino acid sequence expressed by Sequence No. 1 and the 35 kDa protein fragment consisting of amino acid sequence expressed by Sequence No. 2.
  • biomarkers for nonalcoholic fatty liver disease or nonalcoholic steatohepatitis of the present invention includes the partial peptide consisting of amino acid sequence expressed by Sequence No. 3 of inter-alpha-trypsin inhibitor heavy chain H4 precursor.
  • proteins and peptides consisting of amino acid sequences derived from Sequence Nos. 1 through 3 by deletion, exchange, and/or addition of one or a few amino acids can be used as biomarkers and are included in the present invention.
  • the partial peptides that can be used as biomarkers in the present invention include those peptide fragments consisting of not less than 5 amino acid residues arising respectively from inter-alpha-trypsin inhibitor heavy chain H4 precursor consisting of amino acid sequence expressed by Sequence No. 1, the 35 kDa protein fragment consisting of amino acid sequence expressed by Sequence No. 2 of inter-alpha-trypsin inhibitor heavy chain H4 precursor, and the partial peptide consisting of amino acid sequence expressed by Sequence No. 3 of inter-alpha-trypsin inhibitor heavy chain H4 precursor.
  • Non-patent Document 2 The basis for the limitation of peptide fragments consisting of not less than 5 amino acid residues is in the description below in Non-patent Document 2.
  • the document reported that an antibody obtained by using the peptide IRGERA as immunogen, which was the C-terminus (130-135) of histone H3, recognized the peptide IKGERA derived by exchange of K for R and the peptide CGGGERA which was derived by deletion of IR followed by addition of CGG.
  • This demonstrates that the immunogenicity (antigenicity) is recognized by a peptide of not less than 4 amino acid residues.
  • the number of amino acid residue is defined as not less than 5 instead of 4 in the present invention.
  • To make such a low molecular weight peptide as the subject of the present invention is important when the method of detection and differentiation uses immunological means including immunoblot, ELISA and immunoMS.
  • Proteins and partial peptides in glycated form can also be used as biomarkers for detection of nonalcoholic fatty liver disease or nonalcoholic steatohepatitis.
  • An example of such glycated peptides is the peptide of Sequence No. 3.
  • biomarker in the present invention, can be quantified or its presence or absence can be determined qualitatively.
  • Two-dimensional electrophoresis (2-DE) or 2-dimensional chromatography (2-DC) can be used in the present invention to separate biomarkers in biological materials including serum.
  • Known chromatographic methods can be selected from ion-exchange chromatography, reverse-phase chromatography and gel-filtration chromatography. It is also possible to make quantification with the SRM/MRM method in LC-MS/MS technology.
  • the immunoMS method which these inventors have developed, where target protein or peptide is captured by beads (including magnetic ones) with antibody linked to the protein or peptide, eluted from the beads, and determined by mass spectrometry enables convenient determination of presence or absence or the amount of target protein, protein fragment or peptide without the use of 2-DE or chromatography.
  • the kind and amount of a protein in biological materials can be determined by various methods. If target protein (including protein fragment and partial peptide) has been characterized and when an antibody (primary antibody) to it has already been obtained, the following methods can be used:
  • Test serum in a fixed amount (about 1 microliter) after stepwise dilution is dropped onto an appropriate membrane such as of nitrocellulose and dried in air.
  • the membrane is treated with a blocking solution containing a protein such as BSA, washed, reacted with primary antibody, and washed. Thereafter, the membrane is reacted with labeled secondary antibody to detect the primary antibody. The membrane is washed and the label is visualized to measure its density.
  • proteins are transferred onto such an appropriate membrane as of nitrocellulose and their amounts are determined, as in above-mentioned immunoblot, using primary antibody and labeled secondary antibody.
  • Antibody to protein or its partial peptide is fixed to such a plate as a chemically modified microtiter plate. Appropriate amounts of samples after stepwise dilution are applied to the plate and incubated. Proteins and peptides not captured are removed by washing. Next, the plate is incubated with secondary antibody labeled with fluorescent or chemiluminescent substance or enzyme. After addition of respective substrate, fluorescence, chemiluminescence or visible light due to enzyme reaction is measured for evaluation and judgment.
  • a microarray is a general term for devices where solidified materials with affinity for target substances are arrayed on solid support (plate).
  • antibodies or a tamers to proteins and partial peptides are arrayed.
  • a sample of biological material is placed on the microarray for fixation of target proteins or partial peptides and the microarray is then incubated with secondary antibody labeled with fluorescent or chemiluminescent substance or enzyme. After addition of respective substrate, fluorescence, chemiluminescence or visible light due to enzyme reaction is measured.
  • antibody to a specified protein or partial peptide is attached to chemically modified microbeads or plate (protein chip).
  • the microbeads could be magnetic beads. There are no requirements for the material of the plate.
  • the antibody to be used could be (1) an antibody which recognizes the full length form of the specified protein only, (2) an antibody which recognizes a partial peptide only, (3) all of antibodies which recognizes both the specified protein and its partial peptide, or a combination of (1) and (2), (1) and (3), or (2) and (3).
  • Samples after stepwise dilution with original solvent or buffer are added to the microbeads or plate carrying antibody or antibodies and incubated. Those proteins and partial peptides not captured are removed by washing.
  • the protein or partial peptide captured by microbeads or plate is eluted, and analyzed by mass spectrometry with MALDI-TOF-MS SELDI-TOF-MS, etc. Measurements are made with respect to the mass and intensity of the peak due to the protein, protein fragment or partial peptide.
  • a fixed amount of substance serving as the internal standard is added to the original biological material and the intensity of its peak is also measured.
  • the concentration of the target in the original biological material can be calculated from the ratio of peak intensity of the target to the peak intensity of the internal standard. This is called immunoMS method.
  • the present invention includes the method to detect nonalcoholic fatty liver disease or nonalcoholic steatohepatitis from the presence or absence of the above-mentioned biomarker after applying biological material obtained from test subject to 2-DE or surface plasmon resonance.
  • Sequence No. 2 is part of Sequence No. 1.
  • Sequence No. 3 is located at the N-terminus of Sequence No. 2 and the peptide consisting of Sequence No. 3 is detected in the sera of patients with liver disease.
  • the above-described peptide was present in serum in the glycated state shown below.
  • Membrane 0.22 ⁇ m MF-millipore membrane filter
  • TBS 20 mMTris-HCl containing 0.15 MNaCl (pH 7.5)
  • TBSt TBS containing 0.05% Tween 20
  • blocking solution TBSt containing 5% BSA
  • BSA-TBSt TBSt containing 0.1% BSA.
  • the sample from each subject or patient was tested in duplicate.
  • FIG. 1 is a bar graph showing the above-mentioned mean values of dotblot intensity ratio.
  • FL means patients with fatty liver, NS those with NASH, and CH those with chronic hepatitis.
  • the stippled line indicates the intensity ratio of 0.6 as a threshold value.
  • the cases exceeding this threshold value were 0/5 in FL, 8/13 in NASH, and 7/7 in CH. Increased values were seen in high frequency in NASH.
  • the intensity observed in the immunoblot reflects the serum concentration of the 35 kDa protein fragment of inter-alpha-trypsin inhibitor heavy chain H4 precursor. Said 35 kDa protein fragment therefore was demonstrated to be useful in detection of not only chronic hepatitis but of fatty liver disease, particularly NASH.
  • serum samples were treated with Agilent Multiple Affinity Removal System (column size, 4.6 mm ⁇ 100 mm) to remove albumin, IgG, IgA, transferrin, heptoglobin, and antitrypsin.
  • serum 35 ⁇ L was mixed with 175 ⁇ L Buffer A, insoluble materials were eliminated by using 0.45 ⁇ m centrifuge filter and 200 A ⁇ L of it was applied to the system. Thereafter Buffer A was loaded at 0.5 ⁇ L/min for 10 min.
  • the flow-through was concentrated with Microcon 10 (MILLIPORE) and, after addition of 20 mM phosphate buffer (pH 7.0), further concentrated to the final volume of less than 50 ⁇ L.
  • MILLIPORE Microcon 10
  • the protein content of the concentrate was determined, 150 ⁇ g of it was subjected to SDS-PAGE (10% acryl amide gel) followed by Western blotting.
  • SDS-PAGE gel was transferred to PVDF membrane, blocking was carried out with 5% skim milk in TBSt overnight, and the membrane was washed with TBSt and reacted thereafter with the primary antibody (BMPEP1117R, 0.68 ⁇ g/mL) for 1 h followed by washing with TBSt.
  • the membrane was reacted with the secondary antibody (HRP conjugated anti-rabbit IgG, 1:5000, GE Healthcare) and washed with TBSt. Detection was carried out using LAS3000 (Fuji Film).
  • FIG. 2 illustrates the results of the above testing.
  • the sample IDs for FL, NS, CH are identical to those shown in Example 2.
  • a comparison with MW markers reveals that the bands are located at approximately 35 kDa.
  • FIG. 2 also indicates that there is no band that reacts with the primary antibody (BMPEP1117R) other than the bands at approximately 35 kDa.
  • BMPEP1117R the primary antibody
  • each of the immunoblot data obtained in Example 2 equals the sum of the intensities resulting from these bands only.
  • the intensity observed in the immunoblot experiment reflects the total serum concentration of the 35 kDa protein fragment of inter-alpha-trypsin inhibitor heavy chain H4 precursor.
  • Beads to be used for the immunoMS method were prepared as follows: The magnetic beads, Magnosphere MS300/Carboxyl type (JSR Co. Ltd., Tokyo), were supplied in slurry (10 mg beads/mL). MES was used as 0.1 M MES (pH 5.0, pH adjusted with NaOH).
  • EDC means 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and was prepared just before use in the concentration of 10 mg/mL (52.2 mM) in ice-cold MES.
  • One milliliter of dispersed beads slurry (10 mg beads) was transferred into a 2.0 ml-capacity microtube.
  • the magnetic beads were separated from supernatant by precipitation on a magnetic stand and washed with 1.0 mL MES.
  • a solution of antibody, BMPEP1117R was added to the magnetic beads and stirred slowly at room temperature.
  • EDC 100 ⁇ L was added and stirred slowly to allow reaction to take place. Washing with TBSt was repeated 4 times and beads in 1 mL TBSt were stored at 4° C.
  • the measurement using the immunoMS method began with addition of a fixed amount of pure peptide labeled with stable isotope as the internal standard to each serum sample.
  • Two microliter of stable isotope-labeled peptide solution (100 fmol/ ⁇ L) dissolved in 0.1% TFA-50% acetonitrile was added to 25 ⁇ L serum. This was called “A. ”
  • the stable isotope-labeled peptide was obtained by exchanging 12C and 14N of the 6th P of RLAILPASAPPATSNPD respectively with 13C and 15N.
  • the mean m/z [M+H] + for the unlabeled peptide was 1691.93 while that for the labeled peptide was 1697.89.
  • the next step was pretreatment of serum sample. “A” described above was mixed with 475 ⁇ L 0.1% TFA and heated at 100° C. for 15 min. The mixture was cooled in ice, sonicated, and centrifuged. The resulting supernatant was transferred to microcon 10 (MILLIPORE) and centrifuged at 14000 ⁇ g, 4 2 C for 80 min. The filtrate was mixed with 500 ⁇ L 100 mM Tris-HCl (pH 7.5) containing 0.3 M NaCl and 0.2% octylglucoside and the resultant mixture was used as sample solution for the next step.
  • MILLIPORE microcon 10
  • the procedures for immunoprecipitation with the magnetic beads and preparation of samples for mass spectrometry were as follows: The magnetic beads suspension (20 ⁇ L) was added to the sample solution above and the mixture was stirred slowly. Washing and removal of the supernatant was carried out using the magnetic stand. Washing was repeated several times with TBS and finally with 50 mM ammonium bicarbonate (pH 7. 5). Target peptide was eluted from the beads with 50 ⁇ L 2-propanol: H 2 O: formic acid (4:4:1) solution twice. The recovered eluate (approximately 100 ⁇ L) was dried by a vacuum pump.
  • the dried sample was redissolved in 20 ⁇ L 0.095% TFA-5% acetonitrile under sonication (160 W), and applied to C18 tip (PerfectPure C18 Tip, Eppendolf) for adsorption, and the tip was washed with 0.1% TFA. Peptides were eluted with 2 ⁇ L 0.1% TFA-50% acetonitrile, transferred onto the target plate for mass spectrometry, and dried. Next, 1 ⁇ L of matrix solution, i.e., 0.3 mg/mL CHCA in ethanol: acetone (2:1) was dropped onto the dried sample, and the target plate was again dried.
  • matrix solution i.e., 0.3 mg/mL CHCA in ethanol: acetone (2:1) was dropped onto the dried sample, and the target plate was again dried.
  • a MALDI-TOF-MS instrument AXIMA CFR, was used for mass spectrometry. Measurements were done in linear mode. As 200 fmol of the internal standard was contained in each serum sample, the number of fmols in sample could be calculated by multiplying the intensity ratio of sample to internal standard times 200.
  • FIG. 3 shows the results of the immunoMS assay for 2 healthy controls (NR), 5 fatty liver patients (FL) and 13 NASH patients (NS).
  • the mean and standard deviation (bar) are shown for duplicate or triplicate determinations of each sample. IDs that are identical to those in Example 2 are indicated for FL and NS but those for CH do not correspond to each other.
  • the threshold value of 8.3 fmol/ ⁇ L for serum concentration of the glycated peptide is indicated by the stippled line.
  • the number of cases exceeding the threshold/total number of cases was found to be 0/2 for healthy controls, 1/5 for fatty liverpatients and 11/13 forNASH patients. Obviously, the values were high in NASH patients.
  • the mean and standard deviation of serum concentration were as follows (number of subjects in parenthesis): healthy controls, 1.75, 2.47 (2); fatty liver patients, 6.36, 5.18 (5); NASH patients, 17.10, 9.11 (13); chronic hepatitis patients, 17.93, 13.82 (2).
  • FIG. 4 is a scatter diagram of the results for fatty liver and NASH shown in FIG. 3 .
  • the respective mean values are 6.36 fmol/ ⁇ L and 17.10 fmol/ ⁇ L as described above.
  • the p value from t-test being 0.026 indicates that patients with NASH are significantly higher in serum concentration of glycated partial peptide (Sequence No. 3) of inter-alpha-trypsin inhibitor heavy chain H4 precursor than those with fatty liver.
  • FIG. 5 shows the ability of differential diagnosis between fatty liver and NASH in terms of the Receiver Operating Characteristics (ROC) Curve and the corresponding Area under the Curve (AUC).
  • the AUC is 0.862, indicating that the glycated partial peptide of inter-alpha-trypsin inhibitor heavy chain H4 precursor is a useful diagnostic marker differentiating between fatty liver and NASH.
  • the glycated partial peptide (Sequence No. 3) of inter-alpha-trypsin inhibitor heavy chain H4 precursor is expressed as p35 peptide.
  • a sandwich method where the antigen is sandwiched with two antibodies having different epitopes attaching to both ends of it is constructed.
  • the antibodies can be either polyclonal or monoclonal. Described herein is the case for polyclonal antibodies.
  • BMPEP1117R The antibody, BMPEP1117R, was shown in Example 3 ( FIG. 2 ) to react with the N-terminus of the 35 kDa protein fragment (Sequence No. 2) of inter-alpha-trypsin inhibitor heavy chain H4 precursor. To confirm further that BMPEP1117R captures this fragment and that the fragment thus captured reacts with an antibody specific to the C-terminus of the 35 kDa protein fragment (Sequence No. 2), the following experiments were carried out.
  • BMPEP1117C An antibody specific to the C-terminus (herein called BMPEP1117C) was prepared, according to the method described in Example 1, by using partial peptide SATRERRLDYQEGPPGVEIS (217-236) of Sequence No. 2 as the immunogen.
  • the antibody was obtained as the IgG fraction from antiserum.
  • the antibody was cross-linked to Protein G Sepharose using dimethyl pimelimidate (DMP) to yield “antibody beads.”
  • DMP dimethyl pimelimidate
  • a total of 200 ⁇ L supernatant obtained was neutralized with 1 M Tris and concentrated to a volume of 40 ⁇ L using microcon 10 (MILLIPORE) by repeated centrifugation during which the solvent was exchanged to 20 mM phosphate buffer (pH 7.0).
  • MILLIPORE microcon 10
  • the resulting concentrate was subjected to SDS-PAGE with 10% acryl amide gel and Western blotting was carried out by using BMPEP1117C (1:1000) as the primary antibody and HRP conjugated anti-rabbit IgG (1:5000) (GE Healthcare) as the secondary antibody.
  • FIG. 6 shows the results of Western blotting described above.
  • BMPEP1117R captures the 35 kDa protein fragment (Sequence No. 2) of inter-alpha-trypsin inhibitor heavy chain H4 precursor and that the fragment thus captured reacts with the antibody specific to its C-terminus. It is therefore shown that the construction of ELISA for measurement of the 35 kDa protein fragment (Sequence No. 2) of inter-alpha-trypsin inhibitor heavy chain H4 precursor is enabled.
  • nonalcoholic fatty liver disease including nonalcoholic steatohepatitis can be detected by using the biomarkers disclosed in the present invention
  • the invention is applicable to the use in the field of medical diagnosis including that of diagnostic agents.

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PCT/JP2009/004657 WO2010032458A1 (fr) 2008-09-19 2009-09-16 Biomarqueur inédit de la stéatose hépatique non alcoolique et méthode de détection d'une stéatose hépatique non alcoolique au moyen dudit biomarqueur

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