Biomarkers for non-alcoholic fatty liver disease, and methods for detecting non-alcoholic fatty liver disease by using such biomarkers
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 or between patients with fatty liver and 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. Specifically, 35 kDa protein fragment consisting of amino acid sequence expressed by Sequence No. 2 and its partial peptide consisting of amino acid sequence expressed by Sequence No. 3 (including its glycated form) of inter-alpha-trypsin inhibitor heavy chain H4 precursor consisting of amino acid sequence expressed by Sequence No. 1 could be used as biomarkers to detect nonalcoholic fatty liver disease including nonalcoholic steatohepatitis.
1. A method of diagnosing and treating nonalcoholic steatohepatitis in a human patient comprising:
a. generating a rabbit polyclonal antibody by immunizing rabbits with an immunogen, wherein the immunogen consists of SEQ ID NO: 7 conjugated to keyhole limpet hemocyanin (KLH);
b. obtaining a sample from a human patient, wherein said sample is blood or serum;
c. contacting the sample with the rabbit polyclonal antibody generated at step a.;
d. detecting binding between the rabbit polyclonal antibody and at least one biomarker in said sample, wherein the at least one biomarker is selected from the group consisting of:
(i) the protein consisting of SEQ ID NO: 2,
(ii) the peptide consisting of SEQ ID NO: 3, and
(iii) a glycosylated peptide consisting of SEQ ID NO: 3, wherein the glycosylated peptide comprises SEQ ID NO: 3 bound to -GlcNAc-Hex-GlcNAc-Hex;
e. diagnosing the patient as having nonalcoholic steatohepatitis when the at least one biomarker is detected in higher quantity than in a healthy control subject, wherein the healthy control subject does not have nonalcoholic steatohepatitis; and
f. administering treatment to the patient when the patient is diagnosed with nonalcoholic steatohepatitis, wherein said treatment is selected from the group consisting of diet and drug therapy.
2. A method of diagnosing and treating nonalcoholic steatohepatitis in a human patient comprising:
a. generating a rabbit polyclonal antibody by immunizing rabbits with an immunogen comprising an amino acid sequence, wherein the sequence consists of SEQ ID NO: 9;
b. obtaining a sample from a human patient, wherein said sample is blood or serum;
c. contacting the sample with the rabbit polyclonal antibody generated at step a.;
d. detecting binding between the rabbit polyclonal antibody and at least one biomarker in said sample, wherein the at least one biomarker is selected from the group consisting of:
(i) the protein consisting of SEQ ID NO: 1, and
(ii) the protein consisting of SEQ ID NO: 2;
e. diagnosing the patient as having nonalcoholic steatohepatitis when the at least one biomarker is detected in higher quantity than in a healthy control subject,
wherein the healthy control subject does not have nonalcoholic steatohepatitis; and
f. administering treatment to the patient when the patient is diagnosed with nonalcoholic steatohepatitis, wherein said treatment is selected from the group consisting of diet and drug therapy.
3. A method of diagnosing nonalcoholic steatohepatitis in a human patient comprising:
a. generating a rabbit polyclonal antibody by immunizing rabbits with an immunogen consisting of SEQ ID NO: 7 conjugated to KLH;
b. obtaining a sample from a human patient, wherein said sample is blood or serum;
c. contacting the sample with the rabbit polyclonal antibody generated at step a.;
d. detecting binding between the rabbit polyclonal antibody and at least one biomarker in said sample, wherein the at least one biomarker is selected from the group consisting of:
(i) the protein consisting of SEQ ID NO: 2,
(ii) the peptide consisting of SEQ ID NO: 3, and
(iii) a glycosylated peptide consisting of SEQ ID NO: 3, wherein the glycosylated peptide comprises SEQ ID NO: 3 bound to -GlcNAc-Hex-GlcNAc-Hex;
e. diagnosing the patient with nonalcoholic steatohepatitis when the at least one biomarker is detected in higher quantity than in a healthy control subject,
wherein the healthy control subject does not have nonalcoholic steatohepatitis.
4. A method of diagnosing nonalcoholic steatohepatitis in a human patient comprising:
a. generating a rabbit polyclonal antibody by immunizing rabbits with an immunogen comprising a sequence, wherein the sequence consists of SEQ ID NO: 9;
b. obtaining a sample from a human patient, wherein the sample is blood or serum;
c. contacting the sample with the rabbit polyclonal antibody generated at step a.;
d. detecting binding between the rabbit polyclonal antibody and at least one biomarker in said sample, wherein the at least one biomarker is selected from the group consisting of:
(i) the protein consisting of SEQ ID NO: 1, and
(ii) the protein consisting of SEQ ID NO: 2,
e. diagnosing the patient as having nonalcoholic steatohepatitis when the at least one biomarker is detected in higher quantity than in a healthy control subject,
wherein the healthy control subject does not have nonalcoholic steatohepatitis.