Clinical diagnosis of hepatic fibrosis using a novel panel of low abundant human plasma protein biomarkers

Abstract

The inventors have proposed a novel panel of human plasma protein biomarkers for diagnosing hepatic fibrosis and cirrhosis. Presently there is no reliable non-invasive way of assessing liver fibrosis. A 2D-PAGE based proteomics study was used to identify potential fibrosis biomarkers. Plasma from patients with hepatic cirrhosis induced by infection with the hepatitis C virus (HCV) were analysed. Several proteins associated with liver scarring and potentially also related to viral infection were identified. These proteins include 14-3-3 protein zeta/delta, adiponectin, afamin, alpha-1-antitrypsin, alpha-2-HS-glycoprotein, apolipoprotein C-M, apolipoprotein E, C4b-binding protein beta chain, intact/cleaved complement C3dg, corticosteroid-binding globulin, fibrinogen gamma chain, beta haptoglobin at pH 5.46-5.49, haptoglobin-related protein, hemopexin, immunoglobulin J chain, leucine-rich alpha-2-glycoprotein, lipid transfer inhibitor protein, retinol-binding protein 4, serum paraoxonase/arylesterase 1, sex hormone-binding globulin and zinc-alpha-2-glycoprotein.

1 - 19 . (canceled) 
     
     
         20 . A method of diagnosing hepatic fibrosis, comprising:
 a) obtaining a biological sample from a patient in need thereof;   b) determining in the biological sample a level of at least two HF-associated polypeptides, wherein said at least HF-associated polypeptides comprise hemopexin and alpha-2-HS-glycoprotein; and   c) comparing said determined level to a respective control level to determine a positive or a negative diagnosis of said hepatic fibrosis.   
     
     
         21 . The method of  claim 20 , wherein the biological sample is a sample of serum or plasma of the patient. 
     
     
         22 . The method of  claim 20 , wherein the at least two HF-associated polypeptides further comprise at least one of 14-3-3 protein zeta/delta, adiponectin, afamin, alpha-1-antitrypsin, apolipoprotein apolipoprotein E, C4b-binding protein beta chain, intact/cleaved complement C3dg, corticosteroid-binding globulin, fibrinogen gamma chain, beta haptoglobin at pH 5.46-5.49, haptoglobin-related protein, immunoglobulin J chain, leucine-rich alpha-2-glycoprotein, lipid transfer inhibitor protein, retinol-binding protein 4, serum paraoxonase/arylesterase 1, sex hormone-binding globulin, zinc-alpha-2-glycoprotein, inter-α-trypsin inhibitor heavy chain H4 fragments, al antichymotrypsin, apolipoprotein L1, prealbumin, albumin, isoforms of CD5 antigen like protein, beta 2 glycoprotein I, α2 macroglobulin and immunoglobulin components, α1, α2 and α chains of haptoglobin, C3, C4 and factor H-related protein 1, prothrombin, clusterin, and angiotensinogen. 
     
     
         23 . The method of  claim 20 , wherein the at least two HF-associated polypeptides further comprise at least one of zinc-alpha-2-glycoprotein, apolipoprotein E, apolipoprotein L1, clusterin, lipid transfer inhibitor protein, intact/cleaved complement C3dg, and corticosteroid-binding globulin. 
     
     
         24 . The method of  claim 20 , wherein said determining comprises using an agent that is specific to the HF-associated polypeptide. 
     
     
         25 . The method of  claim 24 , wherein the agent is an antibody or a functional equivalent thereof that binds the HF associated peptide. 
     
     
         26 . The method of  claim 24 , wherein said determining is performed using an assay technique. 
     
     
         27 . The method of  claim 26 , wherein said determining is performed using enzyme linked immunosorbent assay, radio-immunoassay, protein dot blot, Western blot, turbidimetry or nephelometry. 
     
     
         28 . The method of  claim 20 , wherein said determining is performed by Multiple Reaction Monitoring using mass spectroscopy. 
     
     
         29 . A method of determining prognosis of hepatic fibrosis, comprising:
 a) obtaining a biological sample from a patient in need thereof;   b) determining in the biological sample a level of at least two HF-associated polypeptides, wherein said at least HF-associated polypeptides comprise hemopexin and alpha-2-HS-glycoprotein; and   c) comparing said determined level to a respective control level to determine a positive or a negative prognosis of said hepatic fibrosis.   
     
     
         30 . The method of  claim 29 , wherein the biological sample is a sample of serum or plasma of the patient. 
     
     
         31 . The method of  claim 29 , wherein the at least two HF-associated polypeptides further comprise at least one of 14-3-3 protein zeta/delta, adiponectin, afamin, alpha-1-antitrypsin, apolipoprotein apolipoprotein E, C4b-binding protein beta chain, intact/cleaved complement C3dg, corticosteroid-binding globulin, fibrinogen gamma chain, beta haptoglobin at pH 5.46-5.49, haptoglobin-related protein, immunoglobulin J chain, leucine-rich alpha-2-glycoprotein, lipid transfer inhibitor protein, retinol-binding protein 4, serum paraoxonase/arylesterase 1, sex hormone-binding globulin, zinc-alpha-2-glycoprotein, inter-α-trypsin inhibitor heavy chain H4 fragments, α1 antichymotrypsin, apolipoprotein L1, prealbumin, albumin, isoforms of CD5 antigen like protein, beta 2 glycoprotein I, α2 macroglobulin and immunoglobulin components, α1, α2 and α chains of haptoglobin, C3, C4 and factor H-related protein 1, prothrombin, clusterin, and angiotensinogen. 
     
     
         32 . The method of  claim 29 , wherein the at least two HF-associated polypeptides further comprise at least one of zinc-alpha-2-glycoprotein, apolipoprotein E, apolipoprotein L1, clusterin, lipid transfer inhibitor protein, intact/cleaved complement C3dg, and corticosteroid-binding globulin. 
     
     
         33 . The method of  claim 29 , wherein said determining comprises using an agent that is specific to the HF-associated polypeptide. 
     
     
         34 . The method of  claim 33 , wherein the agent is an antibody or a functional equivalent thereof that binds the HF associated peptide. 
     
     
         35 . The method of  claim 33 , wherein said determining is performed using an assay technique. 
     
     
         36 . The method of  claim 35 , wherein said determining is performed using enzyme linked immunosorbent assay, radio-immunoassay, protein dot blot, Western blot, turbidimetry or nephelometry. 
     
     
         37 . The method of  claim 29 , wherein said determining is performed by Multiple Reaction Monitoring using mass spectroscopy. 
     
     
         38 . A method of scaling the severity of hepatic fibrosis comprising:
 a) obtaining a biological sample from a patient in need thereof;   b) determining in the biological sample a level of at least two HF-associated polypeptides, wherein said at least HF-associated polypeptides comprise hemopexin and alpha-2-HS-glycoprotein; and   c) comparing said determined level to a respective predetermines level in a population of patients ranging from no fibrosis to cirrhosis.   
     
     
         39 . The method of  claim 38 , wherein the biological sample is a sample of serum or plasma of the patient. 
     
     
         40 . The method of  claim 38 , wherein the at least two HF-associated polypeptides further comprise at least one of 14-3-3 protein zeta/delta, adiponectin, afamin, alpha-1-antitrypsin, apolipoprotein apolipoprotein E, C4b-binding protein beta chain, intact/cleaved complement C3dg, corticosteroid-binding globulin, fibrinogen gamma chain, beta haptoglobin at pH 5.46-5.49, haptoglobin-related protein, immunoglobulin J chain, leucine-rich alpha-2-glycoprotein, lipid transfer inhibitor protein, retinol-binding protein 4, serum paraoxonase/arylesterase 1, sex hormone-binding globulin, zinc-alpha-2-glycoprotein, inter-α-trypsin inhibitor heavy chain H4 fragments, al antichymotrypsin, apolipoprotein L1, prealbumin, albumin, isoforms of CD5 antigen like protein, beta 2 glycoprotein I, α2 macroglobulin and immunoglobulin components, α1, α2 and α chains of haptoglobin, C3, C4 and factor H-related protein 1, prothrombin, clusterin, and angiotensinogen. 
     
     
         41 . The method of  claim 38 , wherein the at least two HF-associated polypeptides further comprise at least one of zinc-alpha-2-glycoprotein, apolipoprotein E, apolipoprotein L1, clusterin, lipid transfer inhibitor protein, intact/cleaved complement C3dg, and corticosteroid-binding globulin. 
     
     
         42 . The method of  claim 38 , wherein said determining comprises using an agent that is specific to the HF-associated polypeptide. 
     
     
         43 . The method of  claim 42 , wherein the agent is an antibody or a functional equivalent thereof that binds the HF associated peptide. 
     
     
         44 . The method of  claim 42 , wherein said determining is performed using an assay technique. 
     
     
         45 . The method of  claim 44 , wherein said determining is performed using enzyme linked immunosorbent assay, radio-immunoassay, protein dot blot, Western blot, turbidimetry or nephelometry. 
     
     
         46 . The method of  claim 38 , wherein said determining is performed by Multiple Reaction Monitoring using mass spectroscopy.