WO2010079253A2 - Bio-markers for diagnosing fibrosis - Google Patents

Abstract

The invention relates to methods for detecting and diagnosing fibrosis and diseases associated with fibrosis, and to methods for determining the effectiveness of a therapy against fibrosis by determining the expression levels of bio-markers, where the one or more bio-markers are selected from the group formed by SLURP1, HAMP, GSN and APOD. The invention also relates to kits comprising reagents suitable for carrying out the methods according to the invention.

Description

 BIOMARKERS FOR THE FIBROSIS DIAGNOSIS

FIELD OF THE INVENTION

The invention relates to the field of diagnosis and, more particularly, to the diagnosis of diseases associated with an increase in deposition of connective tissue in an organ or tissue, preferably in the liver. The diagnosis is carried out using biomarkers whose level of expression correlates with the appearance of fϊbrotic diseases.

TECHNICAL BACKGROUND

Hepatic fibrosis is a central feature of most chronic liver lesions due to metabolic, genetic, viral, and cholestatic diseases. Chronic inflammatory diseases of the liver cause destruction of the parenchyma of the liver and its exchange for scar tissue (fibrosis). Fibrosis is characterized by excess extracellular matrix deposition (ECM) that involves the molecular and histological reorganization of various types of collagen, proteoglycans, structural glycoproteins and hyaluronic acid (hyaluronan). Fibrosis is a brand of liver cirrhosis, which is associated with impaired liver function and significant morbidity and mortality.

The deposition of ECM in the space of Disse (perisinusoidal fibrosis), the generation of subendothelial (incomplete) basal membranes, and the strangulation of hepatocytes by the adjacent matrix impair not only blood flow through the organ, but also biosynthetic function of hepatocytes and the purification capacity of these and other cells, for example of coagulation factors and transport proteins in plasma, hormones, and ammonia. In this way, the diagnosis, follow-up and therapeutic evaluation of phobrosis and phogenesis, that is, the active process of generating new connective tissue in the diseased liver, is of great clinical importance.

The diagnosis of liver fibrosis has been made in the past and is currently practiced mostly through the aggressive puncture biopsy procedure and consecutive histological evaluation based on several numerical scoring systems that produce graduation of necroinflammatory activity and staging (extension) of fibrosis. However, this "gold standard" has many disadvantages in addition to aggressiveness such as sampling error, irreproducible quality of the sample depending on the length and size of the tissue sample and a histological evaluation strictly dependent on the pathologist's experience.

Currently, the most reliable serological method for the evaluation of liver fibrosis uses a panel of 5 markers: α2-macroglobulin, haptoglobulin, apolipoprotein Al, γ-glutamyl transpeptidase, and bilirubin (Imbert-Bismut et al. Lancet 2001; 357: 1069- 1075 and WO0216949). However, this test eliminates the need for biopsy in only 26% of patients and does not accurately predict the presence or absence of fibrosis.

WO0373822 provides a method of diagnosing the presence or severity of liver fibrosis in an individual based on the presence or level of α-MG, HA and TIMP-1.

WO2005116901 discloses several methods to diagnose the presence and / or severity of a liver pathology based on different combinations of markers such as α-2 macroglobulin, hyaluronic acid, apoliprotein Al, N-terminal propeptide of type III collagen, γ-glutamyltranspeptidase, bilirubin , γ-globulins, platelets, prothrombin time, aspartate amino transferase, alanine aminotransferase, urea, sodium, glycemia, triglycerides, albumin, alkaline phosphatases, YKL-40 (human cartilage glycoprotein 39), Tissue matrix metalloproteinase inhibitor 1 (TIMP-I), matrix 2 metalloproteinase (MMP-2) and ferritin. EPl 811041 discloses methods for the diagnosis of fibrosis and / or liver cirrhosis based on the use of markers identified in a rat model for liver fibrosis (rats treated with dimethylnitrosamine) using mRNA expression profiles in micromatrices and quantitative protein profiles of samples of liver and serum.

WO2007003670 discloses methods for the diagnosis of fibrotic alterations by using any of four detectable markers in urine: uromodulin, MAC2BP, AGP 1 and cathepsin A.

WO2008031051 discloses a method for the detection and diagnosis of liver fibrosis using a panel of human serum biomarker proteins. These methods have also been disclosed in Gangadharan et al. [Clinical Chemistry 2007; 53: 1792-1799].

Another non-aggressive alternative approach for the evaluation of liver fibrosis is transient elastography with Fibroscan® (Echosens, Paris, France) that operates by measuring liver stiffness (Sandrin L et al. Ultrasound Med. Biol. 2003; 29: 1705- 1711). A mechanical pulse is generated on the surface of the skin, which spreads through the liver. The speed of the wave is measured by ultrasound. Speed correlates directly with the stiffness of the liver, which in turn reflects the degree of fibrosis - the stiffer the liver is, the greater the degree of fibrosis.

Although there is a significant need for non-aggressive systemic biomarkers, organ-specific and disease for liver fibrosis (and fibrosis of other organs as well), there is currently no individual parameter or combination of them available, which satisfies all the diagnostic criteria required for extended, cost effective, and reliable use. Individual measurements of biochemical markers in serum, plasma or even urine are currently not valid enough to replace liver biopsy.

SUMMARY OF THE INVENTION In a first aspect, the invention relates to the use of at least one biomarker for the detection, diagnosis and evaluation of fibrosis and / or a disease associated with fibrosis or for monitoring the efficacy of a treatment for fibrosis and / or a disease associated with fibrosis in a subject where said biomarker is selected from the group consisting of SLURPl, HAMP, GSN, APOD, SPPl, DEFBl and MASP2.

In a second aspect, the invention relates to a method for the detection, diagnosis and evaluation of fibrosis and / or a disease associated with fibrosis or for monitoring the efficacy of a treatment for fibrosis and / or a disease associated with fibrosis that comprises comparing the expression of one or more biomarkers in a sample of a subject with a predetermined standard for each of said one or more biomarkers; wherein said one or more biomarkers are selected from the group consisting of SLURPl, HAMP, GSN, APOD, SPPl, DEFBl and MASP2; and wherein a significant difference in the expression of said one or more biomarkers in said sample compared to the predetermined standard of each of said one or more biomarkers is indicative of onset, phase, evolution of fibrosis and / or disease associated with fibrosis or of the efficacy of a treatment for fibrosis and / or disease associated with fibrosis.

In yet another aspect, the invention relates to a kit comprising reagents for detecting at least two or more biomarkers selected from the group of SLURPl, HAMP, GSN, APOD, SPPl, DEFBl and MASP2.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Control sample GeI. Stains marked with circles were collected and identified by LC-MS / MS. The spot in the white circle was identified as gelsolin, and the spot in the black circle was identified as apolipoprotein D.

Figure 2. Decreased levels of ApoD and gelsolin proteins identified in cirrhotic urine samples were confirmed by immunoblot. (A) The Urine proteins were separated on an SDS-acrylamide gel and the proteins were transferred on a nitrocellulose membrane. ApoD and gelsolin levels were detected using antibodies specific for these proteins. (B) The intensity of the bands was quantified and represented. The average value of protein levels showed a two-fold decrease in cirrhotic samples for both proteins.

Figure 3. The intensity of hepcidin-25 (peak of 2793 Da), hepcidin-20 (peak of 2193) and SLURP-I (peak of 8855 Da) for cirrhotic, control and low fibrosis samples are represented. The levels of this protein are significantly decreased in cirrhotic patients, on the other hand the low fibrosis group shows an intermediate expression level.

Figure 4. Decreased levels of the SLURPl protein in urine samples of cirrhotic patients with respect to the controls detected by Western Blot. (A) The control urine (C1-C8) and eight urine from cirrhotic patients (P1-P8) were subjected to SDS-acrylamide gel electrophoresis and the proteins were transferred on a nitrocellulose membrane. SLURPl levels were detected using antibodies specific for these proteins. (B) Graph of the optical density corresponding to the SLURP-I bands obtained in the control urine and in cirrhotic patients.

Figure 5. The intensities of Osteopontin (7655 Da peak) and its phosphorylated form (7735 Da peak) for cirrhotic, control and low fibrosis samples are depicted. The levels of this protein are significantly decreased in cirrhotic patients, on the other hand the low fibrosis group shows an intermediate expression level.

Figure 6. The intensities of Defensin Beta 1 (peak of 4623 Da) and MASP2 (peak of 5804 Da) for cirrhotic, control and low fibrosis samples are depicted. The levels of this protein are significantly decreased in cirrhotic patients, on the other hand the low fibrosis group shows an intermediate expression level. DETAILED DESCRIPTION OF THE INVENTION

The authors of the present invention have identified protein biomarkers in human urine samples whose expression correlates with the presence of cirrhosis and liver fibrosis. In particular, the authors have identified gelsolin, apoliprotein D, hepcidin (25 and 20), SLURP-I, Osteopontin (SPPl, OPN or OSTP), Beta 1 Defensin

(DEFBl) and Serine-linked lectin-linked serine protease (MASP2) as proteins whose levels decrease significantly in the urine samples of cirrhotic patients when compared to control samples. These proteins are potential markers of advanced states of liver fibrosis.

Thus, in a first aspect, the invention relates to the use of a biomarker for the detection, diagnosis and evaluation of fibrosis and / or of a disease involving fibrosis or for monitoring the efficacy of a treatment for fibrosis and / or of a disease associated with fibrosis in a subject where said biomarker is selected from the group consisting of SLURPl, HAMP, GSN, APOD, SPPl, DEFBl and MASP2.

The term "biomarker" or, alternatively, "molecular marker", as used herein, is used to refer to a molecule or the expression product of a gene or fragments and variants thereof that show substantial changes in a given disease. and that can be used both for the detection and for the diagnosis of a disease by detecting the appearance of said changes in the biomarker and / or to follow the efficacy of a treatment for that disease by detecting changes in the biomarker as opposed to those that occur in the disease or clinical situation.

In a preferred embodiment, changes in the biomarker are changes in expression levels. The term "expression", as used herein, refers to a process by which a polypeptide is produced from DNA. This process involves the transcription of the gene to a messenger RNA (mRNA), and the translation of this mRNA into the polypeptide. In the context of the invention "changes in the levels of expression "refers to any change in the production of mRNA, polypeptide or both that produces altered relative levels of mRNA, protein or both in a sample with respect to other molecules in the same sample. It will be appreciated that the expression levels of a biomarker can be determined by determining the levels of mRNA in a sample or by determining the levels of the corresponding polypeptide Alternatively, the polypeptide biomarkers can be variants resulting from post-translational modifications, including fragments thereof.

The terms "diagnosis", "detection" and "evaluation" are used interchangeably here, and refers to the assessment of the probability according to which a subject suffers from a disease as well as the assessment of its onset, developmental state , evolution, or its regression, and / or the prognosis of the course of the disease in the future. As those skilled in the art will understand, such assessment, although preferred, may not be correct for 100% of the subjects to be diagnosed. The term, however, requires that a statistically significant part of the subjects can be identified as having the disease or having a predisposition to it. If a part is statistically significant, it can be determined by the person skilled in the art using several well-known statistical evaluation tools, for example, determination of confidence intervals, determination of p-values, Student's t-test, Mann-Whitney test , etc. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80 %, at least 90%, at least 95%. P values are preferably 0.2, 0.1, 0.05.

The expression "follow the effectiveness of a treatment" refers to the assessment of the results of a therapy in a patient suffering from fibrosis or a disease associated with fibrosis. Suitable therapies for the treatment of fibrosis include treatment with PCP inhibitors, IFN-γ or imatinib mesylate (Gleevec). The usefulness of the biomarkers of the invention to track the efficacy of a treatment can also be applied to methods for selecting and screening drugs with potential activity. antifibrosant This process comprises a) administering to the subject (preferably an animal) the drug to be studied; b) at different points of the study (before, during and / or after administration) take biological samples of the animal and determine the marker levels according to the present invention; and c) compare the determinations made in the samples obtained in the different phases of the treatment and compare them to control animals, for example untreated.

The term "SLURP-I", as used herein, refers to the gene known as "containing the secreted domain LY6 / PLAUR 1", also identified by the accession number HGNC ID. 18746 (HUGO Gene Nomenclature Committee, Human Genome Organization), which is located in chromosomal region 8q24.3. The protein encoded by SLURP-I is identified in the Entrez database by GeneID access number: 57152 (updated July 27, 2008) and is also known as "ARS component B" (ARS, ArsB), " urinary antineoplastic protein "(ANUP), MDM," similar to secreted lymphocyte antigen 6 (LY6LS). In a particular embodiment, the expression product of the SLURP1 gene comprises SEQ ID NO: 1, which is a 103 amino acid polypeptide identified with the UniProt ID accession number P55000 (last modified July 22, 2008. Version 75) (SEQ ID NO: 1) as well as orthologs, isoforms and fragments of said polypeptide. In another preferred embodiment, the biomarker is a fragment of said polypeptide comprising amino acids 22-103 (SEQ ID NO: 2), corresponding to the secreted mature polypeptide.

The term "HAMP", as used herein, refers to the "hepcidin antimicrobial peptide" gene, identified by the HGNC ID accession number. 15598, and which is located in chromosomal region 19ql3.1. The "hepcidin antimicrobial peptide"

(HAMP) is identified in the Entrez database with GeneID: 57817 (updated October 12, 2008) and is also known as "liver-expressed antimicrobial peptide" (LEAP-I), hepcidin, HEPC, HFE2B, LEAPl, and "putative liver tumor regressor" (PLTR). In a particular embodiment, the expression product of the HAMP gene comprises any of the sequences SEQ ID NO: 3, which is the 84-amino acid preproprotein identified by the UniProt ID accession number. P81172 (last modified July 22, 2008. Version 82); SEQ ID NO: 4 (amino acids 60-84, known as hepcidin 25 or Hepc25); SEQ ID NO: 5 (amino acids 65-84, known as hepcidin 20 or Hepc20); as well as orthologs, isoforms and fragments of said polypeptide. In a preferred embodiment the HAMP expression product is Hepc25, Hepc20 or both.

The term "GSN", as used herein, refers to the gene "gelsolin (amyloidosis, of the Finnish type), identified by accession number HGNC ID. 4620, and which is located in the chromosomal region 9q33. The expression product of the gene is also identified in the Entrez database as GeneID: 2934 (updated September 28, 2008) or DKFZp313L0718. The protein is known as gelsolin, and alternatively as "actin depolymerizing factor" (ADF), brevina or AGEL Various transcriptional variants for GSN that encode different isoforms have been described In a particular embodiment the GSN expression product comprises any of the sequences SEQ ID NO: 6, which corresponds to the secreted precursor polypeptide identified by UniProt ID. P06396 (last modified October 14, 2008. Version 114) as well as orthologs, isoforms and fragments of said polypeptide.

The term "APOD", as used herein, refers to the "apoliprotein D" gene, also identified by the HGNC accession number: 612, which is located in the chromosomal region 3q26.2-qter. APOD is also identified by "Entrez GeneID: 347" (updated October 12, 2008). In a particular embodiment, the APOD expression product comprises any of the sequences SEQ ID NO: 7, a precursor of the polypeptide identified as UniProt ID. P05090 (last modified July 22, 2008. Version 107) as well as orthologs, isoforms and fragments of said polypeptide.

The term "SPPl", as used herein, refers to the gene "Osteopontin" or "phosphoprotein secreted type 1"; OPN, and known alternatively as BNSP; BSPI; ETA-I;

MGCl 10940, corresponding to the polypeptide identified by the accession number

HGNC: 11255 which is located in the 4q21-q25 chromosomal region. SPPl too is identified by "Entrez Gene ID: 6696" (updated on December 4, 2009). In a particular embodiment, the SPP1 gene expression product comprises SEQ ID NO: 8, which is a 314 amino acid polypeptide identified with the UniProt accession number P 10451 (last modified November 24, 2009. Version 120); as well as orthologs, iso forms and fragments of said polypeptide, including the SPP1 isoforms identified as SEQ ID NO: 8, 9, 10 and 11.

The term "DEFBl", as used herein, refers to the gene "Beta 1 defensin or Defensin beta 1" and alternatively known as BDl; HBDl; DEFB-I; DEFB101; MGC51822, corresponding to the polypeptide identified by HGNC accession number: 2766, which is located in chromosomal region 8p23.2-p23.1. DEFBl is also identified by "Entrez GeneID: 1672" (updated on December 4, 2009). In a particular embodiment, the DEFBl expression product comprises the sequences SEQ ID NO: 9, identified as UniProt P60022.1 (last modified November 3, 2009. Version 72) as well as orthologs, isoforms and fragments of said polypeptide .

The term "MASP2", as used herein, refers to the "Manina-binding lectin serine peptidase 2" and "alternatively, sMAP; MAP 19; MASP-2, also identified by HGNC accession number: 6902, which is located in the chromosomal region Ip36.3-p36.2. MASP2 is also identified by "Entrez GeneID: 10747" (updated on December 4, 2009). In a particular embodiment, the SPP1 expression product comprises the sequence SEQ ID NO: 10, identified as UniProt O00187.3 (last modified November 24, 2009. Version 118); as well as orthologs, isoforms and fragments of said polypeptide, including the isoforms identified in SEQ ID NO: 13 and 14 of the present invention.

The term "fibrosis," as used herein, refers to an excess of extracellular matrix deposition (ECM) that involves molecular and histological reorganization of various types of collagen, proteoglycans, structural glycoproteins and hyaluronic acid.

(hyaluronan). Although fibrosis is a mark of many liver diseases, Other organs may also suffer from excess deposition of connective tissue. In a preferred embodiment, the fibrosis is hepatic fibrosis.

The term "disease associated with fibrosis" refers to any disease where one or more organs suffer from excessive deposition of fibrous connective tissue. These diseases include cystic fibrosis of the pancreas and lungs, injection fibrosis, which can occur as a complication of intramuscular injections, especially in children, endomyocardiac fibrosis, idiopathic pulmonary fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis, a complication of miner's pneumoconiosis, systemic nephrogenic fibrosis, diffuse parenchymal lung disease, post-vasectomy pain syndrome, tuberculosis (TB) can cause fibrosis of the lungs, sickle cell anemia can lead to increase and finally spleen fibrosis and rheumatoid arthritis and cirrhosis , which can cause liver fibrosis. In a preferred embodiment, the disease associated with fibrosis is cirrhosis.

The invention also relates to a method for the detection, diagnosis and evaluation of fibrosis and / or a disease associated with fibrosis or for monitoring the efficacy of a treatment for fibrosis and / or a disease associated with fibrosis comprising comparing the expression of one or more biomarkers in a sample of a subject with a predetermined standard for each of said one or more biomarkers; wherein said one or more biomarkers are selected from the group consisting of SLURPl, HAMP, GSN, APOD, SPPl, DEFBl and MASP2; and wherein a significant difference in the expression of said one or more biomarkers in said sample compared to the predetermined standard of each of said one or more biomarkers is indicative of onset, phase, evolution of fibrosis and / or disease associated with fibrosis or of the efficacy of a treatment for fibrosis and / or disease associated with fibrosis.

The terms and expressions "diagnosis", "follow the efficacy of a treatment", "fibrosis", "disease associated with fibrosis", "biomarker", "SLURPl", "HAMP", "GSN", "APOD", "SPPl "," DEFBl "and" MASP2 "have been previously described in relation to the use of the invention. The method of the invention allows the detection, diagnosis and evaluation of fibrosis and / or a disease associated with fibrosis as well as monitoring the efficacy of a treatment for fibrosis and / or a disease associated with fibrosis. The method of the invention comprises determining the expression of one or more biomarkers selected from the group of SLURPl, HAMP, GSN, APOD, SPPl, DEFBl, MASP2 in a biological sample of an individual.

The term "sample", as used herein, means any biological tissue or fluid taken from a subject. In a preferred embodiment it is biological liquid, for example, blood, serum or, more preferably urine.

The expression of a biomarker of the invention can be evaluated by any of a wide variety of well known methods to detect the expression of a transcribed molecule or its corresponding protein. If the biomarkers are nucleic acid molecules, expression of the marker polynucleotide can be detected using nucleic acid hybridization methods, nucleic acid reverse transcription methods, nucleic acid amplification methods and the like. In a preferred embodiment, expression of a marker gene is evaluated by preparing mRNA / cDNA (i.e., a transcribed polynucleotide) of cells in a sample of a patient, and by hybridizing the mRNA / cDNA with a reference polynucleotide that It is complementary to a polynucleotide comprising the marker gene, and fragments thereof. The cDNA can, optionally, be amplified using any of several polymerase chain reaction methods before hybridization with the reference polynucleotide although it is preferred that it is not amplified.

In a preferred embodiment, the biomarkers are polypeptides, in which case detection can be carried out using immunological methods for the detection of secreted proteins, protein purification methods, protein function or activity assays. In a preferred embodiment, the expression of a marker protein is evaluated using an antibody (eg, a radiolabeled, chromophore-labeled, fluorophore-labeled, or enzyme-labeled antibody), a antibody derivative (for example, an antibody conjugated to a substrate or to the protein or ligand of a protein-ligand pair (e.g., biotin-streptavidin), or an antibody fragment (e.g., a single chain antibody, the hypervariable domain of an isolated antibody, etc.) that specifically binds with a protein corresponding to the marker gene, such as the protein encoded by the open reading frame corresponding to the marker gene or such a protein that has undergone all or part of its modification normal post-translational

The most normal immunoassay is the "adsorption enzyme immunoassay (ELISA)". ELISA is a technique to detect and measure the concentration of an antigen using a labeled (for example enzyme-bound) form of the antibody. There are different ways of

ELISA, which are well known to those skilled in the art. Standard techniques known in the art for ELISA are described in "Methods in Immunodiagnosis", 2 ^a

Edition, Rose and Bigazzi, eds. John Wiley AND Sons, 1980; Campbell et al, "Methods and Immunology", W. A. Benjamín, Inc., 1964; and Oellerich, M. 1984, J. Clin. Chem

Clin. Biochem., 22: 895-904.

In an "ELISA sandwich", an antibody (for example anti-enzyme) is bound to a solid phase (ie, to a microtiter plate) and is exposed to a biological sample containing antigen (for example, enzyme). The solid phase is then washed to remove unbound antigen. A labeled antibody (for example, enzyme bound) is then attached to the bound antigen (if present) forming an antibody-antigen-antibody sandwich. Examples of enzymes that can bind to the antibody are alkaline phosphatase, horseradish peroxidase, luciferase, urease, and B-galactosidase. The enzyme-bound antibody reacts with a substrate to generate a colored reaction product that can be measured.

In a "competitive ELISA", the antibody is incubated with a sample containing antigen (ie, enzyme). The antigen-antibody mixture is then contacted with a solid phase (for example, a microtiter plate) that is coated with antigen (i.e., enzyme). The more antigen present in the sample, the less free antibody will be available to bind to the solid phase. It is added after a labeled secondary antibody (eg, enzyme bound) to the solid phase to determine the amount of primary antibody bound to the solid phase.

In a "immunohistochemical assay" a section of tissue is tested for specific proteins by exposing the tissue to antibodies that are specific to the protein being tested. The antibodies are then visualized by any of a number of methods to determine the presence and amount of the protein present. Examples of the methods used to visualize antibodies are, for example, by means of enzymes bound to the antibodies (for example, luciferase, alkaline phosphatase, horseradish peroxidase, or beta-galactosidase), or chemical methods (for example, DAB chromogen /substratum). The sample is then analyzed microscopically, most preferably by optical microscopy of a sample stained with a dye that is detected in the visible spectrum, using any of a variety of such methods and staining reagents known to the person skilled in the art.

Alternatively, "radioimmunoassays" can be used. A radioimmunoassay is a technique to detect and measure the concentration of an antigen using a labeled form (for example, radioactively or fluorescently labeled) of the antigen. Examples of radioactive labels for antigens include ³ H, ¹⁴ C, and ¹²⁵ I. The concentration of antigen enzyme in a biological sample is measured by causing the antigen in the biological sample to compete with the labeled antigen (eg radioactively) for binding. to an antibody to the antigen. To ensure competitive binding between the labeled antigen and the unlabeled antigen, the labeled antigen is present in a concentration sufficient to saturate the antibody binding sites. The higher the concentration of the antigen in the sample, the lower the concentration of the labeled antigen that will bind to the antibody. In a radioimmunoassay, to determine the concentration of the labeled antigen bound to the antibody, the antigen-antibody complex must be separated from the free antigen. One method of separating the antigen-antibody complex from the free antigen is to precipitate the antigen-antibody complex with anti-isotype antiserum. Another method of separating the antigen-antibody complex from the free antigen is to precipitate the antigen-antibody complex with S. aureus annihilated with formalin. Yet another method to separate the Antigen-antibody complex of the free antigen is to perform a "solid phase radioimmunoassay" where the antibody is bound (for example, covalently) to Sepharose beads, polystyrene wells, polyvinylchloride wells, or microtiter wells. By comparing the concentration of labeled antigen bound to the antibody with a standard curve based on samples having a known antigen concentration, the concentration of antigen in the biological sample can be determined.

An "immunoradiometric assay" (IRMA) is an immunoassay in which the antibody reagent is radioactively labeled. An IRMA requires the production of a multivalent antigen conjugate, by techniques such as conjugation to a protein, for example, rabbit serum albumin (RSA). The multivalent antigenic conjugate must have at least 2 antigen residues per molecule and the antigen residues must be separated at a sufficient distance to allow the binding of at least two antibodies to the antigen. For example, in an IRMA the multivalent antigen conjugate may be attached to a solid surface such as a plastic sphere. Unlabeled "sample" antigen and antibody for the antigen that is radioactively labeled are added to a test tube containing the sphere coated with the multivalent antigen conjugate. The antigen in the sample competes with the multivalent antigen conjugate for the binding sites of the antibody to the antigen. After an appropriate incubation period, unbound reagents are removed by washing and the amount of radioactivity in the solid phase is determined. The amount of radioactive antibody bound is inversely proportional to the concentration of antigen in the sample.

Other techniques can be used to detect a biomarker's protein levels in a biological sample, can be performed according to the preferences of the practitioner, and based on the present disclosure and the type of biological sample (i.e., plasma, urine, sample of tissue etc.). One such technique is immunoblotting (Towbin et al., Proc. Nat. Acad. Sci. 76: 4350 (1979)), where a sample treated in a suitable manner is run on an SDS-PAGE gel before being transferred to a solid support, such as a nitrocellulose filter. Detectable labeled anti-enzyme antibodies can then be used to assess enzyme levels, where the intensity of The detectable brand signal corresponds to the amount of enzyme present. The levels can be quantified, for example by densitometry.

In one embodiment, the levels of biomarkers as disclosed herein, and / or their polypeptides can be detected in a tissue sample by mass spectrometry such as MALDI / TOF (flight time), SELDI / TOF, liquid chromatography. - mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography-mass spectrometry (HPLC-MS), capillary electrophoresis-mass spectrometry, nuclear magnetic resonance spectrometry , or tandem mass spectrometry (for example MS / MS, MS / MS / MS, ESI-MS / MS, etc.). See for example, US patent applications. Nos: 20030199001, 20030134304, 20030077616, which are incorporated herein by reference.

Mass spectrometry methods are well known in the art and have been used to quantify and / or identify biomolecules, such as proteins (see, for example, Li et al. (2000) Tibtech 18: 151-160; Rowley et al . (2000) Methods 20: 383-397; and Kuster and Mann (1998) Curr. Opin. Structural Biol. 8: 393-400). In addition, mass spectrometry techniques have been developed that allow at least partial de novo sequencing of isolated proteins. Chait et al., Science 262: 89-92 (1993); Keough et al, Proc. Nati Acad. Sci. USA. 96: 7131-6 (1999); reviewed in Bergman, EXS 88: 133-44 (2000).

In certain embodiments, a gas phase ion spectrometer is used. In other embodiments, laser desorption / ionization mass spectrometry is used to analyze the sample. Modern laser desorption / ionization ("LDI-MS") mass spectrometry can be carried out in two main variations: matrix-assisted laser desorption / ionization mass spectrometry ("MALDI") and surface-enhanced laser desorption / ionization ("SELDI"). In MALDI, the analyte is mixed with a solution containing a matrix, and a drop of liquid is placed on the surface of a substrate. The matrix solution then co-crystallizes with the biological molecules. The substrate is inserted into the mass spectrometer. The Laser energy is directed to the surface of the substrate where it desorbs and ionizes the biological molecules without significantly fragmenting them. See, for example, US Pat. No. 5118937, and U.S. Pat. No. 5045694.

In SELDI, the surface of the substrate is modified so that it is an active participant in the desorption process. In a variant, the surface is derived with an adsorbent and / or capture reagents that selectively bind to the protein of interest. In another variant, the surface is derived with molecules that absorb energy that are not desorbed when they are reached by the laser. In another variant, the surface is derived with molecules that bind to the protein of interest and that contain a photolytic bond that breaks after laser application. In each of these methods, the bypass agent is generally located at a specific location on the surface of the substrate where the sample is applied. See, for example, US Pat. No. 5719060 and WO 98/59361. The two methods can be combined by, for example, the use of a SELDI affinity surface to capture an analyte and adding a matrix containing liquid to the captured analyte to provide the energy absorbing material.

For additional information regarding mass spectrometers, see, e.g., Principles of Instrumental Analysis, 3rd edition, Skoog, Saunders College Publishing, Philadelphia, 1985.; and Kirk-Othmer Encyclopedia of Chemical Technology, 4 ed. VoI 15 (John Wiley AND Sons, New York 1995), pp. 1071-1094.

The detection of biomarker levels will typically depend on the detection of signal strength. This, in fact, may reflect the amount and character of a polypeptide bound to the substrate. For example, in certain embodiments, the strength of the peak value signal of the spectra of a first sample and a second sample (for example, visually, by computer analysis, etc.) can be compared, to determine the quantities relative of particular biomolecules. Software programs such as the Biomarker Wizard program (Ciphergen Biosystems, Inc., Fremont, Calif.) Can be used as an aid in the analysis of mass spectra. Mass spectrometers and their techniques are well known to those skilled in the art. Alternatively, the levels of a polypeptide biomarker can be determine obtaining a spectrometry spectrum of mass by laser desorption / ionization increased by surface flight time (SELDI-TOF MS) of the sample.

The person skilled in the art will appreciate that the detection, diagnosis and evaluation will be carried out by detecting a decrease in the expression of one or more of the biomarkers of the invention with respect to the reference value. A decrease in expression with the reference value is considered where the difference between the test sample and the reference sample of at least 0.9 times, 0.75 times, 0.2 times, 0.1 times, 0 , 05 times, 0.025 times, 0.02 times, 0.01 times, 0.005 times or even less.

If the method of the invention is carried out for the detection, diagnosis and evaluation of fibrosis and / or a disease associated with fibrosis, then the reference value is the level of expression of the biomarker in a reference sample that is obtained by combining equal amounts of samples from a population of subjects. In general, typical reference samples will be obtained from subjects that are clinically well documented and free of the disease. In such samples, normal (reference) concentrations of the biomarker can be determined, for example by providing the average concentration over the reference population. When determining the reference concentration of the marker, several considerations are taken into account. Among such considerations are the type of sample involved (eg tissue or CSF), age, weight, sex, general physical condition of the patient and the like. For example, equal amounts of a group of at least 2, at least 10, at least 100 to preferably more than 1000 subjects are taken as reference group, preferably classified according to the above considerations, for example of various age categories.

If the method of the invention is carried out to determine the efficacy of a therapy for the treatment of fibrosis or a disease associated with fibrosis, then the reference value used to determine whether a therapy is effective is usually the level of expression of the biomarker or biomarkers under consideration with a patient sample before the start of therapy. According to this method of the invention, the variation with respect to the reference value is an increase in the expression above of the reference value. The level of expression is considered to be increased where the difference between the test sample and the reference sample is at least 1.1 times,

1.5 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times,

80 times, 90 times, 100 times or even more.

It will be understood that the method of the invention can be carried out by determining the level of a variable number of biomarkers. For example, the level (s) of a biomarker, two or more biomarkers, three or more biomarkers or four or more biomarkers as defined in the present invention. The determination of levels of combinations of biomarkers may allow greater sensitivity and specificity in the diagnosis of fibrosis and / or a disease associated with fibrosis or may allow better differentiation of fibrosis from other diseases that may have similar or overlapping biomarkers. Thus, the present invention comprises the simultaneous determination of the following groups of biomarkers:

SLURPl and the SLURPl fragment,

SLURPI and HAMP,

SLURPl and Hepc25,

SLURPl and Hepc20, SLURPI and GSN;

SLURPI and APOD;

SLURPI and SPPl,

SLURPI and DEFBl,

SLURPl and MASP2; SLURPl and HAMP fragment, SLURPl and Hepc25 fragment, SLURPl and Hepc20 fragment, SLURPl and GSN fragment, SLURPl and APOD fragment, SLURP 1 and SPP 1 fragment, SLURPl and DEFBl fragment, SLURPl and MASP2 fragment; HAMP and Hepc25,

HAMP and Hepc20,

HAMP and GSN,

HAMP and APOD, HAMPySPPl,

HAMPyDEFBl,

HAMP and MASP2;

Hepc25 and Hepc20,

Hepc25 and GSN, Hepc25yAPOD,

Hepc25ySPPl,

Hepc25yDEFBl,

Hepc25 and MASP2;

Hepc20 and GSN, Hepc20yAPOD,

Hepc20ySPPl,

Hepc20yDEFBl,

Hepc20 and MASP2

GSN and APOD; GSNySPPl,

GSNyDEFBl,

GSN and MASP2,

APODySPPl,

APODyDEFBl, APOD and MASP2,

SLURPl, SLURPl and HAMP fragment;

SLURPl, SLURPl and Hepc25 fragment;

SLURPl, SLURPl and Hepc20 fragment;

SLURPl, SLURPl and GSN fragment; SLURP 1, SLURP 1 fragment and APOD;

SLURPl, SLURPl and SPPl fragment;

SLURPl, SLURPl and DEFBl fragment; SLURPl, SLURPl and MASP2 fragment;

SLURPl, HAMP and Hepc25;

SLURPl, HAMP and Hepc20;

SLURPl ₅ HAMP and GSN; SLURP 1, HAMP and APOD;

SLURPl ₅ HAMP and SPPl

SLURPl, HAMP and DEFBl

SLURPl, HAMP and MASP2

SLURPl, Hepc25 and Hepc20; SLURP 1, Hepc25 and GSN;

SLURPl, Hepc25 and APOD;

SLURPl, Hepc25 and SPPl

SLURPl, Hepc25 and DEFBl

SLURPl, Hepc25 and MASP2 SLURP 1, Hepc20 and GSN;

SLURPl, Hepc20 and APOD;

SLURPl, Hepc20 and SPPl

SLURPl, Hepc20 and DEFBl

SLURPl, Hepc20 and MASP2 SLURP 1, GSN and APOD;

SLURPI ₅ GSN and SPPl;

SLURPl ₅ GSN and DEFBl;

SLURPl ₅ GSN and MASP2; SLURPl ₅ HAMP and Hepc25 fragment; SLURP 1, HAMP and Hepc20 fragment; SLURPl ₅ HAMP and GSN fragment; SLURPl ₅ HAMP and APOD fragment; SLURPl ₅ HAMP and SPPl fragment; SLURPl ₅ HAMP and DEFBl fragment; SLURP 1, HAMP and MASP2 fragment; SLURPl ₅ Hepc25 and Hepc20 fragment; SLURPl ₅ Hepc25 and GSN fragment; SLURPl, Hepc25 and APOD fragment; SLURPl fragment, Hepc25 and SPPl SLURPl fragment, Hepc25 and DEFBl SLURPl fragment, Hepc25 and MASP2 SLURP fragment 1, Hepc20 and GSN; SLURPl, Hepc20 and APOD fragment; SLURPl fragment, Hepc20 and SPPl SLURPl fragment, Hepc20 and DEFBl SLURPl fragment, Hepc20 and MASP2 SLURP fragment 1, GSN and APOD; SLURPl, GSN and SPPl fragment; SLURPl, GSN and DEFBl fragment; SLURPl, GSN and MASP2 fragment;

HAMP, Hepc25 and Hepc20; HAMP, Hepc25 and GSN;

HAMP, Hepc25 and APOD;

HAMP, Hepc25 and SPPl;

HAMP, Hepc25 and DEFBl;

HAMP, Hepc25 and MASP2; HAMP, Hepc20 and GSN;

HAMP, Hepc20 and APOD;

HAMP, Hepc20 and SPPl;

HAMP, Hepc20 and DEFBl;

HAMP, Hepc20 and MASP2; HAMP, GSN and APOD;

HAMP ₅ GSN and SPPl;

HAMP ₅ GSN and DEFBl;

HAMP, GSN and MASP2;

Hepc25, Hepc20 and GSN; Hepc25, Hepc20 and APOD;

Hepc25, Hepc20 and SPPl;

Hepc25, Hepc20 and DEFBl; Hepc25, Hepc20 and MASP2;

Hepc25, GSN and APOD;

Hepc25, GSN and SPPl;

Hepc25, GSN and DEFBl; Hepc25, GSN and MASP2;

SLURPl, SLURPl fragment, HAMP and Hepc25;

SLURPl, SLURPl fragment, HAMP _and Hepc20;

SLURPl, SLURPl fragment, HAMP and GSN;

SLURPl, SLURPl, HAMP and APOD fragment; SLURP 1, SLURP 1 fragment, HAMP and SPP 1;

SLURPl, SLURPl, HAMP and DEFBl fragment;

SLURPl, SLURPl fragment, HAMP and MASP2;

SLURPl, SLURPl fragment, Hepc25 and Hepc20;

SLURPl, SLURPl fragment, Hepc25 and GSN; SLURP 1, fragment SLURP 1, Hepc25 and APOD;

SLURPl, SLURPl, Hepc25 and SPPl fragment;

SLURPl, SLURPl, Hepc25 and DEFBl fragment;

SLURPl, SLURPl, Hepc25 and MASP2 fragment;

SLURPl, SLURPl fragment, Hepc20 and GSN; SLURP 1, SLURP 1 fragment, Hepc20 and APOD;

SLURPl, SLURPl, Hepc20 and SPPl fragment;

SLURPl, SLURPl, Hepc20 and DEFBl fragment;

SLURPl, SLURPl, Hepc20 and MASP2 fragment;

SLURPl, HAMP, Hepc25 and Hepc20; SLURP 1, HAMP, Hepc25 and GSN;

SLURPl, HAMP, Hepc25 and APOD;

SLURPl, HAMP, Hepc25 and SPPl;

SLURPl, HAMP, Hepc25 and DEFBl;

SLURPl, HAMP, Hepc25 and MASP2; SLURPl, HAMP, Hepc20 and GSN;

SLURPl, HAMP, Hepc20 and APOD;

SLURPl, HAMP, Hepc20 and SPPl; SLURPl, HAMP, Hepc20 and DEFBl;

SLURPl, HAMP, Hepc20 and MASP2;

SLURPl, HAMP, GSN and APOD;

SLURPl, HAMP, GSN and SPPl; SLURP 1, HAMP, GSN and DEFB 1;

SLURPl, HAMP, GSN and MASP2; SLURP1, HAMP, Hepc25 and Hepc20 fragment; SLURPl, HAMP, Hepc25 and GSN fragment; SLURPl, HAMP, Hepc25 and APOD fragment; SLURP 1, HAMP, Hepc25 and SPP 1 fragment; SLURPl, HAMP, Hepc25 and DEFBl fragment; SLURPl, HAMP, Hepc25 and MASP2 fragment; SLURPl, HAMP, Hepc20 and GSN fragment; SLURPl, HAMP, Hepc20 and APOD fragment; SLURP 1, HAMP, Hepc20 and SPP 1 fragment; SLURPl, HAMP, Hepc20 and DEFBl fragment; SLURPl, HAMP, Hepc20 and MASP2 fragment; SLURPl, HAMP, GSN and APOD fragment; SLURPl, HAMP, GSN and SPPl fragment; SLURP 1, HAMP, GSN and DEFB 1 fragment; SLURPl, HAMP, GSN and MASP2 fragment; SLURP1, Hepc25, Hepc20 and GSN fragment; SLURPl, Hepc25, Hepc20 and APOD fragment; SLURPl, Hepc25, Hepc20 and SPPl fragment; SLURP 1, Hepc25, Hepc20 and DEFB 1 fragment; SLURPl, Hepc25, Hepc20 and MASP2 fragment; SLURPl, Hepc20, GSN and APOD fragment; SLURPl, Hepc20, GSN and SPPl fragment; SLURPl, Hepc20, GSN and DEFBl fragment; SLURP 1, Hepc20, GSN and MASP2 fragment;

HAMP, Hepc25, Hepc20 and GSN;

HAMP, Hepc25, Hepc20 and APOD; HAMP, Hepc25, Hepc20 and SPPl;

HAMP, Hepc25, Hepc20 and DEFBl;

HAMP, Hepc25, Hepc20 and MASP2;

Hepc25, Hepc20, GSN and APOD Hepc25, Hepc20, GSN and SPPl

Hepc25, Hepc20, GSN and DEFBl

Hepc25, Hepc20, GSN and MASP2

SLURPl, SLURPl fragment, HAMP, Hepc25 and Hepc20;

SLURPl, SLURPl fragment, HAMP, Hepc25 and GSN; SLURP 1, SLURP 1 fragment, HAMP, Hepc25 and APOD;

SLURPl, SLURPl fragment, HAMP, Hepc25 and SPPl;

SLURPl, SLURPl, HAMP, Hepc25 and DEFBl fragment;

SLURPl, SLURPl fragment, HAMP, Hepc25 and MASP2;

SLURPl, SLURPl, HAMP, Hepc20 and GSN fragment; SLURP 1, SLURP 1 fragment, HAMP, Hepc20 and APOD;

SLURPl, SLURPl, HAMP, Hepc20 and SPPl fragment;

SLURPl, SLURPl, HAMP, Hepc20 and DEFBl fragment;

SLURPl, SLURPl fragment, HAMP, Hepc20 and MASP2;

SLURPl, SLURPl, HAMP, GSN and APOD fragment; SLURP 1, SLURP 1 fragment, HAMP, GSN and SPP 1;

SLURPl, SLURPl, HAMP, GSN and DEFBl fragment;

SLURPl, SLURPl, HAMP, GSN and MASP2 fragment;

SLURPl, SLURPl, Hepc25, Hepc20 and GSN fragment;

SLURPl, SLURPl fragment, Hepc25, Hepc20 and APOD; SLURP 1, SLURP 1 fragment, Hepc20, GSN and APOD; SLURPl, HAMP, Hepc25, Hepc20 and GSN fragment, SLURPl, HAMP, Hepc25, Hepc20 and APOD fragment; SLURPl, HAMP, Hepc25, Hepc20 and SPPl fragment; SLURPl, HAMP, Hepc25, Hepc20 and DEFBl fragment; SLURP1, HAMP, Hepc25, Hepc20 and MASP2 fragment; SLURPl, Hepc25, Hepc20, GSN and APOD fragment; SLURPl, Hepc25, Hepc20, GSN and SPPl fragment; SLURPl, Hepc25, Hepc20, GSN and DEFBl fragment; SLURPl, Hepc25, Hepc20, GSN and MASP2 fragment

HAMP, Hepc25, Hepc20, GSN and APOD;

HAMP, Hepc25, Hepc20, GSN and SPPl; HAMP, Hepc25, Hepc20, GSN and DEFBl;

HAMP, Hepc25, Hepc20, GSN and MASP2;

HAMP, Hepc20, GSN and APOD;

Hepc25, Hepc20, GSN and APOD;

Hepc25, Hepc20, GSN and SPPl; Hepc25, Hepc20, GSN and DEFBl;

Hepc25, Hepc20, GSN and MASP2;

SLURPl, SLURPl fragment, HAMP, Hepc25, Hepc20 and GSN;

SLURPl, SLURPl, HAMP, Hepc25, Hepc20 and APOD fragment;

SLURPl, SLURPl, HAMP, Hepc25, Hepc20 and SPPl fragment; SLURP 1, fragment SLURP 1, HAMP, Hepc25, Hepc20 and DEFB 1;

SLURPl, SLURPl, HAMP, Hepc25, Hepc20 and MASP2 fragment;

SLURPl, HAMP, Hepc25, Hepc20, GSN and APOD;

SLURPl, HAMP, Hepc25, Hepc20, GSN and SPPl;

SLURPl, HAMP, Hepc25, Hepc20, GSN and DEFBl; SLURPl, HAMP, Hepc25, Hepc20, GSN and MASP2;

SLURPl, SLURPl, Hepc25, Hepc20, GSN and APOD fragment;

SLURPl, SLURPl fragment, Hepc25, Hepc20, GSN and SPPl;

SLURPl, SLURPl, Hepc25, Hepc20, GSN and DEFBl fragment;

SLURPl, SLURPl fragment, Hepc25, Hepc20, GSN and MASP2; SLURP 1, SLURP 1 fragment, HAMP, Hepc20, GSN and APOD;

SLURPl, SLURPl, HAMP, Hepc20, GSN and SPPl fragment;

SLURPl, SLURPl fragment, HAMP, Hepc20, GSN and DEFBl;

SLURPl, SLURPl fragment, HAMP, Hepc20, GSN and MASP2;

In addition, the person skilled in the art will appreciate that the methods of the invention can be combined with other methods known in the art for the detection of fibrosis or a disease associated with fibrosis and for the determination of the efficacy of a therapy. for fibrosis or a disease associated with fibrosis. In particular, the biomarkers of the invention can be measured simultaneously with the series of 5 biomarkers defined in WO0216949 (α2-macroglobulin, haptoglobulin, apolipoprotein Al, γ-glutamyl transpeptidase, and bilirubin), with the series of three biomarkers described in WO0373822 ( α-MG, HA and TIMP-I), with the different series of biomarkers described in WO2005116901 (comprising α-2 macroglobulin, hyaluronic acid, apolipoprotein Al, N-terminal propeptide of type III collagen, γ-glutamyltranspeptidase, bilirubin, γ-globulins, platelets, prothrombin time, aspartate amino transferase, alanine aminotransferase, urea, sodium, glycemia, triglycerides, albumin, alkaline phosphatases, YKL-40 (human cartilage glycoprotein 39), Tissue matrix metalloproteinase inhibitor 1 ( TIMP-I), matrix 2 metalloproteinase (MMP-2) and ferritin); with one or more of the markers described in WO2007003670 (which includes uromodulin, MAC2BP, AGPl and cathepsin A), with one or more of the serum biomarkers described in WO2008031051.

In addition, the method of the invention can be used together with other methods to detect fibrosis not based on the determination of expression levels of one or more biomarkers, such as liver biopsy and elastography as described in Sandrin L et al. (Ultrasound Med. Biol. 2003; 29: 1705-1711).

The invention also provides kits comprising reagents for detecting at least two or more biomarkers selected from the group of SLURPl, HAMP, GSN ,, APOD, SPPl, DEFBl, MASP2. The different combinations of biomarkers that can be detected using the kit of the invention have been defined above. In a preferred embodiment, the biomarkers are polypeptides and thus, the reagents that form the kit can be any compound capable of binding with high affinity to the above polypeptide biomarkers. The kit reagents can be, without limitation, any type of antibody or immunoglobulin molecule or fragment thereof such as polyclonal, monoclonal, human or humanized or recombinant antibodies as well as single chain antibodies, for example scFv constructs, or synthetic antibodies as such and that they can belong to any of the following classes of immunoglobulins: IgG, IgM, IgE, IgA, and where where applicable, a subclass of any of the aforementioned classes, for example subclasses of the IgG class such as IgGl, IgG2, IgG2a, IgG2b, IgG3 or IgGM. In addition, the kit may also comprise antibody fragments such as Fv, Fab or F (ab ') ₂ fragments or single chain fragments such as scFv. Double chain fragments such as Fv, Fab or F (ab ') 2 are preferred. Fab and F (ab ') 2 fragments have no Fc fragment contained in intact antibodies. As a beneficial consequence, such fragments are transported faster in the circulatory system, and show less non-specific binding to tissue compared to complete antibody species. Such fragments can be produced from intact antibodies by proteolytic digestion using proteases such as papain (for the production of Fab fragments) or pepsin (for the production of F (ab ') ₂ fragments), or chemical oxidation.

The antibodies can be bound to a solid support forming antibody matrices or protein chips. Protein matrices are solid phase ligand binding assay systems that use immobilized proteins on surfaces that include glass, membranes, microtiter wells, mass spectrometer plates, and balls or other particles. The matrices are highly parallel (multiplexed) and with miniaturized frequencies (microarrays, protein chips). Its advantages include that they are fast and automatable, capable of great sensitivity, economical in reagents, and give plenty of data for a single experiment. Bioinformatic support is important; Data management demands sophisticated software and data comparison analysis. However, the software adapts to that used for DNA matrices, such as much of the hardware and detection systems.

One of the main formats is the capture matrix, in which ligand binding reagents are used, which are normally antibodies but which can also alternatively be protein skeletons, peptides or nucleic acid aptamers, to detect target molecules in mixtures such as plasma or tissue extracts. In diagnosis, capture matrices are used to perform multiple immunoassays in parallel, testing both for several analytes in individual sera for example and testing many serum samples simultaneously. In proteomics, the Capture matrices are used to quantify and compare protein levels in different samples in health and disease, that is, protein expression profile. Different proteins are used than those that bind specific ligands in the matrix format for in vitro functional screening such as protein-protein, protein-DNA, protein-drug, receptor-ligand, enzyme-substrate, etc. The capture reagents themselves are selected and screened against many proteins, optionally in multiplex matrix format against many target proteins.

For matrix construction, protein sources include cell-based expression systems for recombinant proteins, purification of natural sources, in vitro production by cell-free translation systems, and synthetic methods for peptides. Many of these methods are automated for high performance production. For capture matrices and protein function analysis, it is important that the proteins are correctly folded and functional; This is not always the case, for example, where recombinant bacteria proteins are extracted under denaturing conditions. However, denatured protein matrices are useful in the screening of antibodies for cross-reactivity, identification of autoantibodies and selection of ligand-binding proteins.

Protein matrices have been designed as a miniaturization of familiar immunoassay methods such as ELISA and drop transfer, often using fluorescent readers, and facilitated by robotics and high performance detection systems to allow multiple tests to be carried out in parallel. . Physical supports include glass slides, silicone, microwells, nitrocellulose or PVDF membranes, and magnetic microbeads and others. While the most familiar format is protein droplets that leave on planar surfaces, alternative architectures include microfluidic-based CD centrifugation devices (Gyros, Monmouth Junction, NJ) and specialized chip designs, such as built-in microchannels on a plate (for example, The Living Chip ™, Biotrove, Woburn, MA) and tiny 3D posts on a silicone surface (Zyomyx, Hayward CA). Suspended particles are also used as a base for matrices, provided they are coded for identification; The systems include color codes for microballs (Luminex, Austin, TX; Bio-Rad Laboratories), semiconductor nanocrystals (e.g., QDOTS ™, Quantum Dot, Hayward, CA), ball barcode (ULTRAPLEX ™ balls, SmartBead Technologies Ltd, Babraham, Cambridge, RU) and multimetallic microbeads (for example, NANOBARCODES ™ particles, Nanoplex Technologies, Mountain View, CA). The balls are optionally assembled in planar matrices on semiconductor chips (LEAPS ™ technology, BioArray Solutions, Warren, NJ).

Protein immobilization involves both the coupling reagent and the nature of the surface to which it is attached. A good protein matrix support surface is chemically stable before and after coupling procedures, allows good spot morphology, shows minimal non-specific binding, does not contribute to the background in detection systems, and is compatible with different detection systems . The immobilization method used is reproducible, applicable to proteins of different properties (size, hydrophilic, hydrophobic), manageable at high performance and automation, and compatible with the retention of fully functional protein activity. The orientation of the surface bound protein is recognized as an important factor when presenting it to the ligand or substrate in an active state; for capture matrices the most effective binding results are obtained with oriented capture reagents, which generally require site specific protein marking. Both covalent and non-covalent methods of protein immobilization are used and have several pros and cons. Passive adsorption to surfaces is methodologically simple, but allows little quantitative or orientation control. It may or may not alter the functional properties of the protein, and the reproducibility and efficacy are variable. Covalent coupling methods provide stable binding, apply to a range of proteins and have good reproducibility. However, the orientation is variable. In addition, chemical shunt can alter protein function and requires a stable interactive surface. Biological capture methods that use a tag on the protein provide stable binding and bind to the protein specifically and in reproducible orientation, but the biological reagents must first be properly immobilized, and the matrix can require special handling and have variable stability. Several immobilization chemicals and labels for the manufacture of protein matrices have been described. Substrates for covalent bonding include glass slides coated with silane reagents containing amino or aldehyde. In the VERSALINX ™ system (Prolinx, Bothell, WA), reversible covalent coupling is achieved by interaction between the protein derived with phenyldiboronic acid and salicylic hydroxamic acid immobilized on the support surface. This also has low background binding and low intrinsic fluorescence and allows immobilized proteins to retain function. Non-covalent binding of unmodified proteins occurs with porous structures such as HYDROGEL ™ (PerkinElmer, Wellesley, MA), based on a three-dimensional polyacrylamide gel; It has been described that this substrate gives a particularly low background in glass microarrays, and high capacity and retention of protein function. The widely used biological coupling methods are through biotin / streptavidin or hexahistidine / Ni interactions, having modified the protein appropriately. Biotin can be conjugated to a polylysine skeleton immobilized on a surface such as titanium dioxide (Zyomyx, Inc., Hayward, CA) or tantalum pentoxide (Zeptosens, Witterswil, Switzerland). Matrix manufacturing methods include robotic contact printing, inkjet, piezoelectric tapping and photolithography. A number of commercial matrix manufacturers are available [for example, Packard Biosciences, Affymetrix Inc. and Genetix] as well as manual equipment [for example, V and P Scientific]. Bacteria colonies are optionally placed in robotic grids on PVDF membranes for induction of protein expression in situ. In the limit of the spot size and density are the nanomatrices, with spots on the nanometer space scale, which allow thousands of reactions to be carried out on a single chip of less than 1 mm square. BioForce Nanosciences Inc. and Nanolink Inc., for example, have developed commercially available nanomatrices.

The methods of marking and fluorescence detection are widely used. The same instruments that are used for reading DNA microarrays are applicable for protein matrices. For differential presentation, capture matrices (for example, antibodies) are tested with fluorescently labeled proteins from two different cell states, in which the used cell phones are they conjugate directly with different fluorophores (for example, Cy-3, Cy-5) and mix, so that the color acts as a reader for changes in the abundance of the target. The fluorescent reading sensitivity is amplified 10-100 times by tyramide signal amplification (TSA) (PerkinElmer Lifesciences). Planar guided wave technology (Zeptosens) allows ultrasensitive fluorescent detection, with the additional advantage of not having intermediate washing procedures. High sensitivity is achieved with the suspension of balls and particles, using phycoerythrin as a brand (Luminex) or the properties of semiconductor nanocrystals (Quantum Dot). A number of alternative readers have been developed, especially in the area of commercial biotechnology. These include nuclear plasmon resonance adaptations (HTS Biosystems, Intrinsic Bioprobes, Tempe, AZ), rolling circle DNA amplification (Molecular Staging, New Haven, CT), mass spectrometry (Intrinsic Bioprobes; Ciphergen, Fremont, CA), resonance light scattering (Genicon Sciences, San Diego, CA) and atomic force microscopy [BioForce Laboratories].

Capture matrices form the basis of diagnostic chips for the expression profile. They employ high affinity capture reagents, such as conventional antibodies, individual domains, constructed skeletons, peptides or nucleic acid aptamers, to bind and detect specific target ligands in a high yield manner.

Antibody matrices have the required specificity and acceptable background properties, and some are commercially available (BD Biosciences, San Jose, CA; Clontech, Mountain View, CA; BioRad; Sigma, St. Louis, MO). Antibodies to capture matrices are produced either by conventional immunization (polyclonal sera and hybridomas), or as recombinant fragments, normally expressed in E. coli, after selection of phage or ribosome display libraries (Cambridge Antibody Technology, Cambridge , RU; Biolnvent, Lund, Sweden; Affitech, Walnut Creek, CA; Biosite, San Diego, CA). In addition to conventional antibodies, Fab and scFv fragments, individual V domains of camelids or constructed human equivalents (Domantis, Waltham, MA) are optionally useful in matrices. The term "skeleton" refers to ligand-binding protein domains, which are constructed in multiple variants capable of binding to various target molecules with specificity and affinity properties similar to antibody. Variants are produced in a genetic library format and selected against individual targets by phage, bacteria or ribosome presentation. Such skeletons or ligand-binding frameworks include Affybodies based on S. aureus protein A (Affibody, Bromma, Sweden), Trronectins based on fibronectins (Phylos, Lexington, MA) and Anticalins based on lipocalin structure (Pieris Proteolab , Freising- Weihenstephan, Germany). These are used in capture matrices in a manner similar to antibodies and have the advantages of consistency and ease of production.

Non-protein capture molecules, notably single chain nucleic acid aptamers that bind protein ligands with high specificity and affinity, are also used in matrices (SomaLogic, Boulder, CO). The aptamers are selected from oligonucleotide libraries by the Selex ™ method (SomaLogic, Boulder, CO) and their interaction with proteins is increased by covalent binding, by the incorporation of brominated deoxyuridine and UV-activated cross-linking (photoaptamers). Photo-cross-linking to ligands reduces cross-reactivity of aptamers due to specific steric requirements. Aptamers have the advantages of ease of production by automated oligonucleotide synthesis and DNA stability and consistency; in the photoaptamer matrices, universal fluorescent protein dyes are used to detect binding.

Protein analytes that bind to antibody matrices are detected directly or indirectly, for example, through a secondary antibody. Direct marking is used to compare different samples with different colors. Where pairs of antibodies directed to the same protein ligand are available, sandwich immunoassays provide high specificity and sensitivity and are therefore the method of choice for low abundance proteins such as cytokines; They also give the possibility of detection of protein modifications. The methods of Unmarked detection, including mass spectrometry, surface plasmon resonance and atomic force microscopy, prevents ligand alteration. What is required of any method is optimal sensitivity and specificity, with a low background to give a signal at high noise. Since analyte concentrations cover a wide range, sensitivity has to be adapted appropriately. Serial dilution of the sample or the use of antibodies of different affinities are solutions to this problem. The proteins of interest are often those at low concentrations in body fluids and extracts, which require detection in the range of pg or less, such as cytokines or low-expression products in cells. An alternative to a matrix of capture molecules is one made by means of molecular fingerprint technology, in which peptides (for example, from C-terminal regions of proteins) are used as templates to generate structurally complementary, specific cavities sequence in a polymerizable matrix; the cavities can then specifically capture (denatured) proteins that have the appropriate primary amino acid sequence (ProteinPrint ™, Aspira Biosystems, Burlingame, CA).

Another methodology that is useful diagnostically and in the expression profile is the ProteinChip® matrix (Ciphergen, Fremont, CA), in which solid-phase chromatographic surfaces bind to proteins with similar loading or hydrophobicity characteristics of mixtures such as plasma or tumor extracts, and SELDI-TOF mass spectrometry is used for the detection of retained proteins.

Large-scale functional chips have been constructed by immobilizing large numbers of purified proteins and are used to test a wide range of biological functions, such as protein interactions with other proteins, drug-target, enzyme-substrate interactions, etc. They generally require an expression library, cloned in E. coli, yeast or the like from which the expressed proteins are purified, for example, through a His tag and immobilized. Transcription / translation of cell-free proteins is a viable alternative for the synthesis of proteins that do not express well in bacterial systems or other systems in vivo. To detect protein-protein interactions, protein matrices are alternatives in vitro to the system of the two cell-based yeast hybrids and are useful where the latter is deficient, such as interactions involving secreted proteins or proteins with disulfuric bonds. The high-performance analysis of biochemical activities in matrices for yeast protein kinases and for various functions (protein-protein and protein-lipid interactions) of yeast proteome has been described, where a large proportion of all open reading frames was expressed of yeast and immobilized in a microarray. Large-scale proteome chips are also useful in identifying functional interactions, drug screening, etc. (Proteometrix, Branford, CT).

As a two-dimensional presentation of individual elements, a protein matrix is used to screen phage display libraries or ribosomes, to select binding partners, including antibodies, synthetic skeletons, peptides and aptamers. In this way, a library vs. library screening is carried out. The screening of candidate drugs in combinatorial chemical libraries against a matrix of target proteins identified from genomic projects is another application of this approach.

Multiplexed ball assays use a series of spectrally discrete particles that are used to capture and quantify soluble analytes. The analyte is then measured by detection of a fluorescence based emission and flow cytometric analysis. Multiplexed ball assays generate data that is comparable to ELISA based assays, but in a multiplexed or simultaneous manner. The concentration of strangers for the cytometric ball matrix is calculated as with any sandwich test, that is, through the use of known standards and representing the unknown ones against the standard curve. In addition, multiplexed ball assays allow quantification of soluble analytes in samples never before considered due to sample volume limitations. In addition to quantitative data, powerful visual images are generated that reveal unique profiles or signatures that provide the user with additional information at a glance. The kit of the invention may additionally contain instructions for use in determining the protein levels of the biomarkers present in the sample. These instructions can be found in the form of printed material or in the form of electronic support that can store instructions so that they can be read by a subject, such as electronic storage media (optical discs, tapes and the like), optical media (CD- ROM, DVD) and the like. The media may additionally or alternatively contain Internet pages that provide such instructions.

The invention is described here in more detail as the following examples that should be considered as merely illustrative and not limiting the scope of the invention.

EXAMPLES

MATERIALS AND METHODS

SAMPLES

A total of 80 urine samples were obtained from the University Clinic of Navarra. Sample collection was approved by the local Ethics Committee for Clinical Trials of the University Clinic of Navarra, and all patients gave informed consent to participate. Urine samples were from 35 patients with F4 cirrhosis (Metavir index), 10 patients with Fl fibrosis (Metavir index), and 35 healthy controls. The criteria used to classify liver damage was liver biopsy for patients with cirrhosis and fibrosis, and standard blood tests for control individuals. 81.5% of the patients were men and 18.5% women and all were between 38 and 75 years old.

Say

Urine samples were centrifuged at 4000xg for 5 minutes at 4 ⁰ C, and 30 ml supernatants were concentrated using centrifugal filter devices

Amicon Ultra-15 (5000 NMWL) from Millipore. The concentrated proteins were precipitated with the ReadyPrep 2-D Cleanup (Bio-Rad) kit and the resulting precipitates were resuspended in 7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris / HCl pH 8. Protein concentration was determined using the Bradford assay kit (BioRad), using bovine serum albumin as a standard protein. Protein samples (50 μg) were labeled with 400 pmoles of Cy3 and Cy5 fluorescent dyes in N, N-dimethylformamide. A reference sample was prepared by mixing 25 μg of protein from each experimental condition and the resulting set was subsequently labeled with Cy2. Triplicate analyzes were performed alternating fluorescent tide of study samples. The first dimension was performed on an Ettan IPGphor (GE Healthcare) using Immobiline DryStrips pH3-l l NL, 24 cm (GE Healthcare). The samples were loaded at the acid end of the strip with a cup loading device. The isoelectric focusing conditions were those recommended by the manufacturer for the type of strip used. The SDS-PAGE (12.5%) was developed in an Ettan DALTsix (GE Healthcare) at 25 ⁰ C and lW / gel for 12 hours. The images of the gels were captured with a Typhoon Trio (GE Healthcare) at a resolution of 100 μm with λex / λem of 488/520, 532/580, and 633/670 nm for Cy2, Cy3, and Cy5 respectively. Image analysis and statistics were performed with DeCyder Differential version 6.5 software (GE Healthcare) using the Biological Variation Analysis (BVA) module. Only differences with p <0.05 (t test) were accepted.

IDENTIFICATION OF MARKERS BY SAY

The preparative gels were run with 400 μg of protein following the same procedure indicated above. The proteins were visualized by staining with SYPRO Ruby protein gel staining (Bio-Rad) and the images were acquired with a Typhoon Trio using λex / λem 532/560 nm. Differentially represented spots were cut manually and gel samples were processed with a MassPrep (Waters) station. Triptych digestion in the gel was performed with 12.5 ng / µl trypsin in 50 mM ammonium bicarbonate for 12 hours at 37 ^° C. Microcapillary reverse phase CL was performed with a CapLC ™ (Waters) capillary system. Reverse phase separation of the tryptic digested was performed with a fused silica capillary column Atlantis, C 18, 3 μm, 75 μm x 10 cm Nano Ease ™ (Waters) balanced in 5% acetonitrile, 0.2% formic acid . After injection of 6 μl of sample, the column was washed for 5 minutes with the same buffer and the peptides were eluted using a linear gradient of 5-50% acetonitrile in 45 minutes at a constant flow rate of 0.2 μl / min. The column was coupled in line to a Q-TOF Micro (Waters) using a PicoTip nanospray ionization source (Waters). The temperature of the heated capillary was 8O ⁰ C and the potential difference of the spray was 1.8-2.2 kV. MS / MS data was collected in an automated mode dependent on the data. The three most intense ions in each study were sequentially fractionated by collision-induced dissociation (CID) using an isolation width of 2.0 and a relative collision energy of 35%. Data processing was performed with MassLynx 4.0. The database search was done with Phenyx 2.2 (GeneBio, Geneva, Switzerland) against the Uniprot Relay 12.3 knowledge base consisting of UniprotKB / Swiss-Prot Relay 54.3 and UniprotKB / TrEMBL Relay 37.3 with entries 285.335 and 4.932. 421, respectively. The search was enzymatically restricted for trypsin and a failed cut site was allowed. Additional search parameters were as follows: no restriction on molecular weight and isoelectric point; fixed modification, cistern carbamidomethylation; variable modification, methionine oxidation.

SELDI TOF

In the SELDI method, protein solutions are applied to ProteinChip matrix stains, which have been derived with planar chromatographic chemicals. The proteins actively interact with the surface of the chromatographic matrix, and are sequestered according to their potential for surface interaction as well as separated from salts and other contaminants in the sample by subsequent washing in the stain with the appropriate buffer solutions. As in the MALDI technique, the proteins to be analyzed are co-crystallized with UV absorbing components and volatilized by a pulsed UV laser beam. The ionized proteins are then accelerated in an electric field, and the mass to charge ratios of the different ionic species of the proteins can be deduced from their velocity.

Samples were processed and analyzed in duplicate. Urine samples were briefly centrifuged at 10,000 g for 5 minutes at 4 ⁰ C to remove debris and diluted 2: 3 in 9 M urea, 2% CHAPS. All denatured urine samples they were analyzed without further fractionation by dilution 1: 10 with the corresponding binding buffers before applying them to the chip surface. Proteins were analyzed using CMlO (cation exchange), IMAC30 (metal affinity), QlO (anion exchange) and H5 (hydrophobic interaction) chips following the manufacturer's instructions. The following binding buffers were used for each specific type of chip: shallow CMlO buffer (0.1 M sodium acetate, pH 4.0); H50 buffer (the surface was initially loaded with 50% ACN and binding was performed in 10% ACN, 0.1% TFA); QlO buffer (100 mM Tris HCl pH 9); IMAC30 buffer (the surface was initially loaded with 0.1 M cupric sulfate, neutralized with 0.1 M sodium acetate pH 4.0, and the binding was performed in 0.1 M sodium phosphate, 0 sodium chloride , 5 M pH 7.0). The SELDI-TOF profile was performed as follows: the chips were equilibrated twice with 150 μl of the appropriate binding buffer for 5 minutes and loaded twice (30 minutes each load) with 25 μl of sample mixed with 175 μl of buffer of Union. Unbound proteins were removed by washing the chips three times with 150 µl of binding buffer and three times with 200 µl of deionized water. After drying the charged surface, 1 µl of synapine acid (Bio-Rad, Hercules, CA) dissolved in 50% ACN and 0.5% TFA was applied twice and allowed to dry. The analysis of bound proteins / peptides was performed using ProteinChip System, Series 4000 (Bio-Rad, Hercules, CA). The spectra were collected in positive ion mode between the mass range of 1-100 kDa with a focus mass of 48 kDa and 8 kDa. The obtained spectra were processed, normalized and compared with the Ciphergen Express, a data group analysis program, using the default parameters. The statistical analysis chosen was the univariate non-parametric U-Mann-Whitney test. In addition, linear models were used using the LIMMA Bioconductor package that allowed the selection of peptides using a correction of the p-value based on FDR or the B statistic (logarithm of the quotient probability that a peptide is differentially expressed / probability of not being differential). The discriminant capacity of the selected peptides was evaluated with the ROC curve (sensitivity versus specificity) and particularly with the AUC value (area under the curve). Once the most reliable individual markers were established, a multivariate analysis was performed to evaluate the combination that provides the most effective classification of the samples. IMMUNOTRANSFERENCE

Urine samples were mixed with 5x loading buffer (250 mM Tris-HCl pH 6.8, 10% SDS, 50% glycerol, 5% mercaptoethanol, 62.5 mM EDTA, 0 bromine phenol blue, 1%) and 1.2 μg of protein were loaded and run on a 12.5% sodium glycine-dodecyl sulfate-polyacrylamide gel. After electrophoresis, the proteins were transferred to a nitrocellulose membrane by wet transfer (100 V at 4 ⁰ C for 1 hour). After blocking in 5% milk in phosphate buffered saline plus Tween (PBST), the membranes were incubated with a polyclonal rabbit anti-gelsolin antibody (Santa Cruz Biotechnology, Santa Cruz, California), (diluted 1: 1000) and rabbit polyclonal antibody apolipoprotein D (Santa Cruz Biotechnology, Santa Cruz, California) in PBST containing 1% milk, 4 ⁰ C overnight. After washing in PBST the immunoreactive protein bands were visualized by incubating the membranes with anti-rabbit antibody conjugated to peroxidase (diluted 1: 5000 in 5% milk in PBST) for 1 hour at room temperature. Using a Western blotting ECL analysis system, immunoreactive bands were represented on a chemiluminescent film by normal film development.

IDENTIFICATION OF MARKERS BY SELDI-TOF

Retained chromatography (SELDI profile): To test the proteins of interest during purification, the fractions profile was made in ProteinChip CMlO and / or QlO matrices. The matrices were first equilibrated in binding buffer (50 mM Tris, pH 9.0 for QlO matrices and 100 mM Na acetate, pH 4.0 for CMlO matrices). Aliquots of each sample were diluted 10 times in binding buffer and allowed to bind to the surface of the matrix for 30 minutes. The matrix was washed three times with 150 µl of binding buffer, rinsed with deionized water, and allowed to dry before adding matrix. To assess total protein content and purity, 1-2 μL aliquots were also bound to NP20 matrices and allowed to dry before adding the matrix. SPA or CHCA were used as matrices. Anion exchange fractionation: 10 mL of urine were denatured with 16 ml of 50 mM 9M Tris urea, pH 9.0. Q HyperD® F resin (PaIl Corporation) was equilibrated with Ul buffer (1 M urea, 0.2% CHAPS, 50 mM Tris-HCl, pH 9) and incubated with each urine sample. Denatured urine samples were incubated in batches for 1 hour at room temperature with 800 μL of Q HyperD® F resin. The unbound fraction was collected and the resin was washed with 1 mL of 50 mM Tris, pH 9 in 0.1 OGP %. The bound proteins were eluted successively with 1 mL of buffers of pH 7, 6, 5, 4, and 3 in 0.1% OGP and finally with a solution of 33% isopropanol / 17% ACN / 0% TFA ,one%. The profile of an aliquot of each fraction was made on ProteinChip CMlO and / or QlO matrices to be tested for the proteins of interest.

Reverse phase fractionation: 100 μL of Poly-Bio RPC balls were balanced

(BioSepra) with 5% ACN / 0.5% TFA. For the purification and identification of the 2193 and 2793 Da markers, the reduction and alkylation was before the reverse phase chromatography (see the reduction and alkylation section below). For reverse phase chromatography, an appropriate volume of each sample was adjusted to a final concentration of 5% ACN / 0.5% TFA and mixed for 1 hour with the RPC balls at room temperature. The tube was centrifuged and the supernatant was removed by aspiration. The bound proteins were eluted successively with 400 μL of 20% ACN, (25%), 30%, (35%), 40% and 50% in 0.1% TFA (25% ACN and 35% were used only for the purification of the 2193 and 2793 Da peptides). The profile of a 2 μL aliquot of each fraction was made in a ProteinChip NP20 matrix to determine the elution pattern of the markers of interest. The aliquot profile of the selected fractions was also made in a ProteinChip CMlO matrix.

Fractionation using a proteominer system: To enrich the 4.6 kDa peak before purification, fractionation was used as a preliminary step using the proteominer system: 1 ml of Proteominer resin in PBS was equilibrated, 40 ml of urine was added and incubated at 4 ^or C overnight in rotation. The unbound fraction was collected and the resin was transferred to a column for elution. The bound fraction was eluted successively in 1 ml of 1) 2.2 M thiourea, 7.7 M urea, 4.4% CHAPS, 2) 9 M urea, 25 mM citric acid, pH 3.8 and 3) 33.3% isopropyl alcohol, 16.7% acetonitrile, 0.5% trifluoroacetic acid. The column was incubated 10 minutes at room temperature in rotation before collecting each elution to ensure maximum efficiency.

IMAC-Cu fractionation: IMAC HyperCel resin was loaded with 100 mM copper sulfate and equilibrated in PBS, 0.5 M NaCl. The samples were diluted with PBS / 0.5 M NaCl, and incubated with the resin at 4 ° C overnight in rotation. For the purification of the 4.6 kDa peak, the unbound fraction of the proteominer was diluted 1: 3 in PBS / 0.5 M NaCl and incubated with 1.5 ml of copper-charged resin. For the purification of peaks 5.8 and 7.6 / 7.7 kDa, the urine was diluted 1: 2 in PBS / 0.5 M NaCl. Patient samples were pooled (25 ml) and incubated in 1.25 ml of IMAC-Cu resin and the control sample (25 ml) was incubated with 2 ml of IMAC-Cu resin. The unbound fraction was collected and the resins were transferred to two columns, where a wash with PBS / 0.5 M NaCl was performed. Bound proteins were eluted with 1.5 column volumes of 5, 10, 20, 30, 40, 50, 100, and 200 mM imidazole in 50 mM Hepes, pH 7.0. An aliquot of each elution was diluted 1: 10 and its peptide profiles were analyzed on CMlO and QlO chips. Fractions containing 7.6 / 7.7 kDa peaks (20 and 30 mM imidazole) were pooled and diluted 1: 2 in 50 mM Tris-HCl pH 9, and incubated with 0.25 ml of Q resin overnight at 4 ^o C. Elution of bound proteins was performed as indicated in the anion exchange fractionation section.

Resin purification 18: Purification of the 5.8 kDa peptide includes a purification step by hydrophobic exchange in resin 18. This resin was equilibrated in binding buffer ((150 mM NaCl in 100 mM Sodium acetate pH 5) and incubated throughout the overnight at 4 ^o C. Patient urine and control urine was incubated with 100 ul and 200 ul of resin respectively 18. The unbound fraction was collected and the resin bound proteins were eluted successively with 1.5 volumes of the following buffers : El) 4.5 M Urea, E2) 9 M urea, E3) 9 M urea, 0.6% ammonium hydroxide, E4) 9 M urea, 1.2% ammonium hydroxide, and E5) 9 M urea, 2.4% ammonium hydroxide. Fractions from elution with ammonium hydroxide were neutralized with 5M acetic acid. An aliquot of each fraction was diluted 1: 10 and its peptide profiles were analyzed on CMlO chips. Gel electrophoresis: Once it was estimated that the protein of interest was sufficiently enriched and purified, the fractions were concentrated in a CentriVap centrifuge evaporator (Labconco) and redissolved in 20 μL of the appropriate gel loading buffer before gel electrophoresis. 18% acrylamide / Tris SDS-PAGE (Bio-Rad) gels were used. Precision Plus protein standards and / or intended Kaleidoscope (Bio-Rad) standards were included in each gel in order to determine the size of the protein bands. Protein gels were stained using the Colloidal Blue staining kit following the manufacturer's protocol (Invitrogen).

Analytical extraction: The selected protein bands were cut from the polyacrylamide gel with a pasteur pipette and extracted to confirm the m / z of the protein and prepare the sample for digestion. The pieces of gels were washed twice with 200 μL of 50% methanol / 10% acetic acid for 20 minutes, dehydrated with 100 μL of ACN for 15 minutes, and extracted with 70 μL of 50% formic acid / 25% ACN / 15% IPA incubating 2 hours at room temperature with vigorous stirring. The profile of a 1-2 μL aliquot of the extract was made on a ProteinChip NP20 matrix.

Protein Identification:

Identification of peaks 8.85, 2.19 and 2.79 kDa: The pieces of gels were washed twice with 200 μL of 50% methanol / 10% acetic acid for 20 minutes, dehydrated with 100 μL of ACN for 15 minutes, and extracted with 70 μL of 50% formic acid / 25% ACN / 15% IPA by incubating 2 hours at room temperature with vigorous stirring. The profile of a 1-2 μL aliquot of the extract was made on a ProteinChip NP20 matrix.

The 8.85 kDa marker was reduced and rented after gel extraction. The analytical extract was dried in a CentriVap centrifuge concentrator (Labconco), rinsed twice with ammonium hydroxide, and dried. The precipitate was then resuspended in 10 .mu.l of 2.5 mM DTT, 50 mM ammonium bicarbonate pH 8 and incubated at 60 ⁰ C for 15 minutes. An excess of iodoacetamide was then added to the reaction. and incubated for 15 minutes in the dark. The alkylation reaction was quenched with an excess of DTT for 15 minutes in the dark. Before digestion with trypsin, this sample was dried again in the CentriVap. For the enrichment of the 2193 and 2793 Da markers, the samples were reduced and rented before purification. DTT was added to the samples at a concentration of 5 mM and incubated for 1 hour at 22 ^° C. An excess of iodoacetamide was then added to the reaction and incubated for 15 minutes in the dark. The alkylation reaction was quenched with an excess of DTT for 15 minutes in the dark. Digestion in solution for trypsin digested, alkylated dried gels extracts were rehydrated with 20 μL of 50 mM ammonium bicarbonate (pH 8) containing 5 ng / μL of modified trypsin (Roche Applied Science) and incubated for 3 hours at 37 ⁰ C. Identification of proteins by peptide fragmentation using a tandem mass spectrometer. The individual MS and MS / MS spectra were acquired on a tandem mass spectrometer. Equal volumes of trypsin digested and saturated CHCA were mixed and directed to the MALDI target. The digested were also linked to μC18 Zip-Tips (Millipore), eluted in 50% ACN / 0.1% TFA, mixed with an equal volume of and saturated CHCA and directed to the MALDI target. The spectra of 600 to 4000 Da were collected in individual MS mode. After reviewing the spectra, specific ions were selected for MS / MS analysis. The MS / MS spectra were sent to the Mascot database extraction tool (Matrix Sciences) for identification.

Identification of peaks 7.7, 7.6, 5.8 and 4.6 kDa: The corresponding bands of the respective gels were trimmed and 100 µl of 5 mM DTT was added, and they were incubated for 30 minutes at 37 ⁰ C. Then the DTT was removed, 100 µl of iodoacetamide was added and incubated another 30 minutes at room temperature in the dark. The gel bands were washed twice with 50 mM pH8 ammonium bicarbonate and dried on a CentriVap evaporator, for subsequent digestion.

The cut bands were treated to remove Coomassie stain and SDS, incubating successively with 400 µl of 50% methanol / 10% acetic acid (one hour for the first incubation and 30 minutes for the second) and with 400 µl of acetonitrile 50% / 100 mM pH8 ammonium bicarbonate for 30 minutes, and with 100 μl of acetronitrile for 10 minutes. The gel bands were dried on a CentriVap evaporator. For digestion, the dried bands were rehydrated in 20 µl of 50 mM pH8 ammonium bicarbonate with, 5 ng / µl trypsin or 10 ng / µl AspN. Some digestions were attached to Zip / Tips (Millipore), washed twice with 5% acetonitrile / 0.1% trifluoroacetic acid and eluted in 50% acetonitrile / 0.1% trifluoroacetic acid. Identification was performed by peptide fragmentation in a tandem mass spectrometer. Samples were applied on the MALDI spectrometer using the following method: 0.5 µl of 25% saturated CHCA was added to the plate, then 1-2 µl of sample and another 0.5 µl of 25% saturated CHCA were applied. The spectra were collected in the reflectron mode of 500 to 6000 Da. Specific ions were selected for the MS / MS analysis. Fragmentation spectra were sent to the Mascot (Matrix Sciences) database extraction tool for identification.

RESULTS

Say

After sample processing, DIGE gels were obtained from five healthy controls and five cirrhotic patients (Fig. 1). The spots were detected and compared using the DeCyder software with the independent T-test statistical analysis. In this analysis, eighteen differential spots were found with a P value of less than 0.05 and an average ratio greater than 1.5. Seventeen of these proteins decreased in cirrhotic patients and only one of them increased in patients. To identify the differential proteins, a preparative gel was made by combining several control samples, and the proteins were stained with the Sypro Ruby stain

(Biorad). The spots were taken, digested with trypsin and analyzed by

ESI-MS / MS. It was not possible to take some of the differential proteins, due to the low sensitivity of the preparative gel. The peptide profiles and sequences of each spot were analyzed with both Phenix 2.3 and search engines.

ProteinLynx Global Server 2.3 in the Uniprot Global Server database. Two proteins were identified: The apolipoprotein D precursor is produced in various tissues and is secreted to plasma, is involved in the binding and transport of biline.

The plasma depolymerizing gelsolin precursor f is a plasma secreted actin modulating protein that binds actin and f ybronectin. It can promote the assembly of monomers in filaments as well as separate filaments already formed.

The samples used in the 2D-DIGE study were subjected to immunoblot analysis with commercial antibodies for these two proteins, confirming that the data obtained in DIGE analysis were due to a drop in total protein levels in cirrhotic patients (Fig . 2).

SELDI TOF Two studies were performed using the SELDI-TOF technique. The first was carried out in the Spanish National Biotechnology Center (CNB), and the second in the Biomarker Research Center in the Bio-Rad laboratories.

CNB STUDY In this study, 15 samples of liver fibrosis were compared with 15 control cases in CMlO chips (cation exchange), and IMAC-Cu (metal affinity). Spectra analysis gave five significantly different peptides (P <0.01). One of these markers was the 8855 Da peptide with a value of P = 5.7 x 10 ^"4 and an ROC of 0.74.

BIORAD STUDY

The purpose of this study was to obtain additional information, increasing both the number of samples and the types of chips. The number of samples was increased to 35 patients with cirrhosis and 35 healthy controls. In addition, a group of 10 patients with low fibrosis (Metavir Fl index) was added. The number of chips used also increased to four: CMlO (cation exchange), IMAC30 (metal affinity), QlO (anion exchange) and H5 (hydrophobic exchange). The data analysis produced 61 peaks that showed statistically significant differences (P <0.01) between cirrhotic patients and healthy controls. The fibrosis group was too small for a consistent statistical analysis, but provided some information on the specificity of the markers, showing an intermediate behavior between the cirrhotic samples and controls (Figs. 3, 5 and 6).

This second study provided confirmation of four markers detected in the CNB analysis. One of these confirmed markers is the 8855 Da peptide, detected with a value of P = 1, 10 x 10 ^" and an ROC value = 0.74.

In conclusion, the peptide analysis performed in the Bio-Rad laboratories provides, first, confirmation of one of the peptides identified in the previous study, and second, new candidate markers identified by means of the QlO chip.

The prevalence of negative markers found in the DIGE study was confirmed as most of the candidate markers (57 of 61) decreased in patients with cirrhosis.

In addition, another statistical model was applied to the data: the linear model. In this case, the test applied was the LIMMA Bioconductor package. In this analysis, sixteen significantly decreased peptides were found in cirrhotic samples (B> 0).

Peptides that were considered significantly different in both univariate statistical studies were chosen to perform multivariate statistical analysis to improve the success of the classification of the samples by combining individual markers.

Table 1 lists those peptides identified in the two univariate statistical studies that showed significantly different levels of expression between controls and cirrhotic patients. The tabal indicates the protein that has been assigned to each peptide and the ROC p values obtained when discriminating between control and cirrhotic patients. Table 1

The samples used in the SELDI study were subjected to immunoblot analysis with commercial antibodies to the SLURPl protein, confirming that the data obtained in SELDI analysis were due to a drop in total protein levels in cirrhotic patients. Figure 4 shows the decrease in SLURPl protein levels in urine samples from cirrhotic patients with respect to the controls detected by Western Blot.

A useful tool to evaluate the diagnostic capacity of a quantitative test is the so-called ROC curve. It will also help us compare different tests. To obtain the ROC curve, the sensitivity (proportion of true positives) and specificity (proportion of true negatives) are calculated for each of the different values observed in our data and are represented in a graph, with the Sensitivity in the axis of the And, (1 -Specificity) on the X axis. Therefore, the more displaced the ROC curve towards the upper left vertex, the better the discriminatory capacity of the test. Precisely one way to assess this discrimination capacity in a global way is to calculate the area of the polygon that is below the ROC curve, and is called the area under the curve, serving as an index Comparison between diagnostic tests, the larger the area, the better the diagnostic capacity.

Multivariate statistical analysis of the combination of markers 4623/7655/7735/8853/2193/2793/5804 produced a pattern to differentiate healthy cirrhotic individuals, with an ROC value of 0.91.

Multivariate statistical analysis of the combination of markers 4623/7655 / / 8853/2193/2793/5804 produced a pattern to differentiate healthy cirrhotic individuals, with an ROC value of 0.87.

Multivariate statistical analysis of the combination of markers 4623/7655/7735/8853/2193/2793 / produced a pattern to differentiate healthy cirrhotic individuals, with an ROC value of 0.90.

Multivariate statistical analysis of the combination of markers 4623/7655/8853/2793 produced a pattern to differentiate healthy cirrhotic individuals, with an ROC value of 0.87.

Hepcidin is translated as an 84 amino acid peptide and processed to obtain hepcidin-25 (C-terminal residues 60-84 of the peptide) or hepcidin-20 (C-terminal residues 65-84). Hepcidin has been shown to be involved in the maintenance of iron homeostasis. Regulates both intestinal iron absorption and iron storage in macrophages and hepatocytes. Hepcidin increases in response to increased iron levels and inflammation, and decreases in response to hypoxia, anemia and oxidative stress. In addition, hepcidin also has a strong antimicrobial activity against several microorganisms.

SLURP-I has been found to be a marker of late skin differentiation and has been shown to have antitumor activity. It is involved in the maintenance of the physiological and structural integrity of the keratinocyte layers of the skin, defects in SLURP-I are a cause of Meleda Mal. The MASP2 protein is a serine protease that binds to the malignant binding lectin (MBL), causing activation of the complement pathway. MBL is able to bind with repeated structures of sugars present in a wide variety of bacteria and other microorganisms promoting their elimination. Stengaard et al. (New Eng. J. Med. 349: 554-560, 2003) have described that congenital deficiency in the gene encoding MASP2 results in a greater susceptibility to infections and immune diseases.

Defensins function as natural antibiotics that are found on the surface of the skin. They are active against bacteria, fungi and enveloped viruses. Beta-Defensins have 36 to 42 amino acids. Human beta-Defensin 1 (hBD-1) or DEFBl is constitutively expressed at the level of the urinary and respiratory genital tract. It is an antimicrobial peptide involved in the resistance of epithelial surfaces to microbial colonization. These peptides interact with several CDi and lymphocyte receptors, thereby activating adaptive immunity mechanisms.

Osteopontin (SPPl) is a multifunctional glycoprotein with a stimulating effect on fibroblasts and extracellular matrix synthesis. Osteopontin is found in various tissues and has various functions such as the biomineralization of bone tissue and tissue repair, fibrosis and dystrophic calcifications after immunological lesions, it also participates in tumor growth, in the development of cancer and metastasis. It also participates in the initiation of the immune response in the early activation of T lymphocytes and macrophages.

Claims

1. Use of at least one biomarker for the detection, diagnosis and evaluation of fibrosis and / or a disease associated with fibrosis or to follow the efficacy of a treatment for fibrosis and / or a disease associated with fibrosis in a subject where said biomarker is selected from the group consisting of SLURPl, HAMP, GSN, APOD, SPPl, DEFBl and MASP2.

2. Use according to claim 1 wherein the disease associated with fibrosis is cirrhosis.

3. Use according to claim 1 wherein the fibrosis is hepatic fibrosis.

4. A method for the detection, diagnosis and evaluation of fibrosis and / or a disease associated with fibrosis or for monitoring the efficacy of a treatment for fibrosis and / or a disease associated with fibrosis comprising comparing the expression of one or more biomarkers in a sample of a subject with a predetermined standard of each of said one or more biomarkers; wherein said one or more biomarkers is selected from the group consisting of SLURPl, HAMP, GSN, APOD, SPP l, DEFB l and MASP2; and wherein a significant difference in the expression of said one or more biomarkers in said sample compared to the predetermined standard of each said one or more biomarkers is indicative of onset, phase, evolution of fibrosis and / or disease associated with fibrosis or efficacy of a treatment for fibrosis and / or disease associated with fibrosis.

5. A method according to claim 4 wherein a decrease in the expression of one or more biomarkers is indicative that the subject suffers from fibrosis and / or a disease associated with fibrosis.

6. A method according to claims 4 or 5 wherein an increase in expression in one or more biomarkers is indicative that the treatment for fibrosis and / or the disease associated with fibrosis is effective.

7. A method as defined in any of claims 5 to 6 wherein the disease associated with fibrosis is cirrhosis.

8. A method as defined in any of claims 5 to 7 wherein the fibrosis is liver fibrosis.

9. A method as defined in any of claims 5 to 8 wherein the predetermined standard is the level of expression in a reference sample obtained by combining equal amounts of samples from a population of subjects.

10. A method as defined in any of claims 5 to 9 wherein the predetermined standard is the level of the biomarker (s) in a reference sample obtained from the same subject in which the efficacy of the previous therapy is to be evaluated. to the initiation of therapy.

11. A method as defined in any of claims 5 to 10 wherein the sample is urine.

12. A method as defined in claim 1 wherein the expression of the biomarker or bio markers is determined by quantifying the levels of the corresponding protein or proteins.

13. A method as defined in claim 12 wherein the determination of the levels of the protein or proteins is carried out by immunoblotting.

14. A kit comprising reagents for detecting at least two or more biomarkers selected from the group of SLURPl, HAMP, GSN, APOD, SPPl, DEFBl and MASP2.

15. A kit as defined in claim 14 wherein the biomarkers are polypeptides.