Patent Description:
In particular, according to the present invention, biomarkers relating to degradation fragments of Collagen type I, III, IV, V, and VI, elastin, C-reactive protein, and proteoglycans including Biglycan, Decorin, Versican, and Perlecan are found to be useful.

Fibrotic diseases (including those listed in Table <NUM>) are a leading cause of morbidity and mortality, e.g. cirrhosis with <NUM>,<NUM> death per year worldwide<NUM>.

A 'fibrotic disease' is any disease giving rise to fibrosis, whether as a main or a secondary symptom.

Fibrosis is the end result of chronic inflammatory reactions induced by a variety of stimuli including persistent infections, autoimmune reactions, allergic responses, chemical insults, radiation, and tissue injury. Fibrosis is characterized by the accumulation and reorganization of the extracellular matrix (ECM). Despite having obvious etiological and clinical distinctions, most chronic fibrotic disorders have in common a persistent irritant that sustains the production of growth factors, proteolytic enzymes, angiogenic factors, and fibrogenic cytokines, which together stimulate the deposition of connective tissue elements, especially collagens and proteoglycans, which progressively remodel and destroy normal tissue architecture <NUM>, <NUM>. Despite its enormous impact on human health, there are currently no approved treatments that directly target the mechanisms of fibrosis <NUM>.

The key cellular mediator of fibrosis is the myofibroblast, which when activated serves as the primary collagen-producing cell.

Fibrogenesis is a dynamic process involving complex cellular and molecular mechanisms that usually originates from tissue injury <NUM>. Fibrogenesis is the result of an imbalance in normal ECM regulation that alters the concentration of macromolecules leading to increased tissue size and density, with progressively impaired function. These macromolecules are mainly fibrous proteins with structural and adhesive functions, such as collagens and proteoglycans.

Collagens are widely distributed in the human body, i.e. ~ <NUM>% of the protein mass in the human body is composed of collagens. Collagens are responsible for the structural integrity of the ECM of most connective tissues. The ECM content results from a fine balance between synthesis and degradation tightly controlled through regulation of gene expression and protein secretion, but also through endogenous protease inhibition and protein degradation by metalloproteinases and cysteine proteases <NUM>-<NUM>. Table <NUM> lists the major collagen types with their major tissue distribution.

Type I collagen is the most abundant collagen and is found in most connective tissues. It is especially important for the structure of bone and skin where the major collagenous components are type I and III collagens <NUM>.

Collagen type I and III are the major components of liver and lung in a <NUM>:<NUM> ratio in healthy tissue. In addition, collagen type IV and VI are found in the basement membranes in most tissues. The most common localization of type V collagen is within the characteristic collagen fibrils, in association with the collagen type I and III <NUM>.

Some collagens have a restricted tissue distribution: for example, type II, which is found almost exclusively in cartilage <NUM>.

During fibrogenesis the net amount of collagens increases<NUM>-<NUM>. Table <NUM> shows by way of example the collagen increase during liver fibrosis.

The imbalance between synthesis and degradation of ECM during fibrogenesis, results from conversion of the low-density subendothelial matrix into matrix rich in interstitial collagens. The increase in collagen and proteoglycans may be due to one or both of (<NUM>) a decrease in protein production and (<NUM>) impaired protein degradation, and hence less matrix degradation. The decreased protein degradation has recently received increased attention. In the regulation of this process matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play important roles, as well as other proteases and their inhibitors, such as cystein proteases and the cystatins.

MMPs are a large group of endopeptidases, capable of degrading most if not all components of the ECM. Presently, more than <NUM> MMPs have been found. MMPs are characterized by an active site containing a metal atom, typically zinc, and are secreted as zymogens. Different MMPs are expressed in different tissues. In Table <NUM> MMPs in the liver are shown.

TIMPs block MMPs' proteolytic activity by binding in a substrate- and tissue- specific manner to MMP and membrane-type <NUM> metalloproteinase in a trimolecular complex (Table <NUM>). During fibrosis TIMP levels increase dramatically, and MMP levels increase modestly or remain relatively static (except MMP-<NUM>) which in all gives a decrease in degradation of collagens.

A number of biochemical markers have been suggested for fibrotic diseases, although not specific product of the disease. In Table <NUM> is an example of biochemical markers of liver fibrosis used in clinical trial. In addition there are a lot of examples of biomarkers of other fibrotic diseases<NUM>, <NUM>-<NUM>.

<NPL>)<NUM> developed a monoclonal antibody which recognises a neoepitope in type II collgen which is generated by the intrahelical cleavage of collagenases. The neopitope could be detected by ELISA at elevated levels in urine and serum samples from rheumatoid arthritis patients.

<CIT> describes a method for diagnosing the degree of liver fibrosis, comprising the steps of measuring the concentration of type IV collagen high molecular weight form in a sample using an antibody that specifically binds to type IV collagen, and relating the measurement to the degree of liver fibrosis. Again, no use is made of neo-epitopes produced by proteolytic enzymes acting in the body. The sample is actually digested with pepsin, which may obscure the natural pattern of collagen cleavage in the sample.

However, in none of the above mentioned reports is it suggested that measurements of peptide fragments based on antibodies binding to neo-epitopes as now claimed might be useful for the assessment of patients with fibrotic disease.

The present invention provides a method of immunoassay as set out in the claims.

The present invention now provides a method of diagnosis of fibrosis comprising, conducting an immunoassay as described in the claims to measure neo-epitope containing protein fragments naturally present in a patient biofluid sample, and associating an elevation of said measure in said patient above a normal level with the presence of fibrosis, wherein said immunoassay is conducted by a method comprising:
contacting protein fragments naturally present in said sample with an immunological binding partner reactive with a neo-epitope formed by cleavage of a protein by a proteinase and measuring the extent of binding of peptide fragments to said immunological binding partner to measure therein protein fragments comprising said neo-epitope, and wherein said protein is collagen type IV, For these purposes, cardiovascular disease may not be regarded as fibrosis, or the fibrosis detected according to the invention may be other than fibrosis accompanying cardiovascular disease. Optionally, an elevated result in an immunoassay according to this invention is associated with skin fibrosis, lung fibrosis, or liver fibrosis.

The method may comprise the preliminary step of obtaining a patient biofluid sample.

The invention includes a method of immunoassay to measure neo-epitope containing protein fragments naturally present in body fluid sample, wherein said immunoassay is conducted by a method comprising:
contacting protein fragments naturally present in said sample with an immunological binding partner reactive with a neo-epitope formed by cleavage of a protein by a proteinase and measuring the extent of binding of peptide fragments to said immunological binding partner to measure therein protein fragments comprising said neo-epitope, and wherein said protein is collagen type IV.

Said immunological binding partner may have specific binding affinity for peptide fragments comprising a C-terminal neoepitope or an N-terminal neoepitope.

Specific reactivity with or immunological affinity for a neo-epitope will imply that the relevant immunological binding partner is not reactive with intact protein from which the neo-epitope derives. Preferably, said immunological binding partner is not reactive with a neo-epitope sequence, such as a sequence listed below, if the sequence is prolonged past the respective cleavage site.

The term `immunological binding partner' as used herein includes polyclonal and monoclonal antibodies and also specific binding fragments of antibodies such as Fab or F(ab')<NUM>. Thus, said immunological binding partner may be a monoclonal antibody or a fragment of a monoclonal antibody having specific binding affinity.

Preferably, said peptide fragments are fragments of TypeIV. The connective tissue proteins are preferred. Preferably, the neo-epitope sequence to which the immunological binding partner binds is not found in any other protein or is not found in any of the other proteins to which the method of the invention relates.

Several candidate proteases may be responsible for the digestion of proteins in the fibrotic tissues. Most likely, this is the result of the large range of complicated processes resulting in different neo-epitope profiles dependent on the levels of disease.

We have determined that the enzymes listed in the following table cleave type IV collagen at least the following cleavage sites (marked ".

The immunological binding partner may be one specifically reactive with a C-terminal or N-terminal neoepitope formed by cleavage of type IV collagen.

Suitable immunological binding partners may therefore be specifically reactive with any of the following sequences at the N terminal of a peptide:.

or with any of the following sequences at the C-terminal of a peptide:.

Further cleavage sites defining neo-epitopes that may be assayed in a similar manner can be identified by exposing collagen IV to any of the enzymes described herein and isolating and sequencing peptides thereby produced. Furthermore, assays may be based on the neo-epitopes generated adjacent the illustrated cleavage sites, i.e. in the C-terminal sequences that lead up to the N-terminal epitopes given above and the N-terminal sequences that connect to the C-terminal epitopes described.

Assays for more than one of the peptides described above may be conducted separately and their results combined or more than one of the peptides described above may be measured together.

The result of an assay according to the invention may be combined with one or more other measured biomarkers to form a composite index of diagnostic or prognostic value.

Generally, all previously known immunoassay formats can be used in accordance with this invention including heterogeneous and homogeneous formats, sandwich assays, competition assays, enzyme linked assays, radio-immune assays and the like. Thus, optionally, said method is conducted as a competition immunoassay in which said immunological binding partner and a competition agent are incubated in the presence of said sample and the competition agent competes with the peptide fragments in the sample to bind to the immunological binding partner.

Said competition agent may be (<NUM>) a synthetic peptide derived from the sequence of collagen type IV, peptide, or a competition agent derived from (<NUM>) a purified native collagen type IV, cleaved by proteases to reveal said neo-epitope.

One suitable method could be a competition immunoassay using monoclonal antibodies or antibody binding fragments binding to neo-epitopes of collagen type IV. Appropriately selected synthetic peptides coated onto the solid surface of a microtitre plate could compete with the sample for binding to the monoclonal antibodies or binding fragments. Alternatively, purified, native collagen type IV fragments carrying the neo-epitope recognised by the monoclonal antibody or binding fragment could be used on the solid surface. Yet another alternative is to immobilise the monoclonal antibody or binding fragment on the solid surface and then co-incubate the sample with a synthetic peptide appropriately linked to a signal molecule, e.g. horseradish peroxidase or biotin.

The sample may be a sample of serum, blood, plasma or other, e.g. fibrotic tissue biopsy.

Assays may be conducted as sandwich assays using a first immunological binding partner specifically reactive with a said neo-epitope and a second immunological binding partner reactive with the relevant protein to which the neo-epitope belongs. Optionally, said second immunological binding partner is directed to a second neo-epitope of the same protein.

In certain preferred methods the method further comprises comparing the determined level of said binding of said peptide fragments with values characteristic of (a) comparable healthy individuals and/or (b) a pathological fibrotic condition and optionally associating a higher level of the measured peptide (normally indicated by a higher level of binding) with a more severe degree of a said condition.

An aspect of the present invention relates to the development of monoclonal antibodies recognising neo-epitopes as described above, especially for collagen types IV. This can be achieved by immunising mice with synthetic peptides originating from the amino acid sequence of collagen type IV molecules (including the sequences listed above or sequences terminating therein), fusing the spleen-cells from selected mice to myeloma cells, and testing the monoclonal antibodies for binding to neo-epitopes on relevant synthetic peptides. Specificity for neo-epitopes can be ensured by requiring reactivity with a synthetic peptide and a lack of reactivity with either a C-prolongated form of the immunising peptide (for a C-terminal neo-epitope) or an N-terminal prolongated form of the immunising peptide (for an N-terminal neo-epitope). Antibodies for neo-epitopes may also be evaluated to establish a lack of binding capacity to native collagen type IV. Alternatively, specificity for a neo-epitope can be ensured by requiring the reactivity of the antibody to be negatively dependent on the presence of biotin or other functional groups covalently linked to one of the terminal amino acids.

The invention includes an immunological binding partner which is specifically immunoreactive with a neo-epitope formed by cleavage of collagen type IV by a protease at a end-site in any one of the partial sequences of collagen typeIV set out above, and may be for instance a monoclonal antibody or a binding fragment thereof.

The invention includes a cell line producing a monoclonal antibody against a C-terminal or N-terminal neo-epitope formed by cleavage of collagen type IV at the end-sites of sequences in any one of the partial sequences of collagen type IV set out above.

A peptide comprising a C-terminal or N-terminal neo-epitope formed by cleavage of collagen type I, III, IV, V, VI, CRP, vimentin, neurocan, brevican, fibromodulin, serglycins, syndecan, betaglycan, versican, lumican, decorin, perlecan and biglycan in any one of the partial sequences of these proteins set out above is also described, but does not form part of the invention. Such a peptide may be conjugated as a hapten to a carrier for producing an immune response to said peptide, or immobilised to a solid surface or conjugated to a detectable marker for use in an immunoassay.

An isolated nucleic acid molecule coding for a peptide comprising a C-terminal or N-terminal neo-epitope formed by cleavage of collagen type IV in any one of the partial sequences of collagen type IV set out above is also described, but does not form part of the invention.

A vector is also described, but does not form part of the invention. The vector comprises a nucleic acid sequence comprising an expression signal and a coding sequence which codes for the expression of a peptide comprising a C-terminal or N-terminal neo-epitope formed by cleavage of collagen type IV, in any one of the partial sequences of collagen type IV set out above and further includes a host cell transformed with such a vector and expressing a said peptide.

Kits, which can be used conveniently for carrying out the methods described above are also described, but do not form part of the invention. Such kits may include (<NUM>) a microtitre plate coated with synthetic peptide; (<NUM>) a monoclonal antibody or antibody binding fragment of the invention reactive with said synthetic peptide; and (<NUM>) a labelled anti-mouse IgG immunoglobulin. Alternatively, such kits may include (<NUM>) a microtitre plate coated with purified native collagen type IV fragments; (<NUM>) a monoclonal antibody recognising a neo-epitope on collagen type IV fragments and reactive with said purified collagen type IV fragments; and (<NUM>) a labelled anti-mouse IgG immunoglobulin. Alternatively, such kits may include (<NUM>) a microtitre plate coated with streptavidin; (<NUM>) a synthetic peptide linked to biotin; (<NUM>) a monoclonal antibody recognising a neo-epitope on collagen type IV fragments and reactive with said synthetic peptide; and (<NUM>) a labelled anti-mouse IgG immunoglobulin. Yet another alternative could be kits including (<NUM>) a microtitre plate coated with streptavidin; (<NUM>) a synthetic peptide linked to biotin; (<NUM>) a monoclonal antibody recognising a neo-epitope on collagen type IV fragments (and reactive with said synthetic peptide) and conjugated to horseradish peroxidase.

Thus, the invention includes an immunoassay kit comprising an immunological binding partner as described herein, especially in respect of collagen types IV, and a competition agent which binds said immunological binding partner, and optionally one or more of a wash reagent, a buffer, a stopping reagent, an enzyme label, an enzyme label substrate, calibration standards, an anti-mouse antibody and instructions.

The assays described herein are useful in the diagnosis of fibrosis in patients. In addition, the tests are useful for the assessment of disease progression, and the monitoring of response to therapy. The immunological binding partners of the invention may also be used in immunostaining to show the presence or location of collagen type IV cleavage products.

The invention will be further described and illustrated with reference to the following examples and the accompanying drawings, in which:.

Cleavage: Collagen type III isolated from human placenta was dissolved in <NUM> acetic acid (<NUM>/ml). The protein solution was then passed through a filter (Microcon Ultracel YM-<NUM>) to remove fragment contaminations. MMP-<NUM> was preactivated with <NUM>-aminophenylmercuric acetate (APMA, Sigma) at <NUM> for <NUM> hours. After activations, collagen type III and MMP-<NUM> were mixed <NUM>:<NUM> and incubated shaking for <NUM> days at <NUM>.

The solution was analyzed by liquid chromatography/mass spectrometry (LC/MS) and the fragments were identified by performing Mascot Search. The peptide sequences were selected by homology search, ensuring no cross-reactivity to other or related proteins, as well as interspecies cross-reactivity.

Antibody design: The peptide sequences were synthesized and conjugated to ovalbumin (OVA). Mice were immunized ever <NUM>-<NUM> weeks, up to five. Antibody titers were checked by screening peptides, both selection and de-selection. When sufficient antibody titers were achieved, positive mice were selected for fusion, euthanized, and the spleen was disintegrated and B-cells were removed for fusion with myeloma cells. Selections of antibody producing cells were done by culturing and re-seeding the surviving chimera cells in single cell clones. Clones are selected by selection and de-selection peptides followed by native reactivity testing (<FIG>), as neoepitopes are generated by synthetic small peptide sequences, which may not reflect the native proteins. An IgG subtype clone is selected for antibody production. Antibody purification is done by protein-G column.

Assay development: Optimal antibody concentrations are determined by checker-board analysis, with dilutions of antibody coating and screening peptide, in competitions ELISA. The different determination for the collagen degraded by MMP-<NUM> (COS) assay is shown in table <NUM>.

Method: Forty female Sprague-Dawley rats (<NUM> months old) were housed at the animal research facilities at Nordic Bioscience. The experiments were approved by the Experimental Animal Committee of the Danish Ministry of Justice, and were performed according to the European Standard for Good Clinical Practice (<NUM>/<NUM>-<NUM>). The rats were housed in standard type III-H cages at <NUM>-<NUM> with bedding and nest material (Altromin <NUM>; Altromin, Lage, Germany) and purified water (Milli-Q system; Millipore, Glostrup, Denmark) ad libitum. Rats were kept under conditions of a <NUM>-hour light/dark cycle.

Liver fibrosis was induced by common BDL. In short: The rat was anaesthetized, the bile duct found, two ligations were performed around the bile duct followed by dissection between the ligations, the abdomen was closed. In sham operated rats, the abdomen was closed without bile duct ligation.

The rats were divided into <NUM> groups: Group <NUM>(<NUM> BDL and <NUM> sham operated rats) were sacrificed after <NUM> weeks, and Group <NUM> (<NUM> BDL and <NUM> sham operated rats) were sacrificed after <NUM> weeks. On completion of the study period (<NUM>, or <NUM> weeks), after at least <NUM> hours fasting, all surviving animals were asphyxiated by CO<NUM> and sacrificed by exsanguinations.

Blood samples were taken from the retro-orbital sinus of at least <NUM> hours fasting rats under light CO<NUM>/O<NUM> anaesthesia at baseline and at termination. The blood were collected and left <NUM> minutes at room temperature to cloth, followed by centrifugation at <NUM> for <NUM> minutes. All clot-free liquid were transferred to new tubes and centrifuged again at <NUM> for <NUM> minutes. The serum were then transferred to clean tubes and stored at -<NUM>.

COS were measured in x <NUM> diluted serum samples from the rats. Sham and BDL levels were compared by Mann-Whitneys two-tailed nonparametric test (α=<NUM>) of statistical significance assuming normal distribution.

COS levels increased significantly in the BDL groups compared to the Sham-operated animals. The results are shown in <FIG> and b.

COS levels were measured in serum from human with three different fibrotic diseases: Chronic obstructed pulmonary disease (COPD), Scleroderma, and Hepatitis virus C (HCV). The serum samples were retrieved from Sera Laboratories International Ltd (SLI Ltd), UK. COS levels were increased in the three different fibrotic diseases (<FIG>).

Type III collagen (Abcam, Cambridge, UK) was degraded in vitro by activated MMP-<NUM> (Merck KGaA, Darmstadt, Germany) for <NUM> days. Degradation fragments were sequenced by LS-MS/MS and identified by MASCOT search. A specific peptide sequence <NUM>KNGETGPQ was selected for antibody production. The N-terminal of this sequence is residue <NUM> of human collagen type III. The synthetic peptide was conjugated to ovalbumin prior to subcutaneous immunization of <NUM>-<NUM> week old Balb/C mice with about 200µL emulsified antigen and 50µg CO3-610C (KNGETGPQGPGGC-OVA). Consecutive immunizations were performed at two week intervals until stable sera titer levels were reached in Freund's incomplete adjuvant. The mice were bled from the second immunization on. At each bleeding, the serum titer was measured and the mouse with highest anti-serum titer was selected for fusion. After the fourth immunization, this mouse was rested for one month and then boosted intravenously with 50µg CO3-610C in 100µL <NUM>% sodium chloride solution three days before isolation of the spleen for cell fusion.

Monoclonal antibody producing clones were selected using a) immunogenic peptide: KNGETGPQGP-GGC-Ovalbumine (OVA) (<NUM>), b) screening peptide KNGETGPQGP-PG-K-Biotin (<NUM>), c) de-selection peptides KDGETGAAGPPGK-Biotin (<NUM>) representing a type II collagen alpha <NUM> chain, KDGEAGAQGPPGK-Biotin representing a type I collagen alpha <NUM> chain degradation product, purchased from the Chinese Peptide Company, Beijing, China. The ELISA coat plate was obtained from NUNC (Thermofisher, Copenhagen, Denmark). Peptide conjugation reagents and buffers were produced by Pierce (Thermofisher, Copenhagen, Denmark).

Buffer used for dissolving the coating peptide was composed of the following: <NUM> Na<NUM>HPO<NUM>, <NUM><NUM>O, <NUM> KH<NUM>PO<NUM>, <NUM> NaCl, <NUM>,<NUM> KCl, <NUM> EDTA, <NUM>,<NUM> % Tween <NUM>, <NUM> % BSA, <NUM> % sorbitol, pH <NUM>. For a serum assay, buffer containing the following chemicals was used: <NUM> Na<NUM>HPO<NUM>, <NUM><NUM>O, <NUM>,<NUM> KH<NUM>PO<NUM>, <NUM>,<NUM> NaCl, <NUM>,<NUM> KCl, <NUM>,<NUM> % Tween <NUM>, <NUM> % BSA, <NUM>,<NUM> % phenol red, pH <NUM>,<NUM>. A different buffer used for a urine assay contained <NUM> TRIZMA, <NUM>,<NUM> % Tween <NUM>, <NUM>,<NUM> % BSA, <NUM>,<NUM> % Bronidox L5, pH <NUM>,<NUM>. For both serum and urine assays we used a washing buffer composed of <NUM> TRIZMA, <NUM> NaCl, <NUM>,<NUM> % Bronidox L5, <NUM>,<NUM> % Tween <NUM>, and reaction-stopping buffer composed of <NUM>,<NUM> % H<NUM>SO<NUM>. ELISA-plates used for the assay development were Streptavidin-coated from Roche (Hvidovre, Denmark) cat. All ELISA plates were analyzed with the ELISA reader from Molecular Devices, SpectraMax M, (CA.

In preliminary experiments, we optimized the reagents, their concentrations and the incubation periods by performing several checkerboard analyses. A <NUM>-well ELISA plate coated with streptavidin was further coated with <NUM> ng/ml of the synthetic peptide KNGETGPQGP-Biotinylated dissolved in PBS-TBE buffer at <NUM> for <NUM> minutes by constant shaking at <NUM> rpm. After washing with washing buffer, 20µl of sample was added, followed by 100µl of peroxidase conjugated anti-human mAb-NB51-<NUM> CO3-610C solution (<NUM> pg/ml in incubation buffer). The plate was incubated for <NUM> hour at <NUM> during which time it was shaken at <NUM> rpm. This was followed by washing and finally, 100µl tetramethylbenzinidine (TMB) (Kem-En-Tec cat. 438OH) was dispensed and the plate incubated for <NUM> minutes in darkness and shaken at <NUM> rpm. In order to cease the reaction, 100µl of stopping solution was added and the plate analyzed in the ELISA reader at <NUM> with <NUM> as reference.

A standard curve was performed by serial dilution of biotinylated-NB51-<NUM> CO3-610C for a serum assay, and biotinylated-NB51-<NUM> CO3-610C for a urine assay. Standard concentrations were <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> ng/ml.

We designate fragments detected using the immunoassays so obtained as CO3-610C as the amino acid K at the N-terminal of the sequence KNGETGPQGP is amino acid <NUM> of the human collagen III sequence.

<NUM> female Sprague-Dawley rats aged <NUM> months were housed at the animal research facilities at Nordic Bioscience, Copenhagen, Denmark. The experiments were approved by the Experimental Animal Committee of the Danish Ministry of Justice and were performed according to the European Standard for Good Clinical Practice (<NUM>/<NUM>-<NUM>). The rats were housed in standard type III-H cages at <NUM>-<NUM> with bedding and nest material (Altromin <NUM>; Altromin, Lage, Germany) and water ad libitum. Rats were kept under conditions of a <NUM>-hour light/dark cycle.

In <NUM> rats, liver fibrosis was induced by common BDL. The surgical procedure was performed under sterile conditions. The rat was anaesthetized, the bile duct localized and ligated in two places followed by dissection between the ligations, and the abdomen was closed. The other <NUM> rats were subjected to a sham operation, in which the abdomen was closed without bile duct ligation. The rats were then divided into <NUM> groups: Group <NUM> (<NUM> BDL rats and <NUM> sham-operated rats) was sacrificed after <NUM> weeks and Group <NUM> (<NUM> BDL and <NUM> sham-operated rats) was sacrificed after <NUM> weeks. On completion of the study period (<NUM> or <NUM> weeks), after at least <NUM> hours fasting, all surviving animals were asphyxiated by CO<NUM> and sacrificed by exsanguinations.

Blood samples were taken from the retro-orbital sinus of rats after at least <NUM> hours fasting, under light CO<NUM>/O<NUM> anaesthesia, at baseline and at termination. Blood was left <NUM> minutes at room temperature to clot, followed by centrifugation at <NUM> for <NUM> minutes. All clot-free liquid was transferred to fresh tubes and centrifuged again at <NUM> for <NUM> minutes. The serum was then transferred to clean tubes and stored at -<NUM>.

After the rats were put down, their livers were carefully dissected, weighed, fixed in <NUM>% formaldehyde for a minimum of <NUM> hours, cut into appropriate slices and embedded in paraffin. Sections <NUM> thick were cut, mounted on glass slides and stained with Sirius Red. The liver sections were evaluated histologically by assessment of the architecture, presence of inflammation, proliferation of bile ducts and fibrosis. The de novo bile duct formation in the parenchyma was evaluated semi-quantitatively using the following scoring system: normal = <NUM>, mild changes (<NUM>/<NUM> or less of the lobule affected) = <NUM>, moderate changes (between <NUM>/<NUM> and <NUM>/<NUM> of the lobule affected) = <NUM>, and severe changes (<NUM>/<NUM> or more of the lobule affected) = <NUM>. Digital photographs were captured using an Olympus BX60 microscope with x <NUM> and x <NUM> magnification and an Olympus <NUM>-zoom digital camera (Olympus, Tokyo, Japan).

The total collagen concentration was assayed using the commercial QuickZyme Collagen Assay (QuickZyme Bioscience, Leiden, The Netherlands). The concentration of CTX-II was assayed using the commercial Rat CTX-II kit (IDS Nordic, Herlev, Denmark). All samples were assayed in duplicate.

The number of transcripts of type III collagen (Col3a1) in liver tissue samples was determined by quantitative real-time polymerase chain reaction (RT-PCR) using fluorescent reporter probes. The number of Col3a1 copies in the sample was extrapolated from a standard curve obtained using Col3a1 plasmid cDNA Image Clone <NUM> (Geneservice, Cambridge, UK) as dilution standard. Amounts of Col3a1 were normalized with those of housekeeping gene hypoxanthine phosphoribosyltransferase <NUM> (Hprt1). Primers and probes for Col3a1 and Hprt1 mRNAs were designed using NCBI Reference Sequences NM_032085. <NUM> and NM_012583. <NUM> as templates, respectively (TIB Molbiol GmbH, Berlin, Gemany). Total RNA was extracted from frozen liver samples using Absolutely RNA Miniprep kit (Stratagene, La Jolla, CA, USA) following the manufacturer's instructions and its quality assessed in RNA Nano chips using a <NUM> Bioanalyzer instrument (Agilent Technologies, Santa Clara, CA, USA). Immediately after RNA isolation, complementary DNA (cDNA) was synthesised with Transcriptor First Strand cDNA Synthesis kit (Roche, Basel, Switzerland) using 1µg of RNA as the template. For each sample tested, a cDNA synthesis negative control, omitting reverse transcriptase enzyme from the reaction mix, was included. Separate PCR reactions for Col3a1 and Hprt1 were perfomed in a 20µL format using the Lightcycler Faststart DNA Master Plus Hybprobe kit (Roche) according to the manufacturer's instructions. Real time fluorescence data was collected in a Lightcycler <NUM> instrument (Roche).

Tissue was pulverized in excess of liquid nitrogen in a steel mortar. Samples were then transferred into a <NUM> eppendorf tube and left shaking overnight at <NUM> in <NUM> Acetic Acid solution containing protease inhibitor cocktail (Roche). The samples were then sonicated with ultrasounds using <NUM> pulses at <NUM>% amplitude (U50 control, IKA Labortechnik) and left for an additional <NUM> hours at <NUM> after which they were centrifuged for <NUM> minutes at <NUM>,<NUM> rpm. Supernatant was carefully removed, transferred in a new eppendorf and stored at -<NUM>.

Densitometry measurements were performed using UN-SCAN-IT Version <NUM> from Silk Scientific (give city, country).

Histology sections stained with Sirius Red were analyzed using Visiopharm software Version <NUM>. <NUM> (give city, country). Images were acquired using Pixelink PL-A623C microscope digital camera.

<NUM>µg of tissue extract was mixed with loading buffer (Invitrogen LDS 4x, NP0007) containing the reducing agent (NuPAGE, NP0004 from Invitrogen). Samples were then loaded into <NUM>-<NUM>% Bis-Tris gradient gel (NP0332BOX from Invitrogen) and run for <NUM> minutes at 200V. Proteins were then transferred onto a nitrocellulose membrane using the i-Blot transfer system (Invitrogen), blocked with <NUM>% milk in (? Need to spell out?) TTBS overnight at <NUM> degrees. Beta Actin antibody (AbCam ab8229, give company, city country?) was used as a loading control.

Mean values and standard error of the mean (SEM) were calculated using GraphPad Prism (GraphPad Software, Inc. , San Diego, CA, USA) and compared by Student's two-tailed paired t-test (α=<NUM>) of statistical significance assuming normal distribution, or by Mann-Whitney two-tailed non-parametric test (α=<NUM>). The coefficient of correlation (R<NUM>) and the corresponding p-value was determined by linear regression.

Liver appearance: At the time of sacrifice, livers of control animals showed normal gross morphology while livers of BDL animals were enlarged. The liver weights were significantly increased in BDL rats compared to the sham-operated controls (mean weights at sacrifice: <NUM> weeks post-surgery, sham <NUM>; BDL <NUM>; <NUM> weeks post-surgery, sham <NUM>; BDL <NUM>) (<FIG> panel A). Semi-quantitative scoring of liver sections using the <NUM>-<NUM> scale showed significantly more structural changes of the liver at <NUM> weeks compared with <NUM> weeks (<FIG> panel B). <FIG>, panel A shows liver weight in bile duct ligation (BDL)- or sham-operated rats. Data are shown as mean + SEM. [*** , P<<NUM>. Panel B shows scoring of the structural changes in the liver of each group. Data are shown as mean + SEM. **, P=<NUM>. Panel C shows Sirius Red photomicrographs showing the hepatic structure in sham-operated rats, and in BDL rats <NUM> and <NUM> weeks post-surgery. The hepatic structure around the portal tract is clearly disrupted in BDL rats compared with the sham-operated rats. Collagens are highlighted in red. Original magnification was x40.

Under histological examination, the livers of sham-operated animals showed no sign of fibrosis and were microscopically normal (<FIG>). In the BDL livers, a marked ductal proliferation was observed. In the <NUM>-week post-surgery group, the proliferation was located around the portal tract while in the <NUM>-week group the proliferation had spread (<FIG>). Collagen deposition was found around the ductular structures. Inflammation was minimal and confined to the portal tracts. No signs of cholestasis were seen, whether intracellular cholestasis, bile plugs, bile infarctions or hepatocytic rosette formation.

Changes in CO3-610C levels: <FIG> shows in panel A MMP-<NUM> mediated COS degradation serum levels in bile duct ligated (BDL)- or sham-operated rats. Data are shown as mean + standard error of mean. <NUM> weeks post-surgery *** P<<NUM> and <NUM> weeks post-surgery ** P=<NUM>. In panel B are shown CO3-610C delta values (termination-baseline paired), <NUM> weeks post-surgery P<<NUM> and <NUM> weeks post-surgery P=<NUM>. In panel C are shown CTX-II levels in BDL- or sham-operated rats. Data are shown as mean + standard error of mean.

In the BDL groups CO3-610C levels increased significantly compared to sham groups (mean values: <NUM> weeks,post-surgery sham <NUM> ng/ml, BDL <NUM> ng/ml; average increase between the groups was <NUM>%; <NUM> weeks post-surgery, sham <NUM>, BDL <NUM> ng/ml, average increase between the groups was <NUM>%) (<FIG> panels A and B). There were no changes in the sham groups. CTX-II levels indicating collagen type II degradation did not change in the sham or BDL groups (<FIG> panel C).

Type III Collagen Gene Expression: <FIG> shows Type III collagen gene expression in BDL or sham-operated rats. Data are shown as mean + standard of mean; <NUM> weeks post-surgery P<<NUM> and <NUM> weeks post-surgery P=<NUM>
Type III collagen a1 chain mRNA increased significantly in both BDL groups compared with sham-operated rats.

Western Blot and Densitometry: <FIG> shows changes in the expression of CO3-610C in the liver of rats in BDL- and sham-operated groups assessed by A) Western blot <NUM> and <NUM> weeks post-surgery and B) Bands from western blot quantified by densitometry.

Western blot analysis showed very low levels of CO3-610C in sham-operated rats (<FIG> panel A). At and after <NUM> weeks post-surgery CO3-610C levels prominently increased (<FIG> panel A). Results were quantified by densitometry analysis (<FIG> panel B).

Histology image analysis: <FIG> panel A shows in the top row histology sections from BDL- or sham- operated rats stained with Sirius Red. The bottom row shows masked histology sections for quantifying total collagen content (red colour) in the liver. Panel B shows total collagen quantified by Visiopharm software - <NUM> weeks post-surgery P=<NUM>; <NUM> weeks post-surgery P=<NUM>.

Histology sections stained with Sirius Red and enhanced using Visiopharm software showed increasing collagen content over time in BDL-operated rats. (<FIG> panel A). The red color in the mask representing collagen was quantified using the same software (<FIG> panel B) and confirmed a significant increase in total collagen content in BDL-operated rats compared with sham-operated rats (<NUM> weeks post-surgery P=<NUM>; <NUM> weeks post-surgery P=<NUM>).

Correlation: <FIG> panel A shows a correlation of Col3a1 to CO3-610C was found with R<NUM>=<NUM>, P<<NUM>. In panel B, a correlation of CO3-610C to % collagen was found with R<NUM>=<NUM> and P=<NUM>. In panel C a correlation of Co13a1 to % collagen was found with R<NUM>=<NUM>, P<<NUM>.

Correlations were found of the following: Col3a1 mRNA to CO3-610C with R<NUM>=<NUM> and P<<NUM> (<FIG>), and CO3-610C to % collagen quantified by visiopharm with R<NUM>=<NUM> and P=<NUM> (<FIG>), and Col3a1 mRNA to % collagen quantified by visiopharm with R<NUM>=<NUM> and P<<NUM> (<FIG>).

ECM remodelling is an integrated process of tissue development, maintenance and pathogenesis. Proteolytic activity is essential in this process for cell migration, removal of damaged tissue, and sequestering of new proteins, for the correct and optimal tissue orientation and quality (<NUM>:<NUM>). The specific matrix degradation products, neo-epitopes, may be important for the identification of new biochemical markers of liver fibrosis matrix turnover and understanding fibrosis pathogenesis. At present there are no available measuring techniques, nor biochemical markers, that allow for assessment of ECM remodeling in the pathogenesis of fibrosis.

In this example, to investigate the CO3 - 610C marker under in vivo situations, <NUM> months BDL rats were chosen, as they previously have been shown to have a lower collagen remodelling compared to younger rats. The rats are skeletally mature, and the growth plate is almost dormant, thereby contributing to a much lower extent to the overall collagen turnover. This influences the sensitivity and specificity for biomarker. These rats clearly presented with hepatic fibrosis , as evaluated by both quantitative histological analysis, and enlargement with increased weight, thus the model was an appropriate one to look for evidence of ECM remodeling, in particular for evidence of collagen type III in serum.

The present data clearly demonstrate the neo-epitope COS - 610C from MMP-<NUM> mediated collagen type III degradation is a diagnostic biochemical marker for liver fibrosis with an average increases in serum of up to <NUM>% from sham to BDL- operated rats.

To further investigate the biological rationale for the increased COS - 610C marker, we did protein extractions from healthy and diseased livers. By western blotting, we identified a predominant band, suggesting this to be an abundant protein fragment in diseased but not healthy livers. This provides evidence for the pathological accuracy of this novel marker.

To further investigate the pathological turnover representation of the liver, we measured type III collagen mRNA. We found an increase of mRNA in the BDL rats compared to those undergoing the sham operation, which correlates with previous findings. These data strongly suggest that liver fibrosis is not only an accumulation of ECM proteins, but also an accelerated turnover situation, in which both tissue formation and tissue degradation both are highly up regulated. Tissue formation outstrips tissue degradation, leading to an accumulation of scar tissue over time. Previous investigators have used other matrix turnover proteins to assess liver fibrosis, one being the type III collagen formation marker N-terminal type III pro-collagen. This marker represents collagen type III formation and has shown to be increased in liver fibrosis in previous studies.

To further understand and the dynamics of the biochemical makers COS - 610C, we did a range of correlations. Most importantly, there was a significant correlation of COS - 610C to the extent of fibrosis measured in the liver by quantitative histology. The level of liver fibrosis was correlated to the expression levels of the mRNA of collagen type III. Finally, the CO3 - 610C correlated to mRNA of collagen type III in the liver. Taken together, there was a significant correlation of the pathological processes in the liver with the levels of the systemic biochemical markers COS - 610C. In addition the tissue extractions provided evidence that the circulation levels were locally produced.

Human serum samples were obtained from patients with Chronic Obstructive Pulmonary Disease (COPD) (n=<NUM>), scleroderma (n=<NUM>), chronic hepatitis C virus infection (n=<NUM>), and healthy controls (n=<NUM>). The serum samples were tested in the CO3-<NUM> ELISA (see Example <NUM> above) to determine the concentration of CO3-<NUM> fragments. Results are shown in <FIG>. While serum samples from the healthy subjects had concentration of CO3-<NUM> fragments below <NUM> ng/ml, the diseased subjects were found to have elevated levels in circulation suggesting massive tissue remodelling in the affected fibrotic tissues.

Mice were immunized with synthetic peptide KAFVFP (SEQ ID NO1167) conjugated to ovalbumin (KAFVFPKESD-GGC-OVA (SEQ ID NO1049)), spleen cells were used for fusion, and monoclonal antibodies tested for reactivity to biotinylated KAFVFP (SEQ ID NO <NUM>), i.e. (KAFVFPKESD-biotin(SEQ ID NO1049)) immobilized in wells of microtitre plates precoated with streptavidin. Antibodies binding to biotinylated KAFVFPKESD(SEQ ID NO1049), which could be inhibited by co-incubation with KAFVFPKESD (SEQ ID NO1049) but not the elongated peptide RKAFVFPKESD (SEQ ID NO1166), were selected for further characterization. The preferred monoclonal antibody was designated NB94-<NUM>-1A7.

Using a competition ELISA, essentially as described above with biotinylated KAFVFPKESD (SEQ ID NO1049) (used at <NUM> ng/ml) immobilized in the wells of streptavidin-coated microtitre plates, an incubation step (<NUM> minutes at <NUM>) with sample and monoclonal antibody NB94-<NUM>-1A7 followed by a washing step, and then addition of peroxidase-conjugated anti-mouse immunoglobulins. For competition the following material was used in <NUM>-fold dilutions; (<NUM>) the synthetic KAFVFP (SEQ ID NO1167) peptide; (<NUM>) a nonsense peptide (KNEGTG) unrelated to CRP; (<NUM>) a pool of human serum samples; (<NUM>) CRP proteolytically cleaved with MMP3 for <NUM> days, subsequently stopped by addition of EDTA to block protease activity, and stored at -<NUM> until testing; (<NUM>) same as (<NUM>) but using MMP8 instead of MMP3; (<NUM>) same as (<NUM>) except using Cathepsin K (for <NUM> days) instead of MMP3 (and E64 as inhibitor to block Cathepsin K activity).

The data demonstrate that monoclonal antibody NB94-<NUM>-1A7 binds strongly to the synthetic peptide KAFVFPKESD (SEQ ID NO1049), and with CPR cleaved with MMP3 and MMP8. Cleavage of CRP with Cathepsin K release less analyte recognized by monoclonal antibody NB94-<NUM>-1A7. Finally, the data shows that the antibody binds to peptide fragments in human serum confirming the presence of this sequence in circulating peptide fragments.

This study included <NUM> male Wistar rats with fibrosis or cirrhosis and <NUM> male Wistar control rats. To cause them to develop fibrosis or cirrhosis three-month old animals were included in an induction program with carbon tetrachloride (CCl4) and Phenobarbital treatment. CCl<NUM> was administered by inhalation twice weekly and phenobarbital (<NUM>/l) added to the drinking water. Animals were allowed free access to water and food throughout the study.

Liver sections (<NUM>) were stained in <NUM>% Sirius red F3B (Sigma-Aldrich, St. Louis, MO) in saturated picric acid (Sigma-Aldrich). Relative fibrosis area (expressed as a percentage of total liver area) was assessed by analyzing <NUM> fields of Sirius red-stained liver sections per animal. Each field was acquired at 10X magnification [E600 microscope (Nikon) and RT-Slider SPOT digital camera (Diagnostic Instruments, Inc. , Sterling Heights, MI). Results were analyzed using a computerized Bioquant Life Science morphometry system. To evaluate the relative fibrosis area, the measured collagen area was divided by the net field area and then multiplied by <NUM>. Subtraction of vascular luminal area from the total field area yielded the final calculation of the net fibrosis area. From each animal analyzed, the amount of fibrosis as percentage was measured and the average value presented.

Animals were classified into <NUM> different stages of fibrosis and cirrhosis (Group A: moderate fibrosis, group B: advanced fibrosis, Group C: moderate cirrhosis, and Group D: advanced cirrhosis) that were determined by the percentage of Sirius red positive liver area (Group A: <<NUM>%, Group B: <NUM> to10%, Group C: <NUM> to <NUM>% and Group D: ><NUM>%). For this purpose, control and fibrotic/cirrhotic rats were studied considering four different time points during the CCl4 treatment: <NUM>, <NUM>, <NUM> and <NUM> weeks after starting the cirrhosis induction program.

Serum hyaluronan was measured using a sandwich ELISA kit (R&D Systems Inc. , Minneapolis, MN, USA).

Statistical analysis of results was performed by unpaired Student's t tests when appropriate. Data were expressed as mean ±S. , and they were considered significant at a p level of <NUM> or less.

Animals included in this protocol were randomly assigned to one of the following groups: A/ eight weeks of CCl<NUM> treatment, B/ twelve weeks of CCl<NUM> treatment, C/ sixteen weeks of CCl<NUM> treatment and D/ twenty weeks of CCl<NUM> treatment. In parallel, four control groups were studied at the same time points. Thirteen fibrotic rats and seven control rats were included in each group. At the end of the study, rats were placed in standard metabolic cages (Tecniplast Deutschland, Hohenpeissenberg, Germany) during an adaptation period of <NUM> days before proceeding with the twenty-four-hour urine collection. Urinary volumes were determined gravimetrically. During the adaptation period, rats were allowed to get free access to tap water and food. Then, <NUM>-hour urine samples were centrifuged for <NUM> at <NUM>,<NUM> rpm and aliquoted into ten polypropylene tubes (<NUM>µL each). Urine samples were stored at -<NUM> for subsequent analysis.

At scheduled necropsies, rats were weighed, anesthetized with pentobarbital (<NUM>/kl) and decapitated. Blood were collected and allowed to stand at room temperature for <NUM> to allow clotting and then centrifuged for <NUM> at <NUM> rpm. Serum were collected in polypropylene tubes aliquots (<NUM>µl each) and transferred via dry ice to a -<NUM> freezer. Collection of baseline blood samples at the beginning of the CCl<NUM> treatment was not considered in order to avoid additional intervention that may increase the risk of infection and/or introduce modifications in the experimental model that may compromise the evolution of the induced pathophysiological process. For histology and Sirius red staining, half of the left lobe of the liver were placed in <NUM>% neutral buffered formalin for <NUM> hours, embedded in paraffin and sectioned into <NUM>-µm-thick slices. After liver fibrosis quantification, the unused paraffin block material was preserved for biomarker quantification. The other half of the left lobe was flash-frozen in liquid nitrogen and stored for Western blot, RT-PCR or immunohistochemical analysis. Measurements of liver fibrotic area, serum and urine osmolality, Na+ and K+, albumin, creatinine, alanine amino-transferase and lactate dehydrogenase were made according to the Material and Methods section.

Liver collagen was quantified in all study animals by Sirius red staining of liver slices. The final data for each animal was taken as the average of red staining observed in <NUM> consecutive microscope fields (<FIG>).

<FIG> shows representative pictures from two sets of <NUM> images used to quantify collagen accumulation in liver in rat #<NUM> (left) and rat #<NUM> (right) treated with carbon tetrachloride for eight and twenty weeks respectively.

The serum COS marker shows statistically significant increases in both fibrotic and cirrhotic rats compared to control rats. Animals were classified according to a fully automated syrius red staining of the liver procedure used to quantify fibrosis (<FIG>).

<FIG> shows serum CO3 levels in CCl<NUM> inhalation and control rats as performed in Hospital Clinic (Barcelona). Each point represents one animal. Rats were classified according a computerized image analysis method of syrius red staining of the liver used to quantify fibrosis.

When quantitative values of serum CO3 and syrius red staining of the liver were studied in each individual animal, we found a statistically significant correlation between the two variables (R2=<NUM>; n=<NUM>) (<FIG>).

We have compared the levels of CO3-610C with the serological benchmark of liver fibrosis hyaluronic acid (HA). HA levels were quantified with a commercial ELISA kit and results show significant elevations of this ECM component in cirrhotic rats vs. fibrotic animals (<FIG> and <FIG>).

The correlation of CO3 to Sirius red outperformed that of HA. More than seventy percent of the variation in liver fibrosis histological quantification can be explained by the serological measurement of CO3. The remaining thirty percent is due to unknown variables or inherent variability. Instead only <NUM>% of liver fibrosis can be explained by measuring hyaluronic acid (<FIG>).

As expected from the previous result no correlation could be found between CO3 and hyaluronic acid suggesting that they are the result of two independent pathophysiological processes in the development of liver fibrosis (<FIG>).

Mice were treated by application to the skin of PBS or bleomycin. Increasing levels in urine of the MMP-<NUM> mediated collagen III (CO3) degradation fragment CO3-<NUM> were associated with skin fibrosis progression in mice.

<FIG> shows a skin section from a PBS treated mouse at <NUM> weeks of treatment (panel A) and a skin section from Bleomycin treated mouse at <NUM> weeks of treatment (panel B). Skin thickness increase between PBS (n=<NUM>/time point) and Bleomycin (n=<NUM>/time point) treated mice for <NUM> weeks (P = <NUM>), <NUM> weeks (P = <NUM>), <NUM> weeks (P < <NUM>) and <NUM> weeks (P < <NUM>) is plotted in panels C and D. Overall skin thickness increase between PBS (n=<NUM>) and Bleomycin (n=<NUM>) treated mice for the duration of the study (P < <NUM>). Skin width was calculated by Visiopharm software as an overall number per skin section instead of sampling pictures.

<FIG> shows CO3-<NUM> urine assay results which demonstrate a significant increase throughout the time points of the study. The figure shows result per time point (n=<NUM> PBS, n=<NUM> Bleomycin treated per termination point) and collective CO3-<NUM> levels for all time points (n=<NUM> PBS and n=<NUM> Bleomycin treated mice). <NUM> weeks P = <NUM>, <NUM> weeks P < <NUM>, <NUM> weeks P < <NUM>, <NUM> weeks P < <NUM> and overall P < <NUM>.

<FIG> shows a CO3-<NUM> Western blots image with control C and Bleomycin B after <NUM> and <NUM> weeks treatment (panel A). CO3-<NUM> densitometry measurements for all time points (n=<NUM> PBS and n=<NUM> Bleomycin treated per termination point) and collective CO3-<NUM> levels (n=<NUM> PBS and n=<NUM> Bleomycin treated mice) are shown in panel B, demonstating a statistically significant increase of CO3-<NUM> levels (P < <NUM>).

As seen in <FIG>, CO3-<NUM> levels in urine assay were found to be correlated with skin thickness progression, and therefore total collagen deposition r=<NUM>, R2=<NUM>.

As seen in <FIG>,statistically significantcorrelation was found (r = <NUM>, P<<NUM>) between results from the CO3-<NUM> ELISA urine assay and Western blot densitometry measurements.

Claim 1:
A method of immunoassay to measure neo-epitope containing protein fragments naturally present in a body fluid sample, wherein said immunoassay is conducted by a method comprising: contacting protein fragments naturally present in said sample with an immunological binding partner reactive with an N-terminal neo-epitope formed by cleavage of a protein by a proteinase or a C-terminal neo-epitope formed by cleavage of a protein by a proteinase, which immunological binding partner is non-reactive with the intact protein from which the epitope derives and is not reactive with a prolongated version of the neo-epitope sequence in which the neo-epitope sequence is prolonged past the cleavage site, and measuring the extent of binding of peptide fragments to said immunological binding partner to measure therein protein fragments comprising said neo-epitope, wherein said immunological binding partner specifically binds a neo-epitope constituted by an N-terminal amino acid sequence present in peptides produced by cleavage of collagen type IV, said peptides comprising an N-terminal sequence selected from the group consisting of:

<TAB>

or, wherein said immunological binding partner specifically binds a neo-epitope constituted by a C-terminal amino acid sequence present in peptides produced by cleavage of collagen type IV, said peptides comprising an C-terminal sequence selected from the group consisting of:

<TAB>