Patent Publication Number: US-9404932-B2

Title: Pathology biomarker assay

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation-in-part of U.S. application Ser. No. 12/749,652, filed on Mar. 30, 2010, which is a continuation-in-part of PCT/EP2008/064946 filed on Nov. 4, 2008, which claims Convention priority from GB0721713.6 filed in the United Kingdom on Nov. 5, 2007, GB0722748.1 filed in the United Kingdom on Nov. 20, 2007 and GB0802814.4 filed in the United Kingdom on Feb. 15, 2008, and also claims the benefit under 35 U.S.C. §1.119(e) of U.S. Provisional application No. 61/211,467 filed on Mar. 30, 2009 and U.S. Provisional application No. 61/289,081 filed on Dec. 22, 2009. The entire contents of each of the aforementioned patent applications are incorporated herein by this references. 
    
    
     The present invention relates to assays for biomarkers useful in the diagnosis of various diseases including fibrosis diseases and prognosis of its development, including biomarkers indicative of the risk of developing disease, e.g. the risk of developing a fibrosis after a chronic injury. Biomarkers for inflammatory diseases such as ankylosing spondylitis are also described as are biomarkers for cardiovascular diseases (CVD). 
     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, ApoE, lumican, LAMC1, LAMB1, LAMA5 and proteoglycans including Biglycan, Decorin, Versican, and Perlecan are found to be useful. 
     Fibrotic diseases (including those listed in Table 1) are a leading cause of morbidity and mortality, e.g. cirrhosis with 800,000 death per year worldwide 1 . 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Different fibrotic diseases 2   
               
            
           
           
               
               
               
            
               
                   
                 Tissue 
                 Examples of Causes 
               
               
                   
                   
               
               
                   
                 Liver 
                 Viral hepatitis 
               
               
                   
                   
                 Schistosomiasis 
               
               
                   
                   
                 Steatohepatitis (Alcoholic or non-alcoholic) 
               
               
                   
                 Lung 
                 Idiopathic pulmonary fibrosis (IPF) 
               
               
                   
                   
                 Systemic sclerosis (Scleroderma) 
               
               
                   
                 Kidney 
                 Nephrogenic systemic fibrosis (NSF) 
               
               
                   
                   
                 Diabetes 
               
               
                   
                   
                 Untreated hypertension 
               
               
                   
                 Heart 
                 Heart attack 
               
               
                   
                   
                 Hypertension 
               
               
                   
                   
                 Atherosclerosis 
               
               
                   
                   
                 Restenosis 
               
               
                   
                 Eye 
                 Macular degeneration, retinal and vitreal 
               
               
                   
                   
                 retinopathy 
               
               
                   
                 Skin 
                 Systemic sclerosis and scleroderma, keloids, 
               
               
                   
                   
                 hypertrophic scars, burns, genetic factors 
               
               
                   
                   
                 NFS 
               
               
                   
                 Pancreas 
                 Autoimmune/hereditary causes 
               
               
                   
                 Intestine 
                 Crohn&#39;s disease/inflammatory bowl disease 
               
               
                   
                 Brain 
                 Alzheimer&#39;s disease, AIDS 
               
               
                   
                 Bone 
                 Cancer, ageing 
               
               
                   
                 marrow 
               
               
                   
                 Multi- 
                 Surgical complications, chemotherapeutic drug- 
               
               
                   
                 organ 
                 induced fibrosis, radiation-induced fibrosis, 
               
               
                   
                 fibrosis 
                 mechanical injuries 
               
               
                   
                   
               
            
           
         
       
     
     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 3,4 . Despite its enormous impact on human health, there are currently no approved treatments that directly target the mechanisms of fibrosis 5 . 
     The key cellular mediator of fibrosis is the myofibroblast, which when activated serves as the primary collagen-producing cell. 
     Inflammatory Conditions 
     Ankylosing Spondylitis (AS) is a form of chronic inflammation of the spine and the sacroiliac joints. Over time, chronic inflammation of the spine can lead to a complete cementation of the vertebrae ( 110 ), a process with a molecular pathology that still remains to be investigated and fully understood. AS is also a systemic disease, affecting other tissues throughout the body including inflammation in or injury to other joints, as well as to organs, such as eyes, heart, lungs, and kidneys ( 111 ). The inflammation related processes in AS, involving multiple tissues seems to involve molecular processes including increased MMP activity and collagen deposition, seen in most types of connective tissue turnover. 
     Extracellular matrix (ECM) remodeling is a key process of tissue homeostasis ( 112 ,  113 ). Specific proteolytic activities are a prerequisite for a range of cellular functions and interactions within the ECM ( 114 ). These specific activities are precisely coordinated under normal physiological situations, with a specified sequence of events resulting in controlled tissue turnover. In pathological situations, with special emphasis on connective tissue diseases, the normal repair-response relationship is disturbed ( 115 ), leading to excessive remodeling and tissue turnover. The consequence of this ECM remodeling is the release of a range of degradation products of proteins, neoepitopes, generated by the proteases expressed locally in the pathologically affected area. These protein degradation fragments may serve as molecular biomarker targets, as they are more specific for the tissue of origin compared to their intact origins ( 116 ). 
     Endopeptidases, such as matrix metalloproteinases (MMPs), play a major role in the degradation of ECM proteins as collagens and proteoglycans ( 117 ,  118 ). In particular, in connective tissue diseases such as fibrosis, MMP-2 and -9 have been shown to be highly up-regulated ( 119 - 121 ). Recently, neoepitope-based biochemical markers found in urine and serum have received increased attention for their diagnostic and prognostic potential ( 116 ). In particular for slow progressing diseases, such as osteoporosis and osteoarthritis, bone resorption and cartilage degradation markers, based on type I and II collagen degradation products respectively, have been studied extensively ( 122 ). 
     Extracellular Matrix (ECM) 
     Fibrogenesis is a dynamic process involving complex cellular and molecular mechanisms that usually originates from tissue injury 6 . 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. 
     Collagen 
     Collagens are widely distributed in the human body, i.e. ˜30% 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 7-9 . Table 2 lists the major collagen types with their major tissue distribution. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Major collagen types and their tissue distribution. 
               
            
           
           
               
               
               
            
               
                   
                 Collagen 
                   
               
               
                   
                 type 
                 Tissue distribution 
               
               
                   
                   
               
               
                   
                 I 
                 Most connective tissues 
               
               
                   
                 II 
                 Cartilage, vitreous humor 
               
               
                   
                 III 
                 Extensible connective tissues, e.g. liver, skin, 
               
               
                   
                   
                 lung, vascular system 
               
               
                   
                 IV 
                 Basement membranes 
               
               
                   
                 V 
                 Tissues containing collagen I 
               
               
                   
                 VI 
                 Most connective tissues 
               
               
                   
                 VII 
                 Skin, bladder, oral mucosa, umbilical cord, amnion 
               
               
                   
                 VIII 
                 Many tissues, especially endothelium 
               
               
                   
                 XIII 
                 Endothelial cells, skin, eye, heart, skeletal muscle 
               
               
                   
                 XIV 
                 Vessel, bone, skin, cartilage, eye, nerve, tendon, 
               
               
                   
                   
                 uterus 
               
               
                   
                 XXI 
                 Vessel, heart, stomach, kidney, skeletal muscle, 
               
               
                   
                   
                 placenta 
               
               
                   
                   
               
            
           
         
       
     
     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 10 . 
     Collagen type I and III are the major components of liver and lung in a 1:1 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 10 . 
     Some collagens have a restricted tissue distribution: for example, type II, which is found almost exclusively in cartilage 11 . 
     During fibrogenesis the net amount of collagens increases 12-4 . Table 3 shows by way of example the collagen increase during liver fibrosis. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Changes of the composition of collagen from normal to 
               
               
                 cirrhotic human liver  15 . 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                 Distri- 
                 Distri- 
               
               
                   
                   
                 Collagen 
                 Collagen 
                   
                 bution 
                 bution 
               
               
                 Col- 
                   
                 normal 
                 cirrhotic 
                 Times 
                 normal 
                 cirrhotic 
               
               
                 lagen 
                   
                 liver 
                 liver 
                 in- 
                 liver  
                 liver  
               
               
                 type 
                 Chains 
                 (mg/g) 
                 (mg/g) 
                 creased 
                 (%) 
                 (%) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 I 
                 α 1  (I) 
                   
                 16 
                 8 
                 37 
                 42 
               
               
                   
                 α 2  (I) 
                 2 
                   
                   
                   
                   
               
               
                 III 
                 α 1  (III) 
                 2 
                 8 
                 4 
                 37 
                 21 
               
               
                 IV 
                 α 1  (IV) 
                 0.5 
                 7 
                 14 
                 9 
                 18 
               
               
                   
                 α 2  (IV) 
                   
                   
                   
                   
                   
               
               
                 V 
                 α 1  (V) 
                 0.9 
                 7 
                 8 
                 17 
                 18 
               
               
                   
                 α 2  (V) 
                   
                   
                   
                   
                   
               
               
                   
                 α 3  (V) 
                   
                   
                   
                   
                   
               
               
                 VI 
                 α 1  (VI) 
                 0.01 
                 0.1 
                 10 
                 0.2 
                 0.3 
               
               
                   
                 α 2  (VI) 
               
               
                   
               
            
           
         
       
     
     Type V collagen has been documented to be critically important for the formation of collagen fibrils ( 123 ), exemplified by an almost virtual lack of collagen fibril formation in the col5a1−/− mice. In alignment, the heterozygous mice, was associated with a 50% reduction in fibril number and dermal collagen content. This indicates a central role for type V collagen in the life dependent regulation of fibrillogenesis, suggesting this collagen type to be of pivotal interest in many connective diseases. However, there is still a conceptual lack of understanding of the role of type V collagen turnover in connective diseases that may in part may be due to the technical inabilities for investigation of collagen type V degradation and turnover. Interesting, very recently a limited diverse set of proteins were found to bind type V collagen, beginning to elucidate the molecular function of this molecule in more details, of which MMP-2 was one of them ( 124 ). In addition to the molecular characterization, more evidence is emerging that type V collagen directly affects different cellular phenotypes by inducing dynamic motility and other cellular activities, suggesting that this proteins may be more than a passive component of the ECM ( 125 ,  126 ). In direct support of this, we recently described a very strong correlation to liver fibrosis with the formation of type V collagen in two separate animal models of liver fibrosis ( 127 ), suggesting a central role of type V collagen formation in excessive tissue turnover. 
     Type V collagen is a fibril-forming collagen, together with type I, II, III and XI (11), and is formed as heterotrimers of three different α-chains (α1, α2, α3). It typically forms heterofibrils with type I and III collagens and contributes to the organic bone matrix, corneal stroma and the interstitial matrix of muscles, liver, lungs and placenta ( 128 ). Type V collagen mutation results in a range of connective tissue diseases, of which the Ehlers-Danlos syndrome (EDS) the best described. EDS is a heterogeneous group of heritable disorders characterized by joint hypermobility, skin changes (e.g. hyperextensibility, thinness and fragility). The disease can be divided into different subtypes, EDS1 &amp; II. EDS types I and II are characterized by atrophic scars, skin hyper extensibility and joint laxity. It is evident that both subtypes result from mutations in the COL5A1 and COL5A2 genes that encode two of the polypeptide chains of type V collagen ( 129 - 131 ). This highlights the importance of type V collagen in connective tissues diseases. 
     Elastin 
     Elastin is a protein present in many connective tissues, primarily those that are elastic. It has a very high content of the amino acids glycine, valine, alanine, and proline, and has a molecular weight of 64 to 66 kDa. It is organised in an irregular or random coil conformation made up of 830 amino acids. Elastin is made by linking many soluble tropoelastin protein molecules, in a reaction catalyzed by lysyl oxidase, to make a massive insoluble, durable cross-linked array. 
     Elastin serves an important function in arteries as a medium for pressure wave propagation to help blood flow and is particularly abundant in large elastic blood vessels such as the aorta. Elastin is also very important in the lungs, elastic ligaments and the skin. 
     Despite much efforts devoted to the understanding of elastin synthesis and turnover, neo-epitopes originating from the proteolytic cleavage of this matrix molecules have until now not been associated with disease development in fibrosis. 
     Vimentin 
     Vimentin is a member of the intermediate filament family of proteins. Intermediate filaments are an important structural feature of eukaryotic cells. They, along with microtubules and actin microfilaments, make up the cytoskeleton. Although most intermediate filaments are stable structures, in fibroblasts, vimentin exists as a dynamic structure. This filament is used as a marker for mesodermally derived tissues, and as such has been used used as an immunohistochemical marker for sarcomas. 
     Hertig and coworkers (Hertig et al., J Am Soc Nephrol. 2008 August; 19(8):1584-91) investigated if epithelial-to-mesenchymal transition in renal tubular epithelial cells of subjects with chronic allograft nephropathy could predict the progression of fibrosis in the allograft and measured vimentin expression in 83 biopsies from these. They did find an association between elevated vimentin expression and the intestinal fibrosis score at 1 year after surgery. 
     In another study of hepatic fibrosis, Meriden and colleagues (Meriden et al., Clin Gastro &amp; Hepatol 2010; 8:289-296) found a significant association between vimentin expression (in biopsies obtained at F0 stage) and fibrosis progression, with elevated levels predicting rapid progression of the hepatic fibrosis. 
     Accordingly, we wanted to investigate if circulating fragments of vimentin could serve as sensitive and specific biomarkers of fibrosis. 
     Proteoglycans 
     Proteoglycans are a diverse group of macromolecules, which covalently link a variable number of glycosaminoglycan (GAG) side chains to a core protein 16 . These GAGs are polymers of disaccharide repeats (e.g. N-acetyl glucosamine or N-acetyl galactosamine), which are acidic (negatively charged) due to hydroxyl, carboxylated and sulfated side groups on the disaccharide units. This makes them highly hydrophilic, thus aiding the diffusion of water and positive ions (e.g. sodium from extracellular fluids) 17 . Furthermore, GAGS have the ability to form non-covalent links with for example hyaluronic acid chains to form even larger molecular complexes 16 . Table 4 lists the most studied proteoglycans associated with connective tissue. 
                     TABLE 4                  Proteoglycans of the extracellular matrix of connective tissue                             Group   Proteoglycans   Origin   Function               Large   Aggrecan  18     Articular   Extracellular       extracellular       cartilage   matrix stability       proteoglycans       chondrocytes,   (hyaluronan       (aggregating       intervertebral   binding)       and hyaluronan-       disc, nasal       binding)       cartilage           Versican  19,    20     Connective tissue:   Cell-cell and               fibroblast,   cell-matrix               keratinocytes,   interactions               smooth muscle   Binding of sugars               cells, mesangial   in Ca-dependent               cells   manner           Neurocan  21     Nervous tissue   Binds to neural                   cell adhesion                   molecules           Brevican  22     Nervous tissue   Extracellular                   matrix stability       Small Leucine-   Decorin  23     Connective tissue,   Binds to and       rich       cartilage, bone   connect collagen       proteoglycans           molecules (matrix       (collagen-           stabilization and       binding)           thickness)                   Organogenesis                   Binding of TGFβ           Biglycans  24     Capillary   Cell               endothelium, skin   differentiation               (keratinocytes),   Binds and connect               epithelium of   collagen fibrils               kidney           Fibromodulin  17     Connective tissue,   Regulate               bone, cartilage   orientation of                   collagen fibers           Lumican  23     Cornea, muscle,   Controls spacing               cartilage, kidney,   and thickness of               lung, intestine   collagen fibers       Cell-associated   Serglycins  25     Widely distributed   Hemopoietic cell       proteoglycans       to endothelium -   differentiation               intercellular   Adhesion and               compartments   activation of                   lymphoid cells           Syndecans  26     Widely distributed -   Binds collagens,               often cell   fibronectin,               membrane bound   thrombospondin,                   tenascin and bFGF           Betaglycan  27     Widely distributed   TGFβ receptor and                   signaling                   Possible                   reservoir of TGFβ       Basement   Perlecan  28     All basement   Selective barrier       membrane       membranes   for       proteoglycans           macromolecules                   Cell-adhesion                    
C-Reactive Protein
 
     C-reactive protein (CRP) is an acute phase serum protein produced by the liver in response to different clinical conditions such as, inflammation, infection, or trauma 29 . The production of CRP is induced by cytokines such as IL-6, released from the affected or damaged tissues. The physiological role of CRP is yet unknown and discussions on its pro- or anti-inflammatory actions are ongoing. 
     Apolipoprotein E 
     Apolipoprotein E (APOE) is a class of apolipoprotein found in the chylomicron (large lipoprotein particles) and intermediate-density lipoprotein particles that binds to specific receptors on liver cells and peripheral cells. It is essential for the normal catabolism of triglyceride-rich lipoprotein constituents. APOE is 299 amino acids long and transports lipoproteins, fat-soluble vitamins, and cholesterol into the lymph system and then into the blood. It is synthesized principally in the liver, but has also been found in other tissues such as the brain, kidneys, and spleen. APOE is essential for the normal catabolism of triglyceride-rich lipoprotein constituents. APOE was initially recognized for its importance in lipoprotein metabolism and cardiovascular disease. 
     Elastin 
     Elastin is the extracellular matrix molecule responsible for resilience of tissues and was first thought to be restricted to that role. It is now established that elastin degradation may lead to the production of bioactive peptides influencing cell chemotaxis, cell proliferation, and proteases synthesis in a broad panel of normal and tumor cells. 
     LAMC1, LAMA2, LAMB1, and LAMA5 
     Laminins, a family of extracellular matrix glycoproteins, are the major noncollagenous constituent of basement membranes. They have been implicated in a wide variety of biological processes including cell adhesion, differentiation, migration, signaling, neurite outgrowth and metastasis. Laminins are composed of 3 non-identical chains: laminin alpha, beta and gamma (formerly A, B1, and B2, respectively) and they form a cruciform structure consisting of 3 short arms, each formed by a different chain, and a long arm composed of all 3 chains. Each laminin chain is a multidomain protein encoded by a distinct gene. Several isoforms of each chain have been described. Different alpha, beta and gamma chain isomers combine to give rise to different heterotrimeric laminin isoforms which are designated by Arabic numerals in the order of their discovery. The biological functions of the different chains and trimer molecules are largely unknown, but some of the chains have been shown to differ with respect to their tissue distribution, presumably reflecting diverse functions in vivo. 
     LAMC1 (formerly LAMB2) is the laminin subunit gamma-1. 
     LAMA2 is the laminin subunit alpha-2 
     LAMB1 is the laminin subunit beta-1 
     LAMA5 is the laminin subunit alpha-5 
     Proteases 
     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 (1) a decrease in protein production and (2) 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 
     MMPs are a large group of endopeptidases, capable of degrading most if not all components of the ECM. Presently, more than 25 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 5 MMPs in the liver are shown. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 MMPs in the liver 30-32   
               
            
           
           
               
               
               
               
            
               
                 Family 
                 Protease 
                 Source 
                 Substrate 
               
               
                   
               
               
                 Collagenases 
                 MMP-1 
                 HSC 
                 I, II, III, VII, VIII, X, 
               
               
                   
                   
                   
                 gelatin 
               
               
                   
                 MMP-8 
                 Neutrophil 
                 I, II, III, V, VII, X, 
               
               
                   
                   
                   
                 gelatin 
               
               
                   
                 MMP-13 
                 HSC, 
                 I, II, III, VII, X, gelatin 
               
               
                   
                   
                 MFB, KC 
               
               
                 Stromelysins 
                 MMP-3 
                 HSC 
                 III, IV, V, IX, X, XI, 
               
               
                   
                   
                   
                 gelatin, laminin, 
               
               
                   
                   
                   
                 fibronectin, proteoglycans, 
               
               
                   
                   
                   
                 glycoproteins, elastin, pro- 
               
               
                   
                   
                   
                 MMP-1/13 
               
               
                   
                 MMP-10 
                 HSC 
                 III, IV, V, gelatin, 
               
               
                   
                   
                   
                 elastin, aggrecan 
               
               
                   
                 MMP-11 
                 HC 
                 PAI-1, week activity 
               
               
                   
                   
                   
                 against matrix proteins 
               
               
                 Gelatinases 
                 MMP-2 
                 HSC, MBF 
                 I, II, III, IV, V, VII, X, 
               
               
                   
                   
                   
                 XI, gelagin, elastin, 
               
               
                   
                   
                   
                 laminin 
               
               
                   
                 MMP-9 
                 KC, 
                 I, II, III, IV, V, VII, X, 
               
               
                   
                   
                 HSC, HC 
                 XI, gelagin, elastin, 
               
               
                   
                   
                   
                 laminin 
               
               
                   
                 MMP-7 
                 HSC 
                 Entactin, gelatin, elastin, 
               
               
                   
                   
                   
                 fibronectin, vitronectin, 
               
               
                   
                   
                   
                 laminin, fibrinogen 
               
               
                 Metalloelastase 
                 MMP-12 
                 Macrophages 
                 Elastin, gelatins, IV, 
               
               
                   
                   
                   
                 laminin, fibronectin, 
               
               
                   
                   
                   
                 entactin, vitronectin, 
               
               
                   
                   
                   
                 proteoglycan, myelin basic 
               
               
                   
                   
                   
                 protein, α1-antitripsin 
               
               
                 MT-MMPs 
                 MMP-14 
                 HSC, MFB, 
                 I, II, III, gelatin, 
               
               
                   
                   
                 KC 
                 fibronectin, vitronectin, 
               
               
                   
                   
                   
                 laminin, fibrinogen, pro- 
               
               
                   
                   
                   
                 MMP-2, pro-MMP-13 
               
               
                   
                 MMP-15 
                 HC, BDEC 
                 Pro-MMP-2, fibronectin, 
               
               
                   
                   
                   
                 tenascin, laminin, aggrecan, 
               
               
                   
                   
                   
                 perlecan 
               
               
                   
               
            
           
         
       
     
     TIMPs block MMPs&#39; proteolytic activity by binding in a substrate- and tissue-specific manner to MMP and membrane-type 1 metalloproteinase in a trimolecular complex (Table 6). During fibrosis TIMP levels increase dramatically, and MMP levels increase modestly or remain relatively static (except MMP-2) which in all gives a decrease in degradation of collagens. 
                     TABLE 6                  TIMPs in the liver 31                           Name   Sources   Metalloproteinase inhibited               TIMP-1   HSC, MFB, KC, HC   Pro-MMP-9, MMP-1, MMP-2,               MMP-3, MMP-13       TIMP-2   KC, HSC   MT-MMP-1, MT-MMP-2, proMMP-2,               MMP-3, MMP-13, MMP-7       TIMP-3   HC   MT-MMP-1, MT-MMP-2, TACE,               MMP-13                    
Fibroblast Activation Protein
 
     Fibroblast Activation Protein alpha subunit (FAPa or FAP, alpha) is an integral membrane gelatinase belonging to the serine protease family. FAPa is the alpha subunit and DPP4 (CD26) the beta subunit of a heterodimeric membrane-bound proteinase complex also known as 170 kDa Melanoma Membrane Gelatinase, Integral Membrane Serine Proteinase and Seprase. Some cells make only FAPa homodimers, some only DPP4 homodimers. The monomer is inactive. FAP, alpha is selectively expressed in reactive stromal fibroblasts of epithelial cancers, granulation tissue of healing wounds, and malignant cells of bone and soft tissue sarcomas 33 . This protein is thought to be involved in the control of fibroblast growth or epithelial-mesenchymal interactions during development, tissue repair, and epithelial carcinogenesis. It has been shown that expression of FAP increase with the stage of fibrosis 35 . 
     ADAMTS 
     ADAMTS (A Disintegrin And Metalloproteinase with Thrombospondin Motifs) is a family of peptidases, and until now 19 members of this family have been identified in humans. Known functions of the ADAMTS proteases include processing of procollagens and von Willebrand factor as well as cleavage of aggrecan, versican, brevican and neurocan. They have been demonstrated to have important roles in connective tissue organization, coagulation, inflammation, arthritis, angiogenesis and cell migration. 
     Cathepsins 
     There are approximately a dozen members of the cathepsins family of proteases, which are distinguished by their structure, catalytic mechanism, and which proteins they cleave. Most of the members become activated at the low pH found in lysosomes. Thus, the activity of this family lies almost entirely within those organelles (although there are many exceptions). Such as cathepsin K which (among other activities) works extracellularly after secretion by osteoclasts in bone resorption. 
     Cathepsin K (CAT K) and Cathepsin S (CAT S) are both cysteine proteases. 
     Fibrosis Biomarkers 
     A number of biochemical markers have been suggested for fibrotic diseases, although not specific product of the disease. In Table 7 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 12, 36-42 . 
     Table 7 summarizes some of the known markers of liver fibrosis. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                   
                   
                 Chronic 
                   
               
               
                   
                   
                 liver 
               
               
                 Biomarker 
                 Parameters 
                 disease 
                 Reference 
               
               
                   
               
             
            
               
                 One parameter 
                   
                   
                   
               
               
                 CRP 
                   
                 NASH 
                 
                   43 
                 
               
               
                 Hyaluronan 
                   
                 HCV 
                 
                   44-47 
                 
               
               
                 IGF-I 
                   
                 HCV 
                 
                   48 
                 
               
               
                 Leptin 
                   
                 HCV 
                 
                   49 
                 
               
               
                 PIIIP 
                   
                 HCV 
                 
                   50 
                 
               
               
                 Several 
               
               
                 parameters 
               
               
                 MP3 
                 PIIINP, MMP1 
                 HCV 
                 
                   51, 52 
                 
               
               
                 Zheng et al 
                 HA, PIIICP, PIIINP, 
                 Chronic 
                 
                   53 
                 
               
               
                 index 
                 Laminin, C-IV 
                 hepatitis 
               
               
                 Lebensztjen et 
                 Laminin-2, C-IV, MMP2, 
                 HBV 
                 
                   54 
                 
               
               
                 al index 
                 MMP9-TIMP1 index 
               
               
                   
                 Tenascin, hyaluronana, 
                 HBV 
                 
                   55 
                 
               
               
                   
                 Colalegn VI, TIMP-1 
               
               
                 Tsochatzis et 
                 Leptin, adiponectin, 
                 HCV, HBC, 
                 
                   56 
                 
               
               
                 al index 
                 resistin 
                 NASH 
               
               
                 Patel et al 
                 Hyaluronan, TIMP-1, α 2 - 
                 HCV 
                 
                   57 
                 
               
               
                 index 
                 macroglobulin 
               
               
                   
                 TIMP-1, tenascin, collagen 
                 NASH 
                 
                   58 
                 
               
               
                   
                 IV, PIIINP, MMP2, laminin, 
               
               
                   
                 Hyaluronan 
               
               
                 Forns-index 
                 Age, platelet count, γGT, 
                 HCV 
                 
                   51, 59-62 
                 
               
               
                 (76, 77) 
                 cholesterol 
                 HIV/HCV 
               
               
                 FibroTest 
                 Haptoglobin, α 2 - 
                 HCV 
                 
                   45, 51, 60, 61, 
                 
               
               
                 (76, 78) 
                 macroglobulin, 
                 HIV/HCV 
                 
                   63-75 
                 
               
               
                   
                 apolipoprotein A1, γGT, 
                 NAFLD 
               
               
                   
                 bilirubin 
                 NAFLD in 
               
               
                   
                   
                 diabetes 
               
               
                   
                   
                 patients 
               
               
                 Actitest 
                 FibroTest + ALT 
                 HCV 
                 
                   65, 76-78 
                 
               
               
                 APRI (Wai- 
                 AST, platelet count 
                 HIV/HCV 
                 
                   45, 51, 60, 61, 
                 
               
               
                 index) 
                   
                 HCV 
                 
                   64, 66, 79-87 
                 
               
               
                   
                   
                 NAFLD 
               
               
                 Hepascore 
                 Bilirubin, γGT, 
                 HCV 
                 
                   51, 61, 64, 66, 
                 
               
               
                   
                 hyaluronan, α 2 - 
                 HIV/HCV 
                 
                   88 
                 
               
               
                   
                 macroglobulin, age, gender 
               
               
                 FIB-4 
                 Platelet count, AST, ALT, 
                 HIV/HCV 
                 
                   61, 83 
                 
               
               
                   
                 age 
               
               
                 SHASTA 
                 Hyaluronan, albumin, AST 
                 HIV/HCV 
                 
                   61 
                 
               
               
                 Fibroindex 
                 FORN + APRI 
                 HCV 
                 
                   89 
                 
               
               
                 Fibrometer 
                 Platelet count, 
                 HIV/HCV 
                 
                   51, 61, 64, 66, 
                 
               
               
                 test 
                 prothrombin index, AST, α 2 - 
                 HCV 
                 
                   81 
                 
               
               
                   
                 macroglobulin, hyaluronan, 
                 NAFLD 
               
               
                   
                 urea, age 
               
               
                 NFSA 
                 Age, hyperglycaemia, body 
                 NAFLD 
                 
                   81 
                 
               
               
                   
                 mass index, platelets, 
               
               
                   
                 albumin, AST/ALT 
               
               
                 Ultrasound + 
                   
                 HCV 
                 
                   82 
                 
               
               
                 APRI 
               
               
                 Metwally et al 
                 Platelet count, albumin, 
                 HCV 
                 
                   90 
                 
               
               
                 index 
                 AST, history of blood 
               
               
                   
                 transfusion, HBV core 
               
               
                   
                 antibody 
               
               
                 Mohamadnejad 
                 Age, HBV DNA levels, 
                 HCV 
                 
                   91 
                 
               
               
                 et al index 
                 alkaline phosphatase, 
               
               
                   
                 albumin, platelet counts, 
               
               
                   
                 AST 
               
               
                 FibroSpect II 
                 Hyaluronan, TIMP-1, α 2 - 
                 HCV 
                 
                   85, 92, 93 
                 
               
               
                   
                 macroglobulin 
               
               
                 Stepwise 
                 Combination of APRI and 
                 HCV 
                 
                   94 
                 
               
               
                 combination 
                 Fibrotest 
               
               
                 algorithms 
               
               
                 Imbert-Bismut 
                 α 2  macroglobulin, AST, ALT 
                 HCV 
                 
                   95 
                 
               
               
                 index 
                 γGT, total bilirubin, 
               
               
                   
                 albumin, α 1  globulin, α 2   
               
               
                   
                 globulin, β globulin, γ 
               
               
                   
                 globulin, apolipoprotein A 1   
               
               
                 Nunes et al 
                 Age, Platelets, INR, CD4, 
                 HCV/HIV 
                 
                   96 
                 
               
               
                   
                 AST/ALT, Hyaluronan, YKL- 
                 HCV 
               
               
                   
                 40, PIIINP 
               
               
                 Fibroscan+++ 
                 Fibroscan, Fibrotest, 
                 HCV 
                 
                   97 
                 
               
               
                   
                 APRI, 
               
               
                   
               
            
           
         
       
     
     U.S. Pat. No. 5,387,504 describes the neo-epitope VDIPEN released by the action of stromelysin at the aggrecan site N 341 -F 342  and an RIA assay employing a monoclonal antibody specific for this neo-epitope. More generally the use of monospecific antibodies specific for fragments of aggrecan, generated by specific stromelysin cleavage are described. Elevations of stromelysin occur in osteoarthritis, rheumatoid arthritis, atherosclerotic lesions, gout, inflammatory bowel disease (IBD), idiopathic pulmonary fibrosis (IPF), certain cancers, joint injuries, and numerous inflammatory diseases. Stromelysin is reported to be elevated in idiopathic pulmonary fibrosis, and it is alleged that the assay can be conducted on blood or other biological fluids to detect stromelysin cleavage products of aggrecan and that quantitation of such fragments can be used diagnostically in respect of IPF as well as other conditions. However, no evidence for this is provided and there have to our knowledge been no subsequent publications validating this prediction. Such RIA assays have been commercially available for many years and no reports of their successful use in diagnosing or monitoring any fibrotic disease have appeared. 
     U.S. Pat. No. 7,225,080 discloses a method for diagnosis of an inflammatory, a fibrotic or a cancerous disease in a patient by measuring the values of at least four biochemical markers selected from the group consisting of α2-macroglobulin, AST (aspartate aminotransferase), ALT (alanine aminotransferase), GGT (gammaglutamyl transpeptidase), γ-globulin, total bilirubin, albumin, α1-globulin, α2-globulin, haptoglobin, β-globulin, apoA1, IL-10, TGF-β1, apoA2, and apoB in the serum or plasma of said patient, and subsequently combining said values in order to determine the presence of liver fibrosis and/or liver necroinflammatory lesions in said patient. The patent does not teach the quantitative measurement of peptide fragment carrying neo-epitopes generated during fibrotic disease. 
     U.S. Pat. No. 6,060,255 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. 
     U.S. Pat. No. 4,628,027 (Gay) discloses the production of antibodies specific for connective tissue proteins and, more particularly, the production of monoclonal antibodies by fused cell hybrids against human collagens and enzymes involved in collagen degradation. The use of monoclonal antibodies against connective tissue proteins to establish the collagen profile of histological, cytological and biological fluid samples is described. However, the patent does not describe the measurement of connective tissue proteins based on the binding of antibodies to neo-epitopes on said connective tissue proteins. 
     Guañabens N et al, J Bone Miner Res, 1998 98  evaluated the bone turnover markers N-telopeptide of type I collagen (NTX), C-telopeptide of type I collagen (CTX) and N-terminal pro-peptide of collagen type I (PINP) in patients with primary biliary cirrhosis, a disease with increased hepatic fibrosis. The level of NTX, CTX and PINP were elevated in patients compared to controls and correlated with the histological stage of the disease. The antibodies employed in the NTX were raised against a cathepsin K cleaved site in the N-terminal of collagen type I and are dependent on the neoepitope YDGKGVG↓ SEQ ID NO 302. The antibodies employed in the CTX were raised against a cathepsin K cleaved site in the C-terminal of collagen type I and are dependent on the neoepitope EKAHDGGR↓ SEQ ID NO 303. These markers are located in telopeptides of collagen type I and not in the internal part (the triple helical part) of collagen type I. The monoclonal antibodies employed for the PINP assay were raised against an internal epitope in the PINP sequence which is not a neo-epitope. 
     Møller S et al, Gut., 1999 99  demonstrated that the C-terminal cross linked telopeptide of type I collagen (ICTP) was elevated in alcoholic cirrhosis patients compared to controls. The study described showed that a biochemical marker can reflect hepatic fibrosis. The ICTP polyclonal antibody has been raised against trypsin and collagenase cleaved collagen type I. However, the antibodies are not binding to a neo-epitope. 
     Rosen H N et al, Calcif Tissue Int, 2004 100  assessed the bone turnover markers N-telopeptide of type I collagen (NTX) and C-telopeptide of type I collagen (CTX) in women receiving hormone replacement treatment (HRT). In the study it was observed that the bone turnover markers decreased with treatment. The antibodies employed in the NTX were raised against a cathepsin K cleaved site in the N-terminal of collagen type I and are dependent on the neoepitope YDGKGVG↓ SEQ ID NO 302. The antibodies employed in the CTX were raised against a cathepsin K cleaved site in the C-terminal of collagen type I and are dependent on the neoepitope EKAHDGGR↓ SEQ ID NO 303. In contrast to the present invention, these antibodies were used for evaluation of bone metabolism and not fibrosis. 
     Lein M et al, Eur Urol, 2007 101  evaluated the use of the neo-epitope specific bone turnover markers N-telopeptide of type I collagen (NTX) and C-telopeptide of type I collagen (CTX) in prostate cancer patients receiving zoledronic acid. In the study it was observed that the bone turnover markers decreased with treatment. The antibodies employed in the NTX were raised against a cathepsin K cleaved site in the N-terminal of collagen type I and are dependent on the neoepitope YDGKGVG↓ SEQ ID NO 302. The antibodies employed in the CTX were raised against a cathepsin K cleaved site in the C-terminal of collagen type I and are dependent on the neoepitope EKAHDGGR↓ SEQ ID NO 303. In contrast to the present invention, these antibodies were used for evaluation of the bone metabolism during invasion of bone metastases and not fibrosis. 
     PIIINP has been used in a number of studies to assess the severity of fibrotic disease 102  in patients with skin fibrosis following severe burn trauma 103 , for disease progression in noncirrhotic primary biliary cirrhosis 104  in primary biliary cirrhosis and chronic viral hepatitis C 105 . 
     PIIINP and ICTP were measured in patients with fibrosis of the myocardium 106 . 
     Many reports combine a set of biochemical markers to improve the predictive value of the biochemical index. Eleven different serum markers were measured in 205 patients with fibrotic staging from F0 to F4, and the most informative markers were alpha2 macroglobulin, alpha2 globulin (or haptoglobin), gamma globulin, apolipoprotein A1, gamma glutamyltranspeptidase, and total bilirubin 107 . An index of these markers had a negative predictive value (100% certainty of absence of F2, F3, or F4) was obtained for scores ranging from zero to 0.10 (12% [41] of all patients), and high positive predictive value (&gt;90% certainty of presence of F2, F3, or F4) for scores ranging from 0.60 to 1.00 (34% [115] of all patients). 
     WO2010/115749 discloses numerous neoepitopes of some of the proteins described above as fibrosis biomarkers and WO2009/059972 discloses numerous neoepitopes of some of the proteins described above as biomarkers of cardiovascular disease. 
     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 or inflammatory disease. 
    
    
     
       The invention will be further described and illustrated with reference to the following drawing in which: 
         FIG. 1  in panels A and B shows the results of investigating the specificity of a monoclonal antibody according to the invention; and 
         FIG. 2  shows the results of investigating the amount of Collagen V neo-epitope bearing fragments in human serum from ankylosing spondylitis patients. 
     
    
    
     The present invention now provides a method of bioassay comprising, conducting an immunoassay to measure neo-epitope containing protein fragments naturally present in a patient biofluid 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, wherein said neo-epitope is formed by cleavage of a said protein at any one of the cleavage sites shown in the following table: 
     
       
         
           
               
             
               
                 TABLE 8 
               
               
                   
               
             
            
               
                 Collagen Type I 
               
            
           
           
               
               
            
               
                 Protease 
                 Cleavage sites marked ‘.’; SEQ ID NO in ( ) 
               
               
                   
               
               
                 FAP 
                 Q.GA P GLQG M PG.E (1) 
               
               
                   
               
               
                 FAP 
                 D.RGE P G P PGPAGFAGP P GAD.G (2) 
               
               
                   
               
               
                 FAP 
                 D.GVRGLTGPIGPPG P AGA P GD.K (3) 
               
               
                   
               
               
                 FAP 
                 A.GL P GA K GLTGSPGS P G P DGK.T (4) 
               
               
                   
               
               
                 FAP 
                 D.AGPVGPPGP P G PP G P PGPPSAG.F (5) 
               
               
                   
               
               
                 FAP 
                 D.GLNGLPGPIG PP G P RGRTGD.A (6) 
               
               
                   
               
               
                 FAP 
                 S.PGKDGVRGLTGPIGP P GPAGA P .G (7) 
               
               
                   
               
               
                 FAP 
                 L.PGP P G PP GPPGPPGLGGNFAPQ.L (8) 
               
               
                   
               
               
                 FAP 
                 L. P G PP GPPGPPGPPGLGGNFA P Q.L (9) 
               
               
                   
               
               
                 FAP 
                 P.G PP G P PGPPGLGGNFAPQLSY.G (10) 
               
               
                   
               
               
                 FAP 
                 P.GPPG PP G PP GPPGLGGNFAPQL.S (11) 
               
               
                   
               
               
                 FAP 
                 F.PGARGPSG P QG P GG P PGP K GNSG.E (12) 
               
               
                   
               
               
                 FAP 
                 G.L P G PP GPPGPPGPPGLGGNFA P Q.L (13) 
               
               
                   
               
               
                 FAP 
                 G.L P GPPG PP GPPGPPGLGGNFA P Q.L (14) 
               
               
                   
               
               
                 FAP 
                 G.L P G PP GPPGPPGP P GLGGNFA P Q.L (15) 
               
               
                   
               
               
                 FAP 
                 D.AGPVGPPGP P G PP G PP GPPSAGFD.F (16) 
               
               
                   
               
               
                 FAP 
                 D.GLNGLPGPIGPPGPRGRTGDAGPVGP P G PP G PP G PP GPPSAG.F (17) 
               
               
                   
               
               
                 FAP 
                 P.GPPG PP G PP GPPGLGGNFAPQLSYGYDE K STGGISVPGP M GP.S (18) 
               
               
                   
               
               
                 FAP 
                 D.GLNGLPGPIGPPGPRGRTGDAG P VG PP G PP G PP G P PGPPSAGFD.F 
               
               
                   
                 (19) 
               
               
                   
               
               
                 MMP9 + FAP 
                 D.RGE P G P PGPAGFAGP P GAD.G (20) 
               
               
                   
               
               
                 MMP9 + FAP 
                 D.GVRGLTGPIGP P GPAGA P GD.K (21) 
               
               
                   
               
               
                 MMP9 + FAP 
                 D.AGPVGPPG PP G PP GPPGPPSAG.F (22) 
               
               
                   
               
               
                 MMP9 + FAP 
                 D.GLNGL P GPIG PP GPRGRTGD.A (23) 
               
               
                   
               
               
                 MMP9 + FAP 
                 D.AGPVGPPGPPG PP G P PGPPSAGF.D (24) 
               
               
                   
               
               
                 MMP9 + FAP 
                 L. P GPPGPPGPPG PP GLGGNFA P Q.L (25) 
               
               
                   
               
               
                 MMP9 + FAP 
                 P.G PP GPPGP P GLGGNFA P QLSY.G (26) 
               
               
                   
               
               
                 MMP9 + FAP 
                 P.GPPG PP G PP GPPGLGGNFAPQL.S (27) 
               
               
                   
               
               
                 MMP9 + FAP 
                 G.L P G PP GPPGPPGPPGLGGNFA P Q.L (28) 
               
               
                   
               
               
                 MMP9 + FAP 
                 L.PGP P G PP G PP GPPGLGGNFAPQL.S (29) 
               
               
                   
               
               
                 MMP9 + FAP 
                 P.GPPG PP G PP GPPSAGFDFSFLPQPPQE K AHDGGRYYR.A (30) 
               
               
                   
               
               
                 MMP9 + FAP 
                 D.GLNGLPGPIGPPGPRGRTGDAG P VG PP G PP G PP G P PGPPSAGFD.F 
               
               
                   
                 (19) 
               
               
                   
               
               
                 MMP9 
                 G.AAGPPGP.T (31) 
               
               
                   
               
               
                 MMP9 
                 P.VGPVGARGP.A (32) 
               
               
                   
               
               
                 MMP9 
                 P.AGPVGPVGARGP.A (33) 
               
               
                   
               
               
                 MMP9 
                 P.RGL P GP P GA P GP.Q (34) 
               
               
                   
               
               
                 MMP9 
                 G.EAGR P GEAGLPG.A (35) 
               
               
                   
               
               
                 MMP9 
                 Q.DGR P G PP G PP GA.R (36) 
               
               
                   
               
               
                 MMP9 
                 K.DGLNGL P GPIGP P GP.R (37) 
               
               
                   
               
               
                 MMP9 
                 G.AKGE P G P VGVQGP P GP.A (38) 
               
               
                   
               
               
                 MMP9 
                 P.AGARGNDGATGAAGP P GP.T (39) 
               
               
                   
               
               
                 MMP9 
                 P.VGPPG PP G P PGPPGPPSAGF.D (40) 
               
               
                   
               
               
                 MMP9 
                 A.GA P G K DGLNGLPGPIGPPGP.R (41) 
               
               
                   
               
               
                 MMP9 
                 D.AGPVGPPGP P G PP G P PGPPSAG.F (42) 
               
               
                   
               
               
                 MMP9 
                 S.AGAPGKDGLNGL P G P IG PP GP.R (43) 
               
               
                   
               
               
                 MMP9 
                 G.PPGPPGP PG LGGNFA P QLSYG.Y (44) 
               
               
                   
               
               
                 MMP9 
                 P.GP P G PP GPPGPPGLGGNFAPQL.S (45) 
               
               
                   
               
               
                 MMP9 
                 G.L P G PP GPPGPPGPPGLGGNFA P Q.L (46) 
               
               
                   
               
               
                 MMP9 
                 G.ERGPPGP M GPPGLAGPPGESGREGAPGAEGSPG.R (47) 
               
               
                   
               
               
                 FAP 
                 853‘.IGNVGAPGAK’862 (alpha 2 chain) (48) 
               
               
                   
               
               
                 FAP 
                 462‘.AGKEGPVGLP’471 (alpha 2 chain) (49) 
               
               
                   
               
               
                 FAP 
                 249‘.IGSAGPPGFP’258 (alpha 2 chain) (50) 
               
               
                   
               
               
                 FAP 
                 1153‘.DGLNGLPGPI’1162 (alpha 1 chain) (51) 
               
               
                   
               
               
                 FAP 
                 1153‘.D(β)GLNGLPGPI’1162 (alpha 1 chain) (52) 
               
               
                   
               
               
                 FAP 
                 751‘.KGADGSPGKD’760 (alpha 1 chain) (53) 
               
               
                   
               
               
                 FAP 
                 171‘STGGISVPGP.’180 (alpha 1 chain) (54) 
               
               
                   
               
               
                 FAP 
                 167‘.YDEKSTGGIS’176 (alpha 1 chain) (55) 
               
               
                   
               
            
           
           
               
            
               
                 Collagen Type III 
               
            
           
           
               
               
            
               
                 Protease 
                 Cleavage sites marked ‘.’ SEQ ID NO in ( ) 
               
               
                   
               
               
                 FAP 
                 E.AGIPGVPGAK.G (56) 
               
               
                   
               
               
                 FAP 
                 P.KGDPGPPGIP.G (57) 
               
               
                   
               
               
                 FAP 
                 G.PQGPKGDPGPP.G (58) 
               
               
                   
               
               
                 FAP 
                 G.PGMRGMPGSPGGP.G (59) 
               
               
                   
               
               
                 FAP 
                 D.GPPGPAGNTGAPGSPGVSGPKGD.A (60) 
               
               
                   
               
               
                 FAP 
                 H.AGAQGPPGPPGINGSPGGKGEMGPAGIP.G (61) 
               
               
                   
               
               
                 FAP 
                 D.GPRGPTGPIGPPGPAGQPGD.K (62) 
               
               
                   
               
               
                 MMP9 
                 D.GPPGPAGNTGAPGSPGVSGPKGD.A (63) 
               
               
                   
               
               
                 MMP9 
                 P.GSPGPAGQQGAIGSPGPAGPRGP.V (64) 
               
               
                   
               
               
                 MMP9 
                 G.ERGRPGLP.G (65) 
               
               
                   
               
               
                 MMP9 
                 G.KGDRGENGSPG.A (66) 
               
               
                   
               
               
                 MMP9 
                 E.PGKNGAKGEPGP.R (67) 
               
               
                   
               
               
                 MMP9 
                 S.PGERGETGPPGP.A (68) 
               
               
                   
               
               
                 MMP9 
                 P.GGPGADGVPGKDGP.R (69) 
               
               
                   
               
               
                 MMP9 
                 G.PPGKDGTSGHPGP.I (70) 
               
               
                   
               
               
                 MMP9 
                 R.GLPGPPGIKGPAGIPG.F (71) 
               
               
                   
               
               
                 MMP9 
                 L.PGENGAPGPMGPRGAPGE.R (72) 
               
               
                   
               
               
                 MMP9 
                 G.APGAPGGKGDAGAPGERGPP.G (73) 
               
               
                   
               
               
                 MMP9 
                 A.PGLKGENGLPGENGAPGPMGPRGAPG.E (74) 
               
               
                   
               
               
                 MMP9 
                 G.IAGITGARGLAGPPGMPGPRGSPGPQ.G (75) 
               
               
                   
               
               
                 MMP9 
                 G.SPGAKGEVGPAGSPGSNGAPGQRGEPGP.Q (76) 
               
               
                   
               
               
                 MMP9 
                 G.LKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAA.G (77) 
               
               
                   
               
               
                 MMP9 + FAP 
                 D.GPPGPAGNTGAPGSPGVSGPKGD.A (78) 
               
               
                   
               
               
                 ADAMTS-4 
                 267‘.GFDGRNGEKG’276 (79) 
               
               
                   
               
               
                 FAP 
                 533‘.PGMRGMPGSP’542 (alpha 1 chain) (80) 
               
               
                   
               
               
                 Unknown 
                 642‘.GLPGTGGPPG’651 (81) 
               
               
                   
               
               
                 Unknown 
                 652‘ENGKPGEPGP.’661 (82) 
               
               
                   
               
               
                 Unknown 
                 861‘.GERGSPGGPG’870 (83) 
               
               
                   
               
               
                 unknown 
                 899‘PGKDGPPGPA.’908 (alpha 1 chain) (84) 
               
               
                   
               
               
                 MMP9, 
                 442‘.GLPGTGGPPG’451 (85) 
               
               
                 ADAMTS-4 
               
               
                   
               
            
           
           
               
            
               
                 Collagen Type IV 
               
            
           
           
               
               
            
               
                 Protease 
                 Cleavage sites marked ‘.’ SEQ ID NO in ( ) 
               
               
                   
               
               
                 MMP9 
                 333‘.IVIGTGPLGE’342 (alpha 1 chain) (86) 
               
               
                   
               
               
                 MMP9 
                 328‘PGPPGIVIGT.’337 (alpha 1 chain) (87) 
               
               
                   
               
            
           
           
               
            
               
                 Collagen Type V 
               
            
           
           
               
               
            
               
                 Protease 
                 Cleavage sites marked ‘.’ SEQ ID NO in ( ) 
               
               
                   
               
               
                 MMP2 
                 920‘ERGPRGITGK.’929 (alpha 1 chain) (88) 
               
               
                   
               
               
                 MMP2/9 
                 1584‘.RRNIDASQLL’1593 (alpha 1 chain) (89) 
               
               
                   
               
               
                 MMP2/9 
                 229‘.QQGGAGPTGP’238 (alpha 2 chain) (90) 
               
               
                   
               
               
                 MMP2/9 
                 355‘.QRGAHGMPGK’364 (alpha 2 chain) (91) 
               
               
                   
               
               
                 MMP9 
                 525‘.RVGKMGRPGA’534 (alpha 3 chain) (92) 
               
               
                   
               
               
                 MMP2/9 
                 1317‘.HMGREGREGE’1326 (alpha 3 chain) (93) 
               
               
                   
               
            
           
           
               
            
               
                 Collagen Type VI 
               
            
           
           
               
               
            
               
                 Protease 
                 Cleavage sites marked ‘.’ 
               
               
                   
               
               
                 MMP8 
                 873‘.RIAVAQYSDD’883 (94) 
               
               
                   
               
               
                 MMP8 
                 1192‘.QLGTVQQVIS’1202 (95) 
               
               
                   
               
               
                 MMP12 
                 1231‘.RDVVFLIDGS’1241 (96) 
               
               
                   
               
            
           
           
               
            
               
                 ApoE 
               
            
           
           
               
               
            
               
                 Protease 
                 Cleavage sites marked ‘.’ SEQ ID NO in ( ) 
               
               
                   
               
               
                 MMP9 
                 81‘.LMDETM’86 (97) 
               
               
                   
               
               
                 Cat K 
                 246‘RLDEVK.’251 (98) 
               
               
                   
               
               
                 MMP9 
                 101‘TPVAEE.’106 (99) 
               
               
                   
               
               
                 Cat K 
                 126‘.MEEMGS’131 (100) 
               
               
                   
               
            
           
           
               
            
               
                 Biglycan 
               
            
           
           
               
               
            
               
                 Protease 
                 Cleavage sites marked ‘.’ SEQ ID NO in ( ) 
               
               
                   
               
               
                 MMP3 
                 219‘.KLTGIPKDLP’228 (101) 
               
               
                   
               
               
                 MMP3 
                 226‘DLPETLNELH.’235 (102) 
               
               
                   
               
               
                 MMP12 
                 220‘LTGIPKDLPE.’229 (103) 
               
               
                   
               
               
                 MMP13 
                 329‘.RAYYNGISLF’338 (104) 
               
               
                   
               
               
                 MMP13 
                 334‘GISLFNNPVP.’343 (105) 
               
               
                   
               
               
                 MMP12 
                 87‘.EISPDTTLLD’96 (106) 
               
               
                   
               
               
                 MMP12 
                 97‘LQNNDISELR.’106 (107) 
               
               
                   
               
               
                 MMP9 
                 188‘.IEMGGNPLENS’198 (108) 
               
               
                   
               
               
                 MMP9 
                 200‘FEPGAFDGLK.’209 (109) 
               
               
                   
               
               
                 MMP9 
                 148‘.NHLVEIPPNL’157 (110) 
               
               
                   
               
               
                 MMP9 
                 151‘VEIPPNLPSS.’160 (111) 
               
               
                   
               
               
                 MMP9 
                 232‘.NELHLDHNK’ 240 (112) 
               
               
                   
               
               
                 MMP9 
                 344‘.YWEVQPATFR’ 353 (113) 
               
               
                   
               
               
                 MMP9, MMP12 
                 241‘.IQAIELEDLL’ 250 (114) 
               
               
                   
               
            
           
           
               
            
               
                 Decorin 
               
            
           
           
               
               
            
               
                 Protease 
                 Cleavage sites marked ‘.’ SEQ ID NO in ( ) 
               
               
                   
               
               
                 ADAM-TS4 
                 75‘.VPKDLPPDTT’84 (115) 
               
               
                   
               
            
           
           
               
            
               
                 Versican 
               
            
           
           
               
               
            
               
                 Protease 
                 Cleavage sites marked ‘.’ SEQ ID NO in ( ) 
               
               
                   
               
               
                 Cathepsin K 
                 3247‘.YENWRPNQPD’3256 (116) 
               
               
                   
               
               
                 Cathepsin K 
                 3255‘PDSFFSAGED.’3264 (117) 
               
               
                   
               
               
                 Cathepsin K 
                 3221‘.HDYQWIGLN’3229 (118) 
               
               
                   
               
               
                 MMP8 
                 3306‘.KTFGKMKPRY’3316 (119) 
               
               
                   
               
               
                 MMP8 
                 486‘.SVTQIEQIEV’495 (120) 
               
               
                   
               
               
                 MMP8 
                 491‘EQIEVGPLVT.’500 (121) 
               
               
                   
               
            
           
           
               
            
               
                 CRP 
               
            
           
           
               
               
            
               
                 Protease 
                 Cleavage sites marked ‘.’ SEQ ID NO in ( ) 
               
               
                   
               
               
                 Signal peptide 
                 013‘LSHAFG.’018 (122) 
               
               
                   
               
               
                 MMP3/8/9, 
                 011‘TSLSHA.’016 (123) 
               
               
                 Cat S/K 
               
               
                   
               
            
           
           
               
            
               
                 Elastin 
               
            
           
           
               
               
            
               
                 Protease 
                 Cleavage sites marked ‘.’ SEQ ID NO in ( ) 
               
               
                   
               
               
                 Signal peptide 
                 21‘LHPSRP.’26 (124) 
               
               
                   
               
               
                 ADAMTS1/4/8 
                 379‘KAAKYG.’384 (125) 
               
               
                   
               
               
                 MMP8/9 
               
               
                   
               
               
                 MMP9/12 
                 547‘GIGPGG.’552 (126) 
               
               
                   
               
            
           
           
               
            
               
                 Lumican 
               
            
           
           
               
               
            
               
                 Protease 
                 Cleavage sites marked ‘.’ SEQ ID NO in ( ) 
               
               
                   
               
               
                 MMP9 
                 75‘NNQIDHIDEK.’84 (127) 
               
               
                   
               
            
           
           
               
            
               
                 LAMC1 
               
            
           
           
               
               
            
               
                 Protease 
                 Cleavage sites marked ‘.’ SEQ ID NO in ( ) 
               
               
                   
               
               
                 MMP2/9 
                 664‘SAGYLDDVTL.’673 (128) 
               
               
                   
               
               
                 MMP9 
                 1232‘LNRKYEQAKN.’1241 (129) 
               
               
                   
               
            
           
           
               
            
               
                 LAMA2 
               
            
           
           
               
               
            
               
                 Protease 
                 Cleavage sites marked ‘.’ SEQ ID NO in ( ) 
               
               
                   
               
               
                 MMP2 
                 1240‘.QFEGKKLMAY’1249 (130) 
               
               
                   
               
               
                 MMP2/9 
                 2314‘.GLWNFREKEG’2323 (131) 
               
               
                   
               
            
           
           
               
            
               
                 LAMB1 
               
            
           
           
               
               
            
               
                 Protease 
                 Cleavage sites marked ‘.’ SEQ ID NO in ( ) 
               
               
                   
               
               
                 MMP9 
                 209‘ALDPAFKIED.’218 (132) 
               
               
                   
               
               
                 MMP2/9 
                 1629‘.SIESETAASE’1638 (133) 
               
               
                   
               
               
                 MMP2/9 
                 1639‘ETLFNASQRI.’1648 (134) 
               
               
                   
               
            
           
           
               
            
               
                 LAMA5 
               
            
           
           
               
               
            
               
                 Protease 
                 Cleavage sites marked ‘.’ SEQ ID NO in ( ) 
               
               
                   
               
               
                 MMP9 
                 2402‘.NSRNQERLEE’2411 (135) 
               
               
                   
               
               
                 MMP2/9 
                 2901‘EMDTLNEEVV.’2910 (136) 
               
               
                   
               
               
                 MMP9 
                 3056‘DLELADAYYL.’3065 (137) 
               
               
                   
               
            
           
         
       
     
     In amino acid sequences throughout this specification, P indicates hydroxyproline, M indicates oxidised methionine, and K indicates hydroxylysine. 
     The invention includes a method of bioassay comprising, conducting an immunoassay to measure neo-epitope containing protein fragments naturally present in a patient biofluid 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, wherein said protein is ApoE, or a laminin. These proteins are preferably human and the samples are preferably human but the samples and/or the proteins may be mammalian including especially rodent, for instance mouse or rat, or may be dog or primate including monkey. 
     The method may include associating an elevation of said measure in said patient above a normal level with the presence of fibrosis, or inflammatory disease. 
     Optionally, an elevated result in an immunoassay according to this invention is associated with skin fibrosis, lung fibrosis, or liver fibrosis or cardiovascular disease. Optionally, an elevated result in an immunoassay according to the invention may be associated with an inflammatory condition, such as ankylosing spondylitis. The method may comprise the preliminary step of obtaining a patient biofluid sample. 
     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′) 2 . Thus, said immunological binding partner may be a monoclonal antibody or a fragment of a monoclonal antibody having specific binding affinity. 
     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. 
     Collagen Assays 
     Collagen type I 
     We have determined that the enzymes listed in the following table cleave type I collagen at least the following cleavage sites (marked “.”): 
     
       
         
           
               
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Collagen type I cleavage sites. 
                   
               
            
           
           
               
               
            
               
                 Protease 
                 Cleavage sites marked ‘.’ 
               
               
                   
               
            
           
           
               
               
               
            
               
                 FAP 
                 Q.GA P GLQG M PG.E 
                   
               
               
                   
               
               
                 FAP 
                 D.RGE P G P PGPAGFAGP P GAD.G 
               
               
                   
               
               
                 FAP 
                 D.GVRGLTGPIGPPG P AGA P GD.K 
               
               
                   
               
               
                 FAP 
                 A.GL P GA K GLTGSPGS P G P DGK.T 
               
               
                   
               
               
                 FAP 
                 D.AGPVGPPGP P G PP G P PGPPSAG.F 
               
               
                   
               
               
                 FAP 
                 D.GLNGLPGPIG PP G P RGRTGD.A 
               
               
                   
               
               
                 FAP 
                 S.PGKDGVRGLTGPIGP P GPAGA P .G 
               
               
                   
               
               
                 FAP 
                 L.PGP P G PP GPPGPPGLGGNFAPQ.L 
               
               
                   
               
               
                 FAP 
                 L. P G PP GPPGPPGPPGLGGNFA P Q.L 
               
               
                   
               
               
                 FAP 
                 P.GPPG PP G P PGLGGNFAPQLSY.G 
               
               
                   
               
               
                 FAP 
                 P.GPPG PP G PP GPPGLGGNFAPQL.S 
               
               
                   
               
               
                 FAP 
                 F.PGARGPSG P QG P GG P PGP K GNSG.E 
               
               
                   
               
               
                 FAP 
                 G.L P G PP GPPGPPGPPGLGGNFA P Q.L 
               
               
                   
               
               
                 FAP 
                 G.L P GPPGPPGPPG PP GLGGNFA P Q.L 
               
               
                   
               
               
                 FAP 
                 G.L P G PP GPPGPPGP P GLGGNFA P Q.L 
               
               
                   
               
               
                 FAP 
                 D.AGPVGPPGP P G PP G PP GPPSAGFD.F 
               
               
                   
               
               
                 FAP 
                 D.GLNGLPGPIGPPGPRGRTGDAGPVGP P G PP G PP G PP GPPSAG.F 
               
               
                   
               
               
                 FAP 
                 D.GLNGLPGPIGPPGPRGRTGDAGPVGP P G PP G PP G PP GPPSAG.F 
               
               
                   
               
               
                 FAP 
                 P.GPPG PP G PP GPPGLGGNFAPQLSYGYDE K STGGISVPGP M GP.S 
               
               
                   
               
               
                 FAP 
                 D.GLNGLPGPIGPPGPRGRTGDAG P VG PP G PP G PP G P PGPPSAGFD.F 
               
               
                   
               
               
                 MMP9 + FAP 
                 D.RGE P G P PGPAGFAGP P GAD.G 
               
               
                   
               
               
                 MMP9 + FAP 
                 D.GVRGLTGPIGP P GPAGA P GD.K 
               
               
                   
               
               
                 MMP9 + FAP 
                 D.AGPVGPPG PP G PP GPPGPPSAG.F 
               
               
                   
               
               
                 MMP9 + FAP 
                 D.GLNGL P GPIG PP GPRGRTGD.A 
               
               
                   
               
               
                 MMP9 + FAP 
                 D.AGPVGPPGPPG PP G P PGPPSAGF.D 
               
               
                   
               
               
                 MMP9 + FAP 
                 L. P GPPGPPGPPG PP GLGGNFA P Q.L 
               
               
                   
               
               
                 MMP9 + FAP 
                 P.G PP GPPGP P GLGGNFA P QLSY.G 
               
               
                   
               
               
                 MMP9 + FAP 
                 P.GPPG PP G PP GPPGLGGNFAPQL.S 
               
               
                   
               
               
                 MMP9 + FAP 
                 G.L P G PP GPPGPPGPPGLGGNFA P Q.L 
               
               
                   
               
               
                 MMP9 + FAP 
                 L.PGP P G PP G PP GPPGLGGNFAPQL.S 
               
               
                   
               
               
                 MMP9 + FAP 
                 P.GPPG PP G PP GPPSAGFDFSFLPQPPQE K AHDGGRYYR.A 
               
               
                   
               
               
                 MMP9 + FAP 
                 D.GLNGLPGPIGPPGPRGRTGDAG P VG PP G PP G PP G P PGPPSAGFD.F 
               
               
                   
               
               
                 MMP9 
                 G.AAGPPGP.T 
               
               
                   
               
               
                 MMP9 
                 P.VGPVGARGP.A 
               
               
                   
               
               
                 MMP9 
                 P.AGPVGPVGARGP.A 
               
               
                   
               
               
                 MMP9 
                 P.RGL P GP P GA P GP.Q 
               
               
                   
               
               
                 MMP9 
                 G.EAGR P GEAGLPG.A 
               
               
                   
               
               
                 MMP9 
                 Q.DGR P G PP G PP GA.R 
               
               
                   
               
               
                 MMP9 
                 K.DGLNGL P GPIGP P GP.R 
               
               
                   
               
               
                 MMP9 
                 G.AKGE P G P VGVQGP P GP.A 
               
               
                   
               
               
                 MMP9 
                 P.AGARGNDGATGAAGP P GP.T 
               
               
                   
               
               
                 MMP9 
                 P.VGPPG PP G P PGPPGPPSAGF.D 
               
               
                   
               
               
                 MMP9 
                 A.GA P G K DGLNGLPGPIGPPGP.R 
               
               
                   
               
               
                 MMP9 
                 D.AGPVGPPGP P G PP G P PGPPSAG.F 
               
               
                   
               
               
                 MMP9 
                 S.AGAPGKDGLNGL P G P IG PP GP.R 
               
               
                   
               
               
                 MMP9 
                 G.PPGPPG PP GLGGNFA P QLSYG.Y 
               
               
                   
               
               
                 MMP9 
                 P.GP P G PP GPPGPPGLGGNFAPQL.S 
               
               
                   
               
               
                 MMP9 
                 G.L P G PP GPPGPPGPPGLGGNFA P Q.L 
               
               
                   
               
               
                 MMP9 
                 G.ERGPPGP M GPPGLAGPPGESGREGAPGAEGSPG.R 
               
               
                   
               
               
                 FAP 
                 P.IGNVGAPGAK (alpha 1 chain) 
               
               
                   
               
               
                 FAP 
                 P.AGKEGPVGLP (alpha 2 chain) 
               
               
                   
               
               
                 FAP 
                 P.IGSAGPPGFP (alpha 2 chain) 
               
               
                   
               
               
                 FAP 
                 853‘.IGNVGAPGAK’862 (alpha 2 chain) 
               
               
                   
               
               
                 FAP 
                 462‘.AGKEGPVGLP’471 (alpha 2 chain) 
               
               
                   
               
               
                 FAP 
                 249‘.IGSAGPPGFP’258 (alpha 2 chain) 
               
               
                   
               
               
                 FAP 
                 1153‘.DGLNGLPGPI’1162 (alpha 1 chain) 
               
               
                   
               
               
                 FAP 
                 1153‘.D(β)GLNGLPGPI’1162 (alpha 1 chain) 
               
               
                   
               
               
                 FAP 
                 751‘.KGADGSPGKD’760 (alpha 1 chain) 
               
               
                   
               
               
                 FAP 
                 171‘STGGISVPGP.’180 (alpha 1 chain) 
               
               
                   
               
               
                 FAP 
                 167‘.YDEKSTGGIS’176 (alpha 1 chain) 
               
               
                   
               
               
                 FAP 
                 Q.GA P GLQG M PG.E 
               
               
                   
               
               
                 FAP 
                 D.RGE P G P PGPAGFAGP P GAD.G 
               
               
                   
               
               
                 FAP 
                 D.GVRGLTGPIGPPG P AGA P GD.K 
               
               
                   
               
               
                 FAP 
                 A.GL P GA K GLTGSPGS P G P DGK.T 
               
               
                   
               
               
                 FAP 
                 D.AGPVGPPGP P G PP G P PGPPSAG.F 
               
               
                   
               
               
                 FAP 
                 D.GLNGLPGPIG PP G P RGRTGD.A 
               
               
                   
               
               
                 FAP 
                 S.PGKDGVRGLTGPIGP P GPAGA P .G 
               
               
                   
               
               
                 FAP 
                 L.PGP P G PP GPPGPPGLGGNFAPQ.L 
               
               
                   
               
               
                 FAP 
                 L. P G PP GPPGPPGPPGLGGNFA P Q.L 
               
               
                   
               
               
                 FAP 
                 P.GPPG PP G P PGLGGNFAPQLSY.G 
               
               
                   
               
               
                 FAP 
                 P.GPPG PP G PP GPPGLGGNFAPQL.S 
               
               
                   
               
               
                 FAP 
                 F.PGARGPSG P QG P GG P PGP K GNSG.E 
               
               
                   
               
               
                 FAP 
                 G.L P G PP GPPGPPGPPGLGGNFA P Q.L 
               
               
                   
               
               
                 FAP 
                 G.L P GPPGPPGPPG PP GLGGNFA P Q.L 
               
               
                   
               
               
                 FAP 
                 G.L P G PP GPPGPPGP P GLGGNFA P Q.L 
               
               
                   
               
               
                 FAP 
                 D.AGPVGPPGP P G PP G PP GPPSAGFD.F 
               
               
                   
               
               
                 FAP 
                 D.GLNGLPGPIGPPGPRGRTGDAGPVGP P G PP G PP G PP GPPSAG.F 
               
               
                   
               
               
                 FAP 
                 D.GLNGLPGPIGPPGPRGRTGDAGPVGP P G PP G PP G PP GPPSAG.F 
               
               
                   
               
               
                 FAP 
                 P.GPPG PP G PP GPPGLGGNFAPQLSYGYDE K STGGISVPGP M GP.S 
               
               
                   
               
               
                 FAP 
                 D.GLNGLPGPIGPPGPRGRTGDAG P VG PP G PP G PP G P PGPPSAGFD.F 
               
               
                   
               
               
                 MMP9 + FAP 
                 D.RGE P G P PGPAGFAGP P GAD.G 
               
               
                   
               
               
                 MMP9 + FAP 
                 D.GVRGLTGPIGP P GPAGA P GD.K 
               
               
                   
               
               
                 MMP9 + FAP 
                 D.AGPVGPPG PP G PP GPPGPPSAG.F 
               
               
                   
               
               
                 MMP9 + FAP 
                 D.GLNGL P GPIG PP GPRGRTGD.A 
               
               
                   
               
               
                 MMP9 + FAP 
                 D.AGPVGPPGPPG PP G P PGPPSAGF.D 
               
               
                   
               
               
                 MMP9 + FAP 
                 L. P GPPGPPGPPG PP GLGGNFA P Q.L 
               
               
                   
               
               
                 MMP9 + FAP 
                 P.G PP GPPGP P GLGGNFA P QLSY.G 
               
               
                   
               
               
                 MMP9 + FAP 
                 P.GPPG PP G PP GPPGLGGNFAPQL.S 
               
               
                   
               
               
                 MMP9 + FAP 
                 G.L P G PP GPPGPPGPPGLGGNFA P Q.L 
               
               
                   
               
               
                 MMP9 + FAP 
                 L.PGP P G PP G PP GPPGLGGNFAPQL.S 
               
               
                   
               
               
                 MMP9 + FAP 
                 P.GPPG PP G PP GPPSAGFDFSFLPQPPQE K AHDGGRYYR.A 
               
               
                   
               
               
                 MMP9 + FAP 
                 D.GLNGLPGPIGPPGPRGRTGDAG P VG PP G PP G PP G P PGPPSAGFD.F 
               
               
                   
               
               
                 MMP9 
                 G.AAGPPGP.T 
               
               
                   
               
               
                 MMP9 
                 P.VGPVGARGP.A 
               
               
                   
               
               
                 MMP9 
                 P.AGPVGPVGARGP.A 
               
               
                   
               
               
                 MMP9 
                 P.RGL P GP P GA P GP.Q 
               
               
                   
               
               
                 MMP9 
                 G.EAGR P GEAGLPG.A 
               
               
                   
               
               
                 MMP9 
                 Q.DGR P G PP G PP GA.R 
               
               
                   
               
               
                 MMP9 
                 K.DGLNGL P GPIGP P GP.R 
               
               
                   
               
               
                 MMP9 
                 G.AKGE P G P VGVQGP P GP.A 
               
               
                   
               
               
                 MMP9 
                 P.AGARGNDGATGAAGP P GP.T 
               
               
                   
               
               
                 MMP9 
                 P.VGPPG PP G P PGPPGPPSAGF.D 
               
               
                   
               
               
                 MMP9 
                 A.GA P G K DGLNGLPGPIGPPGP.R 
               
               
                   
               
               
                 MMP9 
                 D.AGPVGPPGP P G PP G P PGPPSAG.F 
               
               
                   
               
               
                 MMP9 
                 S.AGAPGKDGLNGL P G P IG PP GP.R 
               
               
                   
               
               
                 MMP9 
                 G.PPGPPG PP GLGGNFA P QLSYG.Y 
               
               
                   
               
               
                 MMP9 
                 P.GP P G PP GPPGPPGLGGNFAPQL.S 
               
               
                   
               
               
                 MMP9 
                 G.L P G PP GPPGPPGPPGLGGNFA P Q.L 
               
               
                   
               
               
                 MMP9 
                 G.ERGPPGP M GPPGLAGPPGESGREGAPGAEGSPG.R 
               
               
                   
               
               
                 FAP 
                 P.IGNVGAPGAK (alpha 1 chain) 
               
               
                   
               
               
                 FAP 
                 P.AGKEGPVGLP (alpha 2 chain) 
               
               
                   
               
               
                 FAP 
                 P.IGSAGPPGFP (alpha 2 chain) 
               
               
                   
               
            
           
         
       
     
     Suitable immunological binding partners may therefore be specifically reactive with any of the following sequences at the N terminal of a peptide: 
     
       
         
           
               
             
               
                 TABLE 10 
               
               
                   
               
               
                 N-terminal sequences of protease generated peptide 
               
               
                 fragments of Collagen type I. (The symbol ‘.’ 
               
               
                 indicates the cleavage site) 
               
               
                 Cleavage sites marked ‘.’/SEQ ID NO ( ) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 .AAGPPG 
                 (138) 
               
               
                   
                   
               
               
                   
                 .AGPVGP 
                 (139) 
               
               
                   
                   
               
               
                   
                 .DGR P G P   
                 (140) 
               
               
                   
                   
               
               
                   
                 .EAGR P G 
                 (141) 
               
               
                   
                   
               
               
                   
                 .GA P GLQ 
                 (142) 
               
               
                   
                   
               
               
                   
                 .GLNGLP 
                 (143) 
               
               
                   
                   
               
               
                   
                 .GLNGL P   
                 (144) 
               
               
                   
                   
               
               
                   
                 .GL P GA K   
                 (145) 
               
               
                   
                   
               
               
                   
                 .GPPG PP   
                 (146) 
               
               
                   
                   
               
               
                   
                 .G PP GPP 
                 (147) 
               
               
                   
                   
               
               
                   
                 .GVRGLT 
                 (148) 
               
               
                   
                   
               
               
                   
                 .L P G PP G 
                 (149) 
               
               
                   
                   
               
               
                   
                 .L P GPPG 
                 (150) 
               
               
                   
                   
               
               
                   
                 .PGARGP 
                 (151) 
               
               
                   
                   
               
               
                   
                 .PGKDGV 
                 (152) 
               
               
                   
                   
               
               
                   
                 .PGP P G P   
                 (153) 
               
               
                   
                   
               
               
                   
                 . P G PP GP 
                 (154) 
               
               
                   
                   
               
               
                   
                 . P GPPGP 
                 (155) 
               
               
                   
                   
               
               
                   
                 .RGE P G P   
                 (156) 
               
               
                   
                   
               
               
                   
                 .RGL P GP 
                 (157) 
               
               
                   
                   
               
               
                   
                 .VGPVGA 
                 (158) 
               
               
                   
                   
               
               
                   
                 .DGLNGL 
                 (159) 
               
               
                   
                   
               
               
                   
                 .AKGE P G 
                 (160) 
               
               
                   
                   
               
               
                   
                 .AGARGN 
                 (161) 
               
               
                   
                   
               
               
                   
                 .VGPPG P   
                 (162) 
               
               
                   
                   
               
               
                   
                 .GA P G K D 
                 (163) 
               
               
                   
                   
               
               
                   
                 .IGSAGP 
                 (164) 
               
               
                   
                   
               
               
                   
                 .AGAPGK 
                 (165) 
               
               
                   
                   
               
               
                   
                 .PPGPPG 
                 (166) 
               
               
                   
                   
               
               
                   
                 .GP P G PP   
                 (167) 
               
               
                   
                   
               
               
                   
                 .YDEKST 
                 (168) 
               
               
                   
                   
               
               
                   
                 .ERGPPG 
                 (169) 
               
               
                   
                   
               
               
                   
                 .IGNVGA 
                 (170) 
               
               
                   
                   
               
               
                   
                 .AGKEGP 
                 (171) 
               
               
                   
                   
               
               
                   
                 .KGADGS 
                 (172) 
               
               
                   
                   
               
               
                   
                 .D(β)GLNGLP 
                 (173) 
               
               
                   
                   
               
            
           
         
       
     
     Alternatively, suitable immunological binding partners may be specifically reactive with any of the following sequences at the C terminal of a peptide: 
                     TABLE 11               C-terminal sequences of protease generated peptide       fragments of Collagen type I (The symbol ‘.’       indicates the cleavage site).       Cleavage sites marked ‘.’/SEQ ID NO ( )                                                AEGSPG.   (174)                       AGA P GD.   (175)                       AGPPGP.   (176)                       AGP P GP.   (177)                       APQLSY.   (178)                       A P QLSY.   (179)                       EAGLPG.   (180)                       GGRYYR.   (181)                       GNFAPQ.   (182)                       GNFA P Q.   (183)                       GPAGA P .   (184)                       GP P GAD.   (185)                       GPPSAG.   (186)                       IGP P GP.   (187)                       IGPPGP.   (188)                       IG PP GP.   (189)                       ISVPGP.   (190)                       LQG M PG.   (191)                       NFAPQL.   (192)                         P GA P GP.   (193)                         P G P DGK.   (194)                       PGP M GP.   (195)                         P G PP GA.   (196)                       P K GNSG.   (197)                       PPSAGF.   (198)                         P QLSYG.   (199)                       PSAGFD.   (200)                       QGP P GP.   (201)                       RGRTGD.   (202)                       VGARGP.   (203)                        
Collagen type III
 
     We have determined that the enzymes listed in the following table cleave type III collagen at least the following cleavage sites (marked *): 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Cleavage sites in collagen type III. 
               
            
           
           
               
               
            
               
                 Protease 
                 Cleavage sites marked ‘.’ 
               
               
                   
               
               
                 FAP 
                 E.AGIPGVPGAK.G 
               
               
                   
               
               
                 FAP 
                 P.KGDPGPPGIP.G 
               
               
                   
               
               
                 FAP 
                 G.PQGPKGDPGPP.G 
               
               
                   
               
               
                 FAP 
                 G.PGMRGMPGSPGGP.G 
               
               
                   
               
               
                 FAP 
                 D.GPPGPAGNTGAPGSPGVSGPKGD.A 
               
               
                   
               
               
                 FAP 
                 H.AGAQGPPGPPGINGSPGGKGEMGPAGIP.G 
               
               
                   
               
               
                 FAP 
                 D.GPRGPTGPIGPPGPAGQPGD.K 
               
               
                   
               
               
                 MMP9 
                 D.GPPGPAGNTGAPGSPGVSGPKGD.A 
               
               
                   
               
               
                 MMP9 
                 P.GSPGPAGQQGAIGSPGPAGPRGP.V 
               
               
                   
               
               
                 MMP9 
                 G.ERGRPGLP.G 
               
               
                   
               
               
                 MMP9 
                 G.KGDRGENGSPG.A 
               
               
                   
               
               
                 MMP9 
                 E.PGKNGAKGEPGP.R 
               
               
                   
               
               
                 MMP9 
                 S.PGERGETGPPGP.A 
               
               
                   
               
               
                 MMP9 
                 P.GGPGADGVPGKDGP.R 
               
               
                   
               
               
                 MMP9 
                 G.PPGKDGTSGHPGP.I 
               
               
                   
               
               
                 MMP9 
                 R.GLPGPPGIKGPAGIPG.F 
               
               
                   
               
               
                 MMP9 
                 L.PGENGAPGPMGPRGAPGE.R 
               
               
                   
               
               
                 MMP9 
                 G.APGAPGGKGDAGAPGERGPP.G 
               
               
                   
               
               
                 MMP9 
                 A.PGLKGENGLPGENGAPGPMGPRGAPG.E 
               
               
                   
               
               
                 MMP9 
                 G.IAGITGARGLAGPPGMPGPRGSPGPQ.G 
               
               
                   
               
               
                 MMP9 
                 G.SPGAKGEVGPAGSPGSNGAPGQRGEPGP.Q 
               
               
                   
               
               
                 MMP9 
                 G.LKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAA.G 
               
               
                   
               
               
                 MMP9 + FAP 
                 D.GPPGPAGNTGAPGSPGVSGPKGD.A 
               
               
                   
               
               
                 ADAMTS-4 
                 267‘.GFDGRNGEKG’276 
               
               
                   
               
               
                 FAP 
                 533‘.PGMRGMPGSP’542 (alpha 1 chain) 
               
               
                   
               
               
                 Unknown 
                 642‘.GLPGTGGPPG’651 
               
               
                   
               
               
                 Unknown 
                 652‘ENGKPGEPGP.’661 
               
               
                   
               
               
                 Unknown 
                 861‘.GERGSPGGPG’870 
               
               
                   
                 697‘GAGPPGPEGG’706 (alpha 1 chain) 
               
               
                   
                 804‘GPPGPAGFPG’813 (alpha 1 chain) 
               
               
                   
                 899‘PGKDGPPGPA.’908 (alpha 1 chain) 
               
               
                   
                 1008‘GEPGRDGNPG’1017 (alpha 1 chain) 
               
               
                   
               
               
                 MMP9, 
                 442‘.GLPGTGGPPG’451 
               
               
                 ADAMTS-4 
               
               
                   
               
            
           
         
       
     
     The immunological binding partner may be one specifically reactive with a C-terminal or N-terminal neoepitope formed by cleavage of type III collagen. 
     Suitable immunological binding partners may therefore be specifically reactive with any of the following sequences at the N terminal of a peptide: 
                     TABLE 13               N-terminal sequences of protease generated peptide       fragments of Collagen type III.       Cleavage sites marked ‘.’/SEQ ID NO ( )                                                .AGAQGP   (204)                       .AGIPGV   (205)                       .APGAPG   (206)                       .ERGRPG   (207)                       .GERGSP   (208)                       .GFDGRN   (209)                       .GGPGAD   (210)                       .GLPGPP   (211)                       .GLPGTG   (212)                       .GPPGPA   (213)                       .GPRGPT   (214)                       .GSPGPA   (215)                       .IAGITG   (216)                       .KGDPGP   (217)                       .KGDRGE   (218)                       .LKGENG   (219)                       .PGENGA   (220)                       .PGERGE   (221)                       .PGKNGA   (222)                       .PGLKGE   (223)                       .PGMRGM   (224)                       .PPGKDG   (225)                       .PQGPKG   (226)                       .SPGAKG   (227)                        
or with any of the following sequences at the C-terminal of a peptide:
 
                     TABLE 14               C-terminal sequences of protease generated peptide       fragments of Collagen type III.       Cleavage sites marked ‘.’/SEQ ID NO ( )                                                AGQPGD.   (228)                       ENGSPG.   (229)                       GDPGPP.   (230)                       GERGPP.   (231)                       GLPGAA.   (232)                       GPAGIP.   (233)                       GPPGIP.   (234)                       GPPGPA.   (235)                       GRPGLP.   (236)                       GSPGGP.   (237)                       GSPGPQ.   (238)                       GVPGAK.   (239)                       KGEPGP.   (240)                       PAGIPG.   (241)                       AGPRGP.   (242)                       PGEPGP.   (243)                       PGKDGP.   (244)                       PRGAPG.   (245)                       RGAPGE.   (246)                       RGEPGP.   (247)                       SGHPGP.   (248)                       SGPKGD.   (249)                       TGPPGP.   (250)                        
Collagen IV
 
     We have determined that the enzymes listed in the following table cleave type IV collagen at least the following cleavage sites (marked “.”): 
     
       
         
           
               
             
               
                 TABLE 15 
               
             
            
               
                   
               
               
                 Cleavage fragments of collagen type IV 
               
            
           
           
               
               
               
            
               
                   
                 Protease 
                 Cleavage sites marked ‘.’ 
               
               
                   
                   
               
               
                   
                 MMP9 
                 333‘.IVIGTGPLGE’342 (alpha 1 chain) 
               
               
                   
                   
               
               
                   
                 MMP9 
                 328‘PGPPGIVIGT.’337 (alpha 1 chain) 
               
               
                   
                   
               
            
           
         
       
     
     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 the following sequence at the N terminal of a peptide: 
                     TABLE 16               N-terminal sequence of protease generated peptide       fragments of Collagen type IV.       Cleavage site marked ‘.’/SEQ ID NO ( )                                                .IVIGTG   (251)                        
or with the following sequence at the C-terminal of a peptide:
 
                     TABLE 17               C-terminal sequences of protease generated peptide       fragments of Collagen type IV.       Cleavage site marked ‘.’/SEQ ID NO ( )                                                GIVIGT.   (252)                        
Collagen V
 
     We have determined that the enzymes listed in the following table cleave type V collagen at least the following cleavage sites (marked “.” or in the absence of a at the end of the sequence): 
     
       
         
           
               
             
               
                 TABLE 18 
               
             
            
               
                   
               
               
                 Cleavage fragments of collagen type V 
               
            
           
           
               
               
               
            
               
                   
                 Protease 
                 Cleavage sites marked ‘.’ 
               
               
                   
                   
               
               
                   
                 MMP2 
                 920‘ERGPRGITGK.’929 (alpha 1 chain) 
               
               
                   
                   
               
               
                   
                 MMP2/9 
                 1584‘.RRNIDASQLL’1593 (alpha 1 chain) 
               
               
                   
                   
               
               
                   
                 MMP2/9 
                 229‘.QQGGAGPTGP’238 (alpha 2 chain) 
               
               
                   
                   
               
               
                   
                 MMP2/9 
                 355‘.QRGAHGMPGK’364 (alpha 2 chain) 
               
               
                   
                   
               
               
                   
                 MMP9 
                 525‘.RVGKMGRPGA’534 (alpha 3 chain) 
               
               
                   
                   
               
               
                   
                 MMP2/9 
                 1317‘.HMGREGREGE’1326 (alpha 3 chain) 
               
               
                   
                   
               
            
           
         
       
     
     The immunological binding partner may be one specifically reactive with a C-terminal or N-terminal neoepitope formed by cleavage of type V collagen. 
     Suitable immunological binding partners may therefore be specifically reactive with any of the following sequences at the N terminal of a peptide: 
                     TABLE 19               N-terminal sequences of protease generated peptide       fragments of Collagen type V.       Cleavage sites marked ‘.’/SEQ ID NO ( )                                                .RRNIDA   (253)                       .QQGGAG   (254)                       .QRGAHG   (255)                       .RVGKMG   (256)                       .HMGREG   (257)                        
or with the following sequence at the C-terminal of a peptide:
 
                     TABLE 20               C-terminal sequence of a protease generated       peptide fragment of Collagen type V.       Cleavage sites marked ‘.’/SEQ ID NO ( )                                                RGITGK.   (258)                        
Collagen VI
 
     We have determined that the enzymes listed in the following table cleave type VI collagen at least the following cleavage sites (marked “.” or in the absence of a ‘.’, at the end of the sequence): 
     
       
         
           
               
             
               
                 TABLE 21 
               
             
            
               
                   
               
               
                 Cleavage fragments of collagen type VI 
               
            
           
           
               
               
               
            
               
                   
                 Protease 
                 Cleavage sites marked ‘.’ 
               
               
                   
                   
               
               
                   
                 MMP8 
                 873‘. RIAVAQYSDD 883’ 
               
               
                   
                   
               
               
                   
                 MMP8 
                 1192‘. QLGTVQQVIS 1202’ 
               
               
                   
                   
               
               
                   
                 MMP12 
                 1231‘.RDVVFLIDGS 1241’ 
               
               
                   
                   
               
            
           
         
       
     
     The immunological binding partner may be one specifically reactive with an N-terminal neoepitope formed by cleavage of type VI collagen. 
     Suitable immunological binding partners may therefore be specifically reactive with any of the following sequences at the N terminal of a peptide: 
                     TABLE 22               N-terminal sequences of protease generated peptide       fragments of Collagen type VI.       Cleavagesites marked ‘.’/SEQ ID NO ( )                                                .RIAVAQ   (259)                       .QLGTVQ   (260)                       .RDVVFL   (261)                        
Proteoglycans
 
     In another aspect of the invention, said peptide fragments are fragments of proteoglycans versican, lumican, biglycan and decorin, which are all identified in fibrotic tissue. 
     Several candidate proteases may be responsible for the digestion of proteoglycans in fibrotic lesions We have determined that the enzymes listed in Table 23 generate biglycan fragments, resulting in at least following cleavage products: 
     Biglycan 
     
       
         
           
               
             
               
                 TABLE 23 
               
             
            
               
                   
               
               
                 Cleavage fragments of biglycan 
               
            
           
           
               
               
               
            
               
                   
                 Protease 
                 Cleavage sites marked ‘.’ 
               
               
                   
                   
               
               
                   
                 MMP3 
                 219‘.KLTGIPKDLP’228 
               
               
                   
                   
               
               
                   
                 MMP3 
                 226‘DLPETLNELH.’235 
               
               
                   
                   
               
               
                   
                 MMP12 
                 220‘LTGIPKDLPE.’229 
               
               
                   
                   
               
               
                   
                 MMP13 
                 329‘.RAYYNGISLF’338 
               
               
                   
                   
               
               
                   
                 MMP13 
                 334‘GISLFNNPVP.’343 
               
               
                   
                   
               
               
                   
                 MMP12 
                 87‘.EISPDTTLLD’96 
               
               
                   
                   
               
               
                   
                 MMP12 
                 97‘LQNNDISELR.’106 
               
               
                   
                   
               
               
                   
                 MMP9 
                 188‘.IEMGGNPLENS’198 
               
               
                   
                   
               
               
                   
                 MMP9 
                 200‘FEPGAFDGLK.’209 
               
               
                   
                   
               
               
                   
                 MMP9 
                 148‘.NHLVEIPPNL’157 
               
               
                   
                   
               
               
                   
                 MMP9 
                 151‘VEIPPNLPSS.’160 
               
               
                   
                   
               
               
                   
                 MMP9 
                 232‘.NELHLDHNK’240 
               
               
                   
                   
               
               
                   
                 MMP9 
                 344‘.YWEVQPATFR’353 
               
               
                   
                   
               
               
                   
                 MMP9, MMP12 
                 241‘.IQAIELEDLL’250 
               
               
                   
                   
               
            
           
         
       
     
     The immunological binding partner may be one specifically reactive with a C-terminal or N-terminal neo-epitope formed by cleavage of type biglycan. 
     Suitable immunological binding partners may therefore be specifically reactive with any of the following at the N terminal of a peptide: 
                     TABLE 24               N-terminal sequences of protease generated peptide       fragments of biglycan.       Cleavage sites marked ‘.’/SEQ ID NO ( )                                                .KLTGIP   (262)                       .RAYYNG   (263)                       .EISPDT   (264)                       .IEMGGN   (265)                       .NHLVEI   (266)                       .NELHLD   (267)                       .YWEVQP   (268)                       .IQAIEL   (269)                        
or with the following sequences in Table 25, at the C-terminal of a peptide:
 
                     TABLE 25               C-terminal sequence of protease generated peptide       fragment of biglycan.       Cleavage sites marked ‘.’/SEQ ID NO ( )                                                TLNELH.   (270)                       PKDLPE.   (271)                       FNNPVP.   (272)                       DISELR.   (273)                       AFDGLK.   (274)                       PNLPSS.   (275)                        
Decorin
 
     
       
         
           
               
             
               
                 TABLE 26 
               
             
            
               
                   
               
               
                 Cleavage fragment of decorin 
               
            
           
           
               
               
               
            
               
                   
                 Protease 
                 Cleavage site marked ‘.’ 
               
               
                   
                   
               
               
                   
                 ADAM-TS4 
                 75‘.VPKDLPPDTT’84 
               
               
                   
                   
               
            
           
         
       
     
     The immunological binding partner may be one specifically reactive with an N-terminal neo-epitope formed by cleavage, of decorin. 
     Suitable immunological binding partners may therefore be specifically reactive with the following at the N terminal of a peptide: 
                     TABLE 27               N-terminal sequence of protease generated peptide       fragments of decorin.       Cleavage sites marked ‘.’/SEQ ID NO ( )                                                .VPKDLP   (276)                        
Versican
 
     
       
         
           
               
             
               
                 TABLE 28 
               
             
            
               
                   
               
               
                 Cleavage fragments of versican 
               
            
           
           
               
               
               
            
               
                   
                 Protease 
                 Cleavage sites marked ‘.’ 
               
               
                   
                   
               
               
                   
                 Cathepsin K 
                 3247‘.YENWRPNQPD’3256 
               
               
                   
                   
               
               
                   
                 Cathepsin K 
                 3255‘PDSFFSAGED.’3264 
               
               
                   
                   
               
               
                   
                 Cathepsin K 
                 3221‘.HDYQWIGLN’3229 
               
               
                   
                   
               
               
                   
                 MMP8 
                 3306‘.KTFGKMKPRY’3316 
               
               
                   
                   
               
               
                   
                 MMP8 
                 486‘.SVTQIEQIEV’495 
               
               
                   
                   
               
               
                   
                 MMP8 
                 491‘EQIEVGPLVT.’500 
               
               
                   
                   
               
            
           
         
       
     
     The immunological binding partner may be one specifically reactive with a C-terminal or N-terminal neo-epitope formed by cleavage of type versican. 
     Suitable immunological binding partners may therefore be specifically reactive with any of the following at the N terminal of a peptide: 
                     TABLE 29               N-terminal sequences of protease generated peptide       fragments of versican.       Cleavage sites marked ‘.’/SEQ ID NO ( )                                                .YENWRP   (277)                       .HDYQWI   (278)                       .KTFGKM   (279)                       .SVTQIE   (304)                        
or with either of the following sequences in Table 30, at the C-terminal of a peptide:
 
                     TABLE 30               C-terminal sequences of protease generated peptide       fragments of versican.       Cleavage sites marked ‘.’/SEQ ID NO ( )                                                FSAGED.   (280)                       VGPLVT.   (281)                        
Lumican
 
     
       
         
           
               
             
               
                 TABLE 31 
               
             
            
               
                   
               
               
                 Cleavage fragment of lumican 
               
            
           
           
               
               
               
            
               
                   
                 Protease 
                 Cleavage sites marked ‘.’ 
               
               
                   
                   
               
               
                   
                 MMP9 
                 75‘NNQIDHIDEK.’84 
               
               
                   
                   
               
            
           
         
       
     
     The immunological binding partner may be one specifically reactive with an N-terminal neo-epitope formed by cleavage of type lumican. 
     Suitable immunological binding partners may therefore be specifically reactive with the following sequence at the C-terminal of a peptide: 
                     TABLE 32               C-terminal sequences of protease generated peptide       fragment of lumican.       Cleavage sites marked ‘.’/SEQ ID NO ( )                                                DHIDEK.   (282)                        
CRP
 
     Several candidate proteases may be responsible for the digestion of CRP in fibrotic tissue the literature reports many different proteases in fibrotic tissue. Most likely, this is the result of the large range of complicated processes eventually leading to fibrosis. However, in our assessment, early phases may consist of a range of MMPs, whereas later stages may rely more on cathepsin K degradation of the matrix, resulting in different neo-epitope profiles dependent on the levels of disease. We have through a range of in vitro cleavages of pure native proteins determined that the enzymes listed in the following tables of cleaved CRP at least following cleavage sites marked ‘.’ in Table 33. 
     
       
         
           
               
             
               
                 TABLE 33 
               
             
            
               
                   
               
               
                 CRP fragments generated by specific proteases. 
               
            
           
           
               
               
               
            
               
                   
                 Protease 
                 Cleavage sites marked ‘.’ 
               
               
                   
                   
               
               
                   
                 Unknown 
                 013‘LSHAFG.’018 
               
               
                   
                   
               
               
                   
                 MMP3/8/9, 
                 011‘TSLSHA.’016 
               
               
                   
                 Cat S/K 
               
               
                   
                   
               
            
           
         
       
     
     Accordingly, in a method of the invention, said peptide fragments preferably comprise a neo-epitope formed by cleavage of CRP by a protease at a site marked by the sign in either one of the above partial sequences of CRP in Table 33. 
     The immunological binding partner may be one specifically reactive with an N-terminal neo-epitope formed by cleavage of CRP. Suitable immunological binding partners may therefore be specifically reactive with either of the following sequences at the C-terminal of a peptide: 
                     TABLE 34               C-terminal sequences of protease generated peptide       fragments of CRP.       Cleavage sites marked ‘.’/SEQ ID NO ( )                                                LSHAFG.   (283)                       TSLSHA.   (284)                        
Elastin
 
     Several candidate proteases may be responsible for the digestion of elastin in fibrotic tissue. We have through a range of in vitro cleavages of pure native proteins determined that the enzymes listed in the following table cleaved elastin at least at the cleavage sites at each end of the following sequences or at the cleavage sites marked ‘.’: 
     
       
         
           
               
             
               
                 TABLE 35 
               
             
            
               
                   
               
               
                 Elastin* fragments generated by specific 
               
               
                 proteases. 
               
            
           
           
               
               
               
            
               
                   
                 Protease 
                 Cleavage sites marked ‘.’ 
               
               
                   
                   
               
               
                   
                 Signal peptide 
                 21‘LHPSRP.’26 
               
               
                   
                   
               
               
                   
                 ADAMTS1/4/8 MMP8/9 
                 379‘KAAKYG.’384 
               
               
                   
                   
               
               
                   
                 MMP9/12 
                 547‘GIGPGG.’552 
               
               
                   
                   
               
               
                   
                 *Aminoacid residue numbers are from the human elastin sequence 
               
            
           
         
       
     
     Accordingly, in a method of the invention, said peptide fragments preferably comprise a neo-epitope formed by cleavage of elastin by a protease at a C-terminal site, in any one of the partial sequences of elastin in Table 35. 
     The immunological binding partner may be one specifically reactive with a C-terminal neo-epitope formed by cleavage of elastin. 
     Suitable immunological binding partners may therefore be specifically reactive with any of the following sequences at the C-terminal of a peptide: 
                     TABLE 36               C-terminal sequences of protease generated peptide       fragments of Elastin.       Cleavage sites marked ‘.’/SEQ ID NO ( )                                                LHPSRP.   (285)                       KAAKYG.   (286)                       GIGPGG.   (287)                        
ApoE
 
     Several candidate proteases may be responsible for the digestion of ApoE in fibrotic tissue. We have through a range of in vitro cleavages of pure native proteins determined that the enzymes listed in the following table cleaved ApoE at least at the cleavage sites at each end of the following sequences or at the cleavage sites marked ‘.’: 
     
       
         
           
               
             
               
                 TABLE 37 
               
             
            
               
                   
               
               
                 Cleavage sites of ApoE 
               
            
           
           
               
               
               
            
               
                   
                 Protease 
                 Cleavage sites marked ‘.’ 
               
               
                   
                   
               
               
                   
                 MMP9 
                 81‘.LMDETM’86 
               
               
                   
                   
               
               
                   
                 Cat K 
                 246‘RLDEVK.’251 
               
               
                   
                   
               
               
                   
                 MMP9 
                 101‘TPVAEE.’106 
               
               
                   
                   
               
               
                   
                 Cat K 
                 126‘.MEEMGS’131 
               
               
                   
                   
               
            
           
         
       
     
     Accordingly, in a method of the invention, said peptide fragments preferably comprise a neo-epitope formed by cleavage of ApoE by a protease at an N- or C-terminal site, or where indicated a site marked by the sign in any one of the partial sequences of ApoE in Table 37. 
     The immunological binding partner may be one specifically reactive with a C-terminal or N-terminal neo-epitope formed by cleavage of ApoE. 
     Suitable immunological binding partners may therefore be specifically reactive with either of the following sequences at the N terminal of a peptide: 
                     TABLE 38               N-terminal sequences of protease generated peptide       fragments of ApoE.       Cleavage sites marked ‘.’/SEQ ID NO ( )                                                .LMDETM   (288)                       .MEEMGS   (289)                        
or with either of the following sequences at the C-terminal of a peptide:
 
                     TABLE 39               C-terminal sequences of protease generated peptide       fragments of ApoE.       Cleavage sites marked ‘.’/SEQ ID NO ( )                                                RLDEVK.   (290)                       TPVAEE.   (291)                        
LAMC1
 
     Several candidate proteases may be responsible for the digestion of LAMC1 in fibrotic tissue. We have through a range of in vitro cleavages of pure native proteins determined that enzymes cleave LAMC1 at least at the cleavage sites at each end of the following sequences or at the cleavage sites marked ‘.’: 
     
       
         
           
               
             
               
                 TABLE 40 
               
             
            
               
                   
               
               
                 Cleavage sites of LAMC1 
               
            
           
           
               
               
               
            
               
                   
                 Protease 
                 Cleavage sites marked ‘.’ 
               
               
                   
                   
               
               
                   
                 MMP2/9 
                 664‘SAGYLDDVTL.’673 
               
               
                   
                   
               
               
                   
                 MMP9 
                 1232‘LNRKYEQAKN.’1241 
               
               
                   
                   
               
            
           
         
       
     
     Accordingly, in a method of the invention, said peptide fragments preferably comprise a neo-epitope formed by cleavage of LAMC1 by a protease at a C-terminal site in either one of the partial sequences of LAMC1 in Table 40. 
     The immunological binding partner may be one specifically reactive with a C-terminal or N-terminal neo-epitope formed by cleavage of LAMC1. 
     Suitable immunological binding partners may therefore be specifically reactive with any of the following sequences at the C-terminal of a peptide: 
                     TABLE 41               C-terminal sequences of protease generated peptide       fragments of LAMC1.       Cleavage sites marked ‘.’/SEQ ID NO ( )                                                LDDVTL.   (292)                       YEQAKN.   (293)                        
LAMA2
 
     Several candidate proteases may be responsible for the digestion of LAMA2 in fibrotic tissue. We have through a range of in vitro cleavages of pure native proteins determined that enzymes cleave LAMA2 at least at the cleavage sites at each end of the following sequences or at the cleavage sites marked ‘.’: 
     
       
         
           
               
             
               
                 TABLE 42 
               
             
            
               
                   
               
               
                 Cleavage sites of LAMA2 
               
            
           
           
               
               
               
            
               
                   
                 Protease 
                 Cleavage sites marked ‘.’ 
               
               
                   
                   
               
               
                   
                 MMP2 
                 1240‘.QFEGKKLMAY’1249 
               
               
                   
                   
               
               
                   
                 MMP2/9 
                 2314‘.GLWNFREKEG’2323 
               
               
                   
                   
               
            
           
         
       
     
     Accordingly, in a method of the invention, said peptide fragments preferably comprise a neo-epitope formed by cleavage of LAMA2 by a protease at an N-terminal site, in either one of the partial sequences of LAMA2 in Table 42. 
     The immunological binding partner may be one specifically reactive with a C-terminal or N-terminal neo-epitope formed by cleavage of LAMA2. 
     Suitable immunological binding partners may therefore be specifically reactive with any of the following sequences at the N terminal of a peptide: 
                     TABLE 43                  N-terminal sequences of protease generated peptide       fragments of LAMA2.                     Protease   Cleavage sites marked ‘.’/SEQ ID NO ( )               MMP2   .QFEGKK (294)               MMP2/9   .GLWNFR (295)                    
LAMB1
 
     Several candidate proteases may be responsible for the digestion of LAMB1 in fibrotic tissue. We have through a range of in vitro cleavages of pure native proteins determined that enzymes cleave LAMB1 at least at the cleavage sites at each end of the following sequences or at the cleavage sites marked ‘.’: 
     
       
         
           
               
             
               
                 TABLE 44 
               
             
            
               
                   
               
               
                 Cleavage sites of LAMB1 
               
            
           
           
               
               
               
            
               
                   
                 Protease 
                 Cleavage sites marked ‘.’ 
               
               
                   
                   
               
               
                   
                 MMP9 
                 209‘ALDPAFKIED.’218 
               
               
                   
                   
               
               
                   
                 MMP2/9 
                 1629‘.SIESETAASE’1638 
               
               
                   
                   
               
               
                   
                 MMP2/9 
                 1639‘ETLFNASQRI.’1648 
               
               
                   
                   
               
            
           
         
       
     
     Accordingly, in a method of the invention, said peptide fragments preferably comprise a neo-epitope formed by cleavage of LAMB1 by a protease at an N- or C-terminal site, in any one of the partial sequences of LAMB1 in Table 44. 
     The immunological binding partner may be one specifically reactive with a C-terminal or N-terminal neo-epitope formed by cleavage of LAMB1. 
     Suitable immunological binding partners may therefore be specifically reactive with the following sequence at the N terminal of a peptide: 
                     TABLE 45               N-terminal sequence of protease generated peptide       fragment of LAMB1.       Cleavage site marked ‘.’/SEQ ID NO ( )                                                .SIESET   (296)                        
or with either of the following sequence at the C-terminal of a peptide:
 
                     TABLE 46               C-terminal sequences of protease generated peptide       fragments of LAMB1.       Cleavage sites marked ‘.’/SEQ ID NO ( )                                                AFKIED.   (297)                       NASQRI.   (298)                        
LAMA5
 
     Several candidate proteases may be responsible for the digestion of LAMA5 in fibrotic tissue. We have through a range of in vitro cleavages of pure native proteins determined that enzymes cleave LAMA5 at least at the cleavage sites at each end of the following sequences or at the cleavage sites marked ‘.’: 
     
       
         
           
               
             
               
                 TABLE 47 
               
             
            
               
                   
               
               
                 Cleavage sites of LAMA5 
               
            
           
           
               
               
               
            
               
                   
                 Protease 
                 Cleavage sites marked ‘.’ 
               
               
                   
                   
               
               
                   
                 MMP9 
                 2402‘.NSRNQERLEE’2411 
               
               
                   
                   
               
               
                   
                 MMP2/9 
                 2901‘EMDTLNEEVV.’2910 
               
               
                   
                   
               
               
                   
                 MMP9 
                 3056‘DLELADAYYL.’3065 
               
               
                   
                   
               
            
           
         
       
     
     Accordingly, in a method of the invention, said peptide fragments preferably comprise a neo-epitope formed by cleavage of LAMA5 by a protease at an N- or C-terminal site, or where indicated a site marked by the sign ‘.’ in any one of the partial sequences of LAMA5 in Table 24. 
     The immunological binding partner may be one specifically reactive with a C-terminal or N-terminal neo-epitope formed by cleavage of LAMA5. 
     Suitable immunological binding partners may therefore be specifically reactive with the following sequences at the N terminal of a peptide: 
                     TABLE 48               N-terminal sequence of protease generated peptide       fragment of LAMA5.       Cleavage sites marked ‘.’/SEQ ID NO ( )                                                .NSRNQE   (299)                        
or with either of the following sequences at the C-terminal of a peptide:
 
     
       
         
           
               
             
               
                 TABLE 49 
               
               
                   
               
               
                 C-terminal sequences of protease generated peptide 
               
               
                 fragments of LAMA5. 
               
               
                 Cleavage sites marked ‘.’/SEQ ID NO ( ) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 LNEEVV. 
                 (300) 
               
               
                   
                   
               
               
                   
                 ADAYYL. 
                 (301) 
               
               
                   
                   
               
            
           
         
       
     
     Further cleavage sites defining neo-epitopes that may be assayed in a similar manner can be identified by exposing collagens, elastin, CRP and proteoglycans or other tissue proteins referred to above 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 (1) a synthetic peptide derived from the sequence of collagen type I, III, IV, V, or VI, or from CRP, or from any of the proteoglycans versican, lumican, decorin and biglycan peptide, ApoE, lumican, LAMC1, LAMB1, or LAMA5 or a competition agent derived from (2) a purified native protein named above 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 an above named protein. 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 protein 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, or plasma or an other type, 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 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. Said condition may be a fibrotic condition or may be an inflammatory condition. 
     An aspect of the present invention relates to the development of monoclonal antibodies recognising neo-epitopes as described above. This can be achieved by immunising mice with synthetic peptides originating from the amino acid sequence of molecules of a named protein (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 protein. 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 the relevant protein by a protease at a end-site in any one of the partial sequences 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 a relevant protein at the end-sites of sequences in any one of the partial sequences set out above. 
     The invention further provides a peptide comprising a C-terminal or N-terminal neo-epitope formed by cleavage of a relevant protein in any one of the partial sequences of these proteins set out above. 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. 
     The invention further comprises an isolated nucleic acid molecule coding for a peptide comprising a C-terminal or N-terminal neo-epitope formed by cleavage of a relevant protein in any one of the partial sequences set out above. 
     The invention further comprises a vector comprising 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 a relevant protein in any one of the partial sequences set out above and further includes a host cell transformed with such a vector and expressing a said peptide. 
     Yet another aspect of the invention relates to kits, which can be used conveniently for carrying out the methods described above. Such kits may include (1) a microtitre plate coated with synthetic peptide; (2) a monoclonal antibody or antibody binding fragment of the invention reactive with said synthetic peptide; and (3) a labelled anti-mouse IgG immunoglobulin. Alternatively, such kits may include (1) a microtitre plate coated with purified native protein fragments; (2) a monoclonal antibody recognising a neo-epitope on the relevant fragments and reactive with said purified protein fragments; and (3) a labelled anti-mouse IgG immunoglobulin. Alternatively, such kits may include (1) a microtitre plate coated with streptavidin; (2) a synthetic peptide linked to biotin; (3) a monoclonal antibody recognising a neo-epitope on protein fragments and reactive with said synthetic peptide; and (4) a labelled anti-mouse IgG immunoglobulin. Yet another alternative could be kits including (1) a microtitre plate coated with streptavidin; (2) a synthetic peptide linked to biotin; (3) a monoclonal antibody recognising a neo-epitope on relevant protein 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 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 or inflammatory conditions 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 the disclosed protein cleavage products. 
     Example 1 
     Identification of Type V Collagen Fragments by Enzyme Cleavage 
     Reagents 
     All reagents used for the experiments were standard high-quality chemicals from companies such as Merck (Whitehouse Station, N.J., USA) and Sigma Aldrich (St. Louis, Mo., USA). The synthetic peptides used for monoclonal antibody production were purchased from the Chinese Peptide Company, Beijing, China. 
     In Vitro Cleavage 
     Purified type V collagen from human placenta (cat. no. ab7537, Abcam, Cambridge, UK) was cleaved with pro-MMP-2 or pro-MMP-9 (cat. no. 444213; 444231; Calbiochem, Merck, Whitehouse Station, N.J., USA). Fifty μg MMP-2 or MMP-9 was activated with 20 μl 1 mM 4-aminophenylmercuric acetate (APMA) in dimethyl sulfoxide and incubated at 37° C. for 3 hours. Type V collagen was delivered dissolved in 0.5M acetic acid. To facilitate MMP cleavage, the protein was dialyzed for two days to remove the acetic acid. The liquid was filtered to remove proteins below 10 kDa (Microcon Ultracel YM-10, cat. no. 42407, Millipore, Billerica, Mass., USA). Each MMP cleavage was performed separately by mixing 100 μg type V collagen and 1 μg of either MMP-2 or MMP-9 in MMP buffer (100 mM Tris-HCl, 100 mM NaCl, 10 mM CaCl 2 , 2 mM Zn acetate, pH 8.0). As control, 100 μg of collagen was mixed with MMP buffer alone. The solutions were incubated for 2 hours at 37° C. The cleavage reaction was stopped using 50 μM ethylenediaminetetraacetic acid (EDTA) to a final concentration of 1 μM. Cleavage was verified by visualization using the SilverXpress® Silver Staining Kit (cat. no. LC6100, Invitrogen, Carlsbad, Calif., USA) according to the manufacturer&#39;s instructions. 
     Peptide Identification 
     Peptide fragments in the in vitro cleaved samples were identified using liquid chromatography (LC) coupled to electrospray ionization (ESI) tandem mass spectrometry (LC-MS/MS). LC-MS samples were ultra-filtrated to remove proteins above 10 kDa, the pH was adjusted to 2.0 using formic acid, and a 4 μL sample was analyzed by LC-MS/MS. LC was performed on a nanoACQUITY HPLC BEH C 18  column (Waters, Milford, Mass., USA) using a formic acid/acetonitrile gradient. MS and MS/MS were performed on a Synapt High Definition Mass Spectrometry quadruple time of flight MS (QUAD-TOF; Waters, Milford, Mass., USA), with an acquisition range of 350-1600 m/z in MS and 50-2000 m/z, in MS/MS. The software “ProteinLynx Global SERVER (PLGS)” (Waters, Milford, Mass., USA) was used to analyze spectra and generate peak lists. To identify peptide, MS and MS/MS data was searched against a type V collagen (FASTA) protein database using the Mascot 2.2 (Matrix Science, Boston, Mass., USA) software with the ESI-QUAD-TOF settings and carbamidomethyl (C), oxidation of methionine (M), oxidation of lysine (K) and oxidation of proline (P) as variable modifications. 
     The six amino acids in the N- or C-terminal of the peptides identified by MS were regarded as a neo-epitope generated by the protease in question. All protease-generated sequences were analyzed for homology and distance to other cleavage sites and tested for homology using NPS@: network protein sequence analysis (Combet C, Blanchet C, Geourjon C, Deleage G. NPS@:network protein sequence analysis. Trends Biochem Sci 2000; 25: 147-50). 
     Example 2 
     Development of an ELISA Assay for Identified Fragments 
     Peptide Conjugation 
     The peptide conjugation was performed using the Maleidide Activated Immunogen Conjugation Kit (Sigma-Aldrich, Mo., USA). Briefly, the cysteine-containing immunogenic neo-epitope (HMGREGREGE-GGC, 400 μl peptide at 5 mg/ml) with one free sulfhydryl (—SH) group was mixed in conjugation buffer with the maleimide-activated ovalbumin (OVA) (180 μl OVA at 10 mg/ml) as a carrier protein with an available maleimide group that could react with sulfhydryl-containing peptides and incubated for 2 hours at room temperature. Conjugated products were cleared of EDTA and sodium azide by desalting or dialysis for two days. For the biotin-conjugated peptides, the biotin-conjugated lysine was added in the solid-phase peptide synthesis procedure. 
     Monoclonal Antibody Development 
     4-6 weeks-old Balb/C mice were immunized subcutaneously with about 200 μl emulsified antigen and 50 μg of the neo-epitope CO5-MMP (HMGREGREGE-GGC-OVA). Consecutive immunizations were performed at 2-week intervals until stable sera titer levels were reached in Freund&#39;s incomplete adjuvant. Blood samples were collected from the 2 nd  immunization. At each blood sampling, the serum titer was determined and the mouse with highest anti-serum titer was selected for fusion. After the 4 th  immunization, this mouse was rested for 1 month and then boosted intravenously with 50 μg CO5-MMP in 100 μl 0.9% sodium chloride solution three days before isolation of the spleen for cell fusion. 
     Fusion and Antibody Screening 
     The fusion procedure performed as described by Gefter et al 132 . Briefly, mouse spleen cells were fused with SP2/0 myeloma fusion partner cells. The hybridoma cells were cloned using a semi-solid medium method and transferred into 96-well microtiter plates for further growth and incubated in a CO 2 -incubater. Standard limited dilution was used to promote monoclonal growth. Supernatants were screened using an indirect ELISA with streptavidin-coated microtitre plates and HMGREGREGE-K-Biotin as a capture peptide. 
     Characterization of Clones 
     Native reactivity and peptide binding of the monoclonal antibodies was evaluated by displacement of native samples (human/rat/mouse serum, plasma and urine) in a preliminary ELISA using 10 ng/mL biotinylated peptide coater on a streptavidin-coated microtitre plate and the supernatant from the growing monoclonal hybridoma. Specificities of the clones to a free peptide (HMGREGREGE), a non-sense peptide, and an elongated peptide ( G HMGREGREG) were tested. Isotyping of the monoclonal antibodies was performed using the Clonotyping System-HRP kit, cat. no. 5300-05 (Southern Biotech, Birmingham, Ala., USA). The selected clones were purified using protein G columns according to manufacturer&#39;s instructions (GE Healthcare Life Science, Little Chalfont, Buckinghamshire, UK). Selected monoclonal antibodies were labeled with horseradish peroxidase (HRP) using the Lightning link HRP labeling kit according to the instructions of the manufacturer (Innovabioscience, Babraham, Cambridge, UK). 
     CO5-MMP ELISA Methodology 
     In preliminary experiments, we optimized the reagents, their concentrations and the incubation periods by performing several checkerboard analyses. The CO5-MMP ELISA was developed as follows: A 96-well streptavidin plate was coated with 5 ng/mL biotinylated synthetic peptide HMGREGREGE-K-Biotin dissolved in assay buffer (25 mM Tris, 1% BSA, 0.1% Tween-20, pH 7.4) and incubated 30 minutes at 20° C. by constant shaking at 300 rpm. Twenty μl of peptide calibrator or sample dissolved in assay buffer were added to appropriate wells, followed by 100 μL of conjugated monoclonal antibody (125 ng/mL) and incubated 1 hour at 4° C. by constant shaking at 300 rpm. Finally, 100 μL tetramethylbenzinidine (TMB) (Kem-En-Tec cat. no. 4380H) was added and the plate was incubated 15 minutes at 20° C. in the dark and shaking at 300 rpm. After each incubation step the plate was washed five times in washing buffer (20 mM Tris, 50 mM NaCl, pH 7.2). The TMB reaction was stopped by adding 100 μl stopping solution (1% HCL) and measured spectrophotometrically at 450 nm with 650 nm as the reference. A standard curve was performed by serial dilution of the CO5-MMP peptide (HMGREGREGE) and plotted using a 4-parametric mathematical fit model. Standard concentrations were 0, 15.625, 31.25, 62.5, 125, 250, 500, 1000 ng/mL. 
     Technical Evaluation 
     From 2-fold dilutions of pooled serum samples, linearity was calculated as a percentage of recovery of the 100% sample. The lower detection limit (LDL) was calculated from 21 determinations of the lowest standard (the zero standard) and calculated as the mean+3× standard deviation. The inter- and intra-assay variation was determined by 10 independent runs of 5° C. samples, with each run consisting of two replicas of double determinations of the samples. Finally, for each assay, a master calibrator prepared from synthetic peptides accurately quantified by amino acid analysis was used for calibration purposes. 
     The analyte stability was determined for three human serum samples for 10 freeze and thaw cycles. 
     Example 3 
     ELISA Characterization 
     The developed CO5-MMP ELISA was evaluated using 20 μl of the samples: intact type V collagen, type V collagen cleaved with MMP-2, type V collagen cleaved with MMP-9, type V collagen cleaved with MMP-13, and an elongated CO5-MMP amino acid sequence ( G HMGREGREG). Cross reactivity was tested using in vitro cleaved collagen type I. 
     Results are shown in  FIG. 1 , panels A and B, showing percent inhibition of the signal of: A) Free-peptide (HMGREGREGE), Intact type V collagen, MMP-9 cleaved type V collagen, MMP-2 cleaved type V collagen; B) Free-peptide (HMGREGREGE), MMP-13 cleaved type V collagen, MMP-9 cleaved type I collagen, elongated peptide ( G HMGREGREG). It can be seen in panel A that the antibody is not substantially reactive with intact collagen type V but is reactive with cleaved collagen type V, and in panel B that the antibody is not reactive with the N-terminal extended peptide. The antibody is thus shown to be specifically reactive with the N-terminal neo-epitope produced by enzyme cleavage. 
     Example 4 
     Clinical Utility 
     Healthy Subjects and AS Patients 
     The biochemical markers were assessed in serum from patients diagnosed with Ankylosing Spondylitis (AS, according to the modified New York criteria) and compared to sex and age matched non-diseased serum samples from the Department of Medicine 3 of the University of Erlangen-Nuremberg. The non-disease group consisted of 21 healthy females and 19 healthy males with a mean age of 43.0 years, range 18 to 66. The AS group consisted of 19 females and 21 males with a mean age of 42.5 years, range 29 to 63 years. 
     Statistics 
     The serum levels of CO5-MMP between the two groups were measured using the ELISA (results shown in  FIG. 2 ) and compared using two-sided non-parametric Mann-Whitney test. In  FIG. 2 , bars indicate mean levels±standard error of the mean (SEM). Area under the curve was measured on ROC. Odds-ratios was extrapolated from contingency table were all subjects were classified as having low (within 2SD of the mean of the normal population) or high (&gt;SD) levels of the biomarker. Results were considered significant when P&lt;0.05. 
     These results strongly suggest that the measured neo-epitope of collagen Type V is a valuable marker for AS. 
     In this specification, unless expressly otherwise indicated, the word ‘or’ is used in the sense of an operator that returns a true value when either or both of the stated conditions is met, as opposed to the operator ‘exclusive or’ which requires that only one of the conditions is met. The word ‘comprising’ is used in the sense of ‘including’ rather than in to mean ‘consisting of’. All prior teachings acknowledged above are hereby incorporated by reference. No acknowledgement of any prior published document herein should be taken to be an admission or representation that the teaching thereof was common general knowledge in Australia or elsewhere at the date hereof. 
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