Cross linked collagen type v assay

Abstract

The present invention relates to a sandwich immunoassay for detecting in a biological sample cross-linked Collagen Type V, and its use in identifying patients with conditions associated with fibrosis, such as ankylosing spondylitis, inflammatory bowel disease, psoriasis and atopic dermatitis. The invention also relates to a kit for performing the sandwich immunoassay.

Description

[0001] Cross linked Collagen Type V assay Technical Field of the Invention The present invention relates to a sandwich immunoassay for detecting in a biological sample cross-linked Collagen Type V, and its use in identifying patients with conditions associated with fibrosis, such as ankylosing spondylitis, inflammatory bowel disease, psoriasis and atopic dermatitis. The invention also relates to a kit for performing the sandwich immunoassay. Type V collagen, a fibrillar collagen Located within the interstitial matrix, home to mesenchymal cells and fibroblast, the fibrillar collagens help to maintain structural tissue integrity, tensile strength, and mechanical and signaling properties [1,2]. The fibrillar collagens encompass type I, II, III, V, XI, XXIV, and XXVII, of which type I, II, and III are viewed as the major fibrillar collagens and the most abundant [2]. Three different α chains of type V collagen exist (α1, α2, and α3), with the heterotypic type V collagen trimer (α12, α2) being the most abundantly expressed, as well as a less abundant homotrimer (α13) [3]. While the collagen fibrils primarily consist of the major collagens, type V collagen forms the core upon which type I and type III collagen attach through the initial formation, and the size of the collagen fibrils is governed by type V collagen non-cartilage tissue [4,5]. Following collagen maturation, type V collagen initiates collagen fibril nucleation upon which type I and type III collagen attach, mediated by enzymatically formed cross-links. The significance of type V collagen in collagen fibril formation is evident in knock-out mice models in which the lack of type V collagen results in insufficient collagen fibril formation despite normal secretion of type I collagen. The lack of type V collagen led to the mice dying during the onset of organogenesis, indicating the requirement of type V collagen for normal fibril formation [6]. The importance of type V collagen is also evident in genetic indications such as Ehlers-Danlos syndrome [7]. Patients present with fragile and soft tissue and impaired wound healing. Moreover, several fibrotic diseases demonstrate significant upregulation of type V collagen [3]. Enzymatic cross-linking The formation of intra- and inter-molecular enzymatic cross-links of the fibrillar collagens is catalyzed by the enzymatic actions of lysyl hydroxylases (LHs), prolyl 3-hydroxylase 3 (P3H3), lysyl oxidase (LOX), and lysyl oxidase-like (LOXLs) enzymes [8]. The enzymatic cross-links are crucial post-translational modifications connecting the fibrillar collagens, strengthening the extracellular matrix in organs [9], blood vessels

[0010] , and the granulation matrix during wound healing [11,12]. Initiated by the hydroxylation of specific lysines within the collagenous helical regions and non-collagenous telopeptides, catalyzed by LHs or P3H3, the hydroxylated or non-hydroxylated lysines are subsequently oxidized by LOXLs [8]. In the extracellular environment, the Cu2+- dependent LOXLs catalyze lysine oxidation in the telopeptides of the fibrillar collagens, including type V collagen

[0013] . However, no conserved cross-linking lysine seems present in the C-terminal telopeptide of type V collagen and potentially only occurs in the N-terminal telopeptide and N-, and C-terminal helix regions

[0014] . Subsequently, hydroxylated and non-hydroxylated lysines in the helical and telopeptidyl regions spontaneously react to form mature trivalent cross-links

[0015] . The functional and mechanical properties of the collagen cross-links are governed by the degree and biochemical nature of the cross-links

[0016] . This is evident when comparing the degree of hydroxylated cross-link species between tissues, with a higher degree of hydroxylation occurring in the tough connective tissue compared to soft connective tissues

[0016] . Differences in the tissue expression of LHs and LOXLs governing the degree of lysine hydroxylation and glycosylations allow for tissue-specific collagen cross-linking rather than collagen specific. The enzymatic cross-linking of fibrillar collagens plays a role under physiological conditions and is also a critical process during fibrosis. Fibrosis results from dysregulated wound healing caused by the inability to resolve tissue insult-induced chronic inflammation, accounting for 45% of deaths in the developed world

[0017] . Continuation of the wound healing process results in the release of multiple pro-fibrotic and pro-inflammatory mediators, inducing activation and differentiation of fibroblasts into myofibroblasts, the primary conductors of tissue fibrosis

[0018] . The activated myofibroblasts deposit excessive fibrillar collagens to the surrounding tissue, and enzymes catalyze collagen cross-linking

[0018] . The deposition of collagen cross-linking enzymes precedes collagen deposition, resulting in an increasingly stiff and non-compliant matrix, which can eventually inhibit tissue and even organ functions

[0019] . Animal models of hepatic [20,21], cardiac

[0022] , lung

[0023] , and peritoneal fibrosis

[0024] have demonstrated the possible influence of increased LOXL expression on matrix stiffness and consequential pro-inflammatory processes. The extensive cross-linking can protect the collagens from proteolysis by blocking the binding sites of the proteases to the collagen α-chains [25,26] and promoting fibrosis through myofibroblast stimulation and recruitment of cells to the site of fibrosis [19,27,28]. With the ability of the heavily cross-linked collagens capable of driving fibrosis independently of inflammation and the lack of approved drugs able to reverse fibrosis, there is an increasing emphasis on the development and assessment of pro-resolution therapy. Resolution and assessment of type V collagen remodeling Long thought to be irreversible, studies have demonstrated how timely and proper intervention, such as direct-acting antivirals for treating hepatitis B or C, can result in tissue regeneration and inflammatory tissue resolution. However, in more complicated fibrotic diseases, a more direct approach targeting the pro-inflammatory extracellular matrix (ECM) may be required, further supported by the ineffectiveness of anti-inflammatory treatment on fibrosis. As the established inflammatory ECM can reactivate fibrosis independently of inflammation, merely halting further collagen deposition may not be sufficient to treat patients with fibrosis. Thus additional options could include inducing fibrosis resolution through proteolytic clearance of the cross-linked collagens. Achieving inflammatory tissue resolution requires fulfillment of the following: (1) cessation of tissue injury, (2) deactivation of pro-inflammatory cells and phenotypic adjustments to, e.g., pro-resolution macrophages, (3) elimination or deactivation of myofibroblast, and (4) degradation and clearance of extracellular matrix components. Pro-resolution therapy aims at promoting degradation and clearance of the established inflammatory ECM, breaking the vicious self-activating feedback loop driven by the heavily cross-linked and non-compliant matrix. Two key processes in fibrosis resolution are the inhibition of further enzymatic cross-linking and enhancing proteolytic degradation of the collagens. LOXL inhibition using the nonspecific inhibitor β-aminopropionitrile or small-molecule inhibitors has demonstrated the ability to mitigate matrix stiffness, reduce collagen cross-linking, and reduce collagen deposition, indicating LOXLs as vital therapeutic targets. Moreover, enhancing collagen degrading proteases such as the matrix metalloproteases (MMP)s can help alleviate fibrosis. A reduction in collagen cross-linking could result in a more accessible matrix for the MMPs, resulting in degradation and clearance of fibrillar collagens, including type V collagen, furthering fibrosis resolution. Multiple MMPs have demonstrated relevance to fibrosis resolution, with the specific MMPs differing between tissues and pathologies, indicating a time and a place for each MMP which may depend on the protease releasing cells. Depending on the time of expression, MMPs can promote fibrosis. However, the MMPs can also promote fibrosis resolution, where MMP-2, MMP-8, MMP-9, and MMP-13 have been shown to induce resolution in liver fibrosis, MMP-1, MMP-3, and MMP-10 catalyzing ECM degradation during wound healing in inflammatory bowel disease, MMP-7 in lung fibrosis resolution, and MMP-2 resolving kidney fibrosis. An essential aspect in the investigation of anti-inflammatory and pro-resolution therapy is sensitive and specific tools for assessing the pharmacodynamics and the achievement of clinical end-points. Current gold standards often involve endoscopies and tissue biopsies, which lack a direct measure of collagen deposition and, more importantly, its resolution. The pro-resolution studies referenced utilize measures of collagen deposition and protease expression as markers for inflammatory resolution. While histological reduction of type I and type III collagen deposition concurrent with increased MMP expression supports the hypothesis of resolution and clearance of the inflammatory ECM, an objective and direct measure is warranted. Representing the primary protein component of the tissue, the remodeling and post-translational modifications (PTMs) of collagen are key aspects in many fibro-inflammatory pathologies in which proteolytic activity release fragments to the circulation. These collagen fragments can act as objective non-invasive biomarkers that reflect the ongoing pathological tissue remodeling through fragment quantification in blood samples

[0318] . While few biomarkers related to type V collagen remodeling exist, there are at least two novel serological biomarkers. The PRO-C5 and C5M biomarkers developed by Nordic Bioscience A / S reflect type V collagen formation and MMP catalyzed degradation, respectively. The biomarkers have demonstrated their use in patients with varying causes of liver disease, patients with ankylosing spondylitis, and patients with IBD. By objectively assessing the C-terminal pro- peptide of type V collagen, a fragment proteolytically released during collagen maturation, the PRO- C5 biomarker evaluates type V collagen formation. Subsequently, during tissue inflammation, the increased activity of the MMPs releases the C5M biomarker from the mature type V collagen. However, none of the current biomarkers of type V collagen can assess the proteolysis of cross- linked type V collagen. The application of such a biomarker would provide an extra level of information, as the fragment would reflect proteolytic degradation as well as enzymatic cross-linking. 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. Fibrotic diseases include those listed in Table A such as inflammatory bowel disease, psoriasis, cirrhosis, and ankylosing spondylitis. Crohn’s disease (CD) and ulcerative colitis (UC) are the two main gastrointestinal disorders of inflammatory bowel disease (IBD) with similar symptoms, such as heightened inflammatory response and structural damage of the intestine. CD can affect the entire gastrointestinal tract, while UC is primarily limited in the colonic mucosa [1]. The cause of IBD is not fully understood, but it is believed to have a genetic basis and an abnormal response of the immune system to environmental factors [2]. The applicant has designed a specific sandwich immunoassay which utilises the neo- epitope of the C-helical region generated by protease cleavage of intact Collagen Type V to detect cross linked Collagen Type V. The levels of the cross-linked degraded Collagen Type V can be used to evaluate patients with fibrotic disease such as inflammatory bowel disease (IBD), ankylosing spondylitis, psoriasis and atopic dermatitis. Summary The present invention is directed to a monoclonal antibody that specifically recognises and binds to a peptide which is a neo-epitope of the C-helical region generated by protease cleavage of intact Collagen Type V. Preferably, said neo-epitope is comprised in amino acid sequence PKGEKGHPGL-COOH(SEQ ID NO:1). The present invention also provides a sandwich immunoassay for detecting in a biological sample cross-linked Collagen Type V (CTX-V) where the cross-linked Collagen Type V comprises at least two strands of the C-helical region of Collagen Type V joined together by inter- strand cross-linking. The method comprises contacting the biological sample comprising the cross- linked Collagen Type V with a first monoclonal antibody bound to a surface, where each strand of Collagen Type V comprised in the cross-linked Collagen Type V has a neo-epitope of the C-helical region generated by protease cleavage of intact Collagen Type V, and adding a second monoclonal antibody. Both monoclonal antibodies are specifically reactive with neo-epitope of the C-helical region generated by protease cleavage of intact Collagen Type V. Preferably said neo-epitope is comprised in amino acid sequence PKGEKGHPGL-COOH(SEQ ID NO:1). The method further comprises determining the amount of binding of the second monoclonal antibody. The present invention is directed further to a kit for use in the sandwich immunoassay as described herein. The kit comprises a solid support to which is bound the first monoclonal antibody as described above and a labelled second monoclonal antibody as described herein. The present invention is also directed to a method for diagnosing a fibrotic disease comprising detecting in a biological sample from a patient cross-linked Collagen Type V (CTX-V) using the immunoassay of the invention. The method comprises contacting the biological sample comprising the cross-linked Collagen Type V with a first monoclonal antibody bound to a surface, where each strand of Collagen Type V comprised in the cross-linked Collagen Type V has a neo- epitope of the C-helical region generated by protease cleavage of intact Collagen Type V, and adding a second monoclonal antibody. Both monoclonal antibodies are specifically reactive with neo- epitope of the C-helical region generated by protease cleavage of intact Collagen Type V. Preferably said neo-epitope is comprised in amino acid sequence PKGEKGHPGL-COOH(SEQ ID NO:1). The method further comprises determining the amount of binding of the second monoclonal antibody. Figures Figure 1: Evaluation of monoclonal antibody specificity. The specificity of the CTX-V monoclonal antibody was tested through its reactivity towards the selection-, elongated-, truncated- peptide and buffer in an indirect competitive ELISA. Figure 2: The relevance of the CTX-V biomarker in IBD patients. The plasma CTX-V levels were plotted for the healthy subjects (HS), Ulcerative colitis (UC), and Crohn’s disease (CD) patients and presented as a Tukey plot. The statistical difference between the plasma levels of HS compared to the levels of UC and CD patients, were done by Kruskal-Wallis using Dunn’s to correct for multiple comparisons. The significance is depicted as, **** p < .0001. Figure 3: Proteolysis of cross-linked type V collagen in an in vitro setting. A) The level of CTX-V measured in the cell supernatants stimulated with or without TGF-β1 is plotted for days 4, 8, and 12. The LLOQ line on the y-axis indicates the lower limit of quantification for the assay. B) The fold-change of CTX-V compared to the uncleaved control is plotted for cleavage with MMP-9 and MMP-13. The statistical difference was calculated by two-way ANOVA (A) or one-way ANOVA (B) using Šídáks to test for multiple comparisons. The significance is depicted as, ** p < .01. Figure 4: CTX-V is upregulated in patients with fibro-inflammatory pathologies. The serum CTX-V levels were plotted for the healthy subjects (HS), ankylosing spondylitis (AS), psoriasis (PSO), and atopic dermatitis (AD) patients and presented as a Tukey plot. The statistical difference between the serum levels of HS compared to the levels of AS, PSO, and AD patients, were done by Kruskal-Wallis using Dunn’s to correct for multiple comparisons. The significance is depicted as, * p < .05, *** p < .001. Description of the Invention As used herein the term “neo-epitope” refers to an epitope formed at a protease cleavage site of Collagen type V, specifically in the C helical region. Preferably the epitope is formed by cleavage with a MMP, such as MMP-9. As used herein the term, the term “competitive ELISA” refers to a competitive enzyme- linked immunosorbent assay. In a “competitive ELISA” the target peptide present in a sample (if any) competes with a known amount of target of peptide (which for example is bound to a fixed substrate or is labelled) for to binding an antibody, and is a technique known to the person skilled in the art. As used herein the term “sandwich immunoassay” refers to the use of at least two antibodies for the detection of an antigen in a sample, and is a technique known to the person skilled in the art. As used herein the term, the term “PRO-C5” refers to the C-terminal pro-peptide of type V collagen released during the maturation of the protein, which generates the N-Terminal epitope TAALGDIMGH (SEQ ID NO: 17). As used herein, the term “C5M” refers to a fragment of type V collagen formed after cleavage by MMP-2 or MMP-9 in the alpha 3 chain comprising the epitope 1317 “HMGREGREGE”1329 (SEQ ID NO: 18). As used herein the term “CTX-V assay” refers to the herein described sandwich immunoassay for detecting and quantifying cross-linked Collagen Type V, also referred to herein as the “target peptide”. Preferably the “target peptide” is an epitope being comprised in the C-terminal amino acid sequence PKGEKGHPGL-COOH (SEQ ID NO:1), formed following cleavage of Collagen Type V with a MMP such as MMP-9 within the C helical region of Collagen Type V (bases 559-1570 of COL5A1 human sequence P20908 https: / / www.uniprot.org / uniprotkb / P20908 / entry.) A monoclonal antibody suitable for use in the method of the invention is disclosed herein and is specifically reactive with a neo-epitope of the C helical region of Collagen Type V, said neo-epitope being comprised in the C-terminal amino acid sequence PKGEKGHPGL-COOH (SEQ ID NO:1). Thus in a first aspect the invention provides a monoclonal antibody that is specifically reactive with a neo-epitope of the C helical region of Collagen Type V, said neo-epitope being comprised in the C-terminal amino acid sequence PKGEKGHPGL-COOH (SEQ ID NO:1). Preferably, the monoclonal antibody does not substantially recognise or bind an elongated version of said C-terminal amino acid sequence which is PKGEKGHPGLIZ -COOH (SEQ ID NO: 2), wherein Z is absent or is one or more amino acids of the sequence of the C helical region of collagen type V following the neo-epitope in the intact Collagen Type V protein. Preferably, the ratio of the affinity of the monoclonal antibody for amino acid sequence PKGEKGHPGL-COOH (SEQ ID NO:1) to the affinity of said monoclonal antibody for elongated amino acid sequence PKGEKGHPGLIZ-COOH (SEQ ID NO: 2) is at least 10 to 1, preferably at least 100 to 1, more preferably at least 1,000 to 1, more preferably at least 10,000 to 1, more preferably at least 100,000 to 1, and most preferably at least 1,000,000 to 1. Preferably, the monoclonal antibody does not recognise or bind a shortened version of said C-terminal amino acid sequence having the amino acid sequence PKGEKGHPG-COOH (SEQ ID NO: 3). Preferably, the ratio of the affinity of the monoclonal antibody for amino acid sequence PKGEKGHPGL-COOH (SEQ ID NO:1) to the affinity of said monoclonal antibody for shortened amino acid sequence PKGEKGHPG-COOH (SEQ ID NO: 3) is at least 10 to 1, preferably at least 100 to 1, more preferably at least 1,000 to 1, more preferably at least 10,000 to 1, more preferably at least 100,000 to 1, and most preferably at least 1,000,000 to 1. As used herein the term “monoclonal antibody” refers to both whole antibodies and to fragments thereof that retain the binding specificity of the whole antibody, such as for example a Fab fragment, F(ab’)2 fragment, single chain Fv fragment, or other such fragments known to those skilled in the art. As is well known, whole antibodies typically have a "Y-shaped" structure of two identical pairs of polypeptide chains, each pair made up of one "light" and one "heavy" chain. The N-terminal regions of each light chain and heavy chain contain the variable region, while the C-terminal portions of each of the heavy and light chains make up the constant region. The variable region comprises three complementarity determining regions (CDRs), which are primarily responsible for antigen recognition. The constant region allows the antibody to recruit cells and molecules of the immune system. Antibody fragments retaining binding specificity comprise at least the CDRs and sufficient parts of the rest of the variable region to retain said binding specificity. Antibodies which retain the same binding specificity may contain the same complementarity-determining regions (CDR). The CDR of an antibody can be determined using methods know in the art such as that described by Kabat et al. Antibodies can be generated from B cell clones as described in the examples. The isotype of the antibody can be determined by ELISA specific for human IgM, IgG or IgA isotype, or human IgG1, IgG2, IgG3 or IgG4 subclasses. Other suitable methods can be used to identify the isotype. In the present invention, the monoclonal antibody may comprise any constant region known in the art. Human constant light chains are classified as kappa and lambda light chains. Heavy constant chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. The IgG isotype has several subclasses, including, but not limited to IgGl, IgG2, IgG3, and IgG4. The monoclonal antibody may preferably be of the IgG isotype, including any one of IgGl, IgG2, IgG3 or IgG4. The amino acid sequence of the antibodies generated can be determined using standard techniques. For example RNA can be isolated from the cells, and used to generate cDNA by reverse transcription. The cDNA is then subjected to PCR using primers which amplify the heavy and light chains of the antibody. For example, primers specific for the leader sequence for all VH (variable heavy chain) sequences can be used together with primers that bind to a sequence located in the constant region of the isotype which has been previously determined. The light chain can be amplified using primers which bind to the 3’ end of the Kappa or Lamda chain together with primers which anneal to the V kappa or V lambda leader sequence. The full length heavy and light chains can be generated and sequenced. Monoclonal antibodies that specifically bind to the C-terminus amino acid sequence PKGEKGHPGL-COOH (SEQ ID No.1) can be generated via any suitable techniques known in the art. For example, the monoclonal antibody may be raised against a synthetic peptide having the amino acid sequence PKGEKGHPGL (SEQ ID No.1), such as for example by: immunizing a rodent (or other suitable mammal) with a synthetic peptide consisting of the sequence PKGEKGHPGL (SEQ ID No.1), which optionally may linked to an immunogenic carrier protein (such as keyhole limpet hemocyanin), isolating and cloning a single antibody producing cell, and assaying the resulting monoclonal antibodies to ensure that they have the desired specificity. An exemplary protocol for producing a monoclonal antibody that that specifically bind to the N-terminus amino acid sequence PKGEKGHPGL (SEQ ID No.1) is described infra. Preferably, the monoclonal antibody or fragment thereof may preferably comprise one or more complementarity-determining regions (CDRs) selected from: CDR-L1: KSSQSLLYSDGKTYLN (SEQ ID No.4) CDR-L2: LVSKLDS (SEQ ID No.5) CDR-L3: WQGTHLPYA (SEQ ID No.6) CDR-H1: DYYMH (SEQ ID No.7) CDR-H2: WIDPENGDREYAPKFQG (SEQ ID No.8) CDR-H3: RGHYEDH (SEQ ID No.9) Preferably the antibody or fragment thereof comprises at least 2, 3, 4, 5 or 6 of the above listed CDR sequences. Preferably the monoclonal antibody or fragment thereof has a light chain variable region comprising the CDR sequences CDR-L1: KSSQSLLYSDGKTYLN (SEQ ID No.4) CDR-L2: LVSKLDS (SEQ ID No.5) CDR-L3: WQGTHLPYA (SEQ ID No.6) Preferably the monoclonal antibody or fragment thereof has a light chain that comprises framework sequences between the CDRs, wherein said framework sequences are substantially identical or substantially similar to the framework sequences between the CDRs in the light chain sequence below (in which the CDRs are shown in bold and underlined, and the framework sequences are shown in italics) KSSQSLLYSDGKTYLNWFLQRPGQSPKRLIYLVSKLDSGVPDRITGTGSGTEFTLKISRVEAEDLG VYYCWQGTHLPYA (SEQ ID No.10) Preferably the monoclonal antibody or fragment thereof has a heavy chain variable region comprising the CDR sequences CDR-H1: DYYMH (SEQ ID No.7) CDR-H2: WIDPENGDREYAPKFQG (SEQ ID No.8) CDR-H3: RGHYEDH (SEQ ID No.9) Preferably the monoclonal antibody or fragment thereof has a heavy chain that comprises framework sequences between the CDRs, wherein said framework sequences are substantially identical or substantially similar to the framework sequences between the CDRs in the heavy chain sequence below (in which the CDRs are shown in bold and underlined, and the framework sequences are shown in italics) DYYMHWVKQRPEQGLEWIGWIDPENGDREYAPKFQGKATMTADTSSNTAYLQISSLTSEDTAVY YCNMRGHYEDH (SEQ ID No.11) As used herein, the framework amino acid sequences between the CDRs of an antibody are substantially identical or substantially similar to the framework amino acid sequences between the CDRs of another antibody if they have at least 70%, 80%, 90% or at least 95% similarity or identity. The similarity or identity may be measured over the entire length of each intervening framework sequence. The similar or identical amino acids may be contiguous or non-contiguous. The framework sequences may contain one or more amino acid substitutions, insertions and / or deletions. Amino acid substitutions may be conservative, by which it is meant the substituted amino acid has similar chemical properties to the original amino acid. A skilled person would understand which amino acids share similar chemical properties. For example, the following groups of amino acids share similar chemical properties such as size, charge and polarity: Group 1 Ala, Ser, Thr, Pro, Gly; Group 2 Asp, Asn, Glu, Gln; Group 3 His, Arg, Lys; Group 4 Met, Leu, Ile, Val, Cys; Group 5 Phe Thy Trp. A program such as the CLUSTAL program to can be used to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or similarity (identity plus conservation of amino acid type) for an optimal alignment. A program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. Both types of analysis are contemplated in the present invention. Identity or similarity is preferably calculated over the entire length of the framework sequences. In certain preferred embodiments, the monoclonal antibody or fragment thereof may comprise the light chain variable region sequence: DVVMTQTPLTLSVTIGQPASISCKSSQSLLYSDGKTYLNWFLQRPGQSPKRLIYLVSKLDSGVPD RITGTGSGTEFTLKISRVEAEDLGVYYCWQGTHLPYAFGGGTKLEIK (SEQ ID No.12) (CDRs bold and underlined; Framework sequences in italics) and / or the heavy chain variable region sequence: EVQLQQSGAELVRSGAAVKLSCTASGFNIEDYYMHWVKQRPEQGLEWIGWIDPENGDREYAPKF QGKATMTADTSSNTAYLQISSLTSEDTAVYYCNMRGHYEDHWGQGTSLTVSS (SEQ ID No.13) (CDRs bold and underlined; Framework sequences in italics) In a second aspect, present invention relates to a sandwich immunoassay for detecting in a biological sample cross-linked Collagen Type V, said cross-linked Collagen Type V comprising at least two strands of the C helical region of Collagen Type V joined together by inter-strand cross- linking, said method comprising: contacting said biological sample comprising said cross-linked Collagen Type V with a first monoclonal antibody bound to a surface, where each strand of Collagen Type V comprised in the cross-linked Collagen Type V has a neo-epitope of the C-helical region generated by protease cleavage of intact Collagen Type V, and adding a second monoclonal antibody; and determining the amount of binding of said second monoclonal antibody; wherein both said first monoclonal antibody and said second monoclonal antibody are specifically reactive with a neo-epitope of the C helical region of Collagen Type V, said neo-epitope being comprised in a C-terminal amino acid sequence PKGEKGHPGL-COOH (SEQ ID NO:1). Preferably the sandwich immunoassay of the second aspect utilises a monoclonal antibody of the first aspect of the invention. Therefore preferred features of the monoclonal antibody of the first aspect, are also preferred features of the second aspect. The herein described sandwich immunoassay uses antibodies which bind to the same epitope as both catcher and detector antibody, i.e. the first and second antibody, therefore a double strand peptide (i.e. cross-linked) can be recognized by the assay. As both monoclonal antibodies bind to the same epitope, if the second monoclonal antibody binds, then at least two linked strands of the peptide must be present. Preferably, the sandwich immunoassay is used to quantify the amount of cross-linked Collagen Type V in a biofluid, wherein said biofluid may be, but is not limited to, serum, plasma, urine, amniotic fluid, tissue supernatant or cell supernatant. The human biofluid sample may be a sample from a human patient having medical signs or symptoms indicative of a fibrotic disease. Examples of fibrotic disease are shown in the Table A below: Ti Li Lu Ki Preferably the fibrotic disease is selected from ankylosing spondylitis, psoriasis, atopic dermatitis or inflammatory bowel disease. Preferably the biofluid sample is a sample from a human patient having medical signs or symptoms indicative of Crohn’s disease (CD) or ulcerative colitis (UC). Preferably the biofluid sample is a sample from a human patient having medical signs or symptoms indicative of active inflammatory bowel disease, for example active Crohn’s disease (CD) or active ulcerative colitis (UC). The sandwich immunoassay may be, but is not limited to, a radioimmunoassay, fluorescence immunoassay, or an enzyme-linked immunosorbent assay. As used herein the term “ELISA” (enzyme-linked immunosorbent assay) refers to an immunoassay in which the target peptide present in a sample (if any) is detected using antibodies linked to an enzyme, such as horseradish peroxidase or alkaline phosphatase. The activity of the enzyme is then assessed by incubation with a substrate generating a measurable product. The presence and / or amount of target peptide in a sample can thereby be detected and / or quantified. ELISA is a technique known to those skilled in the art. The sandwich immunoassay can detect and determine the amount of binding between said monoclonal antibody and peptides in the sample. The amount of binding can be correlated with values associated with normal healthy subjects and / or values associated with known disease severity and / or values obtained from said patient at a previous time point and / or with a predetermined cut-off value. As used herein the term “amount of binding” refers to the quantification of binding between monoclonal antibody and target peptide, which said quantification is determined by comparing the measured values of target peptide in the biofluid samples against a calibration curve, wherein the calibration curve is produced using standard samples of known concentration of the target peptide. The term "specifically bind" as used herein means that the antibody binding is selective for the antigen and that this binding can be distinguished from unwanted or non-specific interactions. The ability of a monoclonal antibody to bind to a specific epitope or peptide sequence can be measured either through an enzyme-linked immunosorbent assay (ELISA) as described herein or other techniques familiar to one of skill in the art, e.g. surface plasmon resonance (SPR) technique (analyzed e.g. on a BIAcore instrument) and traditional binding assays. The extent of binding of a monoclonal antibody to an unrelated protein is less than about 10% of the binding of the monoclonal antibody to the epitope or peptide as measured, e.g., by ELISA. “Affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an epitope binding region of an antibody) and its binding partner (e.g., an epitope or antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., an antigen binding moiety and an antigen). The affinity of a molecule for its partner can generally be represented by the dissociation constant (Kd), which is the ratio of dissociation and association rate constants (koir and kon, respectively). Thus, equivalent affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same. The dissociation constant represents the concentration of the antigen at which half of the binding sites on the antibody are occupied. A lower Kd indicates a higher binding affinity between the antibody and antigen, while a higher Kd reflects weaker binding. Several methods are available to measure the Kd of an antibody, including surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and fluorescence-based assays. In certain aspects, a monoclonal antibody that binds to the epitope or peptide has a dissociation constant (KD) of < 1 pM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g.108M or less, e.g. from 108M to 1013M, e.g., from 109M to 1013M). As used herein the term “predetermined cut-off value” means an amount of binding that is determined statistically to be indicative of a high likelihood of a disease or a particular severity thereof (or prognosis therefor) in a patient, in that a measured value of the target peptide in a patient sample that is at or above the statistical cut-off value corresponds to at least a 70% probability, preferably at least an 75% probability, more preferably at least an 80% probability, more preferably at least an 85% probability, more preferably at least a 90% probability, and most preferably at least a 95% probability of the presence of said disease or said particular severity thereof. As used herein, the term “values associated with normal healthy subjects” means standardised quantities of binding determined by the method described supra for samples from subjects considered to be healthy, i.e. without disease; and the term “values associated with known disease severity” means standardised quantities of binding determined by the method described supra for samples from patients known to have disease of a known severity. In the specific assay disclosed herein which measures in biofluids target peptides having the C-terminus amino acid sequence PKGEKGHPGL-COOH (SEQ. ID No.1), the calibration curve is produced using standard samples of known concentration of a calibration peptide having the C-terminus amino acid sequence PKGEKGHPGL (SEQ. ID No.1), and which may in particular consist of the amino acid sequence PKGEKGHPGL (SEQ. ID No.1). The values measured in the biofluid samples are compared to the calibration curve to determine the actual quantity of target peptide in the sample. In a preferred embodiment, the second monoclonal antibody may be labeled in order to determine the amount of binding of said second monoclonal antibody. Preferably, the second monoclonal antibody may be an enzyme-linked antibody. The enzyme may be, but is not limited to, horseradish peroxidase (HRP). Preferably, the second monoclonal antibody may be radiolabeled or linked to a fluorophore. Although these are preferred labels to be used with the invention, it is envisaged that any suitable labeling system may be employed, such as, but not limited to, DNA reporters or electrochemiluminescent tags. Alternatively, a further labeled antibody which recognises the second monoclonal antibody may be used to determine the amount of binding of said second monoclonal antibody. The further labeled antibody may be labeled using a label as described above. In a preferred embodiment of the invention, the sandwich immunoassay may further comprise correlating the quantity of cross-linked Collagen Type V determined by said method with standard fibrotic disease samples of known disease severity to evaluate the severity of a fibrotic disease. Such a fibrotic disease may be, but is not limited to, inflammatory bowel disease (IBD), ankylosing spondylitis, psoriasis or atopic dermatitis. The method may be an immunoassay method for diagnosing and / or monitoring and / or assessing the likelihood of an fibrotic disease in a patient, the method comprising carrying out the method of immunoassay of the second aspect of the invention on a biofluid sample obtained from said patient with the monoclonal antibody, detecting and determining the amount of binding between the monoclonal antibodies and peptides in the sample, and correlating said amount of binding with values associated with normal healthy subjects and / or values associated with known disease severity and / or values obtained from said patient at a previous time point. Preferably the fibrotic disease is an inflammatory bowel disease, ankylosing spondylitis, atopic dermatitis or psoriasis. Preferably, the inflammatory bowel disease is Crohn’s disease or ulcerative colitis. Preferably the inflammatory bowel disease is active inflammatory bowel disease, for example active Crohn’s disease (CD) or active ulcerative colitis (UC). In a further aspect, the present invention provides a method of treating a fibrotic disease in a patient in need thereof, the method comprising: (a) carrying out a method of immunoassay in accordance with the second aspect of the present invention on a blood, serum or plasma sample from a patient in order to detect whether the patient has a fibrotic disease and / or assess the severity of a fibrotic disease in the patient; and (b) administering to the patient a medicament for the treatment of said fibrotic disease if it is determined in step (a) that the patient has said fibrotic disease or a particular severity thereof. Preferably the fibrotic disease is an inflammatory bowel disease such as Crohn’s disease or ulcerative colitis, ankylosing spondylitis, atopic dermatitis or psoriasis. The inflammatory bowel disease is preferably active inflammatory bowel disease e.g. active Crohn’s disease or active ulcerative colitis. Suitable treatments for inflammatory bowel disease include anti-inflammatories e.g. aminosalicylates; immunosuppressants, corticosteroids, and antibodies such as infliximab or adalimumab. Suitable treatments for ankylosing spondylitis include physiotherapy, pain killers including paracetamol, codeine and non-steroidal anti-inflammatory drugs such as ibuprofen, diclofenac, etoricoxib, and naproxen; monoclonal antibody treatment such as secukinumab and ixekizumab; corticosteroids, Disease-modifying anti-rheumatic drugs (DMARDs) and surgery. Suitable treatments for psoriasis include topical corticosteroids, emollients, vitamin D analogues, calcineurin inhibitors, coal tar, and dithranol. Suitable treatments for atopic dermatitis include topical and / or oral corticosteroids, emollients, topical calcineurin inhibitors such as pimecrolimus or tacrolimus, wet dressings, light therapy and antihistamines. In another aspect, the present invention relates to a kit for use in the sandwich immunoassay as described herein, the kit comprising a solid support to which is bound a first monoclonal antibody as described above; and a labelled second monoclonal antibody as described above. The kit may be for use in diagnosing or predicting the risk of an fibrotic disease, preferably in conjunction with the methods according to the second aspect of the invention. Preferably the fibrotic disease is an inflammatory bowel disease such as Crohn’s disease or ulcerative colitis, ankylosing spondylitis, atopic dermatitis or psoriasis. The inflammatory bowel disease is preferably active inflammatory bowel disease e.g. active Crohn’s disease or active ulcerative colitis. In a further aspect, the sandwich immunoassay described herein may be used in a method for evaluating the efficacy of a drug targeting cross-linking enzymes such as LHs, P3H3 lysyl oxidases (LOXs), and LOXL enzymes. For example the method can be used to evaluate drugs such as an antagonist drug targeting LHs, P3H3, LOXs or LOXLs. Accordingly, the present invention also relates to a method for evaluating the efficacy of an antagonist drug targeting a cross-linking enzyme selected from a LH, a P3H3, a LOX or a LOXL, wherein said method comprises using the sandwich immunoassay described herein to quantify the amount of CTX-V in at least two biological samples, said biological samples having been obtained from a subject at a first time point and at least one subsequent time point during a period of administration of the antagonist drug to said subject, and wherein a reduction in the quantity of CTX-V from said first time point to said at least one subsequent time point during the period of administration of the antagonist drug is indicative of an efficacious antagonist drug targeting said cross-linking enzyme. Preferably, the method quantifies the efficaciousness of the antagonist drug. Preferably, the method evaluates the efficacy of an antagonist drug targeting a LH or LOXL. Example 1 Sandwich assay development Reagents All reagents used in the experiments were high-quality chemicals from Merck (Whitehouse Station, NJ, USA) and Sigma (St. Louis, MO, USA). Synthetic peptides used for monoclonal antibody production, assay development, and validation were 1) Immunogenic peptide: Keyhole Limpet Hemocyanin (KLH)-CGG-PKGEKGHPGL (SEQ ID No. 14), 2) Coating peptide: Biotin- PKGEKGHPGL (SEQ ID No. 15), 3) Selection peptide: PKGEKGHPGL (SEQ ID No. 1) or CPKGEKGHPGL(SEQ ID No. 16) x CPKGEKGHPGL (dimer linked by an N-terminal disulphide bridge), 4) Elongated peptide: PKGEKGHPGLI (SEQ ID No.2), 5) Truncated peptide: PKGEKGHPG (SEQ ID No.3). The dimeric selection peptide was used for assay development and validation. All synthetic peptides were purchased from Genscript (Piscataway, NJ, USA). Monoclonal antibody production and clone characterization Monoclonal antibodies were generated in Balb / C mice as

[0030] targeting the neo-epitope (1478'- PKGEKGHPGL -'1487 (SEQ ID No.1)) located in the C-helical region of type V collagen. Briefly, 200 µL of emulsified antigen and 100 µg immunogenic peptide with Sigma adjuvant System were injected. We repeated the intravenous injection of immunogenic peptide (100 μg in 100 μL 0.9% NaCl ) after one month, isolating the splenocytes after 72 h for cell fusion in SP2 / 0 myeloma cells

[0031] . The supernatants were screened for reactivity against the selection peptide and the elongated and truncated peptides in an indirect competitive enzyme-linked immunosorbent assay (ELISA) using streptavidin precoated plates (Roche, Hvidovre, Denmark, cat. No 11940279), coated with 4 ng / mL of the coating peptide. Clones were selected by testing the antibodies' selective and unique reactivity towards the selection peptide or the immunogenic peptide. The elongated and truncated peptides were not detected (Figure 1). The selected antibody was isotyped using the SBA Clonotyping™ System-HRP (Southern Biotech, Birmingham, AL, USA), and purified using a protein- G column from GE healthcare Life Sciences (Little Chalfont, Buckinghamshire, UK). The selected antibody was sequenced and the CDRs determined. The sequence of the chains are as follows (CDRs in bold; Framework sequence in Italics; Constant region underlined): Light chain: Amino acid sequence (238 aa) DVVMTQTPLTLSVTIGQPASISCKSSQSLLYSDGKTYLNWFLQRPGQSPKRLIYLVSKLDSGVPD RITGTGSGTEFTLKISRVEAEDLGVYYCWQGTHLPYAFGGGTKLEIKRADAAPTVSIFPPSSEQLT Heavy chain: Amino acid sequence (464 aa): The selected antibody was used in the sandwich immunoassay described below for both the capture antibody and detection antibody. The capture antibody was generated by mixing 110 µL Na2CO3 / NaHCO3buffer, pH 9.6, 1 mL (1mg / mL) of antibody, and 13.3 µL of biotinamidohexanoic acid N-hydroxysuccinimide ester and the solution incubated at 20°C for 1 hour with end-over-end rotation. Subsequently, 110 µL of 0.2 M ethanolamine, pH 8.0, was added to the solution and incubated as before. The solution was dialyzed overnight in a Zeba 7k MWCO desalting column (Thermo Scientific, Waltham, MA, USA, cat No. 89889) submerged in 1x PBS at 4°C. A portion of the solution was labeled with horseradish peroxidase using a kit from Sigma (cat 11829696001) according to the manufacturers' instructions to be used as the detection antibody. CTX-V direct sandwich ELISA protocol Streptavidin precoated 96-well plates (Roche Diagnostic's, Hvidovre, Denmark, cat. No.11940279) were coated with 0.250 µg / mL capture antibody diluted 1 / 200 in assay buffer (50 mM PBS, 1% BSA, 0.1% Tween-20, 150 mM NaCl, pH 7.4) and incubated 30 min at 20°C rotating 300 rounds-per- minute. Twenty µL of standard, control, or sample were added, followed by 100 µL of incubation buffer (25 mM PBS, 1% BSA, 0.1% Tween-20, 75 mM NaCl, 5% Liquid II, pH.7.4). Plates were then placed at 20°C under rotation for one hour. Following sample incubation, plates were coated with 0.500 µg / mL detection antibody diluted 1 / 100 in incubation buffer for one hour. Plates were washed using a 25mM TRIZMA, 50mM NaCl, 0.036% Bronidox L5, 0.1% Tween 20 buffer between each incubation. Subsequently, 100 µL chemiluminescence substrate was added to the wells, incubated for 3 minutes at 20°C in the dark, and measured at 470 nm. With the results obtained from a 1.5-fold serial dilution of the dimeric selection peptide, a standard curve was plotted using a 4-parametric mathematical fit model. Unknown sample measurements were interpolated with the standard curve to obtain the concentration (ng / mL) of CTX-V. Technical validation The lower limit of detection (LLOD) was determined from 21 zero samples (i.e., incubation buffer). LLOD was calculated as the mean+3× standard deviation (SD). The upper limit of quantification was determined from the highest point of the standard curve, with a recovery percentage <20% determined on ten independent runs. Inter- and intra-assay variation was determined by ten independent runs of five quality control samples in double determination, with a minimum of three healthy human serum or plasma EDTA samples (Valley Biomedical, Winchester, VA, USA). The acceptance criteria for inter-and intra-assay variation were 15% and 10%, respectively. Assay linearity, specificity, accuracy, and interference were determined by calculating the percentage recovery with a validation criterion of 100% ± 20% from the reference sample. Using the undiluted sample as a reference, the linearity was evaluated by a 1:2-fold dilution of four healthy serum and human plasma EDTA samples. Spiking two healthy human serum or plasma EDTA samples and calculating the recovery between the actual and theoretical measurements determined assay accuracy. Interference was evaluated by spiking healthy human serum or plasma EDTA samples with a known concentration of biotin (low=5 ng / mL, high 100 ng / mL), hemoglobin (low=0.078 mM, high=0.155 mM), or lipid (low=2.42 mM, high=5.49 mM). The recovery was calculated between the unspiked and the low or high interferent samples. Analyte and reagent stability The analyte stability was determined by calculating the percentage recovery of three healthy human serum and plasma EDTA samples from the non-stressed sample. Samples underwent five freeze and thaw cycles CTX-V technical performance No reactivity towards the elongated or truncated peptides was observed, demonstrated by the lack of signal inhibition. An increasing signal inhibition was observed with increasing concentrations of the selection peptide and immunogenic peptide, demonstrating the specificity of the monoclonal antibody towards the selected neo-epitope of CTX-V (Figure 1). The technical validation and analysis and reagent stability were acceptable based on the validation criteria (Table 2). T T L U I I L A I A A Example 2 Analysis of samples from patients with Inflammatory Bowel Disease Clinical cohorts The study subjects were enrolled in a prospective observational study at Odense University hospital (ClinicalTrials.gov ID: NCT02612103), previously described in

[0029] . The analysis conducted here included patients with UC (n = 47) and CD (n = 39), and healthy subjects (n = 32). Informed consent from each patient was collected before blood sampling. The Regional Ethics Committee of Southern Denmark (journal number: S-20150107) approved the study and was conducted according to the Declaration of Helsinki. Patient demographics are presented in Table 1. The biomarkers levels of the UC, CD, and healthy subjects were evaluated at baseline. Statistical analysis Categorial patient characteristics are presented as frequency (percentage), with continuous variables as mean ± SD. Statistical differences in categorial patient characteristics were calculated using Fisher's exact test for two groups or Chi-square test for more than two groups. Differences in continuous variables, including biomarker levels between healthy subjects and patients with UC or CD, were done by Kruskal-Wallis applying Dunn's test correcting for multiple comparisons. The biomarker data is depicted as ng / mL and plotted as Tukey plots. Receiver operating characteristic (ROC) analysis was performed to evaluate the diagnostic potential of the CTX-V biomarker. All statistical analyses were performed in GraphPad Prism v.9.1.1 (Graph Pad Software, La Jolla, CA, USA) or MedCalc v.19.3 (MedCalc Software, Ostend, Belgium). Asterisks indicate the following: *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; ns=non-significant difference. RESULTS Cohort demographics Patients with UC and CD have increased proteolytic degradation of cross-linked type V collagen The plasma CTX-V levels were significantly elevated in patients with UC or CD, compared to the healthy subjects (all: p < 0.0001, differences in means [3.87 ng / mL and 2.19 ng / mL]) (Figure 2). Quantifying proteolytic and cross-linked type V collagen can identify patients with UC or CD Performing ROC analysis, the discriminative capacity of CTX-V was assessed, demonstrating an area under the curve between healthy subjects and patients with UC or CD of 0.99 (sensitivity: 100%, specificity: 96.87%) and 0.99 (sensitivity: 100%, specificity: 96.87%), respectively. Table 3 summarizes the results of the ROC analysis. T S C ≥ > > > > ≥ > > > > A R Example 3 – Analysis of samples from patients with Ankylosing spondylitis, psoriasis and atopic dermatitis Clinical cohorts Commercial serum samples was obtained from patients diagnosed with ankylosing spondylitis (AS) (n = 19), psoriasis (PSO) (n = 9), atopic dermatitis (AD) (n = 10), and healthy subjects (HS) (n = 32). Samples from patients with AS, PSO, and AD were obtained from Protogenex (Inglewood, California, USA), whereas samples from the HS were obtained from Valley Biomedical (Winchester, Virginia, USA). Patient demographics are presented in Table 4. Scar-in-a-Jar, a fibrotic in vitro model Human primary intestinal fibroblasts purchased from Cell biologics (Chicago, Illinois, USA) (cat. no. H-6025) were grown to confluence and seeded in passage 6-8 at a density of 30,000 cells / well in 48-well plates in a high serum medium (10% fetal bovine serum (FBS) (cat. no. F7524, Sigma- Aldrich, St. Louise, Missouri, USA) in Dulbecco's modified eagle medium (DMEM) + Glutamax (cat. no.31966, Gibco, Life Technologies, Carlsbad, California, USA)) on day -2. On day -1, the cells were serum-starved in a low serum medium (0.4% FBS DMEM), avoiding interference with biomarker measurements. Upon induction of fibrogenesis on day 0, cells were cultured in a low serum medium containing ficoll-70 (112.5 mg / mL, cat. no. F2878, Sigma-Aldrich, St. Louise, Missouri, USA) and -400 (75 mg / mL, cat. no. F4375, Sigma-Aldrich, St. Louise, Missouri, USA), supplemented with 1.0% L-ascorbic acid, phosphate magnesium salt, n-hydrate (cat. no. 013– 19,641, Wako, Osaka, Japan) without or with stimulation of 20 ng / mL TGF-β1 (cat no.100-B-010 / CF, R&D system, Minneapolis, Minnesota, USA). Cell culturing occurred by incubation at 37°C with 95% O2and 5% CO2for 12 days, exchanging for a freshly prepared medium without or with TGF-β1 stimulation at days 0, 4, and 8, saving the supernatants at days 4, 8, and 12 for subsequent CTX-V measurements. Three replicates were performed without and with stimulation. Cell-matrix cleavage On day 12, having removed the supernatant, the scar-in-a-jar (SIAJ) cell model wells were washed twice in PBS and stored at -20°C. For the in vitro cleavages of the matrix-containing wells, the plates were thawed and washed gently with the digestion buffer (50 mM Tris-HCl, 200 mM NaCl, 10 mM CaCl2, 100 uM ZnCl, pH 7.5). Pro-forms of MMP-9 (cat. no.911-MP-010, R&D system, Minneapolis, Minnesota, USA) and MMP-13 cat. no.511-MM-010, R&D system, Minneapolis, Minnesota, USA) were activated by dilution in the digestion buffer to 100 ng / µL pro-protease and 1 mM 4- Aminophenylmercuric acetate (APMA, cat. no. A9563, Sigma-Aldrich, St. Louise, Missouri, USA), incubating pro-MMP-9 for 24 hours and pro-MMP-13 for two hours at 37°C. 500 µL of 0.5 µg activated MMP diluted in the digestion buffer was added to the wells, incubating the plates at 37°C for 72 hours. Subsequently, the proteases were inhibited by adding 1 µM EDTA to the wells. The cleavage supernatants were stored at -20°C until CTX-V measurements. Wells incubated only containing the respective digestion buffer were used as a control. Statistical analysis The biomarker data of the SIAJ cell model was analysed by two-way ANOVA comparing data of non- stimulated and stimulated cells, applying Šídák 's test correcting for multiple comparisons. Data are presented as the mean ± SD. Statistical analysis of the CTX-V fold-change of the cell-matrix cleavage was done using the exact biomarker measurements (ng / mL) of the three replicates comparing with and without active MMP by one-way ANOVA correcting for multiple comparisons by Šídák. The fold change of CTX-V was calculated based on the respective media controls of the SIAJ and cell-matrix cleavage experiments. RESULTS Cohort demographics Table PSO, Clinic Age, m Gende BMI (k Smok Abbre body *Demo In vitro generation of the CTX-V fragment Analyzing the supernatants of the human primary intestinal fibroblast stimulated with or without TGF- β1 did not demonstrate any proteolytic release of the CTX-V fragment. However, following incubation with activated MMP-9 resulted in a 3.2-fold increase (p < 0.01) of CTX-V compared to the uncleaved control (Figure 3). Proteolysis of cross-linked type V collagen in fibro-inflammatory pathologies Measuring the serum levels of CTX-V in patients with AS, PSO, and AD and comparing them with the levels of HS, patients suffering from AS, PSO, or AD had significantly elevated biomarker levels (AS: p < 0.001, PSO: p < 0.001, AD: p < 0.05) (Figure 4).

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Claims

Claims1. A monoclonal antibody that specifically recognises and binds to a neoepitope of the C-helical region generated by protease cleavage of intact Collagen Type V, wherein said neo-epitope is comprised in amino acid sequence PKGEKGHPGL-COOH(SEQ ID No. 1).

2. The monoclonal antibody of claim 1 , wherein the monoclonal antibody is a monoclonal antibody raised against a synthetic peptide having the C-terminus amino acid sequence PKGEKGHPGL- COOH (SEQ ID No. 1).

3. The monoclonal antibody of claim 1 or claim 2, wherein the antibody does not specifically recognise or bind to a peptide having the C-terminus amino acid sequencePKGEKGHPGLIZ (SEQ ID No. 2), wherein Z is absent or is one or more amino acids of the sequence of the C helical region of collagen type V.

4. The monoclonal antibody of any preceding claim, wherein the antibody does not specifically recognise or bind to a peptide having the C-terminus amino acid sequencePKGEKGHPG (SEQ ID No. 3).

5. A sandwich immunoassay for detecting in a biological sample cross- linked Collagen Type V, said cross-linked Collagen Type V comprising at least two strands of the C- helical region of Collagen Type V joined together by inter-strand cross-linking, said method comprising: contacting said biological sample comprising said cross-linked Collagen Type V with a first monoclonal antibody bound to a surface, wherein each strand of Collagen Type V comprised in the cross-linked Collagen Type V has a neo-epitope of the C-helical region generated by protease cleavage of intact Collagen Type V; adding a second monoclonal antibody; anddetermining the amount of binding of said second monoclonal antibody; wherein both said first monoclonal antibody and said second monoclonal antibody are specifically reactive with neo-epitope of the C-helical region generated by protease cleavage of intactCollagen Type V, and said neo-epitope is comprised in amino acid sequence PKGEKGHPGL-COOH(SEQ ID NO: 1).

6. The sandwich immunoassay of claim 5, wherein the monoclonal antibody does not substantially recognise or bind an elongated version of said C-terminal amino acid sequence which is PKGEKGHPGLIZ -COOH (SEQ ID NO: 2), wherein Z is absent or is one or more amino acids of the sequence of the C helical region of collagen type V. 7 . The sandwich immunoassay of claim 5 or 6, wherein the sandwich immunoassay is used to quantify the amount of cross-linked Collagen Type V in a biological sample.

8. The sandwich immunoassay of claim 7, further comprising correlating the quantity of cross-linked Collagen Type V determined by said method with standard fibrotic disease samples of known disease severity to evaluate the severity of an fibrotic disease.

9. The sandwich immunoassay of claim 8, wherein the fibrotic disease is inflammatory bowel disease (IBD), ankylosing spondylitis, psoriasis or atopic dermatitis.

10. The sandwich immunoassay of any one of claims 7 to 9, wherein the biological sample is a biofluid.

11. The sandwich immunoassay of claim 10, wherein said biofluid is serum, plasma, urine, amniotic fluid, tissue supernatant or cell supernatant.

12. The sandwich immunoassay of any one of claims 5 to 11 , wherein the sandwich immunoassay is a radioimmunoassay, fluorescence immunoassay, or an enzyme-linked immunosorbent assay.

13. The sandwich immunoassay of any one of claims 5 to 12, wherein the second monoclonal antibody is labeled.

14. The sandwich immunoassay of claim 13, wherein the second monoclonal antibody is an enzyme-linked antibody.

15. The sandwich immunoassay of claim 14, wherein the enzyme is horseradish peroxidase (HRP).

16. The sandwich immunoassay of claim 13, wherein the second monoclonal antibody is radiolabeled or linked to a fluorophore.

17. The sandwich immunoassay of any one of claims 5 to 12, wherein a further labeled antibody which recognises the second monoclonal antibody is used to determine the amount of binding of said second monoclonal antibody.

18. A kit for use in a sandwich assay, the kit comprising: a solid support to which is bound the first monoclonal antibody as defined in any one of claims 1 to 4; and the second monoclonal antibody as defined in any one of claims 1 to 4, said second monoclonal antibody comprising a label.

19. A method for diagnosing an fibrotic disease, the method comprising;i) measuring the levels of CTX-V, in a biofluid sample from a patient using the sandwich immunoassay of any one of claims 5 to 17; ii) comparing the levels of CTX-V in a biofluid sample from a healthy patient with the levels of CTX-V in a biofluid sample from a patient thought to have an fibrotic disease, iii) determining if the patient thought to have an fibrotic disease does have an fibrotic disease based on the comparison of step ii).

20. The method of claim 19 further comprising iv) treating the patient thought to have an fibrotic disease with a medicament to treat the fibrotic disease.21 . A method for evaluating the efficacy of an antagonist drug targeting a cross linking enzyme selected from a LH, a P3H3, a LOX or a LOXL wherein said method comprises using the sandwich immunoassay of any one of claims 5 to 17 to quantify the amount of CTX-V in at least two biological samples, said biological samples having been obtained from a subject at a first time point and at at least one subsequent time point during a period of administration of the antagonist drug to said subject, and wherein a reduction in the quantity of CTX-V from said first time point to said at least one subsequent time point during the period of administration of the antagonist drug is indicative of an efficacious antagonist drug targeting LOXs.

22. The method of claim 21 , wherein the method evaluates the efficacy of an antagonist drug targeting LH or LOXL.

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