Crosslinked V-type collagen assay

Abstract

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

Description

Technical Field

[0001] Technical Field to Which the Invention Belongs The present invention relates to a sandwich immunoassay for detecting cross-linked type V collagen in biological samples, and its use in the identification of patients having conditions associated with fibrosis such as ankylosing spondylitis, inflammatory bowel disease, psoriasis, and atopic dermatitis. The present invention also relates to a kit for performing the sandwich immunoassay.

Background Art

[0002] Type V collagen, which is fibrillar collagen Fibrillar collagen is present within the extracellular matrix localized in mesenchymal cells and fibroblasts and serves to maintain the structural integrity, tensile strength, mechanical properties, and signaling properties of tissues [1, 2]. Fibrillar collagen includes types I, II, III, V, XI, XXIV, and XXVII, among which types I, II, and III are regarded as the major fibrillar collagens and are the most abundant [2]. Type V collagen has three different α-chains (α1, α2, and α3), and the heterotypic V-type collagen trimer (α12, α2) is most abundantly expressed, and although present in small amounts, there is also a homotrimer (α13) [3]. Collagen fibrils consist mainly of the major collagens, but type V collagen forms a nucleus to which type I and type III collagens bind via initial formation, and the size of the collagen fibrils is governed by type V collagen in non-cartilaginous tissues [4, 5]. After collagen maturation, type V collagen initiates the nucleation of collagen fibrils, onto which type I and type III collagens bind via enzymatically formed cross-links.

[0003] The importance of type V collagen in collagen fiber formation is evident in knockout mouse models, where collagen fiber formation is insufficient despite normal secretion of type I collagen when type V collagen is deficient. Deficiency of type V collagen caused mice to die early in organogenesis, indicating that type V collagen is required for normal fibrillogenesis [6]. The importance of type V collagen is also evident in genetic syndromes such as Ehlers-Danlos syndrome [7]. Patients present with fragile and soft tissues and impaired wound healing. Furthermore, several fibrotic diseases show marked upregulation of type V collagen [3].

[0004] Enzymatic crosslinking The formation of intermolecular and intramolecular enzymatic cross-links of fibrillar collagen is catalyzed by the enzymatic action of lysyl hydroxylase (LH), prolyl 3-hydroxylase 3 (P3H3), lysyl oxidase (LOX), and lysyl oxidase-like (LOXL) enzymes [8]. Enzymatic cross-linking is an important post-translational modification that connects fibrillar collagen and strengthens the extracellular matrix of organs [9], blood vessels

[10] , and granulation matrices during wound healing [11, 12]. Hydroxylation of specific lysines in the collagen helical region and non-collagen telopeptides, catalyzed by LH or P3H3, is initiated, and then hydroxylated or non-hydroxylated lysines are oxidized by LOXL [8]. In the extracellular environment, Cu 2+Dependency LOXL catalyzes the lysine oxidation in the telopeptides of fibrillar collagen containing type V collagen

[13] . However, it is not thought that there are conserved cross-linked lysines in the C-terminal telopeptide of type V collagen, and it may occur only in the N-terminal telopeptide and the N-terminal and C-terminal helical regions

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

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

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

[16] . Differences in the tissue expression of LH and LOXL, which govern the degree of lysine hydroxylation and glycosylation, allow for tissue-specific rather than collagen-specific collagen cross-links.

[0005] Enzymatic cross-linking of fibrillar collagen plays a role under physiological conditions and is also an important process during fibrosis. Fibrosis results from a dysregulation of wound healing due to the inability to resolve chronic inflammation caused by tissue damage and accounts for 45% of deaths in developed countries

[17] . As the wound healing process continues, multiple fibrosis-promoting mediators and inflammatory mediators are released, inducing the activation of fibroblasts and their differentiation into myofibroblasts, which are the main drivers of tissue fibrosis

[18] . Activated myofibroblasts deposit excessive fibrillar collagen in the surrounding tissue, and enzymes catalyze the cross-linking of collagen

[18] . The deposition of collagen cross-linking enzymes precedes the deposition of collagen, resulting in a progressively stiffer and less compliant matrix that can ultimately inhibit tissue and even organ function

[19] . In animal models of liver fibrosis [20, 21], cardiac fibrosis

[22] , pulmonary fibrosis

[23] , and peritoneal fibrosis

[24] , increased LOXL expression has been shown to affect matrix stiffness and the resulting inflammatory processes. Extensive cross-linking protects collagen from proteolysis by blocking the binding sites of proteases to collagen α-chains [25, 26] and promotes fibrosis through the stimulation of myofibroblasts and the recruitment of cells to the fibrotic site [19, 27, 28]. Since severely cross-linked collagen has the ability to promote fibrosis independently of inflammation and there are no approved drugs that can reverse fibrosis, the development and evaluation of anti-inflammatory therapies are becoming increasingly important.

[0006] Resolution and evaluation of type V collagen remodeling Although it has long been thought to be irreversible, studies have demonstrated how timely and appropriate interventions, such as antiviral drugs that act directly on the treatment of hepatitis B or C, can lead to tissue regeneration and the resolution of inflamed tissues. However, in more complex fibrotic diseases, a more direct approach targeting the inflammatory extracellular matrix (ECM) may be required, which is further supported by the ineffectiveness of anti-inflammatory treatments for fibrosis. Since established inflammatory ECM can reactivate fibrosis independently of inflammation, simply preventing further collagen deposition may not be sufficient for the treatment of fibrotic patients. Therefore, inducing the resolution of fibrosis by proteolytic clearance of cross-linked collagen is also included as a further option. To achieve the resolution of inflamed tissues, the following must be satisfied: (1) cessation of tissue damage, (2) inactivation of inflammatory cells and phenotypic adjustment, for example, to anti-inflammatory macrophages, (3) removal or inactivation of myofibroblasts, and (4) degradation and clearance of extracellular matrix components. Anti-inflammatory therapies aim to promote the degradation and clearance of established inflammatory ECM, breaking the vicious self-activation feedback loop promoted by severely cross-linked and incompatible matrices. Two important processes in fibrosis resolution are the inhibition of further enzymatic cross-linking and the promotion of collagen proteolysis. Inhibition of LOXL using the non-specific inhibitor β-aminopropionitrile or small molecule inhibitors has shown the ability to relax matrix height, reduce collagen cross-linking, and decrease collagen deposition, indicating that LOXL is an important therapeutic target.

[0007] Furthermore, enhancing collagen-degrading proteases such as matrix metalloproteinase (MMP) can help alleviate fibrosis. If the cross-linking of collagen decreases, it becomes a more accessible matrix for MMP, and as a result, fibrillar collagen, including type V collagen, can be degraded and removed, promoting the resolution of fibrosis. Although multiple MMPs have been shown to be involved in the resolution of fibrosis, specific MMPs vary depending on the tissue and pathological condition, indicating that the timing and location of each MMP differ depending on the protease-releasing cells. Depending on the expression timing, MMP can promote fibrosis. However, MMP-2, MMP-8, MMP-9, and MMP-13 have been shown to induce the resolution of liver fibrosis, MMP-1, MMP-3, and MMP-10 catalyze ECM degradation in the wound healing process of inflammatory bowel disease, MMP-7 induces the resolution of pulmonary fibrosis, and MMP-2 induces the resolution of renal fibrosis.

[0008] An essential aspect in studying anti-inflammatory and resolution therapies is a sensitive and specific tool for evaluating pharmacodynamic effects and achieving clinical endpoints. Current gold standards often include endoscopy and tissue biopsy, but these do not allow for direct measurement of collagen deposition, and more importantly, they have insufficient resolution. Resolution research utilizes measurements of collagen deposition and protease expression as markers of inflammation resolution. The histological decrease in the deposition of type I and type III collagen accompanied by increased expression of MMP supports the hypothesis of the resolution and clearance of inflammatory ECM, but objective and direct indicators are needed. Since collagen is a major protein component of tissues, collagen remodeling and post-translational modification (PTM) are important aspects in many fibrotic inflammatory pathologies, and fragments are released into the circulation by proteolytic activity. These collagen fragments can function as objective non-invasive biomarkers reflecting ongoing pathological tissue remodeling through fragment quantification in blood samples

[0318] .

[0009] There are few biomarkers related to the remodeling of type V collagen, but there are at least two new serological biomarkers. The PRO-C5 and C5M biomarkers developed by Nordic Bioscience A / S reflect the formation of type V collagen and the MMP-catalyzed degradation, respectively. These biomarkers have been shown to be used in patients with liver diseases, ankylosing spondylitis, and IBD for various reasons. The PRO-C5 biomarker evaluates the formation of type V collagen by objectively assessing the C-terminal propeptide of type V collagen, which is a fragment released by proteolysis during collagen maturation. Then, during tissue inflammation, the activity of MMP increases, and the C5M biomarker is released from mature type V collagen. However, none of the current type V collagen biomarkers can evaluate the proteolysis of cross-linked type V collagen. By applying such a biomarker, the fragment reflects not only enzymatic cross-linking but also proteolysis, so more advanced information can be obtained. "Fibrotic disease" refers to any disease that causes fibrosis, whether it is a main symptom or a secondary symptom. Fibrosis is the final result of a chronic inflammatory response induced by various stimuli, including persistent infection, autoimmune reaction, allergic response, chemical injury, 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 two major gastrointestinal diseases of inflammatory bowel disease (IBD) and have similar symptoms such as enhanced inflammatory response and structural damage to the intestine. CD affects the entire gastrointestinal tract, while UC is mainly limited to the large intestine mucosa [1]. The cause of IBD is not fully understood, but it is thought to have an abnormal immune response to genetic basis and environmental factors [2]. Summary of the Invention

[0010] The applicant designed a specific sandwich immunoassay to detect cross-linked type V collagen using neoepitopes in the C-helical region generated by protease cleavage of intact collagen type V. The level of cross-linked degraded collagen type V can be used to evaluate patients with fibrotic diseases such as inflammatory bowel disease (IBD), ankylosing spondylitis, psoriasis, and atopic dermatitis.

[0011] Overview The present invention relates to a monoclonal antibody that specifically recognizes and binds to a peptide that is a neoepitope in the C-helical region generated by protease cleavage of intact collagen type V. Preferably, the neoepitope is contained in the amino acid sequence PKGEKGHPGL-COOH (SEQ ID NO: 1).

[0012] The present invention also provides a sandwich immunoassay for detecting cross-linked type V collagen (CTX-V) in a biological sample, wherein the cross-linked type V collagen comprises at least two chains of the C-helical region of type V collagen linked together by interchain cross-links. The method includes contacting a biological sample containing cross-linked type V collagen with a first monoclonal antibody bound to a surface, wherein each chain of type V collagen contained in the cross-linked type V collagen has a neoepitope in the C-helical region generated by protease cleavage of intact collagen type V, and adding a second monoclonal antibody. Both monoclonal antibodies exhibit specific reactivity with the neoepitope in the C-helical region generated by protease cleavage of intact collagen type V. Preferably, the neoepitope is contained in the amino acid sequence PKGEKGHPGL-COOH (SEQ ID NO: 1). The method further includes determining the amount of bound second monoclonal antibody.

[0013] Furthermore, the present invention relates to a kit for use in the sandwich immunoassay described herein. This kit includes a solid-phase support to which the above-described first monoclonal antibody is bound, and a labeled second monoclonal antibody described herein.

[0014] Also, the present invention relates to a method for diagnosing a fibrotic disease, which includes detecting cross-linked type V collagen (CTX-V) in a biological sample of a patient using the immunoassay of the present invention. This method includes contacting a biological sample containing cross-linked type V collagen with a first monoclonal antibody bound to a surface, where each chain of type V collagen contained in cross-linked type V collagen has a neoepitope of the C-helical region generated by protease cleavage of intact type V collagen, and adding a second monoclonal antibody. Both monoclonal antibodies show specific reactivity with the neoepitope of the C-helical region generated by protease cleavage of intact type V collagen. Preferably, the neoepitope is contained in the amino acid sequence PKGEKGHPGL-COOH (SEQ ID NO: 1). This method further includes determining the amount of binding of the second monoclonal antibody.

Brief Description of the Drawings

[0015]

Figure 1

Figure 2

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DETAILED DESCRIPTION OF THE INVENTION

[0016] Description of the Invention As used herein, the term "neoepitope" refers to a protease cleavage site of type V collagen, specifically an epitope formed in the C-helical region. Preferably, the epitope is formed by cleavage with an MMP such as MMP-9.

[0017] As used herein, the term "competitive ELISA" refers to a competitive enzyme-linked immunosorbent assay. In a "competitive ELISA," the target peptide (if present) in a sample competes with a known amount of peptide target (e.g., bound to a fixed substrate or labeled) for binding to an antibody, which is a technique known to those skilled in the art.

[0018] 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, which is a technique known to those skilled in the art.

[0019] As used herein, the term "PRO-C5" refers to the C-terminal propeptide of type V collagen that is released during protein maturation, thereby generating the N-terminal epitope TAALGDIMGH (SEQ ID NO: 17).

[0020] As used herein, the term "C5M" refers to a fragment of type V collagen formed after cleavage by MMP-2 or MMP-9 of the α3 chain containing the epitope 1317 "HMGREGREGE" 1329 (SEQ ID NO: 18).

[0021] As used herein, the term "CTX-V assay" refers to the sandwich immunoassay described herein for detecting and quantifying cross-linked type V collagen, referred to herein as the "target peptide". Preferably, the "target peptide" is an epitope contained in the C-terminal amino acid sequence PKGEKGHPGL-COOH (SEQ ID NO: 1) formed after cleavage of type V collagen by an MMP such as MMP-9 within the C-helical region of type V collagen (COL5A1 human sequence P20908 https: / / www.uniprot.org / uniprotkb / P20908 / entry bases 559-1570).

[0022] Monoclonal antibodies suitable for use in the methods of the present invention are disclosed herein and exhibit specific reactivity with neoepitopes in the C-helical region of type V collagen, where the neoepitopes are contained in the C-terminal amino acid sequence PKGEKGHPGL-COOH (SEQ ID NO: 1). Thus, in a first aspect, the present invention provides a monoclonal antibody that exhibits specific reactivity with a neoepitope in the C-helical region of type V collagen, where the neoepitopes are contained in the C-terminal amino acid sequence PKGEKGHPGL-COOH (SEQ ID NO: 1).

[0023] Preferably, the monoclonal antibody does not substantially recognize or bind to the long-chain type of the C-terminal amino acid sequence of PKGEKGHPGLIZ-COOH (SEQ ID NO: 2) (in the sequence, Z is absent or one or more amino acids of the sequence of the C-helical region of type V collagen after the neoepitope in the intact type V collagen protein).

[0024] Preferably, the ratio of the affinity of the monoclonal antibody for the amino acid sequence PKGEKGHPGL-COOH (SEQ ID NO: 1) to the affinity of the monoclonal antibody for the long-chain 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 1000 to 1, more preferably at least 10000 to 1, more preferably at least 100000 to 1, and most preferably at least 1000000 to 1.

[0025] Preferably, the monoclonal antibody does not recognize or bind to the shortened type of the C-terminal amino acid sequence having the amino acid sequence PKGEKGHPG-COOH (SEQ ID NO: 3).

[0026] Preferably, the ratio of the affinity of the monoclonal antibody for the amino acid sequence PKGEKGHPGL-COOH (SEQ ID NO: 1) to the affinity of the monoclonal antibody for the 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 1000 to 1, more preferably at least 10000 to 1, more preferably at least 100000 to 1, and most preferably at least 1000000 to 1.

[0027] As used herein, the term "monoclonal antibody" refers to both a complete antibody and fragments of a complete antibody that retain the binding specificity of the complete antibody, such as Fab fragments, F(ab’)2 fragments, single-chain Fv fragments, or other such fragments known to those of skill in the art. As is well known, a complete antibody generally has a "Y-shaped" structure of two identical pairs of polypeptide chains, each pair consisting of one "light" chain and one "heavy" chain. The N-terminal region of each of the light and heavy chains contains a variable region, while the C-terminal portion of each of the heavy and light chains constitutes a constant region. The variable region contains three complementarity-determining regions (CDRs) that are primarily responsible for antigen recognition. The constant region enables the antibody to mobilize cells and molecules of the immune system. Antibody fragments that retain binding specificity include at least the CDRs and the remaining portion of the variable region sufficient to retain said binding specificity. Antibodies that retain the same binding specificity may contain the same complementarity-determining regions (CDRs). The CDRs of an antibody can be determined using methods known in the art, such as those described by Kabat et al.

[0028] Antibodies can be generated from B cell clones as described in the Examples. The isotype of an antibody can be determined by ELISA specific for human IgM, IgG, or IgA isotypes, or for human IgG1, IgG2, IgG3, or IgG4 subclasses. Other suitable methods can be used to identify the isotype. In the present invention, monoclonal antibodies may contain any constant region known in the art. Human constant light chains are classified as κ and λ light chains. Heavy chain constant chains are classified as μ, δ, γ, α, or ε, and define the antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. The IgG isotype has several subclasses including, but not limited to, IgG1, IgG2, IgG3, and IgG4. Monoclonal antibodies are preferably IgG isotype antibodies containing any one of IgG1, IgG2, IgG3, or IgG4.

[0029] The amino acid sequence of the generated antibody can be determined using standard techniques. For example, RNA can be isolated from cells and used to generate cDNA by reverse transcription. Next, PCR can be performed on this cDNA using primers that amplify the heavy and light chains of the antibody. For example, a primer specific for the leader sequence for the entire VH (heavy chain variable) sequence can be used together with a primer that binds to a sequence located in the constant region of a previously determined isotype. The light chain can be amplified using a primer that binds to the 3' end of the κ or λ chain together with a primer that anneals to the Vκ or Vλ leader sequence. Full-length heavy and light chains can be produced and sequenced.

[0030] Monoclonal antibodies that specifically bind to the C-terminal amino acid sequence PKGEKGHPGL-COOH (SEQ ID NO: 1) may be prepared by any suitable technique known in the art. For example, monoclonal antibodies can be prepared by immunizing rodents (or other suitable mammals) with a synthetic peptide consisting of the amino acid sequence PKGEKGHPGL (SEQ ID NO: 1) (optionally linked to an immunogenic carrier protein such as keyhole limpet hemocyanin), isolating and cloning a single antibody-producing cell, and performing an assay to confirm that the resulting monoclonal antibodies have the desired specificity, etc., such that they are specific for the synthetic peptide having the amino acid sequence PKGEKGHPGL (SEQ ID NO: 1). An exemplary protocol for producing monoclonal antibodies that specifically bind to the N-terminal amino acid sequence PKGEKGHPGL (SEQ ID NO: 1) is described below.

[0031] Preferably, the monoclonal antibody or its fragment preferably 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) and may contain one or more complementarity determining regions (CDRs) selected from the group consisting of:

[0032] Preferably, the antibody or fragment thereof comprises at least two, three, four, five or six of the CDR sequences listed above.

[0033] Preferably, the monoclonal antibody or fragment thereof has a light chain variable region comprising the following CDR sequences: CDR-L1: KSSQSLLYSDGKTYLN (SEQ ID NO: 4) CDR-L2: LVSKLDS (SEQ ID NO: 5) CDR-L3: WQGTHLPYA (SEQ ID NO: 6)

[0034] Preferably, the monoclonal antibody or fragment thereof has a light chain comprising a framework sequence between the CDRs, wherein the framework sequence is substantially identical or substantially similar to the framework sequence between the CDRs of the following light chain sequence (in the sequence, the CDRs are shown in bold and underlined, and the framework sequences are shown in italics).

Chemical Structure

[0035] Preferably, the monoclonal antibody or fragment thereof has a heavy chain variable region comprising the following CDR sequences: CDR-H1: DYYMH (SEQ ID NO: 7) CDR-H2: WIDPENGDREYAPKFQG (SEQ ID NO: 8) CDR-H3: RGHYEDH (SEQ ID NO: 9)

[0036] ​​Preferably, the monoclonal antibody or its fragment has a heavy chain containing a framework sequence between the CDRs, wherein the framework sequence is substantially identical or substantially similar to the framework sequence between the CDRs of the following heavy chain sequence (in the sequence, the CDRs are shown in bold and underlined, and the framework sequences are shown in italics). [Chemical formula]

[0037] As used herein, when the framework amino acid sequence between the CDRs of one antibody and the framework amino acid sequence between the CDRs of another antibody have at least 70%, 80%, 90%, or at least 95% similarity or identity, the framework amino acid sequence between the CDRs of one antibody is substantially identical or substantially similar to the framework amino acid sequence between the CDRs of the other antibody. Similarity or identity can be measured over the entire length of each intervening framework sequence. Similar or identical amino acids may or may not be contiguous.

[0038] The framework sequence may include one or more amino acid substitutions, insertions, and / or deletions. The amino acid substitutions may be conservative, which means that the substituted amino acid has similar chemical properties to the original amino acid. One of ordinary skill in the art will understand which amino acids share similar chemical properties. For example, the following groups of amino acids have 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.

[0039] To compare amino acid sequences, programs such as the CLUSTAL program can be used. This program compares amino acid sequences and finds the optimal alignment by appropriately inserting spaces into either sequence. For the optimal alignment, the identity or similarity of amino acids (identity and conservation of amino acid type) can be calculated. Programs such as BLASTx align the longest stretches of similar sequences and assign values to the matches. In this way, it is possible to obtain a comparison in which several similar regions are found, each with a different score. In the present invention, both types of analysis are contemplated. Identity or similarity is preferably calculated over the entire length of the framework sequence.

[0040] In certain preferred embodiments, the monoclonal antibody or fragment thereof has the following light chain variable region sequence: [Chemical formula] (CDRs are in bold and underlined; framework sequences are in italics) and / or the following heavy chain variable region sequence: [Chemical formula] (CDRs are in bold and underlined; framework sequences are in italics) may be included.

[0041] In a second aspect, the present invention is a sandwich immunoassay for detecting crosslinked type V collagen in a biological sample, wherein the crosslinked type V collagen comprises at least two chains of the C-helical region of type V collagen linked together by interchain crosslinks, and the method Contacting the biological sample containing the cross-linked type V collagen with a first monoclonal antibody bound to a surface, wherein each chain of type V collagen contained in the cross-linked type V collagen has a neoepitope of the C-helical region generated by protease cleavage of intact type V collagen; and Adding a second monoclonal antibody; and Determining the amount of binding of the second monoclonal antibody comprising both the first monoclonal antibody and the second monoclonal antibody exhibit specific reactivity with the neoepitope of the C-helical region of type V collagen, the neoepitope is contained in the C-terminal amino acid sequence PKGEKGHPGL-COOH (SEQ ID NO: 1), relating to a sandwich immunoassay.

[0042] Preferably, the sandwich immunoassay of the second aspect utilizes the monoclonal antibody of the first aspect of the present invention. Thus, the preferred features of the monoclonal antibody of the first aspect are also the preferred features of the second aspect.

[0043] In the sandwich immunoassay described herein, since antibodies that bind to the same epitope are used as both the capture antibody and the detection antibody, i.e., the first antibody and the second antibody, double-stranded peptides (i.e., cross-linked type) can be recognized by the assay. Since both monoclonal antibodies bind to the same epitope, if the second monoclonal antibody binds, there should be at least two peptide linkage chains present.

[0044] Preferably, the sandwich immunoassay is used to quantify cross-linked type V collagen in a biofluid, and the biofluid may be, but is not limited to, serum, plasma, urine, amniotic fluid, tissue supernatant or cell supernatant.

[0045] A human biological fluid sample may be a sample from a human patient having a medical sign or symptom that is an indicator of a fibrotic disease. Examples of fibrotic diseases are shown in Table A below.

[0046] [Table 1]

[0047] Preferably, the fibrotic disease is selected from ankylosing spondylitis, psoriasis, atopic dermatitis or inflammatory bowel disease. Preferably, the biological fluid sample is a sample from a human patient having a medical sign or symptom that is an indicator of Crohn's disease (CD) or ulcerative colitis (UC). Preferably, the biological fluid sample is a sample from a human patient having a medical sign or symptom that is an indicator of active inflammatory bowel disease, such as active Crohn's disease (CD) or active ulcerative colitis (UC).

[0048] The sandwich immunoassay may be, but is not limited to, a radioimmunoassay, a fluorescence immunoassay, or an enzyme-linked immunosorbent assay.

[0049] As used herein, the term "ELISA" (enzyme-linked immunosorbent assay) refers to an immunoassay in which a target peptide (if any) present in a sample is detected using an antibody linked to an enzyme such as horseradish peroxidase or alkaline phosphatase. Next, the activity of the enzyme is evaluated by incubation with a substrate that generates a measurable product. Thereby, the presence and / or amount of the target peptide in the sample can be detected and / or quantified. ELISA is a technique known to those skilled in the art.

[0050] The sandwich immunoassay can detect and determine the amount of binding between the monoclonal antibody and the peptide in the sample. The amount of binding can be correlated with a value associated with a normal healthy subject, and / or a value associated with the severity of a known disease, and / or a value obtained from the patient at a previous time point, and / or a predetermined cut-off value.

[0051] As used herein, the term "amount of binding" refers to the quantification of the binding between the monoclonal antibody and the target peptide, and the quantification is determined by comparing the measured value of the target peptide in the biological fluid sample aliquot with a calibration curve, which is created using standard samples with known concentrations of the target peptide.

[0052] As used herein, the term "specifically binds" means that the binding of an antibody is selective for an 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 using the enzyme-linked immunosorbent assay (ELISA) described herein or other techniques well known to those of skill in the art, such as surface plasmon resonance (SPR) technology (e.g., analyzed on a BIAcore instrument) and conventional binding assays. The degree of binding of a monoclonal antibody to a protein unrelated to the monoclonal antibody is less than about 10% of the binding of the monoclonal antibody to the epitope or peptide, as measured, for example, by ELISA. "Affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., the epitope-binding region of an antibody) and its binding partner (e.g., an epitope or antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antigen-binding site and an antigen). The affinity of a molecule for its partner can generally be expressed as a dissociation constant (Kd), which is the ratio of the dissociation and association rate constants (koff and kon, respectively). Thus, equivalent affinities can be composed of different rate constants as long as the ratio of the rate constants is the same. The dissociation constant represents the concentration of antigen at which half of the antibody binding sites are occupied. The lower the Kd, the higher the binding affinity between the antibody and the antigen, and the higher the Kd, the weaker the binding. Several methods are available for measuring the Kd of an antibody, including surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and fluorescence-based assays. In certain embodiments, a monoclonal antibody that binds to an 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., 10 8 M or less, e.g., 10 8 M to 10 13 M, e.g., 10 9 M to 10 13 M).

[0053] As used herein, the term "predetermined cut-off value" means a combined amount that is statistically determined to indicate a high likelihood of a disease or a particular severity thereof (or its prognosis) in a patient, and a measured value of a target peptide in a patient sample that is above the statistical cut-off value corresponds to the presence of the disease or a particular severity thereof with a probability of at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.

[0054] As used herein, the term "value related to normal healthy subjects" means a standardized combined amount determined by the above method for samples of healthy subjects, i.e., subjects considered to be disease-free, and the term "value related to known disease severity" means a standardized combined amount determined by the above method for samples of patients known to have a disease of known severity.

[0055] In a specific assay disclosed herein for measuring a target peptide having the C-terminal amino acid sequence PKGEKGHPGL-COOH (SEQ ID NO: 1) in a biological fluid, a calibration curve is created using standard samples of calibration peptides of known concentration having the C-terminal amino acid sequence PKGEKGHPGL (SEQ ID NO: 1) (in particular, which may consist of the amino acid sequence PKGEKGHPGL (SEQ ID NO: 1)). The value measured in the biological fluid sample is compared with the calibration curve to determine the actual amount of the target peptide in the sample.

[0056] In a preferred embodiment, to determine the binding amount of the second monoclonal antibody, the second monoclonal antibody may be labeled.

[0057] Preferably, the second monoclonal antibody may be an enzyme-conjugated antibody. The enzyme may be, but is not limited to, horseradish peroxidase (HRP).

[0058] Preferably, the second monoclonal antibody may be radiolabeled or may be conjugated to a fluorophore.

[0059] These are preferred labels for use in the present invention, although it is contemplated that any suitable labeling system may be employed, such as, but not limited to, DNA reporters or electrochemiluminescent tags.

[0060] Alternatively, the amount of the second monoclonal antibody bound may be measured using a further labeled antibody that recognizes the second monoclonal antibody. The further labeled antibody may be labeled using the above labels.

[0061] In a preferred embodiment of the present invention, the sandwich immunoassay may further comprise correlating the amount of cross-linked type V collagen determined by the method with a standard fibrotic disease sample of known disease severity to assess the severity of the fibrotic disease. Such fibrotic diseases may include, but are not limited to, inflammatory bowel disease (IBD), ankylosing spondylitis, psoriasis or atopic dermatitis.

[0062] The above method may be an immunoassay method for the diagnosis and / or monitoring and / or assessment of the likelihood of a fibrotic disease in a patient, the method comprising performing an immunoassay method of the second aspect of the present invention on a biological fluid sample obtained from the patient using a monoclonal antibody, detecting and determining the amount of binding between the monoclonal antibody and the peptide in the sample, and correlating the amount of binding with a value associated with a normal healthy subject and / or a value associated with the severity of a known disease and / or a value obtained from the patient at a previous time point. Preferably, the fibrotic disease is 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 an active inflammatory bowel disease, such as active Crohn's disease (CD) or active ulcerative colitis (UC).

[0063] In a further aspect, the present invention is a method of treating a fibrotic disease in a patient in need thereof, comprising: (a) performing an immunoassay method according to a second aspect of the present invention on a blood, serum or plasma sample derived from the patient to detect whether the patient has a fibrotic disease and / or to assess the severity of the fibrotic disease in the patient; and (b) if in step (a) it is determined that the patient has a fibrotic disease or a particular severity thereof, administering to the patient an agent for treating the fibrotic disease is provided.

[0064] 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 an inflammatory bowel disease, for example, active Crohn's disease or active ulcerative colitis.

[0065] Suitable treatments for inflammatory bowel disease include anti-inflammatory drugs such as aminosalicylates; immunosuppressive drugs, corticosteroids, and antibodies such as infliximab or adalimumab. Suitable therapeutic agents for ankylosing spondylitis include physical therapy, paracetamol, codeine and analgesics including non-steroidal anti-inflammatory drugs such as ibuprofen, diclofenac, etoricoxib, and naproxen; monoclonal antibody therapies such as secukinumab and ixekizumab; corticosteroids, disease-modifying anti-rheumatic drugs (DMARDs) and surgery. Suitable treatments for psoriasis include topical corticosteroids, emollients, vitamin D analogs, 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, phototherapy and antihistamines.

[0066] In another aspect, the present invention relates to a kit for use in the sandwich immunoassay described herein, the kit comprising a solid support to which the above-described first monoclonal antibody is bound; and the above-described labeled second monoclonal antibody.

[0067] This kit may preferably be for use in the diagnosis or prediction of the risk of fibrotic diseases, together with a method according to the second aspect of the present 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 an active inflammatory bowel disease, for example, active Crohn's disease or active ulcerative colitis.

[0068] In a further aspect, the sandwich immunoassay described herein can be used in a method for evaluating the efficacy of a drug that targets a cross-linking enzyme such as LH, P3H3 lysyl oxidase (LOX), and LOXL enzyme. For example, this method can be used to evaluate a drug such as an antagonist that targets LH, P3H3, LOX or LOXL.

[0069] Thus, the present invention also relates to a method for evaluating the efficacy of an antagonist that targets a cross-linking enzyme selected from LH, P3H3, LOX or LOXL, the method comprising quantifying CTX-V in at least two biological samples using the sandwich immunoassay described herein, the biological samples being obtained from the subject at a first time point and at least one subsequent time point during the period of administration of the antagonist to the subject, and a decrease in the amount of CTX-V from the first time point to the at least one subsequent time point during the period of administration of the antagonist is an indicator of an effective antagonist that targets the cross-linking enzyme.

[0070] Preferably, this method quantifies the efficacy of the antagonist.

[0071] Preferably, this method evaluates the effectiveness of an antagonist targeting LH or LOXL.

Example

[0072] Example 1 Development of Sandwich Assay Reagents All reagents used in this study were high-quality chemicals manufactured by Merck (Whitehouse Station, NJ, USA) and Sigma (St. Louis, MO, USA). The 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 disulfide bridge), 4) long-chain peptide: PKGEKGHPGLI (SEQ ID NO: 2), 5) C-terminally 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).

[0073] Production of Monoclonal Antibodies and Characterization of Clones Monoclonal antibodies targeting the neoepitope (1478’-PKGEKGHPGL-’1487 (SEQ ID NO: 1)) located in the C-helical region of type V collagen were generated in Balb / C mice as described in

[30] . Briefly, 200 μL of emulsified antigen and 100 μg of immunogenic peptide were injected together with Sigma adjuvant system. One month later, intravenous injection of the immunogenic peptide (100 μg in 100 μL of 0.9% NaCl) was repeated, and spleen cells were isolated 72 hours later for cell fusion with SP2 / 0 myeloma cells

[31] . The supernatant was screened for reactivity against the selected peptide, long-chain peptide, and truncated-terminal peptide using an indirect competitive enzyme-linked immunosorbent assay (ELISA) with streptavidin pre-coated plates (Roche, Vizual, Denmark, catalog number 11940279) coated with 4 ng / mL of coating peptide. Clones were selected by examining the selective and unique reactivity of the antibodies against the selected peptide or immunogenic peptide. The long-chain peptide and truncated-terminal peptide were not detected (Figure 1). The selected antibodies were examined for isotype 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).

[0074] The nucleotide sequence of the selected antibody was determined and the CDRs were determined.

[0075] The sequences of the chains are as follows (CDRs are in bold; framework sequences are in italics; constant regions are underlined):

[0076] Light chain: Amino acid sequence (238 aa)

[0077]

Chemical formula

[0078] CDR-L1: KSSQSLLYSDGKTYLN (SEQ ID NO: 4) CDR-L2: LVSKLDS (SEQ ID NO: 5) CDR-L3: WQGTHLPYA (SEQ ID NO: 6)

[0079] Heavy chain: Amino acid sequence (464 aa)

Chemical formula

[0080] CDR-H1: DYYMH (SEQ ID NO: 7) CDR-H2: WIDPENGDREYAPKFQG (SEQ ID NO: 8) CDR-H3: RGHYEDH (SEQ ID NO: 9)

[0081] The selected antibody was used in the following sandwich immunoassay for both the capture antibody and the detection antibody.

[0082] The capture antibody was generated by mixing 110 μL of Na2CO3 / NaHCO3 buffer, pH 9.6, 1 mL (1 mg / mL) of antibody, and 13.3 μL of biotinamidocaproic acid N-hydroxysuccinimide ester, and incubating the solution at 20 °C for 1 hour with inversion and rotation. Next, 110 μL of 0.2 M ethanolamine, pH 8.0 was added to this solution and incubated as above. The solution was dialyzed overnight at 4 °C in a Zeba 7k MWCO desalting column (Thermo Scientific, Waltham, MA, USA, catalog number 89889) immersed in 1×PBS. A portion of the solution was labeled with horseradish peroxidase using a kit from Sigma (catalog 11829696001) according to the manufacturer's instructions and used as the detection antibody.

[0083] CTX-V Direct Sandwich ELISA Protocol A streptavidin pre-coated 96-well plate (Roche Diagnostic’s, Visby, Denmark, catalog number 11940279) was coated with 0.250 μg / mL of capture antibody diluted 1 / 200 with assay buffer (50 mM PBS, 1% BSA, 0.1% Tween-20, 150 mM NaCl, pH 7.4) and incubated at 20 °C for 30 minutes at 300 revolutions per minute. After adding 20 μL of standard, control, or sample, 100 μL of incubation buffer (25 mM PBS, 1% BSA, 0.1% Tween-20, 75 mM NaCl, 5% Liquid II, pH 7.4) was added. Next, the plate was placed under rotation at 20 °C for 1 hour. After sample incubation, the plate was coated with 0.500 μg / mL of detection antibody diluted 1 / 100 with incubation buffer for 1 hour. Between each incubation, the plate was washed using 25 mM TRIZMA, 50 mM NaCl, 0.036% Bronidox L5, 0.1% Tween 20 buffer. Then, 100 μL of chemiluminescent substrate was added to the wells, incubated at 20 °C for 3 minutes in the dark, and measured at 470 nm. Using the results obtained from a 1.5-fold serial dilution solution of the dimeric selected peptide, a standard curve was plotted using a 4-parameter mathematical fitting model. The measured values of the unknown samples were interpolated with the standard curve to determine the concentration (ng / mL) of CTX-V.

[0084] Technical Validation The lower limit of detection (LLOD) was determined from 21 zero samples (i.e., incubation buffer). The LLOD was calculated as the mean value + 3 × standard deviation (SD). The upper limit of quantification was determined from the highest point of the standard curve with a recovery rate < 20% determined in 10 independent runs. Inter-assay and intra-assay variability were determined by measuring 5 quality control samples 2 times in 10 independent runs using at least 3 samples of healthy human serum or plasma EDTA samples (Valley Biomedical, Winchester, VA, USA). The acceptance criteria for inter-assay and intra-assay variability were 15% and 10%, respectively. Assay linearity, specificity, accuracy, and interference were determined by calculating the recovery rate based on the validation criteria of 100% ± 20% from reference samples. Undiluted samples were used as references, and linearity was evaluated by 1:2 fold dilution of 4 samples of healthy human serum and human plasma EDTA samples. Assay accuracy was determined by adding 2 samples of healthy human serum or plasma EDTA samples and calculating the recovery rate between the actual measured value and the theoretical measured value. Interference was evaluated by adding biotin at known concentrations (low concentration = 5 ng / mL, high concentration = 100 ng / mL), hemoglobin (low concentration = 0.078 mM, high concentration = 0.155 mM), or lipid (low concentration = 2.42 mM, high concentration = 5.49 mM) to healthy human serum or plasma EDTA samples. The recovery rate was calculated between the non-added sample and the low or high concentration interference samples.

[0085] Stability of Analytes and Reagents The stability of the analyte was determined by calculating the recovery rate from non-stressed samples of 3 samples of healthy human serum and plasma EDTA samples. The samples were subjected to 5 freeze-thaw cycles.

[0086] Technical Performance of CTX-V No reactivity against long-chain peptides and truncated peptides was observed, which was indicated by the lack of signal inhibition. As the concentrations of the selected peptide and the immunogenic peptide increased, an increase in signal inhibition was observed, indicating the specificity of the monoclonal antibody against the selected neoepitope of CTX-V (Figure 1). The technical verification and analysis, and the stability of the reagents were acceptable based on the validation criteria (Table 2).

[0087]

Table 2

[0088] Example 2 Analysis of Samples from Patients with Inflammatory Bowel Disease Clinical Cohort The study subjects were registered in a prospective observational study at Odense University Hospital (ClinicalTrials.gov ID: NCT02612103) and have been previously described in

[29] . The analysis performed here included UC patients (n = 47), CD patients (n = 39), and healthy subjects (n = 32). Informed consent was obtained from each patient before blood sampling. The Regional Ethics Committee of Southern Denmark (journal number: S-20150107) approved this study, which was conducted in accordance with the Declaration of Helsinki. The patient demographics are shown in Table 1. Biomarker values for UC, CD, and healthy subjects were evaluated at baseline.

[0089] Statistical Analysis Patient characteristics by category were presented as frequency (percentage), and continuous variables were shown as mean ± SD. Statistical differences in patient characteristics by category were calculated using Fisher's exact test for two groups and chi-square test for three or more groups. The difference in continuous variables, including biomarker values, between healthy controls and UC or CD patients was determined by the Kruskal–Wallis test with Dunn's test for multiple comparison correction. Biomarker data were expressed in ng / mL and plotted using Tukey plots. To evaluate the diagnostic ability of the CTX-V biomarker, receiver operating characteristic (ROC) analysis was performed. All statistical analyses were conducted using 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 = no significant difference.

[0090] Results Demographics of the Cohort

[0091]

Table 3

[0092] Proteolysis of crosslinked type V collagen is increased in UC and CD patients Plasma CTX-V levels were significantly elevated in UC or CD patients compared to healthy controls (all: p < 0.0001, difference in mean values [3.87 ng / mL and 2.19 ng / mL]) (Figure 2). By quantifying proteolytically and cross-linked type V collagen, UC or CD patients can be identified. When ROC analysis was performed to evaluate the discriminatory ability of CTX-V, the area under the curve between healthy controls and UC or CD patients was 0.99 (sensitivity: 100%, specificity: 96.87%) and 0.99 (sensitivity: 100%, specificity: 96.87%), respectively. The results of the ROC analysis are summarized in Table 3.

[0093]

Table 4

[0094] Example 3 - Analysis of Samples from Patients with Ankylosing Spondylitis, Psoriasis and Atopic Dermatitis Clinical Cohort Commercially available serum samples were obtained from patients diagnosed with ankylosing spondylitis (AS) (n = 19), psoriasis (PSO) (n = 19), atopic dermatitis (AD) (n = 10), and healthy controls (HS) (n = 32). Samples from AS, PSO, and AD patients were obtained from Protogenex (Inglewood, CA, USA), and HS samples were obtained from Valley Biomedical (Winchester, VA, USA). Patient demographics are shown in Table 4.

[0095] Scar-in-a-Jar, a fibrosis in vitro model Human primary intestinal fibroblasts (Catalog No. H-6025) purchased from Cell Biologics (Chicago, Illinois, USA) were grown to confluence and seeded at a density of 30,000 cells / well in rows 6 - 8 of a 48-well plate in high serum medium (10% fetal bovine serum (FBS) (Catalog No. F7524, Sigma-Aldrich, St. Louis, Missouri, USA), Dulbecco's Modified Eagle Medium (DMEM) + Glutamax (Catalog No. 31966, Gibco, Life Technologies, Carlsbad, California, USA)) 2 days prior. One day prior, the cells were serum-starved in low serum medium (0.4% FBS DMEM) to avoid interference with biomarker measurements. On day 0 when fibroblast formation was induced, the cells were cultured with or without stimulation by 20 ng / mL TGF-β1 (Catalog No. 100-B-010 / CF, R&D system, Minneapolis, Minnesota, USA) in low serum medium containing 1.0% L-ascorbic acid, magnesium phosphate salt, n-hydrate (Catalog No. 013-19,641, Wako, Osaka, Japan), 1.0% Ficoll-70 (112.5 mg / mL, Catalog No. F2878, Sigma-Aldrich, St. Louis, Missouri, USA), and -400 (75 mg / mL, Catalog No. F4375, Sigma-Aldrich, St. Louis, Missouri, USA). The cells were incubated at 37 °C, 95% O2, and 5% CO2 for 12 days, with the medium replaced with freshly prepared medium without or with TGF-β1 stimulation on days 0, 4, and 8, and the supernatants on days 4, 8, and 12 were stored for subsequent CTX-V measurements. Three replicates were performed for both without stimulation and with stimulation.

[0096] Cell-matrix cleavage On the 12th day, the supernatant was removed, the Ska-in ajar (SIAJ) cell model wells were washed twice with PBS, and stored at -20 °C. For in vitro cleavage of the matrix-containing wells, the plates were thawed and gently washed with digestion buffer (50 mM Tris-HCl, 200 mM NaCl, 10 mM CaCl2, 100 μM ZnCl, pH 7.5). The pro-forms of MMP-9 (Catalog number 911-MP-010, R&D system, Minneapolis, Minnesota, USA) and MMP-13 (Catalog number 511-MM-010, R&D system, Minneapolis, Minnesota, USA) were digested in digestion buffer with 100 ng / μL of pro-protease and 1 mM of 4-aminophenylacetic acid (APMA, Catalog number A9563, Sigma-Aldrich, St. Louis, Missouri, USA), and pro-MMP-9 was activated by incubating at 37 °C for 24 hours and pro-MMP-13 was activated by incubating at 37 °C for 2 hours. 500 μL of 0.5 μg of activated MMP diluted with digestion buffer was added to the wells, and the plates were incubated at 37 °C for 72 hours. Then, protease was inhibited by adding 1 μM EDTA to the wells. The cleaved supernatant was stored at -20 °C until CTX-V measurement. Wells incubated with only each digestion buffer were used as controls.

[0097] Statistical Analysis Biomarker data of the SIAJ cell model were analyzed by two-way ANOVA comparing data of unstimulated and stimulated cells, and Sidak's test was applied to correct for multiple comparisons. Data are shown as mean ± SD. Statistical analysis of the fold change in CTX-V of cell-matrix cleavage was performed by one-way ANOVA using exact biomarker measurements (ng / mL) of 3 replicates compared with the presence or absence of active MMP, and Sidak was used to correct for multiple comparisons. The fold change in CTX-V was calculated based on the respective medium controls of the SIAJ experiment and the cell matrix cleavage experiment.

[0098] Results Demographics of the Cohort

[0099]

Table 5

[0100] In vitro generation of CTX-V fragments Analysis of the supernatants of human primary intestinal fibroblasts stimulated or not stimulated with TGF-β1 also showed no proteolytic release of the CTX-V fragment. However, after incubation with activated MMP-9, a 3.2-fold increase in CTX-V (p < 0.01) was observed compared to the non-cleaved control (Figure 3).

[0101] Proteolysis of crosslinked type V collagen in fibroinflammatory pathologies After measuring the serum levels of CTX-V in AS, PSO, and AD patients and comparing them with the levels of HS, the biomarker levels were significantly elevated in AS, PSO, or AD patients (AS: p < 0.001, PSO: p < 0.001, AD: p < 0.05) (Figure 4).

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Claims

1. A monoclonal antibody that specifically recognizes and binds to a neoepitope of a C-helical region generated by protease cleavage of intact V-type collagen, wherein the neoepitope is contained in the amino acid sequence PKGEKGHPGL-COOH (SEQ ID NO: 1), said monoclonal antibody.

2. The monoclonal antibody according to claim 1, wherein the monoclonal antibody is a monoclonal antibody against a synthetic peptide having a C-terminal amino acid sequence PKGEKGHPGL-COOH (SEQ ID NO: 1).

3. The monoclonal antibody according to claim 1 or 2, wherein the monoclonal antibody does not specifically recognize or bind to a peptide having a C-terminal amino acid sequence PKGEKGHPGLIZ (SEQ ID NO: 2) (in the sequence, Z is absent or is one or more amino acids of the sequence of the C-helical region of V-type collagen).

4. The monoclonal antibody according to any one of claims 1 to 3, wherein the monoclonal antibody does not specifically recognize or bind to a peptide having a C-terminal amino acid sequence PKGEKGHPG (SEQ ID NO: 3).

5. A sandwich immunoassay for detecting cross-linked type V collagen in a biological sample, wherein the cross-linked type V collagen comprises at least two strands of the C-helical region of type V collagen linked together by intermolecular cross-links, the sandwich immunoassay comprising: contacting the biological sample containing the cross-linked type V collagen with a first monoclonal antibody bound to a surface, wherein each strand of type V collagen contained in the cross-linked type V collagen has a neoepitope of a C-helical region generated by protease cleavage of intact type V collagen; adding a second monoclonal antibody; and determining the amount of binding of the second monoclonal antibody wherein both the first monoclonal antibody and the second monoclonal antibody exhibit specific reactivity with a neoepitope of a C-helical region generated by protease cleavage of intact type V collagen, and the neoepitope is contained in the amino acid sequence PKGEKGHPGL-COOH (SEQ ID NO: 1), said sandwich immunoassay.

6. The sandwich immunoassay according to claim 5, wherein the monoclonal antibody does not substantially recognize or bind to a long-chain type of the C-terminal amino acid sequence represented by PKGEKGHPGLIZ-COOH (SEQ ID NO: 2) (in the sequence, Z is absent or is one or more amino acids of the sequence of the C-helical region of type V collagen).

7. The sandwich immunoassay according to claim 5 or 6, wherein the sandwich immunoassay is used for quantifying cross-linked type V collagen in a biological sample.

8. The sandwich immunoassay according to claim 7, further comprising correlating the amount of cross-linked type V collagen determined by the sandwich immunoassay with a standard fibrotic disease sample of a known disease severity in order to evaluate the severity of a fibrotic disease.

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

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

11. The sandwich immunoassay according to claim 10, wherein the biological fluid is serum, plasma, urine, amniotic fluid, tissue supernatant or cell supernatant.

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

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

14. The sandwich immunoassay according to claim 13, wherein the second monoclonal antibody is an enzyme-conjugated antibody.

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

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

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

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

19. A method for diagnosing a fibrotic disease, comprising: i) measuring the CTX-V level in a biological fluid sample of a patient using the sandwich immunoassay according to any one of claims 5 to 17; ii) comparing the CTX-V level in a biological fluid sample of a healthy subject with the CTX-V level in a biological fluid sample of a patient suspected of having a fibrotic disease; iii) determining, based on the comparison of step ii), whether the patient suspected of having a fibrotic disease has a fibrotic disease said method.

20. iv) treating the patient suspected of having a fibrotic disease with an agent for treating the fibrotic disease further comprising the method according to claim 19.

21. A method for evaluating the efficacy of an antagonist targeting a cross-linking enzyme selected from LH, P3H3, LOX or LOXL, comprising: said method comprising quantifying CTX-V in at least two biological samples using the sandwich immunoassay according to any one of claims 5 to 17, said biological samples being obtained from the subject at a first time point and at least one subsequent time point during the period of administration of the antagonist to the subject, wherein a decrease in the amount of CTX-V from the first time point to the at least one subsequent time point during the period of administration of the antagonist is an indicator of an effective antagonist targeting LOX, said method.

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

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