Endotrophin assay

Abstract

The present invention relates to immunoassays for detecting the whole endotrophin peptide. The assay can be used to detect and / or monitor a disease in a patient and / or assessing the likelihood of, or the severity of a disease in a patient. The disease may be selected from cardiovascular disease, idiopathic pulmonary fibrosis (IPF) or chronic kidney disease. The cardiovascular disease may in particular be heart failure, and especially heart failure with preserved ejection fraction. The immunoassay may be for assessing the likelihood of adverse outcomes of the cardiovascular disease, chronic kidney disease and all cause mortality.

Description

[0001]ENDOTROPHIN ASSAY Field of the Invention The present invention relates to immunoassays for detecting the whole endotrophin peptide. The assay can be used to detect and / or monitor a disease in a patient and / or assessing the likelihood of, or the severity of a disease in a patient. The disease may be selected from cardiovascular disease, idiopathic pulmonary fibrosis (IPF) or chronic kidney disease. The cardiovascular disease may in particular be heart failure, and especially heart failure with preserved ejection fraction. The immunoassay may be for assessing the likelihood of adverse outcomes of the cardiovascular disease, chronic kidney disease and all cause mortality. Background The burden of heart failure (HF) has increased dramatically over the last several years. Approximately half of HF is secondary to HF with preserved ejection fraction (HFpEF), which is anticipated to represent an even larger proportion of the total burden of HF as the population ages. Despite multiple phase-III randomized controlled trials over the last few decades, a pharmacologic intervention proven to provide a clear benefit for this patient population remains to be identified. The heterogeneity of the HFpEF syndrome has been identified as an important barrier to demonstrating the effectiveness of candidate pharmacologic interventions. Given the heterogenous nature of HFpEF, different degrees of contribution from various pathophysiological processes may unfavorably influence average responses to pharmacologic therapies tested in clinical trials. Therefore, the availability of simple, non-invasive biomarkers capable of readily identifying relevant underlying specific biologic processes that can be targeted with pharmacologic interventions represents a promising approach to enhance our clinical and therapeutic approach to HFpEF. The hetererogeneity of HFpEF also has important implications for the differential prognosis of individual patients. The ability to more effectively risk-stratify HFpEF patients is greatly needed. Novel risk-stratification markers may not only improve our ability to prognosticate HFpEF patients in clinical practice, but would be of great value to inform enrollment of high-risk individuals in future trials. Myocardial fibrosis is thought to play a role in the pathophysiology of HFpEF. Increased fibrosis results from an excess of formation relative to degradation of collagen, ultimately leading increased interstitial collagen deposition in the interstitium. Increased myocardial extracellular matrix deposition has been demonstrated in HFpEF in autopsy specimens and in vivo studies and has been shown to correlate with left ventricular (LV) passive stiffening and diastolic dysfunction in this condition. Myocardial fibrosis may also contribute to reduced coronary flow reserve, ventricular dyssynchrony and a propensity to arrhythmia. Given the role of myocardial fibrosis in HFpEF, simple fibrotic biomarkers that reflect the underlying dynamic process of fibrosis progression or regression of fibrosis would be highly valuable. The extracellular volume fraction (ECVF), an index of myocardial fibrosis measured by cardiac magnetic resonance imaging, has been reported to predict adverse outcomes in patients with HFpEF, or at risk for HFpEF. Although MRI will likely have an important role in the assessment of myocardial fibrosis in preclinical studies, early-phase research in humans and some clinical settings, its cost and availability are likely to limit or preclude its use in global phase-III trials and in clinical practice. In addition, many patients with HFpEF are not candidates for ECVF measurements due to claustrophobia or advanced renal disease. Thus, the search for circulating biomarkers of tissue fibrosis remains an area of great interest. Collagen Type VI is a unique extracellular collagen which can form an independent microfibrillar network in the basement membrane of cells. It can interact with other matrix proteins including collagens, biglycan, and proteoglycans. In muscle, type VI collagen is part of the sarcolemma and is involved in anchoring the muscle fiber into the intramuscular extracellular matrix, and so is involved in force transmission. Moreover, mutations in type VI collagen can cause Bethlem myopathy and Ullrich congenital muscular dystrophy. It has been reported that the C-terminal amino acid sequence of the type VI collagen α3 chain is cleaved off from the mature type VI microfibril after secretion. However, Type VI collagen is not just involved in muscles and muscle loss. The microflamentous interstitial type VI collagen, a triple helical molecule composed of the constituent chains α1(VI), α2(VI), and α3(VI), is expressed in most connective tissues and prominently in adipose tissue, where it anchors cells through its interconnections with other ECM proteins. During the formation of microfilaments, the triple-helical core of type VI collagen is proteolytically released from the pro-peptide, and cleavage of the C-terminal pro-peptide of the α3(VI) chain generates a 77 amino acid molecule endotrophin, an adipokine. The amino acid sequence of endotrophin is: TEPLALTETDICKLPKDEGTCRDFILKWYYDPNTKSCARFWYGGCGGNENKFGSQKECEKVCA PVLAKPGVISVMGT (SEQ ID No: 14). PRO-C6 is a biomarker for formation of collagen type VI and endotrophin release, comprising a C-terminal epitope of the C5 domain of the α3 chain of type VI collagen that is cleaved off when a novel collagen type VI molecule assembles in the extracellular matrix, and which C-terminal epitope is also a C-terminal epitope of the bioactive fragment endotrophin. The PRO-C6 biomarker, and a PRO-C6 assay (specifically, a PRO-C6 ELISA) are described in WO2016 / 156526. The assay utilizes a monoclonal antibody that specifically binds to the C- terminus 10 amino acid sequence of the C5 domain of the α3 chain of collagen type VI. Endotrophin’s role as a pro-fibrotic, pro-inflammatory and pro-tumorigenic molecule has been observed in preclinical models of breast cancer and liver fibrosis. PRO-C6 has been established as a prognostic biomarker for mortality and disease progression in chronic kidney disease and diabetic kidney disease patients32-34and as a predictive marker for response to glucose lowering therapy in diabetic patients35. As described in WO2020 / 245404, PRO-C6 has also been used as a marker for patients with heart failure with preserved ejection fraction (HFpEF) and found to be effective diagnostic and prognostic biomarkers of heart failure. Summary The present inventors have developed a sandwich immunoassay which uses the PRO-C6 antibody which binds to the last 10 amino acids of the COL6A3 chain sequence (KPGVISVMGT (SEQ ID No: 1)), and an ETP antibody which binds to the first 10 amino acids of the endotrophin sequence (TEPLALTETD (SEQ ID No: 27)). The assay detects whole endotrophin molecules, not just the C terminus as detected by the PRO-C6 assay. The entire endotrophin molecule (Endotrophin biomarker) is an improved biomarker compared to PRO-C6 alone. The assay was found to be more effective than the PRO-C6 assay in separating different group of patients with heart failure. In addition, the assay was also able to identify patients with idiopathic pulmonary fibrosis with greater sensitivity than PRO-C6. The new immunoassay gave higher hazard ratios for mortality and cardiovascular events than PRO-C6, and a similar hazard ratio for kidney failure endpoint. Accordingly, in a first aspect the present invention provides a sandwich immunoassay method for detecting the Endotrophin biomarker in a biofluid sample, wherein said method comprises: (iii) contacting said biofluid sample from a patient with a first monoclonal antibody bound to a surface; (ii) adding a second monoclonal antibody; and (iii) determining the amount of binding of said second monoclonal antibody; wherein one of the first monoclonal antibody and second monoclonal antibody specifically binds the C-terminal epitope of the C5 domain of the α3 chain of type VI collagen and the other of the first monoclonal antibody and second monoclonal antibody specifically binds the N-terminal epitope of endotrophin. The first monoclonal antibody binds and captures the target peptide present in the sample. The second monoclonal antibody then binds to the target peptide which has been bound by the first monoclonal antibody. The amount of second monoclonal antibody bound to the peptide can then be detected and / or quantified. Preferably the monoclonal antibody which specifically binds the C-terminal epitope of the C5 domain of the α3 chain of type VI collagen specifically binds to the C-terminus amino acid sequence KPGVISVMGT (SEQ ID No: 1) (also referred to herein as the “PRO-C6 sequence”, or simply “PRO-C6”). Preferably said monoclonal antibody does not specifically bind to an elongated version of said C-terminus amino acid sequence which is KPGVISVMGTA (SEQ ID No: 2), and / or does not specifically bind to a truncated version of said C-terminus amino acid sequence which is KPGVISVMG (SEQ ID No: 3). Preferably, the ratio of the affinity of said antibody for the C-terminus amino acid sequence KPGVISVMGT (SEQ ID No: 1) to the affinity of said antibody for the elongated C-terminus amino acid sequence KPGVISVMGTA (SEQ ID No: 2), and / or for the truncated C-terminus amino acid sequence KPGVISVMG (SEQ ID No: 3), is at least 10 to 1, and more preferably is at least 50 to 1, at least 100 to 1, at least 500 to 1, at least 1,000 to 1, at least 10,000 to 1, at least 100,000 to 1, or at least 1,000,000 to 1. As used herein the term “C-terminus” refers to a C-terminal peptide sequence at the extremity of a polypeptide, i.e. at the C-terminal end of the polypeptide, and is not to be construed as meaning in the general direction thereof. Preferably the monoclonal antibody which specifically binds the N-terminal epitope of endotrophin specifically binds to the N-terminus amino acid sequence TEPLALTETD (SEQ ID No: 27) (also referred to herein as “ETP sequence” or “ETP”). Preferably said monoclonal antibody does not specifically bind to an elongated version of said N-terminus amino acid sequence which is STEPLALTETD (SEQ ID No: 28), and / or does not specifically bind to a truncated version of said N-terminus amino acid sequence which is EPLALTETD (SEQ ID No: 29). Preferably, the ratio of the affinity of said antibody for the N-terminus amino acid sequence TEPLALTETD (SEQ ID No: 27) to the affinity of said antibody for the elongated N-terminus amino acid sequence STEPLALTETD (SEQ ID No: 28) and / or for the truncated N-terminus amino acid sequence EPLALTETD (SEQ ID No: 29), is at least 10 to 1, and more preferably is at least 50 to 1, at least 100 to 1, at least 500 to 1, at least 1,000 to 1, at least 10,000 to 1, at least 100,000 to 1, or at least 1,000,000 to 1. As used herein the term “N-terminus” refers to a N-terminal peptide sequence at the extremity of a polypeptide, i.e. at the N-terminal end of the polypeptide, and is not to be construed as meaning in the general direction thereof. The patient biofluid sample may be, but is not limited to, blood, serum, plasma, urine or amniotic fluid. Preferably the biofluid is serum or plasma. 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. In the methods of the present invention, a monoclonal antibody comprising any constant region known in the art can be used. In the case of mouse antibodies and human antibodies, the constant light chains are classified as either kappa or 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 in the case of humans and IgGl, IgG2a, IgG2b, IgG2c and IgG3 in the case of mice. The monoclonal antibody may preferably be of the IgG isotype, including any one of the IgG subclasses. The CDR of an antibody can be determined using methods known 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 or murine IgM, IgG or IgA isotype, or IgG subclasses such as human IgG1, IgG2, IgG3 or IgG4 or murine IgGl, IgG2a, IgG2b, IgG2c and IgG3. 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 KPGVISVMGT (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 KPGVISVMGT (SEQ ID No. 1), such as for example by: immunizing a rodent (or other suitable mammal) with a synthetic peptide consisting of the sequence KPGVISVMGT (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. The monoclonal antibody that specifically binds to the PRO-C6 sequence may preferably comprises one or more complementarity-determining regions (CDRs) selected from: CDR-L1: RSSQRIVHSNGITFLE (SEQ ID No: 4) CDR-L2: RVSNRFS (SEQ ID No: 5) CDR-L3: FQGSHVPLT (SEQ ID No: 6) CDR-H1: DFNMN (SEQ ID No: 7) CDR-H2: AINPHNGATSYNQKFSG (SEQ ID No: 8) CDR-H3: WGNGKNS (SEQ ID No: 9). Preferably the antibody comprises at least 2,3,4,5 or 6 of the above listed CDR sequences. Preferably the monoclonal antibody light chain variable region comprises the CDR sequences: CDR-L1: RSSQRIVHSNGITFLE (SEQ ID No: 4) CDR-L2: RVSNRFS (SEQ ID No: 5) and CDR-L3: FQGSHVPLT (SEQ ID No: 6). Preferably the monoclonal antibody light chain 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) RSSQRIVHSNGITFLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAED LGLYYCFQGSHVPLT (SEQ ID No: 10). Preferably the monoclonal antibody heavy chain variable region comprises the CDR sequences: CDR-H1: DFNMN (SEQ ID No: 7) CDR-H2: AINPHNGATSYNQKFSG (SEQ ID No: 8) and CDR-H3: WGNGKNS (SEQ ID No: 9). Preferably the monoclonal antibody heavy chain 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) DFNMNWVKQSHGKSLEWIGAINPHNGATSYNQKFSGKATLTVDKSSSTAYMELNSLTSDDSA Monoclonal antibodies that specifically bind to the N-terminus amino acid sequence TEPLALTETD (SEQ ID No: 27) 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 TEPLALTETD (SEQ ID No: 27), such as for example by: immunizing a rodent (or other suitable mammal) with a synthetic peptide consisting of the sequence TEPLALTETD (SEQ ID No: 27), 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. The ETP binding monoclonal antibody, that specifically binds to the N-terminus amino acid sequence TEPLALTETD (SEQ ID No: 27), may preferably comprises one or more complementarity- determining regions (CDRs) selected from: CDR-L1a: RSSQSLVHSNGDTFLH (SEQ ID No: 15) CDR-L2a: QVSNRFS (SEQ ID No: 16) CDR-L3a: SQSTHVPWT (SEQ ID No: 17) CDR-H1a: RHAMS (SEQ ID No: 18) CDR-H2a: ISSGGTYTYYPDSEKG (SEQ ID No: 19) CDR-H3a: GVYYGGDQFDT (SEQ ID No: 20). Preferably the antibody comprises at least 2,3,4,5 or 6 of the above listed CDR sequences. Preferably the monoclonal antibody light chain variable region comprises the CDR sequences: CDR-L1a: RSSQSLVHSNGDTFLH (SEQ ID No: 15) CDR-L2a: QVSNRFS (SEQ ID No: 16) CDR-L3a: SQSTHVPWT (SEQ ID No: 17) Preferably the ETP binding monoclonal antibody light chain 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) RSSQSLVHSNGDTFLHWYLQKPGQSPKLLIYQVSNRFSGVPDRFSGSGSGTDFTLRINRVEAE DLGVYFCSQSTHVPWT (SEQ ID No: 21). CDR-H1a: RHAMS (SEQ ID No: 18) CDR-H2a: ISSGGTYTYYPDSEKG (SEQ ID No: 19) CDR-H3a: GVYYGGDQFDT (SEQ ID No: 20). Preferably the ETP binding monoclonal antibody heavy chain 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) RHAMSWVRQTLEKRLEWVASISSGGTYTYYPDSEKGRFTISRDNAKNTLYLQMSGLRSEDTA MYYCTRGVYYGGDQFDT (SEQ ID No: 22). 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 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 that specifically binds to the PRO-C6 sequence may comprise the light chain variable region sequence: DVVMTQTPLSLPVNLGDQASISCRSSQRIVHSNGITFLEWYLQKPGQSPKLLIYRVSNRFSGVP DRFSGSGSGTDFTLKISRVEAEDLGLYYCFQGSHVPLTFGAGTRLELK (SEQ ID No: 12) and / or the heavy chain variable region sequence: EVQLQQSGPVMVKPGTSVKTSCKASGYTFTDFNMNWVKQSHGKSLEWIGAINPHNGATSYN QKFSGKATLTVDKSSSTAYMELNSLTSDDSAVYYCARWGNGKNSWGQGTTLTVSS (SEQ ID No: 13) (CDRs bold and underlined; Framework sequences in italics) In certain preferred embodiments, the monoclonal antibody that specifically binds to the ETP epitope (the N-terminus amino acid sequence TEPLALTETD (SEQ ID No: 27)) may comprise the light chain variable region sequence: DVVMTQTPLSLPVSLGDQASISCRRSSQSLVHSNGDTFLHWYLQKPGQSPKLLIYQVSNRFSG VPDRFSGSGSGTDFTLRINRVEAEDLGVYFCSQSTHVPWT FGGGTKLEIKR (SEQ ID No: 23) and / or the heavy chain variable region sequence: EVMLVESGGGLVKPGGSLKLSCAASGFTFSRHAMSWVRQTLEKRLEWVASISSGGTYTYYPD SEKGRFTISRDNAKNTLYLQMSGLRSEDTAMYYCTRGVYYGGDQFDTWGQGTVLTVSS (SEQ ID No: 24) (CDRs bold and underlined; Framework sequences in italics) 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. The sandwich immunoassay may be, but is not limited to, a radioimmunoassay, fluorescence immunoassay, or an enzyme-linked immunosorbent assay. The first monoclonal antibody may also be known as the catcher antibody. As used herein a “catcher” antibody is an antibody that is capable of binding to the target peptide which is bound to a solid surface. The solid surface may be a surface of a well, or a bead or other particle. When the solid surface is brought into contact with a sample, the target peptide in the sample is bound by the catcher antibody. The second monoclonal antibody may also be known as the detector antibody. As used herein a “detector antibody” is an antibody that is capable of binding to a different epitope of the target peptide to the catcher antibody. When the detector antibody comes into contact with the target peptide / catcher antibody complex or peptide which has been bound by the catcher antibody, it also binds to the target peptide. The presence of the bound detector antibody can be detected by either a label on the detector antibody or by using a further antibody which can bind to the detector antibody, which itself is labelled. Once the target peptide has been contacted with and bound by the first (or catcher) antibody, there is a wash step to remove any unbound peptide and impurities present. The wash step may occur before the addition of the detector antibody, so the only target peptide present is that bound by the catcher antibody. The second (or detector) antibody is added to detect the amount of bound peptide. Any unbound detector antibody is removed by a wash step prior to determining the amount of detector antibody bound by the target peptide. There may be a single wash step after the addition of the detector antibody which removes both peptides not bound by the capture antibody (which may or may not be bound by the detector antibody) and any unbound detector antibody. Thus, the method may comprise the following steps: (a) Contacting a sample containing the target peptide with a first monoclonal antibody bound to a surface; (b) Optional wash step (c) Adding a second monoclonal antibody which specifically binds to the target peptide at a different epitope to the first monoclonal antibody (d) Removal of any unbound second monoclonal antibody (e) Determining the amount of binding of the second monoclonal antibody Alternatively, once the target peptide has been contacted and bound by the capture antibody, there may be a washing step to remove any unbound peptide, followed by an elution step to release the bound target peptide. The eluted or released peptide can then be contacted with the detector antigen and the amount of binding of the second antibody determined. Thus the immunoassay may comprise the following steps: (a) Contacting a sample containing the target peptide with a first monoclonal antibody bound to a surface; (b) Wash step (c) Elution step to release bound target peptide; (d) Adding a second monoclonal antibody which binds to the target peptide at a different epitope to the first monoclonal antibody to the eluted peptide (e) Wash step (f) Determining the amount of binding of the second monoclonal antibody The herein described sandwich immunoassay uses an antibody which binds to the PRO-C6 eptiope (KPGVISVMGT) and one which binds to the ETP epitope (TEPLALTETD (SEQ ID No: 27)). The PRO-C6 antibody may be the catcher antibody with the ETP antibody being the detector antibody. Alternatively, the PRO-C6 antibody may be the detector antibody with the ETP antibody being the catcher antibody. Therefore, the whole endotrophin peptide, which contains both ETP and PRO-C6 epitopes is detected by the assay. As both monoclonal antibodies bind to the same peptide, if the second monoclonal antibody binds to a peptide bound by the first monoclonal antibody, then the entire endotrophin peptide must be present. The sandwich immunoassay can detect and determine the amount of binding between said second monoclonal antibody and entire endotrophin peptide 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. An elevated level of binding and so increased amount of the Endotrophin biomarker is indicative of the presence of a disease or increased disease severity. Thus, the sandwich immunoassay may further comprise correlating the quantity of whole endotrophin determined by said method with standard disease samples of known disease severity to evaluate the severity of the disease. Such diseases may be, but not limited to, cardiovascular disease, IPF or kidney disease. The cardiovascular disease may in certain embodiments be heart failure. In particular, the cardiovascular disease may be heart failure with a preserved ejection fraction (HFpEF). The kidney disease may in certain embodiments be IgA nephropathy (IgAN). 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 method may be an immunoassay method for detecting and / or monitoring and / or assessing the likelihood of a disease in a patient, the method comprising carrying out the method of immunoassay of the invention on a biofluid sample obtained from said patient with the monoclonal antibodies, detecting and determining the amount of binding between the monoclonal antibodies and whole endotrophin 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. The disease may be selected from cardiovascular disease, IPF or kidney disease. The cardiovascular disease may in certain embodiments be heart failure. In particular, the cardiovascular disease may be heart failure with a preserved ejection fraction (HFpEF). The kidney disease may in certain embodiments be IgA nephropathy (IgAN). The method may in certain embodiments be a method for assessing the severity of a disease in a patient that comprises assessing the likelihood of patient mortality and / or hospitalization as a result of chronic kidney disease, cardiovascular disease and / or a composite of adverse cardiovascular events. Cardiovascular events include cardiovascular mortality, ischemic cardiovascular disease, stroke and heart failure. In a further aspect, the present invention provides a method of treating a disease in a patient in need thereof, the method comprising: (a) carrying out a method of immunoassay in accordance with the present invention on a blood, serum or plasma sample from a patient in order to detect whether the patient has a disease and / or assess the severity of a disease in the patient; and (b) administering to the patient a medicament for the treatment of said disease if it is determined in step (a) that the patient has said disease or a particular severity thereof. The disease may be selected from cardiovascular disease, IPF or kidney disease. The cardiovascular disease may in certain embodiments be heart failure. In particular, the cardiovascular disease may be heart failure with a preserved ejection fraction (HFpEF), including asymptomatic heart failure with a preserved ejection fraction and symptomatic heart failure with a preserved ejection fraction, or heart failure with reduced ejection fraction. The kidney disease may in certain embodiments be IgA nephropathy (IgAN). As used herein the term “values associated with normal healthy subjects and / or values associated with known disease severity” means standardised quantities determined by the method described supra for subjects considered to be healthy, i.e. without the relevant disease (i.e. without a cardiovascular disease, or IPF or kidney disease) and / or standardised quantities determined by the method described supra for subjects known to have a disease (i.e. cardiovascular disease, or IPF or kidney disease) of a known severity. In some embodiments of the method according to the first aspect, the amount of binding of the second monoclonal antibody are compared with one or more predetermined cut-off values. As used herein the “ predetermined cut-off value” means an amount of binding that is determined statistically to be indicative of a high likelihood of disease i.e. cardiovascular disease, or IPF or kidney disease in a patient, or a particular level of severity thereof (or prognosis thereof), in that a measured value of biomarker binding in a patient sample that is at or above the statistical cutoff value corresponds to at least a 70% probability, preferably at least an 80% probability, preferably at least an 85% probability, more preferably at least a 90% probability, and most preferably at least a 95% probability of the presence or likelihood of a disease (i.e. cardiovascular disease, or IPF or kidney disease) or of a particular level of severity of the disease. 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 (koff 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). In the specific assay disclosed herein which measures in biofluids target peptides having the C- terminus amino acid sequence KPGVISVMGT (SEQ. ID No. 1) and the N-terminus sequence TEPLALTETD(SEQ ID No: 27), the calibration curve is produced using standard samples of known concentration of a calibration peptide having the C-terminus amino acid sequence KPGVISVMGT (SEQ. ID No.1) and the N-terminus sequence TEPLALTETD (SEQ ID No: 27) and which may in particular consist of the endotrophin peptide. 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 binds 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 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 second monoclonal antibody as described above, which is preferably labelled. The kit may be for use in detecting or predicting the risk of a disease, preferably in conjunction with the methods according to the invention. Such diseases may be, but not limited to, cardiovascular disease, IPF or kidney disease. The cardiovascular disease may in certain embodiments be heart failure. In particular, the cardiovascular disease may be heart failure with reduced ejection fraction (HFrEF) or heart failure with a preserved ejection fraction (HFpEF), including asymptomatic heart failure with a preserved ejection fraction and symptomatic heart failure with a preserved ejection fraction. The invention also provides a monoclonal antibody that specifically binds to the N-terminal epitope of endotrophin comprising or consisting of the amino acid sequence TEPLALTETD (SEQ ID No: 27). Preferably said monoclonal antibody does not specifically bind to an elongated version of said N-terminus amino acid sequence which is STEPLALTETD (SEQ ID No: 28), and / or to a truncated version of said N-terminus amino acid sequence which is EPLALTETD (SEQ ID No: 29). The invention will now be described in the examples below which refer to the following figures: Figure 1 shows the levels of Endotrophin biomarker and PRO-C6 biomarker in a panel of patients with heart failure. Figure 2 shows the correlation between Endotrophin biomarker and PRO C6 biomarker levels in patients with heart failure with preserved ejection fraction and heart failure with reduced ejection fraction. Figure 3 shows the levels of Endotrophin biomarker and PRO-C6 biomarker in a panel of patients with idiopathic pulmonary fibrosis. Figure 4 shows the correlation between Endotrophin biomarker and PRO C6 biomarker levels in patients in the BNP study cohort. Figure 5 shows the levels of Endotrophin biomarker in a panel of patients with IgA nephropathy. Figure 6 shows the correlation between circulating endotrophin and fibrosis levels evaluated in histology. Figure 7 shows the ETP antibody selectivity. The presently disclosed embodiments are described in the following Examples, which are set forth to aid in the understanding of the disclosure, and should not be construed to limit in any way the scope of the disclosure as defined in the claims which follow thereafter. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the described embodiments, and are not intended to limit the scope of the present disclosure nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric. Example 1 - Antibody development for PRO -C6 A monoclonal antibody specific for PRO-C6 was developed as described in WO 2016 / 156526 (Nordic Bioscience, incorporated herein by reference) using the last 10 amino acids of the type VI collagen α3 chain (i.e. the C-terminus sequence3168’KPGVISVMGT’3177(SEQ ID No: 1)) as an immunogenic peptide. Briefly, 4-6-week-old Balb / C mice were immunized subcutaneously with 200μl emulsified antigen with 60μg of the immunogenic peptide. Consecutive immunizations were performed at 2-week intervals in Freund's incomplete adjuvant, until stable sera titer levels were reached, and the mice were bled from the 2nd immunization on. At each bleeding, the serum titer was detected and the mouse with highest antiserum titer and the best native reactivity was selected for fusion. The selected mouse was rested for 1 month followed by intravenous boosting with 50μg of immunogenic peptide in 100μl 0.9% sodium chloride solution 3 days before isolation of the spleen for cell fusion. Mouse spleen cells were fused with SP2 / 0 myeloma fusion partner cells. The fusion cells were raised in 96-well plates and incubated in the CO2-incubator. Here standard limited dilution was used to promote monoclonal growth. Cell lines specific to the selection peptide and without cross- reactivity to either elongated peptide (KPGVISVMGTA (SEQ ID No: 2), Chinese Peptide Company, China) or truncated peptide (KPGVISVMG (SEQ ID No: 3), American Peptide Company, USA) were selected and sub-cloned. At last the antibodies were purified using an IgG column. The antibodies generated were sequenced and the CDRs determined. The sequence of the chains are as follows (CDRs underlined and in bold; C-terminus Constant region in italics): Heavy Chain Sequence (mouse IgG1 isotype) EVQLQQSGPVMVKPGTSVKTSCKASGYTFTDFNMNWVKQSHGKSLEWIGAINPHNGATSYN QKFSGKATLTVDKSSSTAYMELNSLTSDDSAVYYCARWGNGKNSWGQGTTLTVSSAKTTPPS VYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTV PSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPK VTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFK CRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWN GQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG K (SEQ ID No:27) CDR-H1: DFNMN (SEQ ID No: 7) CDR-H2: AINPHNGATSYNQKFSG (SEQ ID No: 8) CDR-H3: WGNGKNS (SEQ ID No: 9) Light Chain Sequence (mouse Kappa isotype) DVVMTQTPLSLPVNLGDQASISCRSSQRIVHSNGITFLEWYLQKPGQSPKLLIYRVSNRFSGVP DRFSGSGSGTDFTLKISRVEAEDLGLYYCFQGSHVPLTFGAGTRLELKRADAAPTVSIFPPSSE QLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEY ERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID No: 28) CDR-L1: RSSQRIVHSNGITFLE (SEQ ID No: 4) CDR-L2: RVSNRFS (SEQ ID No: 5) CDR-L3: FQGSHVPLT (SEQ ID No: 6) Development of ETP antibody A monoclonal antibody specific for ETP epitope was developed using the first 10 amino acids of the endotrophin peptide (i.e. the N-terminus sequence TEPLALTETD (SEQ ID No: 27)) as an immunogenic peptide. Briefly, 4-6-week-old Balb / C mice were immunized subcutaneously with 200μl emulsified antigen with 60μg of the immunogenic peptide. Consecutive immunizations were performed at 2-week intervals in Freund's incomplete adjuvant, until stable sera titer levels were reached, and the mice were bled from the 2nd immunization on. At each bleeding, the serum titer was detected and the mouse with highest antiserum titer and the best native reactivity was selected for fusion. The selected mouse was rested for 1 month followed by intravenous boosting with 50μg of immunogenic peptide in 100μl 0.9% sodium chloride solution 3 days before isolation of the spleen for cell fusion. Mouse spleen cells were fused with SP2 / 0 myeloma fusion partner cells. The fusion cells were raised in 96-well plates and incubated in the CO2-incubator. Here standard limited dilution was used to promote monoclonal growth. Cell lines specific to the selection peptide and without cross- reactivity to the truncated peptide (EPLALTETD American Peptide Company, USA) were selected and sub-cloned. At last the antibodies were purified using an IgG column. The antibodies generated were sequenced and the CDRs determined. (CDRs underlined and in bold; N-terminus signal peptide and C-terminus Constant region in italics): Heavy Chain Sequence (mouse IgG2b isotype) MNFGLSLIFLVLVLKGVQCEVMLVESGGGLVKPGGSLKLSCAASGFTFSRHAMSWVRQTLEKR LEWVASISSGGTYTYYPDSEKGRFTISRDNAKNTLYLQMSGLRSEDTAMYYCTRGVYYGGDQ FDTWGQGTVLTVSSAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSS SVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPC KECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTA QTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILP PPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTS KWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK (SEQ ID No: 25) CDR-H1a: RHAMS (SEQ ID No: 18) CDR-H2a: ISSGGTYTYYPDSEKG (SEQ ID No: 19) CDR-H3a: GVYYGGDQFDT (SEQ ID No: 20). Light Chain Sequence (mouse Kappa isotype) MKLPVRLLVLMFWIPASSSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGDTFLHWYLQK PGQSPKLLIYQVSNRFSGVPDRFSGSGSGTDFTLRINRVEAEDLGVYFCSQSTHVPWTFGGGT KLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQ DSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID No: 26) CDR-L1a: RSSQSLVHSNGDTFLH (SEQ ID No: 15) CDR-L2a: QVSNRFS (SEQ ID No: 16) CDR-L3a: SQSTHVPWT (SEQ ID No: 17) Specific binding of the ETP antibody to the standard peptide (TEPLALTETD (SEQ ID No: 27)) and lack of cross-reactivity to the truncated peptide (EPLALTETD (SEQ ID No: 29)) was confirmed using a competitive ELISA. Briefly, streptavidin-coated plates coated with 4 ng / mL biotin-labelled coating peptide (standard peptide labelled with biotin at its C-terminus) were prepared. Various concentrations of standard peptide or truncated peptide were added to each well followed immediately by 2ng / ml of HRP-labelled monoclonal antibody (the ETP antibody), and the plates were incubated. After incubation and washing, 3,3’,5,5’-tetramethylbenzidine (TMB) was added. To stop the enzyme reaction of TMB, diluted sulphuric acid was added, and the absorbance was measured at 450nm with 650nm as the reference using an ELISA reader. The results are shown in Figure 7. Endotrophin sandwich assay The assay employs the PRO-C6 antibody, binding to the last 10 amino acids of the COL6A3 chain sequence (KPGVISVMGT) as the detector, and the ETP antibody, binding the first 10 amino acids of the endotrophin sequence (TEPLALTETD (SEQ ID No: 27)) as the capture antibody. The standard peptide is the whole endotrophin sequence: TEPLALTETDICKLPKDEGTCRDFILKWYYDPNTKSCARFWYGGCGGNENKFGSQKECEKVCA PVLAKPGVISVMGT (SEQ ID No: 14). The ETP antibody was used as the catcher antibody and labelled with biotin. The PRO-C6 antibody was labelled with horse radish peroxidase (HRP) and used as the detector antibody. The assay procedure was follows: 1. Coat streptavidin plates with catcher Ab 500ng / ml, 100 µL / well in 50mM PBS-BTB 8g / L NaCl, pH 7.4 for 30min at 18-22°C on a microtiter plate mixing apparatus (300 rpm) 2. Wash 5 times with washing buffer (20 mM TRIS, 50 mM NaCl, pH 7.2) 3. Add 20 µL standards / samples / controls to the plate, followed by adding 125 ng / ml, 100 µL detector antibody in 50 mM PBS-BTB, 8g / L NaCl + 5% liquid II (Osteocalcin EIA Puf-Liq, Roche Diagnostics), pH 7.4) and incubate for 20±1 hour at 2-8°C on a microtiter plate mixing apparatus (300 rpm) 4. Wash 5 times with washing buffer 5. Add 100 µL 3,3',5,5'-tetramethylbenzidine (TMB) (ThermoFisher Scientific) and incubate 15 min in darkness at 18-22°C on a microtiter plate mixing apparatus (300 rpm) 6. Add 100 µL stopping solution (0.1% sulphuric acid) 7. Read absorbance at 450 nm with 650 nm as reference on a plate reader. Clinical samples Heart Failure (HF) The Endotrophin immunoassay was used to assess serum samples from a panel of patients (a) with heart failure with preserved ejection fraction (HFpEF), who were either asymptomatic (HFpEF B, n=30) or symptomatic (HFpEF C, n=30), (b) with heart failure with reduced ejection fraction (HFrEF, n=60) and (c) in age- and sex-matched controls (n=30). The measurements were compared with those performed with the PRO-C6 assay as described in WO2016 / 156526. (Figures 1 and 2). The Endotrophin immunoassay shows a higher precision in separating the patients in the different groups and a narrower distribution of the data in the different groups. Idiopathic pulmonary fibrosis (IPF) The Endotrophin immunoassay was used to assess serum samples from a cohort of patients with idiopathic pulmonary fibrosis (n=20) and in age and sex-matched healthy controls (n=12). Also in this case, Endotrophin immunoassay had a numerically higher AUC than PRO-C6 (AUC: 0.90 (0.77-1.00), p=0.0002 vs AUC: 0.83 (0.66-1.00), p=0.002). The correlation between the markers was: rho=0.49, p=0.004. (Figure 3). BNP study cohort The Endotrophin immunoassay was used to assess serum samples from the BNP study (Unrecognized cardiovascular disease in T2D patients – screening using NT-proBNP), which included 200 participants with type 2 diabetes (T2D) recruited between January 2007 and February 2008 from the outpatient clinic at Steno Diabetes Center, Copenhagen. Inclusion criteria were: diagnosis of T2D, no history of Coronary artery disease (CAD) or symptoms suggestive of CVD, and persistent urinary albumin excretion ratio (UAER) >30 mg / 24 h (in 2 / 3 consecutive measurements). Exclusion criteria were: normal UAER or nonpersistent elevated UAER, symptoms of HF, angina pectoris, ischemic CVD or diagnosed MI, ischemic heart failure (HF), malignant arrhythmia, and atrial fibrillation. The clinical characteristics of the cohort are summarized in the table below: Participants were traced through the Danish National Death Register and the Danish National Health Register on 1 January 2014. Definition of endpoints: 1) All-cause mortality 2) CV events (included CV mortality, stroke, ischemic CVD, and HF). For patients with multiple events, only the first was included. 3) Kidney endpoint (defined as decline in eGFR >30% evaluated as change from baseline to the last available measurement). Data on chronic kidney disease (CKD) progression were available for 175 (88.4%) participants. Median (range) follow-up time was 6.5 (0.3–7.2) years. The hazard ratio for mortality was calculated using Cox proportional hazard regression analysis for doubling of PRO-C6 or Endotrophin. The Endotrophin immunoassay presented higher hazard ratios for mortality and CV events than PRO-C6 immunoassay, and similar hazard ratio for the kidney endpoint. The correlation between Endotrophin biomarker and PRO C6 biomarker levels in this patient cohort is shown in Figure 4. IgA nephropathy (IgAN) The Endotrophin immunoassay was used to assess serum samples from a cohort of patients with IgA nephropathy (n=98) and in healthy controls (n=64). Levels of fibrosis were evaluated in histology from kidney biopsy taken at the time of blood sampling. Levels of the Endotrophin biomarker were elevated in serum of patients with IgA nephropathy (Figure 5), and they correlated with levels of tubulointerstitial fibrosis in histology (Figure 6).

Claims

Claims 1. A method of sandwich immunoassay for detecting the endotrophin peptide in a biofluid sample, wherein said method comprises: (i) contacting said biofluid sample from a patient with a first monoclonal antibody bound to a surface; (ii) adding a second monoclonal antibody; and (iii) determining the amount of binding of said second monoclonal antibody; wherein one of the first monoclonal antibody and second monoclonal antibody specifically binds the C-terminal epitope of the C5 domain of the α3 chain of type VI collagen and the other of the first monoclonal antibody and second monoclonal antibody specifically binds the N-terminal epitope of endotrophin.

2. The method of claim 1 wherein the first monoclonal antibody specifically binds the N- terminal epitope of endotrophin and the second monoclonal antibody specifically binds the C- terminal epitope of the C5 domain of the α3 chain of type VI collagen.

3. The method of claim 1 or claim 2 wherein the monoclonal antibody that specifically binds the C-terminal epitope of the C5 domain of the α3 chain of type VI collagen specifically binds to a C-terminus amino acid sequence KPGVISVMGT (SEQ ID No: 1).

4. The immunoassay method of claim 3 wherein said monoclonal antibody does not specifically bind to an elongated version of said C-terminus amino acid sequence which is KPGVISVMGTA (SEQ ID No: 2), or to a truncated version of said C-terminus amino acid sequence which is KPGVISVMG (SEQ ID No: 3).

5. The method of any preceding claim, wherein the antibody that specifically binds the N- terminal epitope of endotrophin specifically binds to the N-terminus amino acid sequence TEPLALTETD (SEQ ID No: 27).

6. The method of claim 5 wherein said monoclonal antibody does not specifically bind to a truncated version of said N-terminus amino acid sequence which is EPLALTETD (SEQ ID No: 29).

7. The method of any preceding claim, wherein said biofluid is serum or plasma.

8. The method of any preceding claim wherein the sandwich immunoassay is used to quantify the amount of endotrophin peptide in a biological sample.

9. The method of claim 8, further comprising correlating said amount of binding of said second antibody as determined in step (iii) 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 a predetermined cut-off value, preferably wherein the disease is selected from cardiovascular disease, idiopathic pulmonary fibrosis (IPF) or kidney disease.

10. The method of Claim 9, wherein the cardiovascular disease is heart failure.

11. The method of Claim 9, wherein the cardiovascular disease is heart failure with a preserved ejection fraction (HFpEF) including asymptomatic heart failure with a preserved ejection fraction and symptomatic heart failure with a preserved ejection fraction, or heart failure with reduced ejection fraction.

12. The method of Claim 9, wherein the method is a method for assessing the severity of a disease in a patient that comprises assessing the likelihood of patient mortality and / or hospitalization as a result of the disease and / or a composite of adverse cardiovascular events.

13. The sandwich immunoassay of any preceding claim, wherein the sandwich immunoassay is a radioimmunoassay, fluorescence immunoassay, or an enzyme-linked immunosorbent assay.

14. The sandwich immunoassay of any preceding claim, wherein the second monoclonal antibody is labeled.

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

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

17. The sandwich immunoassay of claim 14, wherein the second monoclonal antibody is radiolabeled or linked to a fluorophore.

18. The sandwich immunoassay of any one of claims 1 to 13, wherein a further labeled antibody which binds the second monoclonal antibody is used to determine the amount of binding of said second monoclonal antibody.

19. A kit for use in a sandwich assay, the kit comprising: a solid support to which is bound the first monoclonal antibody; and the second monoclonal antibody preferably comprising a label; wherein one of the first monoclonal antibody and second monoclonal antibody specifically binds the C-terminal epitope of the C5 domain of the α3 chain of type VI collagen and the other of the first monoclonal antibody and second monoclonal antibody specifically binds the N-terminal epitope of endotrophin.

20. A monoclonal antibody that specifically binds to the N-terminal epitope of endotrophin comprising the amino acid sequence TEPLALTETD (SEQ ID No: 27).

21. The monoclonal antibody of claim 20, wherein said monoclonal antibody is a monoclonal antibody raised against a synthetic peptide having the N-terminus amino acid sequence TEPLALTETD (SEQ ID No: 27).

22. The monoclonal antibody of claim 20 or claim 21 wherein said monoclonal antibody does not specifically bind to an elongated version of said N-terminus amino acid sequence which is STEPLALTETD (SEQ ID No: 28), and / or to a truncated version of said N-terminus amino acid sequence which is EPLALTETD (SEQ ID No: 29).

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