Anastellin biomarker

Abstract

To provide a competitive immunoassay for detecting anastellin in a biological sample, and a method for identifying patients having conditions associated with idiopathic pulmonary fibrosis (IPF).SOLUTION: The present invention relates to: a monoclonal antibody that specifically recognizes anastellin neoepitope N-terminal amino acid sequence PIQWNAPQPS (SEQ ID NO:1), which is a part of a cryptic domain in a first type III domain of fibronectin; and an immunoassay method. The method comprises: i) contacting a monoclonal antibody that specifically binds to the N-terminal amino acid sequence PIQWNAPQPS (SEQ ID NO:1) with a patient sample; and ii) detecting and determining a level of binding between the monoclonal antibody and a peptide in the sample.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a competitive immunoassay for detecting anasterin in a biological sample and its use in identifying patients with a condition related to idiopathic pulmonary fibrosis (IPF). The present invention also relates to a kit for carrying out the competitive immunoassay.

[0002] IPF is a progressive interstitial lung disease with a significant impact on health-related quality of life and disease burden, with a life expectancy of 2–3 years from diagnosis if left untreated.[1] Currently, only two anti-fibrotic treatment options, nintedanib and pirfenidone, are approved by the FDA, neither of which can completely halt disease progression.

[0003] IPF is characterized by scarring of the lung interstitium due to deposition and remodeling of extracellular matrix (ECM) proteins [2]. A ubiquitous and abundant component of the ECM is the high molecular weight, dimeric, glycoprotein fibronectin (FN), approximately 500–600 kDa, consisting of three overall domain units; 12 type I, 2 type II, and 15–17 type III domains [3]. Each domain is a module of two antiparallel β-sheets, in which the type III domain can extend and partially unfold when an external force is applied from the surrounding ECM and cellular environment [4]. Upon mechanical stretching of FN, cryptic domains can become exposed and available for protein interactions or cleavage. Anastellin is part of the cryptic domain in the first type III domain and has been shown to be a site of biological activity [5]. The stretching of FN, allowing its interaction with other FN molecules, and the resulting exposure of anasterlin, have been shown to induce FN fibrillogenesis, generating the so-called superfibronectin, which has been found to have enhanced adhesive properties [6]. Furthermore, anasterlin peptide fragments have been shown to selectively inhibit lysophospholipid signaling by the Ras / ERK pathway [7]. Accordingly, anasterlin has been shown to be able to inhibit tumor growth and metastasis in preclinical rodent models of human cancer, and to inhibit endothelial cell microvascular proliferation in vitro [8]

[10] . Despite the effects that anasterlin has on FN fibrillogenesis, the role of anasterlin in fibrosis is largely unknown and still requires investigation. Moreover, the relationship between circulating anasterlin and disease states or clinical parameters has not been investigated so far.

[0004] Applicants have designed a specific competitive immunoassay, FN-ANA, that utilizes a neoepitope of anastelin. The levels of anastelin-derived neoepitopes can be used to assess patients with fibrosis, particularly IPF. Summary of the Invention

[0005] The present invention is directed to a monoclonal antibody that specifically recognizes and binds to a peptide that is a neoepitope of the N-terminus of anasterin within the domain of the FN protein. Preferably, said neoepitope has the amino acid sequence NH 2 -PIQWNAPQPS (SEQ ID NO: 1). Thus, in a first aspect, the present invention provides a monoclonal antibody that specifically binds to the free N-terminal amino acid sequence PIQWNAPQPS (SEQ ID NO: 1) (i.e. the FN-ANA target sequence).

[0006] In a second aspect, the present invention also provides an immunoassay for detecting anastellin (FN-ANA) in a biological sample, the method comprising contacting a patient sample with a monoclonal antibody that specifically binds to a neoepitope of anastellin, and detecting and determining the amount of binding between the monoclonal antibody and a peptide in the sample, where the neoepitope consists of the N-terminal sequence of anastellin.

[0007] In a preferred embodiment, the immunoassay method comprises: i) contacting the patient sample with a monoclonal antibody that specifically binds to the N-terminal amino acid sequence PIQWNAPQPS (SEQ ID NO:1) (also referred to herein as "FN-ANA" or "FN-ANA target sequence"); and ii) detecting and determining the amount of binding between said monoclonal antibody and the peptide in the sample; Includes.

[0008] In a preferred embodiment, the immunoassay method is an immunoassay method for detecting and / or monitoring a particular level of IPF or its severity in a patient, which method further comprises correlating said amount of binding with a value associated with normal healthy subjects and / or a value associated with a known disease severity and / or a value obtained from said patient at a previous time point and / or a predetermined cut-off value.

[0009] In a preferred embodiment, the patient sample is a human biological fluid sample. Preferably, the sample is a blood-based sample, such as blood (whole blood), plasma or serum.

[0010] In a third aspect, the present invention provides a method of treating fibrosis, particularly IPF, in a patient in need thereof, the method comprising: (a) performing an immunoassay method for detecting a particular level of fibrosis or its severity according to the first aspect of the invention on a sample from a patient; and (b) if the patient is determined in step (a) to have fibrosis or a particular level thereof, administering to the patient a therapy for the treatment of the fibrosis. Includes.

[0011] In a fourth aspect, the present invention is further directed to a kit for use in an immunoassay as described herein. The kit comprises a monoclonal antibody as described above.

[0012] Description of the invention A monoclonal antibody suitable for use in the method of the present invention is disclosed herein, which specifically reacts with a neoepitope of anastelin, said neoepitope being comprised in the N-terminal amino acid sequence PIQWNAPQPS (SEQ ID NO: 1). When the monoclonal antibody is a monoclonal antibody that specifically binds to the N-terminal amino acid sequence PIQWNAPQPS (SEQ ID NO: 1), the monoclonal antibody preferably does not substantially recognize or specifically bind to a peptide having the N-terminal amino acid sequence HPIQWNAPQPS (SEQ ID NO: 2) (i.e., an extended version of the FN-ANA target sequence extended at the N-terminus by the addition of a histidine residue) and / or does not substantially recognize or specifically bind to a peptide having the N-terminal amino acid sequence IQWNAPQPS (SEQ ID NO: 3) (i.e., a shortened version of the FN-ANA target sequence shortened by the removal of the first proline residue). Preferably, the ratio of the affinity of the antibody for the FN-ANA target sequence versus the affinity of the antibody for extended and / or shortened versions of the target sequence is at least 10:1, preferably at least 100:1, more preferably at least 1,000:1, more preferably at least 10,000:1, more preferably at least 100,000:1, and most preferably at least 1,000,000:1.

[0013] The term "specifically binds" as used herein means that the antibody binding is selective for the antigen (i.e., epitope) and that this binding can be distinguished from unwanted or non-specific interactions. The ability of a monoclonal antibody to bind to a particular epitope or peptide sequence can be measured either through enzyme-linked immunosorbent assay (ELISA) as described herein or other techniques well known to those skilled in the art, such as surface plasmon resonance (SPR) technology (e.g., analyzed 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 its epitope or peptide, as measured, for example, by ELISA. "Affinity" refers to the strength of the totality 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 otherwise indicated, 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 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 the dissociation and association rate constants (koff and k, respectively). Thus, equivalent affinities may include different rate constants as long as the ratio of the rate constants remains the same. The dissociation constant represents the concentration of antigen at which half of the binding sites on the antibody are occupied. A lower Kd suggests a higher binding affinity between the antibody and the antigen, while a higher Kd reflects a 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 embodiments, a monoclonal antibody that binds to an epitope or peptide has a 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~10 13 M, for example 10 9 M~10 13 It has a dissociation constant (Kd) of 1 M.

[0014] As used herein, the term "monoclonal antibody" refers to both whole antibodies and fragments thereof that retain the binding specificity of the whole antibody, such as, for example, Fab fragments, F(ab')2 fragments, single-chain Fv fragments, 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 being composed of one "light chain" and one "heavy chain". The N-terminal region of each light and heavy chain comprises the variable region, while the C-terminal portion of each heavy and light chain constitutes 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. An antibody fragment that retains the binding specificity comprises at least the CDRs and sufficient remaining portions of the variable region 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.

[0015] Antibodies can be generated from B cell clones as described in the Examples. The isotype of the antibody can be determined by ELISA specific for human IgM, IgG or IgA isotypes, or human IgG1, IgG2, IgG3 or IgG4 subclasses. Other suitable methods can be used to identify the isotype.

[0016] The amino acid sequence of the antibody produced can be determined using standard techniques. For example, RNA can be isolated from cells and used to generate cDNA by reverse transcription. The cDNA is then subjected to PCR using primers that amplify the heavy and light chains of the antibody. For example, a primer specific to the leader sequence for all VH (variable heavy) sequences can be used along with a primer that binds to a sequence located in the constant region of a predetermined isotype. The light chain can be amplified using a primer that binds to the 3' end of the kappa or lambda chain along with a primer that anneals to the Vkappa or Vlamda leader sequence. Full-length heavy and light chains can be produced and sequenced.

[0017] Where the monoclonal antibody specifically binds to the N-terminal amino acid sequence PIQWNAPQPS (SEQ ID NO:1), the monoclonal antibody may be raised against, for example, a synthetic peptide having the N-terminal amino acid sequence PIQWNAPQPS (SEQ ID NO:1). For example, the monoclonal antibody may be raised by: (a) immunizing a rodent (or other suitable mammal) with a synthetic peptide comprising the N-terminal sequence PIQWNAPQPS (SEQ ID NO:1), where the peptide may optionally be linked at its C-terminus to an immunogenic carrier protein (such as keyhole limpet hemocyanin); (b) isolating and cloning cells that produce a single antibody; and (c) assaying the resulting monoclonal antibodies to ensure that they have the desired specificity. Representative protocols for the development, production and characterization of suitable monoclonal antibodies are described in the Examples section below.

[0018] In certain representative embodiments, when the monoclonal antibody is a monoclonal antibody that specifically binds to the N-terminal amino acid sequence PIQWNAPQPS (SEQ ID NO:1), the monoclonal antibody preferably has the structure: CDR-L1: RSSQSLVHSNGNTYLH (SEQ ID NO: 16) CDR-L2: KVSNRFS (SEQ ID NO: 17) CDR-L3: SQSTHVPYT (SEQ ID NO: 18) CDR-H1: DYEMH (SEQ ID NO: 19) CDR-H2: AIHPGRGAAAYNQKFKD (SEQ ID NO: 20) CDR-H3: SEEYGNYEDAMDY (SEQ ID NO: 21) It may comprise one or more complementarity determining regions (CDRs) selected from:

[0019] Preferably, the monoclonal antibody comprises at least two, three, four, five or six of the above listed CDR sequences.

[0020] Preferably, the monoclonal antibody has the CDR sequences: CDR-L1: RSSQSLVHSNGNTYLH (SEQ ID NO: 16) CDR-L2: KVSNRFS (SEQ ID NO: 17) and CDR-L3: SQSTHVPYT (SEQ ID NO: 18) The light chain variable region comprises:

[0021] Preferably, the monoclonal antibody has a light chain comprising framework sequences between the CDRs that are substantially identical or substantially similar to the framework sequences between the CDRs in the light chain sequence below (the CDRs are shown in bold and underlined, and the framework sequences are shown in italics): [ka]

[0022] Preferably, the monoclonal antibody has the CDR sequences: CDR-H1: DYEMH (SEQ ID NO: 19) CDR-H2: AIHPGRGAAAYNQKFKD (SEQ ID NO: 20) and CDR-H3: SEEYGNYEDAMDY (SEQ ID NO: 21) The heavy chain variable region comprises:

[0023] Preferably, the monoclonal antibody has a heavy chain comprising framework sequences between the CDRs that are substantially identical or substantially similar to the framework sequences between the CDRs in the heavy chain sequence below (the CDRs are shown in bold and underlined, and the framework sequences are shown in italics): [ka]

[0024] Preferably, the monoclonal antibody comprises the light chain variable region sequence: [ka]

[0025] (CDRs are bold and underlined; framework sequences are italics) and / or the heavy chain variable region sequence: [ka]

[0026] (CDRs are bold and underlined; framework sequences are italics) Includes.

[0027] 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 sequence may contain one or more amino acid substitutions, insertions and / or deletions. The amino acid substitutions may be conservative, meaning that the substituted amino acid has similar chemical properties as the original amino acid. A person skilled in the art will 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.

[0028] Programs such as the CLUSTAL program can be used to compare amino acid sequences. This program finds the optimal alignment by comparing amino acid sequences and inserting spaces in either sequence as appropriate. For optimal alignment, it is possible to calculate amino acid identity or similarity (identity plus conservation of amino acid types). Programs such as BLASTx align the longest stretches of similar sequences and assign a value to the fit value. It is therefore possible to obtain a comparison in which several regions of similarity are found, each with a different score. Both types of analysis are envisaged in the present invention. Identity or similarity is preferably calculated over the entire length of the framework sequences.

[0029] As used herein, the term "N-terminus" refers to an N-terminal peptide sequence at the end of a polypeptide, i.e., at the N-terminus of the polypeptide, and is not to be construed as implying a general orientation thereof. As used herein, the term "C-terminus" refers to a C-terminal peptide sequence at the end of a polypeptide, i.e., at the C-terminus of the polypeptide, and is not to be construed as implying a general orientation thereof.

[0030] As used herein, the terms "peptide" and "polypeptide" are used interchangeably.

[0031] Antibodies according to the first aspect of the invention are particularly suitable for use in carrying out the immunoassay methods according to the second and third aspects of the invention. Preferred embodiments and features of antibodies for use in the immunoassay methods according to the second and third aspects will therefore be apparent from the above discussion of preferred embodiments of monoclonal antibodies according to the first aspect.

[0032] The immunoassay can detect and determine the amount of binding between the monoclonal antibody and the peptide in the sample, which can be correlated with values ​​associated with normal healthy subjects and / or values ​​associated with known disease severity and / or values ​​obtained from the patient at a previous time point and / or a pre-determined cut-off value.

[0033] In a preferred embodiment, the immunoassay is a competitive assay or a sandwich assay. The immunoassay may be, for example, a radioimmunoassay or an ELISA. Such assays are techniques known to those skilled in the art. The most preferred immunoassay is a competitive ELISA.

[0034] As used herein, the term "ELISA" (Enzyme-Linked Immunosorbent Assay) refers to an immunoassay in which a target peptide (if present) present in a sample is detected using an antibody linked to an enzyme, such as horseradish peroxidase or alkaline phosphatase. The activity of the enzyme is then assessed by incubation with a substrate that produces a measurable product. The presence and / or amount of the target peptide in the sample can thereby be detected and / or quantified. ELISA is a technique known to those skilled in the art.

[0035] As used herein, the term "competitive ELISA" refers to competitive enzyme-linked immunosorbent assay, a technique known to those of skill in the art.

[0036] 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 those of skill in the art.

[0037] Preferably, an immunoassay is used to quantify the amount of anasterin in a biological fluid, which may be, but is not limited to, serum, plasma, bronchoalveolar lavage fluid, saliva, urine, amniotic fluid, tissue supernatant or cell / tissue supernatant.

[0038] The human biological fluid sample may be a sample from a human patient having medical signs or symptoms suggestive of a fibrotic disease, in particular IPF.

[0039] As used herein, the term "amount of binding" refers to the quantification of the binding between the antibody and the peptide in the patient sample. The quantification may be determined, for example, by comparing the measured value of binding in the patient sample to a standard curve generated using the measured value of binding in a standard sample containing a known concentration of the peptide to which the antibody specifically binds, to determine the amount of the peptide to which the antibody specifically binds in the patient sample. Any suitable analytical method can be used to measure the amount of binding. For example, an ELISA method can be used, in which spectrophotometric analysis is used to measure the amount of binding both in the patient sample and when generating the standard curve.

[0040] As used herein, the term "predetermined cutoff value" refers to an amount of binding that is statistically determined to be indicative of a high likelihood of a disease (e.g., IPF) or a particular severity thereof in a patient, where a measured value of a target peptide in a patient sample that is equal to or greater than the statistical cutoff value corresponds to at least a 70% probability of the presence of said disease or a particular severity thereof, preferably at least a 75% probability, more preferably at least an 80% probability, more preferably at least an 85% probability, more preferably at least a 90% probability, and most preferably at least a 95% probability.

[0041] As used herein, the term "value associated with a normal healthy subject" refers to the normalized amount of binding determined by the methods described above for samples from subjects who are considered to be healthy, i.e., disease-free (i.e., free of IPF); and the term "value associated with a known disease severity" refers to the normalized amount of binding determined by the methods described above for samples from patients known to have disease of known severity (i.e., IPF).

[0042] In certain assays disclosed herein for measuring a target peptide having the N-terminal amino acid sequence PIQWNAPQPS (SEQ ID NO:1) in a biological fluid, a calibration curve is generated using standard samples of known concentrations of a calibration peptide having the N-terminal amino acid sequence PIQWNAPQPS (SEQ ID NO:1), which may in particular consist of the amino acid sequence PIQWNAPQPS (SEQ ID NO:1). The values ​​measured in the biological fluid sample are compared to the calibrator to determine the actual amount of the target peptide in the sample.

[0043] In a preferred embodiment, the monoclonal antibody may be labeled to determine the amount of binding of the monoclonal antibody.

[0044] Preferably, the monoclonal antibody is an enzyme-linked antibody, which may be, but is not limited to, horseradish peroxidase (HRP).

[0045] Preferably, the monoclonal antibody is radiolabeled or linked to a fluorophore.

[0046] Although these are the preferred labels for use in the present invention, it is anticipated that any suitable labeling system may be used, including but not limited to DNA reporters or electrochemiluminescent tags.

[0047] Alternatively, a second labeled antibody that recognizes the first monoclonal antibody may be used to determine the amount of binding of the first monoclonal antibody. The additional labeled antibody may be labeled using a label as described above.

[0048] In a preferred embodiment of the present invention, the competitive immunoassay may further comprise correlating the amount of anasterin determined by the above method with a standard fibrotic disease sample of known disease severity to assess the severity of the fibrotic disease.Such a fibrotic disease may be, but is not limited to, IPF.

[0049] The method may be an immunoassay method for diagnosing and / or monitoring and / or assessing possible fibrotic disease in a patient, the method comprising carrying out the immunoassay method of the second aspect of the invention on a biological fluid sample obtained from said patient using a monoclonal antibody, detecting and determining the amount of binding between the monoclonal antibody and the peptide in the sample, and correlating said amount of binding with values ​​associated with normal healthy subjects and / or with values ​​associated with known disease severity and / or with values ​​obtained from said patient at previous time points. Preferably, the fibrotic disease is IPF.

[0050] The method can be used to monitor the response of a patient to treatment. Samples can be taken when treatment is started and analyzed using the immunoassay of the second embodiment. The amount of binding between monoclonal antibody and peptide in the sample can be correlated with the value obtained from the patient at a given time point, for example before treatment.

[0051] In a third aspect, the present invention provides a method of treating fibrosis, particularly IPF, in a patient in need thereof, the method comprising: (a) performing an immunoassay method for detecting a particular level of fibrosis or its severity according to the second aspect of the invention on a sample from a patient; and (b) if it is determined in step (a) that the patient has fibrosis or a particular level thereof, administering to the patient a therapy for the treatment of the fibrosis. Includes.

[0052] Preferred embodiments of the method according to the third aspect will be apparent from the above discussion of preferred embodiments of the method according to the second aspect.

[0053] The treatment can be any treatment suitable for treating the fibrotic disease in question, such as IPF. The treatment can include or consist of, for example, one or more drugs, one or more lifestyle changes, one or more surgeries, or a combination thereof. The drug can be formulated for topical, inhaled, or systemic administration. The topical drug can be formulated, for example, as a cream, foam, gel, lotion, or ointment for administration. The systemic drug can be formulated, for example, for enteral or parenteral administration. The surgery can be curative, preventive, palliative, and / or restorative. The inhaled drug includes aerosolized liquids or fine powders that can be administered using an inhaler, or a metered-dose device such as a nebulizer.

[0054] Suitable treatments for IPF include nintedanib and pirfenidone.

[0055] In a fourth aspect, the present invention provides a monoclonal antibody according to the first aspect of the invention, and -Streptavidin-coated well plates - Biotinylated peptide PIQWNAPQPS-Li-biotin (SEQ ID NO: 4), where Li is an optional linker - a proofreading protein containing the N-terminal amino acid sequence PIQWNAPQPS (SEQ ID NO: 1) -Antibody Biotinylation Kit -Antibody HRP labeling kit -Antibody Radiolabeling Kit The present invention provides an immunoassay kit comprising at least one of the following:

[0056] The immunoassay kit according to the fourth aspect of the invention is particularly suitable for use in carrying out the immunoassay methods according to the second and third aspects of the invention. Further preferred embodiments and features of the immunoassay kit according to the fourth aspect will therefore be apparent from the above discussion of preferred embodiments of the methods according to the second and third aspects.

[0057] The kit may be for use in diagnosing or predicting the risk of a fibrotic disease, preferably in conjunction with the method according to the second aspect of the invention. Preferably, the fibrotic disease is IPF.

[0058] The invention will now be illustrated in the following examples which refer to the following figures. [Brief description of the drawings]

[0059] [Figure 1] Figure 1 shows sequence alignment analysis for FN-ANA across species. Arrows indicate the cleavage site and start of the FN-ANA neoepitope for different species, italicized and underlined letters indicate the first 10 amino acids from the neoepitope start, and bold highlights the differences between human and other species investigated. [Diagram 2]Figure 2 shows the specificity of the FN-ANA epitopes. The mAbs raised against the FN-ANA neoepitopes were specific for the intended antigen (selected peptides with neoepitope sequences) and the assay did not react with the shortened, extended, nonsense-coated, or nonsense-selected peptides at different concentrations. The specificity of the immunoassay was investigated using colorimetric detection, and the optical density (OD) was measured at a wavelength of 450 nm with 650 nm as background reference. [Diagram 3] Figure 3 shows the characterization of FN-ANA in healthy controls and IPF subjects. 3A+C) Age-related scatter plots of FN-ANA serum levels and 3B+D) Violin plots of comparison of FN-ANA serum levels in women and men. Correlations were investigated by Spearman's rank correlation coefficient and comparisons between groups were performed by Mann-Whitney U test. CI, confidence interval; IPF, idiopathic pulmonary fibrosis; ns, not significant. [Figure 4] Figure 4 shows a direct comparison of FN-ANA vs. FN-EDB in healthy controls and IPF. Figure 4A) Violin plot of FN-ANA serum levels in healthy controls and IPF subject groups. Figure 4B) ROC curve of FN-ANA in healthy controls vs. IPF subjects. Figure 4C) Violin plot of FN-EDB serum levels in healthy controls and IPF subject groups. Figure 4D) ROC curve of FN-EDB in healthy controls vs. IPF subjects. AUC, area under the curve; CI, confidence interval; IPF, idiopathic pulmonary fibrosis; ROC, receiver operating characteristic *p<0.05, ****p<0.0001 [Diagram 5] Figure 5 shows the characteristics of FN-ANA and FN-EDB with respect to IPF clinical parameters. Figure 5A) Box plot of FN-ANA serum levels in IPF subjects with and without antifibrotic treatment. Figure 5B) Scatter plot of FN-ANA serum levels and forced vital capacity (FVC; % predicted). Figure 5C) Box plot of FN-EDB serum levels in IPF subjects with and without antifibrotic treatment. Figure 5D) Scatter plot of FN-EDB serum levels and FVC (% predicted).

[0060] material and method All reagents used were high quality chemicals from Merck (Whitehouse Station, NJ, USA) and Sigma (St. Louis MO, USA) unless otherwise specified. Synthetic peptides (Table 1) were obtained from GenScript (Piscataway, NJ, US) and were of high quality purity. [Table 1]

[0061] Identification of neoepitope targets The first 10 amino acids of the anastellin N-terminus within the FN protein were considered as a potential neoepitope as mentioned in Uniprot.com [Identifier: PRO_0000390479], and the first 6 amino acids were predicted to be important for antibody-antigen recognition. For this reason, the first 6 amino acids [PIQWNA] ​​(SEQ ID NO: 9) were analyzed for uniqueness by protein blast using NPS@:Network Protein Sequence Analysis Tool

[11] to ensure no cross-reactivity with other proteins, followed by species homology analysis using UniProt sequence alignment tool

[12] . The potential neoepitope generated at the anastellin N-terminus was named FN-ANA, and antibody production and subsequent assay development were initiated.

[0062] Production and selection of monoclonal antibodies Monoclonal antibodies (mAbs) were raised against the FN-ANA neoepitope sequence 626↓627PIQWNAPQPS (SEQ ID NO: 1) using the following method. Five female Balb / C mice aged 6-7 weeks were immunized subcutaneously with 200 μL of emulsified antigen and 100 μg of immunogenic peptide (PIQWNAPQPS-GGC-KLH (SEQ ID NO: 6)) using Stimmune (Thermo Fisher). Immunizations were repeated every 2 weeks until stable serum antibody titer levels were reached. Mice with the highest serum titers and showing the best selectivity for the selected peptide (PIQWNAPQPS (SEQ ID NO: 1)) and not the extended peptide (HPIQWNAPQPS (SEQ ID NO: 2)) were selected for fusion and rested for one month. The selected mice were then boosted intravenously with 50 μg of immunogenic peptide in 100 μL of 0.9% NaCl solution 3 days before isolation of the spleen for cell fusion. The spleen cells of the mice were fused with SP2 / 0 myeloma cells to generate hybridoma cells as described by Gefter et al.

[13] . The hybridoma cells were seeded into each well in a 96-well microtiter plate for further growth using limiting dilution to promote the growth of single clones. Indirect ELISA performed in streptavidin-coated 96-well microtiter plates using a biotin-labeled peptide (PIQWNAPQPS-K-biotin (SEQ ID NO: 5)) as the coating peptide was used to screen the supernatants from the hybridoma clones for reactivity to identify the best mAb-producing clones. The best clones were selected based on specificity to the selected peptides and lack of reactivity to the deselected peptides (extended, truncated, and nonsense peptides; see Table 1). Supernatants were collected and mAbs were purified using HiTrap affinity columns (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK), after which mAbs were labeled with HRP using the Roche Peroxidase Labeling Kit (catalogue no. 11829696001, Merck).Finally, antibody isotypes were determined using isotype-specific antisense primers or universal primers according to the technical manual of the SMARTScribe™ Reverse Transcriptase Kit (Takara, Cat. No: 639537). All procedures were carried out according to the manufacturer's instructions.

[0063] The isotype, sequence and CDRs of this monoclonal antibody were determined. The sequences of the chains are as follows (CDRs are underlined and in bold; N-terminal signal peptide and C-terminal constant region are in italics):

[0064] [ka] [ka]

[0065] FN-ANA assay development The final FN-ANA assay parameters were empirically determined through several optimization experiments investigating different reagents, concentrations, incubation times and temperatures in addition to testing reactivity against selected and deselected peptides (Table 1) and human sera. The final FN-ANA competitive ELISA procedure was performed with chemiluminescence detection and determined as follows: 100 μL of 3 ng / mL coating peptide (PIQWNAPQPS-K-biotin (SEQ ID NO: 5)) in coating buffer (50 mM Tris-buffered saline (TBS), pH 8.0 with 1% (w / v) bovine serum albumin, 0.1% (w / v) Tween-20, and 0.36% (v / v) Bronidox (BTB), 4 g / L NaCl, 0.20 g / L KCl) was added to each well of a 96-well streptavidin-coated microtiter white plate (catalog no. 655995, Greiner Bio-one, Taufkirchen, Germany), which was then incubated at 20° C. for 30 minutes with orbital shaking at 300 RPM. After incubation, the plates were emptied and washed five times with wash buffer (25 mM Tris, 3.0 g / L NaCl, 0.1% (w / v) Tween-20, pH 7.2). A 10-point standard curve using two-fold dilutions was prepared from the selected peptide (PIQWNAPQPS (SEQ ID NO: 1)) at a starting concentration of 200 ng / mL in incubation buffer (50 mM TBS-BTB, 4.0 g / L NaCl, 0.20 g / L KCl, 5% Liquid II (catalog no. 11941836001, Roche), pH 8.0). 20 μl / well of standards, assay controls, and serum samples were added to appropriate wells in duplicate, followed by 100 μL / well of HRP-labeled mAb at 6 ng / mL in incubation buffer, after which the plates were incubated at 4° C. for 20 hours with orbital shaking at 300 RPM. The next day, chemiluminescent substrate (catalog no. 11582950001, Roche Diagnostics) was prepared according to the manufacturer's instructions and mixed by rotation in the dark at room temperature for 20 min before analyzing the plates.Following a wash step, 100 μL / well of chemiluminescent material was added and the plate was incubated in the dark for a total of 3 min with orbital shaking at 300 RPM for 1 min, after which luminescence was detected on a SpectraMax i3x microplate reader (Molecular Devices, Sunnyvale CA, USA) with an integration time of 1 s at all wavelengths as per the microplate reader settings. Data analysis was performed with SoftMax Pro version 7.1.0 software (Molecular Devices) using a 10-point calibration curve plotted using a 4-parameter logistic curve fit of selected peptide standards.

[0066] Technical evaluation Antibody specificity was assessed using synthetic peptides and determined as the percentage signal inhibition of 2-fold diluted selected and deselected peptides (extended, truncated, and nonsense) in addition to the nonsense coating (peptide details listed in Table 1) using the procedure described above with the following exception: instead of adding a chemiluminescent substrate, the colorimetric substrate 3,3',5,5'-tetramethylbenzidine One (catalog no. 4380H, Kem-En-Tec, Taastrup, Denmark) was added to each well after the incubation step with HRP-labeled FN-ANA antibody. Plates were incubated for 15 min in the dark with orbital shaking at 300 RPM, after which the incubation was stopped by adding 100 μL / well of 0.1% (v / v) sulfuric acid. The absorbance was measured at a wavelength of 450 nm with a reference of 650 in a microplate reader (VersaMax, Molecular Devices, Sunnyvale, CA, USA).

[0067] Ten independent assay runs were used to establish the IC50 (50% inhibitory concentration) and the upper limit of quantification (ULOQ), which was determined as the highest concentration of selected peptide achieving recovery within 100±20% of the nominal concentration. The lower limit of quantification (LLOQ) of the analyte was determined for serum samples by triplicate evaluation of five independent runs using four human serum samples covering the lower range of the calibration curve. The CV% of the samples with 95% confidence intervals were plotted against the measured sample concentrations, and a power regression model was used to estimate the LLOQ as the lowest analyte concentration with a CV% equal to 20%. The limit of blank (LOB) for the assay was established as the mean signal of 60 measurements of the blank sample (i.e., incubation buffer) plus three times the standard deviation (SD). Intra- and inter-assay variability was determined from ten independent runs of eight human serum samples and two assay control samples (i.e., low and high concentrations of selected peptide) with a CV% ≤ 15% as the acceptance criterion. The minimum required dilution (MRD) and assay linearity of diluted samples were determined by two-fold dilutions of four human serum samples quantified in three independent runs, and the recovery of diluted samples was calculated as the recovery of the undiluted sample with the recovery within 100±20% as the acceptance threshold of the dilution.

[0068] Analyte stability was determined in human serum samples stressed by up to five freeze / thaw cycles or by storage at 4°C or 20°C for 2, 4, 24, or 48 hours. Recovery was calculated using an unstressed sample as a reference. Analyte assay precision was determined in three human serum samples spiked with a two-fold dilution of the selected peptide and three human serum samples spiked with a two-fold dilution of another human serum sample with a known high concentration of the analyte. Recovery was calculated for the spiked samples using the theoretical total amount of the analyte in the sample as a reference. Analytical interference was assessed by spiking low / high concentrations of lipids (1.5 / 5.0 mg / mL), hemoglobin (2.5 / 5.0 mg / mL), or biotin (5.0-100 ng / mL) into three human serum samples with known concentrations and calculating the recovery in the spiked samples using the unspiked sample as a reference. All tests used duplicate measurements of samples unless otherwise stated.

[0069] Clinical evaluation The clinical relevance of the FN-ANA assay was evaluated in serum samples from 98 IPF and 133 healthy control subjects. Serum samples were performed in duplicate and values ​​outside the measurement range were assigned to LLOQ (lower limit of quantification) or ULOQ (upper limit of quantification) values, as appropriate. Healthy controls were obtained from a commercial vendor (Discovery Life Sciences, Los Osos, CA). IPF patients had prevalent illness at the time of blood sample collection and were part of a larger study previously described

[14] . Inclusion criteria for IPF subjects were age >18 years and IPF diagnosis, and exclusion criteria were inability to complete the questionnaire due to language or intellectual barriers. Clinical parameters were collected at the same time as blood collection.

[0070] Comparison of FN-ANA with different assays targeting the type III domain of FN The utility of FN-ANA was compared with a different immunoassay specific for another type III domain of FN, the EDB domain (FN-EDB). The FN-EDB immunoassay was compared directly with FN-ANA and the diagnostic performance was evaluated in a cohort of IPF patients (n=98) and healthy controls (n=133). Furthermore, associations with clinical parameters were investigated and compared for the two assays. The systematic procedure of FN-EDB was similar to that of FN-ANA, except for the amino acid sequence of the synthetic peptide used to coat the plates (TGLEPGIDYD-K-biotin (SEQ ID NO: 10)), the assay control (TGLEPGIDYD (SEQ ID NO: 11)), and the standard peptide (TGLEPGIDYD (SEQ ID NO: 11)) to generate the calibration curve. In addition, the coating buffer (25 mM PBS-BTB, 8 g / L NaCl, pH 7.4) and incubation buffer (25 mM PBS-BTB, 8 g / L NaCl, 5% Liquid II (catalog no. 11941836001, Roche), pH 7.4) were different. The neoepitope specificity of the mAb used in the FN-EDB immunoassay was determined as 1324↓1325TGLEPGIDYD (SEQ ID NO: 11) within the FN sequence.

[0071] statistical analysis Biomarker serum levels in IPF and healthy control subjects were analyzed by Mann-Whitney U test. Group separation was assessed by area under the curve (AUC) with receiver operating characteristic (ROC). Correlation between biomarker serum levels and clinical parameters was assessed by Spearman rank correlation coefficient, and statistical significance between genders was assessed by Mann-Whitney U test. Recovery rates (REC%), interquartile ranges (IQR) of biomarker measurements, and estimated 95% confidence intervals (95%CI) are displayed where appropriate. P values ​​less than 0.05 were considered statistically significant. Statistical analysis and graphing were performed using GraphPad Prism version 9.5.0 (GraphPad Software Inc., La Jolla, CA).

[0072] Ethics statement All animals were treated according to animal welfare guidelines. Antibody production in mice was approved by the Danish National Agency (Inspectorate of Animal Experiments) under approval number 2013-15-2934-00956. Collection and collection of human serum complied with international ethical guidelines for the handling of human samples and patient information. All patients signed informed consent and the study was approved by the local ethical committee and conformed to the 1975 Declaration of Helsinki.

[0073] result FN-ANA Technology Assessment and Characterization The first six amino acids of the N-terminus of anastellin were investigated for uniqueness from other proteins and, based on that uniqueness, were selected as a neoepitope representing FN remodeling and anastellin release. Furthermore, sequence conservation of the anastellin N-terminus was investigated by comparison of the first 10 amino acids between human, mouse, rat, and bovine (Figure 1). The sequences had 100% conservation between all species compared for the first four amino acids, with the first difference at the fifth amino acid for the bovine sequence. The mouse and rat sequences had 100% conserved alignment from the neoepitope start to the seventh amino acid.

[0074] For the generation of FN-ANA mAbs, mice were immunized with immunogenic synthetic peptides carrying the first 10 amino acids from the neoepitope start of the human sequence. The resulting mAbs with the highest selectivity to the selected peptides and the best intrinsic reactivity and stability were selected for assay development. The FN-ANA assay was found to react only with the intended peptides (selection and coating peptides) and not with the deselected peptides (truncated, extended, nonsense coating and nonsense peptides), suggesting that the mAb-antigen recognition of the intended neoepitope is highly specific (Figure 2).

[0075] A summary of the technical evaluation of the final FN-ANA assay is listed in Table 2. Briefly, the LOB was determined to be 1.47 ng / mL and the measurement range of analyte quantification in human serum was determined to be 1.6-100 ng / mL while achieving low intra-assay (4.1%) and inter-assay (9.3%) variations in analytical measurements. The linearity of the analyte in serum had good precision for analyte recovery when investigated by the mean recovery within the acceptable range of 1:2 (102.5%) and 1:4 dilutions (110.0%), as well as synthetic peptide recovery in serum (114.9%) and serum recovery in serum (105.4%). The analyte stability was acceptable for at least five freeze-thaw cycles of serum samples (97.9%) and for long-term storage of samples at different temperatures and times up to 4 hours at 20°C (93.5%) and 48 hours at 4°C (90.6%), suggesting preservation of neoepitope integrity. No interference was observed in serum samples spiked with hemoglobin or lipids, or with up to 80 ng / mL biotin. [Table 2]

[0076] Clinical evaluation of FN-ANA and comparison with FN-EDB After assay development and technical evaluation were completed, clinical evaluation of the FN-ANA was performed in a cohort of 98 prevalent IPF patients and 133 healthy control subjects with the demographics summarized in Table 3 .

[0077] [Table 3]

[0078] No association of FN-ANA was observed for age or sex in healthy controls or IPF subjects (Figure 3). Serum FN-ANA levels were significantly elevated in IPF patients (median 5.68 [IQR 4.80-6.83] ng / mL) when compared with healthy control subjects (median 3.09 [IQR 2.34-4.21] ng / mL; p<0.0001) (Figure 4A). Separation between groups was further investigated by ROC curve analysis, which showed good separation of IPF and healthy controls with an AUC of 0.878 (95% CI 0.83-0.92, p<0.0001) (Figure 4B). In comparison, FN-EDB serum levels were also significantly different when comparing healthy controls (median 29.7 [IQR 21.72-38.24] ng / mL) with IPF (median 31.38 [IQR 25.79-46.84] ng / mL, p=0.0116) (Figure 4C), but to a lesser extent than FN-ANA, which had very similar mean levels for the two groups. Thus, ROC curve analysis achieved statistical significance when separating healthy controls from IPF subjects, but had a lower AUC of 0.598 (95% CI 0.52-0.67, p=0.0117).

[0079] FN-ANA serum levels were significantly lower in IPF subjects who received antifibrotic treatment (mean 5.55 ng / mL) compared to those who did not (mean 6.78 ng / mL, p=0.0471) (Figure 5A). Furthermore, FN-ANA serum levels were negatively correlated with the clinically relevant lung function measure FVC (FVC; r=-0.288; p=0.0040) (Figure 5B). Contrary to this, FN-EDB serum levels were not significantly different between IPF subjects with (mean 38.24 ng / mL) or without (mean 37.69 ng / mL, p=ns) antifibrotic treatment, and levels did not correlate with FVC (FVC; r=-0.007, p=ns) (Figure 5C+Figure 5D).

[0080] Consideration A novel competitive chemiluminescence immunoassay, the FN-ANA assay, was developed to quantify the free N-terminal anasterin neoepitope in serum. The assay is specific for this neoepitope and is accurate, precise, and robust. Furthermore, FN-ANA is a relevant biomarker for IPF.

[0081] Increased expression and deposition of different fibronectin domains have been associated with prior fibrosis

[15] -

[17] . Anastellin domains have not previously been quantified in serum or proposed as a biomarker for IPF. The serological biomarker FN-ANA was able to separate IPF subjects from healthy subjects with higher accuracy than another type III domain biomarker of FN; FN-EDB (AUC of 0.878 vs. 0.598). Anastellin has previously been speculated to be suitable as a potential treatment for cancer due to its inhibition of preclinical tumor growth, angiogenesis, and metastasis [7], [8],

[10] . The antifibrotic effect of anastellin has not been previously explored or shown in either a preclinical or clinical setting, and its potential association with clinical parameters related to fibrosis. This study showed that circulating anastellin levels assessed by the FN-ANA biomarker were significantly associated with FVC, an endpoint commonly used in clinical trials for IPF. Interestingly, FN-EDB did not correlate with FVC levels. Moreover, FN-ANA may be a potential pharmacodynamic biomarker, as serum levels are low in IPF subjects receiving antifibrotic therapy, suggesting a potential effect of treatment on circulating anastellin levels. In comparison, the FN-EDB biomarker showed no association with treatment status, thus highlighting the advantage of FN-ANA over other FN type III domain biomarkers.

[0082] In conclusion, the present invention provides a novel method for quantifying circulating anasterlin in serum by measuring a unique fragment of the free N-terminus of anasterlin. The FN-ANA assay is robust and provides a biomarker for IPF with diagnostic and pharmacodynamic capabilities that exceed alternative FN biomarkers, as well as association with disease severity as determined by pulmonary function measurements.

[0083] In this specification, unless expressly indicated otherwise, the word "or" is used in the sense of an operator that returns a true value if either or both of the stated conditions are met, as opposed to the operator "exclusive or", which requires that only one of the conditions be met. The word "comprising" is used in the sense of "including or consisting of". All prior teachings acknowledged above are incorporated herein by reference. Admission of any previously published document herein should not be construed as an admission or representation that the teachings were common general knowledge in Australia or anywhere else at the date hereof.

[0084] References [1] TM Maher et al., “Global incidence and prevalence of idiopathic pulmonary fibrosis,” Respir Res, vol. 22, no. 1, p. 197, Jul. 2021, doi: 10.1186 / s12931-021-01791-z. [2] FJ Martinez et al., “Idiopathic pulmonary fibrosis,” Nat Rev Dis Primers, vol. 3, no. 1, p. 17074, 2017, doi: 10.1038 / nrdp.2017.74. [3] C. J. Dalton and C. A. Lemmon, “Fibronectin: Molecular Structure, Fibrillar Structure and Mechanochemical Signaling,” Cells, vol. 10, no. 9. 2021. Doi: 10.3390 / cells10092443. [4] G. Baneyx, L. Baugh, and V. Vogel, “Fibronectin extension and unfolding within cell matrix fibrils controlled by cytoskeletal tension,” Proceedings of the National Academy of Sciences, vol. 99, no. 8, pp. 5139-5143, 2002. [5] C. Cho, R. Kelsh-Lasher, A. Ambesi, and P. J. McKeown-Longo, “Cryptic activity within the Type III1 domain of fibronectin regulates tissue inflammation and angiogenesis,” Curr Top Pept Protein Res, vol. 16, p. 37, 2015, Accessed: Aug. 29, 2022. [Online]. Available: / pmc / articles / PMC4981920 / [6] A. Morla, E. Ruoslahti, E. J. Fogerty, V. E. Koteliansky, and D. E. Mosher J Biol Chem, “A fibronectin self-assembly site involved in fibronectin matrix assembly: reconstruction in a synthetic peptide,” J Cell Biol, vol. 118, no. 2, p. 421, Jul. 1992, doi: 10.1083 / JCB.118.2.421. [7] A. Ambesi and P. J. McKeown-Longo, “Anastellin, the angiostatic fibronectin peptide, is a selective inhibitor of lysophospholipid signaling,” Molecular Cancer Research, vol. 7, no. 2, pp. 255-265, 2009. [8] M. Yi and E. Ruoslahti, “A fibronectin fragment inhibits tumor growth, angiogenesis, and metastasis,” Proceedings of the National Academy of Sciences, vol. 98, no. 2, pp. 620-624, 2001. [9] R. Pasqualini, S. Bourdoulous, E. Koivunen, V. L. Woods Jr, and E. Ruoslahti, “A polymeric form of fibronectin has antimetastatic effects against multiple tumor types,” Nat Med, vol. 2, no. 11, pp. 1197-1203, 1996.

[10] D. M. Neskey, A. Ambesi, K. M. Pumiglia, and P. J. McKeown-Longo, “Endostatin and Anastellin inhibit distinct aspects of the angiogenic process,” Journal of Experimental & Clinical Cancer Research, vol. 27, pp. 1-8, 2008.

[11] C. Combet, C. Blanchet, C. Geourjon, and G. Deleage, “NPS@: Network Protein Sequence Analysis,” Trends Biochem Sci, vol. 25, no. 3, pp. 147-150, Mar. 2000, doi: 10.1016 / S0968-0004(99)01540-6.

[12] A. Bateman et al., “UniProt: the universal protein knowledgebase,” Nucleic Acids Res, vol. 45, no. D1, pp. D158-D169, Jan. 2017, doi: 10.1093 / NAR / GKW1099.

[13] M. L. Gefter, D. H. Margulies, and M. D. Scharff, “A simple method for polyethylene glycol-promoted hybridization of mouse myeloma cells,” Somatic Cell Genet, vol. 3, no. 2, pp. 231-236, Mar. 1977, doi: 10.1007 / BF01551818.

[14] T. S. Prior et al., “Clusters of comorbidities in idiopathic pulmonary fibrosis,” Respir Med, vol. 185, Aug. 2021, doi: 10.1016 / J.RMED.2021.106490.

[15] A. F. Muro et al., “An essential role for fibronectin extra type III domain A in pulmonary fibrosis,” Am J Respir Crit Care Med, vol. 177, no. 6, pp. 638-645, 2008.

[16] C. M. Novak et al., “Alveolar macrophages drive lung fibroblast function in cocultures of IPF and normal patient samples,” American Journal of Physiology-Lung Cellular and Molecular Physiology, vol. 324, no. 4, pp. L507-L520, 2023.

[17] C. Upagupta, C. Shimbori, R. Alsilmi, and M. Kolb, “Matrix abnormalities in pulmonary fibrosis,” European Respiratory Review, vol. 27, no. 148, 2018.

Claims

1. A monoclonal antibody that specifically recognizes the N-terminal amino acid sequence PIQWNAPQPS (SEQ ID NO: 1).

2. The monoclonal antibody of claim 1, which specifically binds to the N-terminal amino acid sequence PIQWNAPQPS (sequence number 1) and does not specifically bind to a peptide having the N-terminal amino acid sequence HPIQWNAPQPS (sequence number 2).

3. The monoclonal antibody of claim 1 or 2, which specifically binds to the N-terminal amino acid sequence PIQWNAPQPS (sequence number 1) and does not specifically bind to a peptide having the N-terminal amino acid sequence IQWNAPQPS (sequence number 3).

4. A monoclonal antibody described in any one of claims 1 to 3, which is raised against a synthetic peptide having the N-terminal amino acid sequence PIQWNAPQPS (SEQ ID NO: 1).

5. 1. A method of immunoassay comprising: i) contacting the patient sample with a monoclonal antibody that specifically binds to the N-terminal amino acid sequence PIQWNAPQPS (SEQ ID NO:1); and ii) detecting and determining the amount of binding between said monoclonal antibody and peptide in said sample. A method comprising:

6. 1. An immunoassay method for detecting and / or monitoring a particular level of fibrotic disease or its severity in a patient, comprising: iii) correlating said amount of binding with values ​​associated with normal healthy subjects and / or with values ​​associated with known disease severity and / or with values ​​obtained from said patient at previous time points and / or with predefined cut-off values. The method of claim 5 further comprising:

7. The method of claim 6, wherein the fibrotic disease is idiopathic pulmonary fibrosis.

8. 8. The method of any one of claims 5 to 7, wherein the patient sample is selected from blood, plasma or serum.

9. The method of any one of claims 5 to 8, wherein the monoclonal antibody specifically binds to the N-terminal amino acid sequence PIQWNAPQPS (sequence number 1) and does not specifically bind to a peptide having the N-terminal amino acid sequence HPIQWNAPQPS (sequence number 2).

10. The method of any one of claims 5 to 9, wherein the monoclonal antibody specifically binds to the N-terminal amino acid sequence PIQWNAPQPS (sequence number 1) and does not specifically bind to a peptide having the N-terminal amino acid sequence IQWNAPQPS (sequence number 3).

11. 11. The method of any one of claims 5 to 10, wherein the monoclonal antibody is raised against a synthetic peptide having the N-terminal amino acid sequence PIQWNAPQPS (SEQ ID NO:1).

12. The method of any one of claims 5 to 11, wherein the immunoassay is a competitive assay or a sandwich assay.

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

14. A monoclonal antibody according to any one of claims 1 to 4, and - Streptavidin-coated well plates - Biotinylated peptide PIQWNAPQPS-L-biotin (SEQ ID NO: 4) (where L is an optional linker) - Secondary antibodies for use in sandwich immunoassays - a proofreading protein comprising the N-terminal amino acid sequence PIQWNAPQPS (SEQ ID NO: 1) -Antibody Biotinylation Kit -Antibody HRP labeling kit - Antibody Radiolabeling Kit An immunoassay kit comprising at least one of the above.

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