Immunoassay for detecting N-terminal peptides of proteolytic cleavage products of cartilage intermediate lamina protein-1

Abstract

Disclosed are monoclonal antibodies that bind to the N-terminal amino acid sequence of cartilage intermediate lamina protein-1 (SLNPDTGLWE (SEQ ID NO: 1), also referred to as "CILP-M"), which is generated by cleavage with MMP-1, MMP-8, and MMP-12. Also disclosed are immunoassay methods and kits for detecting CILP-M in patient samples. The methods and kits are used to detect and monitor joint disease and to assess the likelihood that a patient will respond to drug treatment.

Description

[Technical Field]

[0001] The present invention relates to monoclonal antibodies that bind to the N-terminal sequence of cartilage intermediate lamina protein-1 generated by cleavage with MMP-1, MMP-8, and MMP-12. The present invention also relates to an immunoassay method for detecting the N-terminal sequence of cartilage intermediate lamina protein-1 in a patient-derived sample, such as whole blood, plasma, or serum, and an immunoassay kit suitable for carrying out the method. [Background technology]

[0002] Articular cartilage is a heterogeneous tissue composed of cells organized within a matrix. The matrix is ​​formed by chondrocytes, and its main components are fibrils of collagens, such as types I and III collagen, and large aggregates of proteoglycans (Reference 1). Other non-collagenous extracellular matrix proteins, such as cartilage intermediate lamina protein (CILP), are also present in the cartilage matrix (Reference 2). CILP-1 was the first glycoprotein identified in articular cartilage and is thought to play a role in cartilage scaffolding (Reference 2). It is secreted by articular chondrocytes (Reference 2) and is a factor in maintaining cartilage homeostasis by slowing the turnover of matrix proteins for remodeling (Reference 3). A key feature of rheumatoid arthritis is a partial alteration in cartilage turnover, leading to dysregulation of cartilage composition. During cartilage erosion, CILP-1 is secreted by articular chondrocytes and accumulates in the cartilage extracellular matrix (Reference 2). CILP-1 has been shown to be cleaved in vitro by various human proteases to generate various fragments (Ref. 4). CILP synthesis is increased in cartilage in early osteoarthritis (OA) (Ref. 5) and has been associated with musculoskeletal disorders (Refs. 6, 7). Other studies have shown that CILP-1 is highly expressed in intervertebral discs, and its expression is increased in lumbar discopathy (Ref. 8). It has also been suggested that an immune response to CILP may be one of the causes of inflammatory joint destruction present in rheumatoid arthritis (RA) and OA (Ref. 9). Furthermore, CILP mRNA expression was observed in synovial tissue from OA patients (Ref. 10), indicating that its expression is not limited to articular cartilage. These findings suggest that CILP protein may play a role in cartilage structure and rheumatic diseases. Summary of the Invention

[0003] Applicants have now discovered that CILP-1 is cleaved by MMP-1, MMP-8, and MMP-12 to generate a neoepitope having the N-terminal sequence SLNPDTGLWE (SEQ ID NO: 1) (referred to herein as "CILP-M" and / or the "target sequence"). Applicants have also discovered that CILP-M is generated in vivo and released into the circulation, and have developed an immunoassay targeting CILP-M. Furthermore, Applicants have evaluated the biological value of CILP-M in joint-related diseases (RA, AS, and OA) in two exploratory patient studies and investigated the prognostic potential of CILP-M in anti-TNF-α treatment, demonstrating that measured levels of CILP-M in serum samples reflect disease activity.

[0004] Thus, in a first aspect, the present invention provides a monoclonal antibody that specifically binds to the N-terminal amino acid sequence SLNPDTGLWE (SEQ ID NO: 1).

[0005] The term "N-terminus" as used herein refers to the N-terminal amino acid sequence at the extremity of a polypeptide, i.e., the amino acid sequence at the N-end of a polypeptide, and should not be construed as implying a general orientation thereof. As used herein, the terms "peptide" and "polypeptide" are used interchangeably.

[0006] 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 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 consisting of two identical paired polypeptide chains, each of which is composed of one "light" chain and one "heavy" chain. The N-terminal regions of each of the light and heavy chains comprise the variable region, while the C-terminal portions of each of the heavy and light chains constitute the constant region. The variable regions contain three complementarity-determining regions (CDRs), which are primarily responsible for antigen recognition. The constant region enables the antibody to recruit cells and molecules of the immune system. Antibody fragments that retain binding specificity contain at least the CDRs and a sufficient portion of the remainder of the variable region to retain binding specificity.

[0007] In the present invention, monoclonal antibodies containing any constant region known in the art can be used. In the case of mouse and human antibodies, constant light chains are classified as kappa and lambda light chains. Constant heavy chains are classified as mu, delta, gamma, alpha, or epsilon, defining the antibody isotype as IgM, IgD, IgG, IgA, or IgE, respectively. The IgG isotype has several subclasses, including IgG1, IgG2, IgG3, and IgG4 in humans, and several subclasses, including IgG1, IgG2a, IgG2b, IgG2c, and IgG3 in mice, but is not limited to these. The monoclonal antibody preferably belongs to the IgG isotype, including any one of the IgG subclasses (e.g., IgG1, IgG2, IgG3, or IgG4 in the case of human antibodies).

[0008] The CDRs of an antibody can be determined using methods known in the art, such as those described by Kabat et al. Antibodies can be generated from B cell clones as described in those examples. The isotype of an antibody can be determined by ELISA specific for the IgM, IgG, or IgA isotype (human or mouse) or subclass (human or mouse). The amino acid sequence of the generated antibody can be determined using standard techniques. For example, RNA can be isolated from cells and used to generate cDNA by reverse transcription. The cDNA can then be subjected to PCR using primers that amplify the heavy and light chains of the antibody. For example, primers specific to the leader sequences of all VH (variable heavy) sequences can be used together with primers that bind to sequences 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 together with a primer that anneals to the leader sequence of Vkappa or Vlamda. Full-length heavy and light chains can be generated and sequenced.

[0009] In a preferred embodiment, the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence WSLNPDTGLWE (SEQ ID NO: 2). In a preferred embodiment, the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence LNPDTGLWE (SEQ ID NO: 3). In a preferred embodiment, the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence NPDTGLWE (SEQ ID NO: 4).

[0010] In a preferred embodiment, the monoclonal antibody is raised against a synthetic peptide having the N-terminal amino acid sequence SLNPDTGLWE (SEQ ID NO: 1). For example, monoclonal antibodies can be raised by (a) immunizing a rodent (or other suitable mammal) with a synthetic peptide comprising the N-terminal amino acid sequence SLNPDTGLWE (SEQ ID NO: 1), optionally linked to an immunogenic carrier protein (such as keyhole limpet hemocyanin) at its C-terminus; (b) isolating and cloning a single antibody-producing cell; and (c) assaying the resulting monoclonal antibodies to determine that they have the desired specificity.

[0011] In certain exemplary embodiments, the monoclonal antibody may preferably comprise one or more complementarity determining regions (CDRs) selected from the following: CDR-L1: KASQNVGTDVV (SEQ ID NO: 5) CDR-L2: SASYRYS (SEQ ID NO: 6) CDR-L3: QHYDNYPLT (SEQ ID NO: 7) CDR-H1: NYYIH (SEQ ID NO: 8) CDR-H2: WISPGIVDTEYNEKFKN (SEQ ID NO: 9) CDR-H3: RSAGSYGDFDY (SEQ ID NO: 10) Preferably, the monoclonal antibody comprises at least two, three, four, five or six of the above CDR sequences.

[0012] Preferably, the monoclonal antibody has a light chain variable region comprising the following CDR sequences: CDR-L1: KASQNVGTDVV (SEQ ID NO: 5) CDR-L2: SASYRYS (SEQ ID NO: 6), and CDR-L3: QHYDNYPLT (SEQ ID NO: 7)

[0013] Preferably, the monoclonal antibody has a light chain comprising inter-CDR framework sequences that are substantially identical to or substantially similar to the inter-CDR framework sequences in the light chain sequence below (CDRs are shown in bold and underlined, framework sequences are shown in italics):

[0014] [ka]

[0015] Preferably, the monoclonal antibody has a heavy chain variable region comprising the following CDR sequences: CDR-H1: NYYIH (SEQ ID NO: 8) CDR-H2: WISPGIVDTEYNEKFKN (SEQ ID NO: 9), and CDR-H3: RSAGSYGDFDY (SEQ ID NO: 10)

[0016] Preferably, the monoclonal antibody has a heavy chain comprising inter-CDR framework sequences that are substantially identical to or substantially similar to the inter-CDR framework sequences of the heavy chain sequence below (CDRs are shown in bold and underlined, framework sequences are shown in italics):

[0017] [ka]

[0018] Preferably, the monoclonal antibody comprises the light chain variable region sequence:

[0019] [ka]

[0020] (CDRs are bold and underlined; framework sequences are italic) and / or a heavy chain variable region sequence:

[0021] [ka]

[0022] (CDRs are bold and underlined; framework sequences are italic) Includes:

[0023] As used herein, the amino acid sequence of a framework present between the CDRs of an antibody is "substantially identical" or "substantially similar" to the amino acid sequence of a framework present between the CDRs of another antibody if it has at least 70%, 80%, 90%, or at least 95% similarity or identity to the amino acid sequence of the framework present between the CDRs of the other antibody. The similar or identical amino acids may be contiguous or non-contiguous.

[0024] The framework sequences may contain one or more amino acid substitutions, insertions, and / or deletions. Amino acid substitutions may be conservative, meaning that the substituted amino acid has similar chemical properties to the original amino acid. Those 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 in terms of size, charge, polarity, etc.: 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.

[0025] Programs such as the CLUSTAL program can be used to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by appropriately inserting spaces within either sequence. For optimal alignment, it is possible to calculate amino acid identity or similarity (identity as well as conservation of amino acid type). Programs such as BLASTx align the longest stretch of similar sequences and assign values ​​to matching positions. In this way, a comparison can be made, and several regions of similarity, each with a different score, will be found. It is contemplated that these two types of analysis can be used in the present invention. Identity or similarity is preferably calculated over the entire length of the framework sequence.

[0026] In a second aspect, the present invention provides an immunoassay method comprising the steps of: i) contacting a sample from a patient with a monoclonal antibody according to the first aspect of the present invention; ii) detecting binding between the monoclonal antibody and the peptide in the sample and determining the amount of binding.

[0027] In a preferred embodiment, the method is for detecting and / or monitoring a disease in a subject and / or assessing the likelihood that the subject will respond to treatment with a disease therapeutic agent, further comprising the steps of: iii) correlating the determined amount of binding with values ​​associated with normal healthy individuals and / or with values ​​associated with known disease severity and / or with values ​​obtained from the subject at previous time points and / or with predetermined cut-off values.

[0028] In a preferred embodiment, the disease is a joint disease. Preferably, the joint disease is rheumatoid arthritis, ankylosing spondylitis, or osteoarthritis. In a preferred embodiment, the treatment with a disease therapeutic agent is an anti-TNF-α therapy. In a preferred embodiment, the patient sample is selected from blood, serum, or plasma. Preferably, the sample is serum or plasma. In a preferred embodiment, the immunoassay is a competitive assay or a sandwich assay. The immunoassay may be, for example, a radioimmunoassay or an enzyme-linked immunosorbent assay (ELISA). Such assays are techniques known to those skilled in the art.

[0029] As used herein, the term "amount bound" refers to the quantification of binding between an antibody and a peptide in a patient sample. This quantification may be determined, for example, by comparing the measured binding in the patient sample to a calibration curve generated using measurements of binding in standard samples containing known concentrations of the peptide to which the antibody specifically binds, to determine the amount of peptide in the patient sample to which the antibody specifically binds. In the examples provided below, an ELISA method is used, which utilizes spectrophotometric analysis to measure both the amount of binding in the patient sample and the amount of binding when generating the calibration curve. However, any suitable analytical method may be used.

[0030] As used herein, the term "predetermined cutoff value" refers to a statistically determined amount of binding that indicates a high likelihood that a subject has a disease or a particular severity of the disease, in which a measurement of the target peptide in a subject's sample that is equal to or greater than the statistical cutoff value corresponds to at least a 70% probability, 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 of the presence of the disease or of the disease being of a particular severity.

[0031] As used herein, the term "value associated with a normal healthy individual" refers to a normalized binding amount determined by the above method for a sample obtained from a subject who is considered healthy, i.e., disease-free; and the term "value associated with known disease severity" refers to a normalized binding amount determined by the above method for a sample obtained from a subject known to have a disease of known severity, e.g., a disease with a known likelihood of responding to treatment.

[0032] In a third aspect, the present invention provides a method for treating a joint disease in a patient in need thereof, the method comprising the steps of: (a) performing an immunoassay according to the second aspect of the invention on a sample obtained from a patient to detect whether the patient has a joint disease and / or to predict whether the patient will respond to treatment for a joint disease; and (b) if it is determined in step (a) that the patient has a joint disease or that the patient is likely to respond to treatment for the joint disease, administering to the patient a drug for treating the joint disease.

[0033] The drug may be any drug suitable for treating the target joint disease. The drug may, for example, include or consist of one or more topical drugs, one or more systemic drugs, or a combination thereof. Topical drugs may be formulated, for example, in the form of a cream, foam, gel, lotion, or ointment, for application to one or more areas of the joint requiring treatment. Systemic drugs may, for example, be formulated in a form for administration via the gastrointestinal tract or parenterally.

[0034] For example, if the joint disease is osteoarthritis, applicable topical agents may be selected from nonsteroidal anti-inflammatory drugs (e.g., diclofenac or ketoprofen) or topical capsaicin; applicable systemic agents may be selected from analgesics (e.g., paracetamol or opioids); anti-TNF-α therapy (e.g., etanercept, infliximab, golimumab, adalimumab or apremilast); glucocorticoids (e.g., hydrocortisone or triamcinolone), or platelet-rich plasma.

[0035] Where the joint disease is rheumatoid arthritis, applicable medications may, for example, be topical medications, which may be selected from nonsteroidal anti-inflammatory drugs (e.g., diclofenac or ketoprofen) or topical capsaicin; applicable systemic medications may be selected from disease-modifying antirheumatic drugs (e.g., methotrexate, sulfasalazine, leflunomide, hydroxychloroquine); anti-TNF-α therapy (e.g., etanercept, infliximab, golimumab, adalimumab or apremilast); antibody therapy (e.g., rituximab or tocilizumab); analgesics (e.g., paracetamol or opioids); and glucocorticoids (e.g., hydrocortisone or triamcinolone).

[0036] When the joint disease is ankylosing spondylitis, applicable drugs may be selected from, for example, anti-inflammatory drugs (e.g., ibuprofen, phenylbutazone, diclofenac, indomethacin, naproxen, or COX-2 inhibitors), disease-modifying antirheumatic drugs (e.g., methotrexate, sulfasalazine, leflunomide, hydroxychloroquine), anti-interleukin-6 inhibitors (e.g., tocilizumab or rituximab); anti-interleukin-17A inhibitors (e.g., secukinumab and ixekizumab); Janus kinase inhibitors (e.g., tofacitinib); and anti-TNF-α therapy (e.g., etanercept, infliximab, golimumab, adalimumab, or apremilast).

[0037] In a particular embodiment, step (a) of the method may comprise performing an immunoassay according to the first aspect of the invention on a sample obtained from the patient to determine whether the patient is likely to respond to treatment for the joint disease, and step (b) of the method may comprise administering to the patient a drug for treating the joint disease only if it is determined in step (a) that the patient is likely to respond to treatment. For example, the method may include administering an agent if the likelihood of responding to the treatment is at or above a particular level. In particular, in one embodiment, the joint disease is ankylosing spondylitis, and step (b) includes administering anti-TNF-α therapy to the patient if step (a) determines that CILP-M in the patient's sample is at a level associated with the likelihood of responding to the anti-TNF-α therapy, and also includes administering anti-TNF-α therapy to the patient if it is determined that the patient is likely to respond to the treatment.

[0038] In a fourth aspect, the present invention provides an immunoassay kit comprising a monoclonal antibody according to the first aspect of the invention and at least one of the following: - streptavidin-coated well plates; - biotinylated peptide SLNPDTGLWE-L-biotin (SEQ ID NO: 15), where L is an optional linking group; - Secondary antibodies used in sandwich immunoassays; - a calibration protein comprising the N-terminal amino acid sequence SLNPDTGLWE (SEQ ID NO: 1); - Antibody biotinylation kit; - Antibody HRP labeling kit; - antibody radiolabeling kit; and - Assay visualization kit.

[0039] The immunoassay kit according to the fourth aspect of the invention is particularly suitable for use in carrying out the method according to the second aspect of the invention. Accordingly, further preferred embodiments and features of the immunoassay kit according to the fourth aspect will be apparent from the above discussion of preferred embodiments of the method according to the second aspect. [Brief explanation of the drawings]

[0040] [Figure 1] Figure 1: Alignment and specificity of the CILP-M assay. Figure 1A) Sequence alignment of the target sequence of human CILP-M (SEQ ID NO: 21) with those of mouse (SEQ ID NO: 22), bovine (SEQ ID NO: 23), and rat (SEQ ID NO: 24). The target sequence is highlighted, and protease cleavage is indicated by an arrow. Figure 1B) Specificity of the CILP-M assay. Reactivity to the standard peptide (SLNPDTGLWE (SEQ ID NO: 1)), truncated peptide (NPDTGLWE (SEQ ID NO: 4)), extended peptide (WSLNPDTGLWE (SEQ ID NO: 2)), and nonsense standard peptide and coaguloside (DSGPEYADVV (SEQ ID NO: 16)). The signal is shown in relative light units per second (RLU) as a function of the standard peptide.

[0041] [Figure 2] Figure 2: In vitro cleavage of human articular cartilage by enzymes. CILP-M levels during cleavage of human articular cartilage by MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP12, MMP13, ADAMTS4, and ADAMTS5 were measured using a CILP-M competitive chemiluminescence immunoassay.

[0042] [Figure 3-1]Figure 3: CILP-M levels and associated receiver operating characteristic curves in the discovery and validation cohorts. Figure 3A) CILP-M levels in the discovery cohort, which included healthy donors (n=13), rheumatoid arthritis patients (RA, n=18), ankylosing spondylitis patients (AS, n=14), and osteoarthritis patients (OA, n=8). Figure 3B) ROC curves evaluating the ability of CILP-M to discriminate between healthy controls and each of RA, OA, and AS in the discovery cohort.

[0043] [Figure 3-2] Figure 3C) CILP-M levels in the validation cohort, which included healthy donors (n = 105), RA patients (n = 23), and AS patients (n = 89). Figure 3D) ROC curve analysis evaluating the ability of CILP-M to discriminate between healthy controls and RA / AS patients in the validation cohort. Data were analyzed using ANCOVA (adjusted for age and sex in the validation cohort) and area under the receiver operating characteristic curve (AUROC) analysis.

[0044] [Figure 4] Figure 4: CILP-M levels stratified into responders (n=26) and non-responders (n=9) to anti-TNF-α therapy. Response to anti-TNF-α treatment in AS patients at baseline (week 0) and week 12. Data are presented as Tukey box plots, and response to anti-TNF-α was predicted using a logistic regression model based on a 50% reduction in the BASDAI index at 3 months after treatment. [Example]

[0045] The embodiments disclosed herein are described in the following examples. These examples are presented to aid in understanding the present disclosure and should not be construed in any way to limit the scope of the disclosure as defined in the claims that follow. The examples set forth below are presented 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 disclosure, nor are they intended to imply that the experiments described below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0046] Materials and Methods All reagents used in the experiments were high quality chemicals from Merck (Whitehouse Station, NJ, USA) and Sigma (St. Louis MO, USA) unless otherwise stated. All synthetic peptides used for antibody production and assay validation were purchased from GenScript (Piscataway, NJ, US) (Table 1).

[0047] [Table 1]

[0048] Monoclonal antibody development, production and characterization The amino acid sequence 700'↓SLNPDTGLWE '710 (SEQ ID NO: 1) was used to generate monoclonal antibodies (mAbs). Immunization was initiated by subcutaneous injection of 200 μl of emulsified antigen containing 100 μg of immunogenic peptide (SLNPDTGLWE-GGC-KLH) into 4-6 week-old Balb / C mice using Stimune (Thermo Fisher Scientific). Immunizations were repeated every 2 weeks until serum antibody titers stabilized. Mice with the highest serum antibody titers were selected for cell fusion and allowed to rest for one month. Three days before removal of the spleen for cell fusion, the mice were boosted with 100 μl of 0.9% NaCl solution containing 50 μg of immunogenic peptide intravenously. To generate hybridoma cells, mouse splenocytes were fused with mouse SP2 / 0 myeloma cells as described by Gefter et al. (13). Clones were then cultured in 96-well microtiter plates and subjected to limiting dilution to promote monoclonal proliferation. Supernatants were screened for reactivity by indirect ELISA on streptavidin-coated plates. SLNPDTGLWE-K-Biotin (SEQ ID NO: 18) was used as the screening peptide, and the standard peptide SLNPDTGLWE (SEQ ID NO: 1) was used to further test the specificity of the clones. Supernatants were collected from the hybridoma cells and purified using a HiTrap affinity column (GE Healthcare Life Sciences, Little Chalfront, Buckinghamshire, UK) according to the manufacturer's instructions, and the antibody isotype was determined using a Rapid ELISA Mouse Monoclonal Antibody Isotyping Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

[0049] Native reactivity was assessed using human serum, citrated plasma, heparinized plasma, EDTA plasma, and rat serum purchased from a commercial supplier (Valley Biomedical, Winchester, VA). mAbs were selected that specifically recognized the standard peptide (SLNPDTGLWE (SEQ ID NO: 1)) but not the one-amino acid-extended sequence (WSLNPDTGLWE (SEQ ID NO: 2)) or the one-amino acid-truncated sequence (NPDTGLWE (SEQ ID NO: 14)).

[0050] CILP-M assay development The development of the competitive chemiluminescence immunoassay (CLIA) included preliminary optimization experiments in which several tests were performed to analyze the reagents, concentrations, incubation times, and incubation temperatures. The procedure for the CILP-M competitive ELISA was as follows: A 96-well streptavidin-coated white microplate (Greiner Bio-One, Kremsmunster, Austria) was coated with 2.5 ng / mL of biotinylated synthetic peptide (SLNPDTGLWE-K-biotin) (SEQ ID NO: 18) in assay buffer (10 mM phosphate-buffered saline (PBS), 1% bovine serum albumin, 0.1% Tween-20, 0.36% bronidox, 4 g / L NaCl, pH 7.4 at 20°C) and incubated for 30 min at 20°C with shaking (300 rpm) in the dark. Next, 20 μL / well of selected peptide (100 ng / mL) and sample were added to the appropriate wells, and 100 μL / well of HRP-labeled antibody diluted to a concentration of 100 ng / mL in assay buffer was added and incubated for 1 hour at 20°C with shaking (300 rpm) in the dark. After each incubation step, the wells were washed five times with standard wash buffer (20 mM Tris, 50 mM NaCl, pH 7.2). A working solution of chemiluminescent substrate (Roche, BM Chemiluminescence ELISA substrate (POD), Basel, Switzerland) was mixed 15 min before use, and 100 μL / well was added to the plate and incubated for 3 min at 20°C with shaking (300 rpm) in the dark. Relative light units were measured at all wavelengths within 5 min using a microplate luminometer reader (Spectramax M5, Molecular Devices, CA, USA). The standard curve was calculated using a four-parameter logistic curve fit [Y = (AD) / (1 + (x / C) ∧ B) + D, where R > 0.9. Data were analyzed using SoftMax Pro version 7.0.3 software.

[0051] Technical evaluation Two-fold dilutions of four human serum samples were used to assess the linearity of the immunoassay, which was calculated as the percent recovery of the undiluted samples. Antibody specificity was calculated as the percent signal inhibition by two-fold dilutions of the standard peptide (SLNPDTGLWE) (SEQ ID NO: 11), the extended peptide (WSLNPDTGLWE) (SEQ ID NO: 2), the truncated peptide (LNPDTGLWE) (SEQ ID NO: 3), and the nonsense peptide (DSGPEYADVV) (SEQ ID NO: 16).

[0052] Intra- and inter-assay variation was determined by 10 independent measurements of five quality controls and two duplicate measurements of two kit controls. The accuracy of the assay was measured in healthy human serum samples spiked with the standard peptide and in serum samples containing known high concentrations of CILP-M, and was calculated as the percent recovery between the measured and predicted concentrations of the standard peptide and the percent recovery between the measured and predicted concentrations of serum samples containing high concentrations of CILP-M spiked with the concentration of the analyte in the serum. Analytical interferences were measured by spiking serum samples with known concentrations of low and high hemoglobin (2.50 / 5 mg / mL), hyperlipidemia (1.50 / 5 mg / mL), and biotin (3 / 9 ng / mL). Percent recoveries were calculated using normal serum samples as a reference. Normal reference levels were 0-10 mg / dL (0-0.00161 mmol / L), <150 mg / dL (<1.6935 mmol / L), and 0.221-3.004 ng / mL for hemoglobin, hyperlipidemia, and biotin, respectively. Interferences were calculated as the percent recovery of the analyte in non-spiked serum. The measurement range was defined as the range between the lower limit of the measurement range (LLMR) and the upper limit of the measurement range (ULMR), determined from 10 independent measurements using a standard peptide. Measurements below the LLMR or above the UMLR were assigned a value of LLMR / UMLR, respectively. 50 (50% inhibitory concentration) was determined from the standard curve.

[0053] Analyte stability was investigated by performing temperature studies and by performing repeated freeze-thaw cycling studies on serum samples. In the temperature study, three human serum samples were incubated at 4°C or 20°C for 0, 2, 4, 24, and 48 hours, and then the CILP-M levels in the samples at different incubation times and temperatures were measured. Recovery rates were evaluated using the 0-hour sample as a reference. The effect of four cycles of freeze-thaw cycling was evaluated for three serum samples, and freeze-thaw recovery was calculated using the zero-cycle sample as a reference. Each sample was measured in duplicate.

[0054] In vitro cleavage Articular cartilage biopsies from patients with osteoarthritis who underwent knee replacement surgery were obtained from Gentofte Hospital, Denmark. Human cartilage collection and recovery followed international ethical guidelines for the handling of human samples and human subject information. All participants signed informed consent, and the study was approved by the local ethics committee. A broad range of enzymes known to cleave human articular cartilage were tested (4). The enzymes used were MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, MMP-12, MMP-13, ADAMTS4, and ADAMTS5. Cartilage digestion was performed as follows: 30 mg of pulverized cartilage sample was placed in 250 μL of digestion buffer as previously described (4) with 1 μg of each enzyme. Digestion was performed for 24 h per experiment. The reaction was stopped by adding 5 mM EDTA. Cleavage products were measured using the CILP-M ELISA.

[0055] Biological evaluation of CILP-M ELISA The bioavailability of the CILP-M ELISA was evaluated using serum samples from discovery and validation cohorts. The discovery cohort was obtained from a commercial vendor, Proteogenex (Culver City, CA). The discovery cohort included healthy donors (n = 13), patients diagnosed with RA (n = 18), patients diagnosed with AS (n = 14), and patients diagnosed with OA (n = 8). The validation cohort was collected at the University of Alberta, Canada, and included serum samples from patients diagnosed with RA (n = 23) and AS (n = 89). The results were compared with age-matched healthy donor samples (n = 105) obtained from a commercial vendor, BioIVT (Westerbury, NY, USA). Each AS patient in the validation cohort was monitored for the Bass AS Disease Activity Index (BASDAI) and modified Stork AS Spinal Cord Score (mSASSS). After informed consent and approval by the local ethical committee, samples were collected from both cohorts in accordance with the Declaration of Helsinki of 1975. Serum samples were obtained and stored at -80°C until use.

[0056] ethical statement All animals were handled in accordance with animal welfare guidelines. The production of monoclonal antibodies in mice was approved by the Danish National Authority (Animal Experimentation Inspectorate) under approval number 2013-15-2934-00956.

[0057] statistical analysis For all statistical analyses performed, a p value of less than 0.05 was considered significant. Baseline characteristics were expressed as number (frequency) and percentage for categorical variables and as mean (standard deviation) for continuous variables. The Kruskal-Wallis rank test was used to test for differences at baseline between participant groups. One-way analysis of covariance at baseline was performed for both studies. Area under the receiver operating characteristic curve (AUROC) analysis was performed to examine the discriminatory accuracy of CILP-M between patients with AS or RA and healthy controls at baseline. Age and sex were adjusted for in the validation cohort analysis. In the validation study, a logistic regression model was used to predict response to anti-TNF-α treatment (Enbrel, Humira, Remicade, or Simponi) in AS patients based on a 50% reduction in the BASDAI index 3 months after treatment (patients had not received prior biologic treatment). Statistical analyses and graphing were performed using GraphPad Prism version 9 (GraphPad Software, Inc., La Jolla, CA) and R studio version 4.2.1 (R Foundation for Statistical Computing, Vienna, Austria. URL https: / / www.R-project.org).

[0058] 〔result〕 Technical evaluation and characterization of CILP-M Monoclonal antibody clone NB326#76 13B5-1C6-2C4 showed the best native reactivity, peptide affinity, and stability for the assay and was selected for assay development. The isotype, sequence, and CDRs of this monoclonal antibody were determined. The peptide chain sequences are as follows (CDRs are underlined and bold; N-terminal signal peptide and C-terminal constant region are italicized):

[0059] Heavy chain sequence (mouse IgG1 isotype)

[0060] [ka]

[0061] Light chain sequence (mouse kappa isotype)

[0062] [ka]

[0063] The technical evaluation of CILP-M is summarized in Table 2. Briefly, the measurement range (LLOQ-ULOQ) was determined to be 0.54-30.00 ng / mL. Inter- and intra-assay variations were 10.3% and 7.2%, respectively, and linearity was observed in the range from undiluted to 2-fold dilutions in human serum. Analyte stability was good both during long-term storage at 4°C / 20°C (95% and 92%, respectively) and during three freeze-thaw cycles (92-119%). Hemoglobin, hyperlipidemia, and biotin did not interfere with CILP-M measurement in human serum.

[0064] [Table 2]

[0065] Alignment of the human target sequence of CILP-M (SEQ ID NO: 21) with the corresponding mouse (SEQ ID NO: 22), bovine (SEQ ID NO: 23), and rat (SEQ ID NO: 24) target sequences using Uniprot revealed that the corresponding mouse and bovine target sequences were 100% aligned with the human sequence, whereas the rat sequence had one mismatch at position 5 (Fig. 1A). To assess the specificity of the CILP-M assay, mAbs were tested against extended peptides, truncated peptides, nonsense standard peptides, and nonsense coaters, and showed no reactivity to these peptides (Fig. 1B).

[0066] To identify the enzyme responsible for the production of CILP-M, human articular cartilage was digested with various enzymes, and it was observed that CILP-M is mainly produced by MMP-1, MMP-8, and MMP-12 (Fig. 2).

[0067] Baseline demographic and clinical characteristics Tables 3 and 4 show the statistical characteristics of both cohorts. In the discovery cohort, there was a significant difference in age between the groups (p<0.0001). OA patients were, on average, 30 years older than the healthy donors, RA, and AS in this cohort, which was a significant difference. There was no significant difference in gender distribution between the groups. In the validation cohort, there was also a significant difference in age between the groups (p<0.0001). RA patients were, on average, 15 years older than the healthy donors and AS. There were also more males in the RA and AS groups compared to the healthy donors (p<0.0001).

[0068] [Table 3]

[0069] [Table 4]

[0070] CILP-M is upregulated in rheumatic diseases in the discovery and validation cohorts CILP-M levels were measured in two independent cohorts. The discovery cohort consisted of healthy donors and patients diagnosed with RA, AS, and OA. In this cohort, patients with RA, AS, and OA showed significantly higher CILP-M levels than healthy donors (P = 0.001, P = 0.0007, and P = 0.006, respectively; Figure 3A). No differences were observed between the patient groups. The diagnostic performance (AUROC) of CILP-M was 0.966 (95% CI = 0.910-1.000, p < 0.0001) for patients with RA compared with healthy donors, 0.962 (95% CI = 0.896-1.000, p < 0.0001) for patients with AS compared with healthy donors, and 0.971 (95% CI = 0.911-1.000, p < 0.0004) for patients with OA compared with healthy donors, as shown in Figure 3B.

[0071] The validation cohort included healthy donors and patients diagnosed with RA or AS. In this population, patients with RA and AS confirmed elevated CILP-M levels in rheumatic diseases (both p<0.0001, Figure 3C). The diagnostic ability (AUROC) of CILP-M was 0.936 (95% CI = 0.894-0.978, p<0.0001) for patients with RA compared with healthy donors, and 0.955 (95% CI = 0.926-0.984, p<0.0001) for patients with AS compared with healthy donors, as shown in Figure 3D.

[0072] In the validation cohort, 67 AS patients received anti-TNF-α therapy, and 35 of the 67 patients underwent BASDAI measurements before and after treatment. To investigate response to treatment based on a 50% reduction in BASDAI, the 35 patients for whom BASDAI information was available underwent CILP-M level measurements. It was found that the likelihood of responding to anti-TNF-α treatment tended to increase with increasing baseline CILP-M units.

[0073] [Essay] Applicant developed and characterized a competitive ELISA for detecting CILP-M using a monoclonal antibody targeting an MMP-generated neoepitope of CILP-1. Key findings from the experiment include: 1) a technically robust and specific assay was developed for the CILP-M neoepitope generated primarily by MMP1, MMP8, and MMP12; 2) CILP-M can be measured in human serum, and CILP-M levels were elevated in patients with RA, AS, and OA, as demonstrated in discovery and validation cohorts; and 3) CILP-M demonstrated the ability to distinguish between anti-TNF-α responders and non-responders.

[0074] The CILP-M assay was characterized as technically stable and accurate, demonstrating acceptable dilution recovery, interference, and stability tests, with acceptable inter- and intra-assay variability of 10.3% and 7.2%, respectively. The CILP-M target epitope was discovered by mass spectrometry analysis of human articular cartilage in a previous study (Ref. 4), but in that study it was primarily generated by ADAMTS5. To determine which proteases generated the CILP-M cleavage site, we tested a series of proteases. In contrast to previous findings, our in vitro cleavage demonstrated that the CILP-M epitope was generated by MMP-1, MMP-8, and MMP-12. Destruction and fusion of articular cartilage are important features of rheumatic diseases (Refs. 11-13). Serine proteases and MMPs are highly expressed in the extracellular matrix during joint injury and contribute to cartilage degradation (Ref. 13). ADAMTS4 and ADAMTS5 are thought to be the major aggrecan-degrading enzymes in cartilage (Ref. 3), whereas MMPs degrade all components of the extracellular matrix (ECM) (Ref. 14). MMP-1 and MMP-8 are the most important collagenases involved in the degradation of endochondral collagen, and both have been known for a very long time (Ref. 14). MMP-1 is primarily produced by synovial cells lining the joint and plays a dominant role in OA by regulating the collagen degradation process (Ref. 15). High levels of CILP-1 have been detected in synovial tissue (Ref. 10) and can be cleaved by MMP-1. MMP-8 is expressed at sites of inflammation and has been associated with a wide range of inflammatory diseases (Refs. 13, 16). MMP-12 is secreted by inflammatory macrophages (Ref. 17), and increased MMP-12 expression in macrophages has been shown to exacerbate the progression of inflammatory arthritis in rabbits (Ref. 18).

[0075] To the best of our knowledge, this study is the first to explore cleavage products of CILP-1 in rheumatic diseases and the first to detect CILP-1 epitopes in blood samples. CILP-M levels were found to be elevated in RA, AS, and OA compared with healthy donors. Previous studies have linked CILP-1 to cartilage degenerative diseases, particularly OA (References 6 and 7). Anti-CILP antibodies have also been shown to be found in OA and RA patients (Reference 19), suggesting that an autoimmune response to CILP is involved in the pathogenesis of OA and RA. To the best of our knowledge, this is the first study to investigate the role of CILP-1 in patients with AS. The presented findings are consistent with previous studies demonstrating advanced extracellular matrix remodeling in the cartilage of AS patients (20, 21). However, CILP-1 has also been implicated in pulmonary hypertension and myocardial fibrosis (22, 23), suggesting that it is not specific to articular cartilage.

[0076] Given the need for better tools to monitor response to treatment in AS patients, we also investigated whether CILP-M could predict response to anti-TNF-α and observed that patients who responded to anti-TNF-α treatment had higher CILP-M levels before treatment.

[0077] In conclusion, a novel neoepitope biomarker, CILP-M, measuring MMP-1-, MMP-8-, and MMP-12-mediated fragments of CILP-1 was developed and validated for use in human serum samples. CILP-M was upregulated in two independent cohorts of RA and AS patients. Based on these results, CILP-M may be useful for assessing cartilage remodeling in degenerative joint diseases.

[0078] In this specification, unless otherwise specified, the word "or" is used to mean an operator that returns a true value if one or both of the stated conditions are met, as opposed to the "exclusive or" operator, which requires that only one of several conditions be met. The word "comprising" is used to mean "including, or consisting of." All prior teachings acknowledged above are incorporated herein by reference. Any acknowledgment in this specification of a prior-published document should not be taken as an admission or representation that the teaching of that document was common general knowledge in Australia or elsewhere at the time of this specification.

[0079] [References] 1. Sophia Fox AJ, Bedi A, Rodeo SA. Basic science of articular cartilage: structure, composition, and function. Sports Health [Internet] SAGE Publications; 2009 [cited 2022 Mar 9];1:461. Available from: / pmc / articles / PMC3445147 / 2. Lorenzo P, Bayliss MT, Heingold D. A novel cartilage protein (CILP) present in the intermediate region of human articular cartilage increases with age * . 1998 [cited 2022 Jul 19]; Available from: http: / / www.jbc.org 3. Goldring MB, Marcu KB. Cartilage homeostasis in normal subjects and in rheumatic diseases. Arthritis Res Ther [Internet] BioMed Central; 2009 [cited 2022 Jul 19];11:224. Available from: / pmc / articles / PMC2714092 / 4. Zane EY, Brittain IJ, Laska DA, Mitchell PG, Smel EU, Kalsdal MA, et al. Characterization of metalloprotease cleavage products of human articular cartilage. Arthritis Rheum [Internet] Arthritis Rheum; 2008 [cited 2022 Jul 19];58:2420-31. Available from: https: / / pubmed.ncbi.nlm.nih.gov / 18668564 / 5. Lorenzo P, Bayliss MT, Heingold D. Altered patterns and synthesis of extracellular matrix macromolecules in early osteoarthritis. Matrix Biol 2004;23:381-91.

[0080] 6. Valdes AM, Hart DJ, Johns KA, Sardulescu G, Swarbrick P, Doyle D V., et al. Collaborative study of candidate genes associated with the prevalence and progression of knee osteoarthritis. Arthritis Rheum [Internet] John Wiley & Sons, Ltd; 2004 [cited 2022 Jul 19];50:2497-507. Available from: https: / / onlinelibrary.wiley.com / doi / full / 10.1002 / art.20443 7. Valdes AM, Van Oyen M, Hart DJ, Sardulescu GL, Loughlin J, Doherty M, et al. Reproducible genetic associations between candidate genes and clinical knee osteoarthritis in men and women. Arthritis Rheum [Internet] John Wiley & Sons, Ltd; 2006 [cited 2022 Jul 19];54:533-9. Available from: https: / / onlinelibrary.wiley.com / doi / full / 10.1002 / art.21621 8. Seki S, Kawaguchi Y, Chiba K, Mikami Y, Kizawa H, Ooya T, et al. A functional SNP in CILP, which encodes a cartilage intermediate layer protein, It is associated with susceptibility to lumbar disc disease. Nat Genet [Internet] Nature Publishing Group; 2005 [cited 2022 Jul 19];37:607-12. Available from: https: / / u-toyama.elsevierpure.com / en / publications / a-functional-snp-in-cilp-encoding-cartilage-intermediate-layer-pr 9. Tsuruha JI, Masuko-Hongou K, Kato T, Sakata M, Nakamura H, Nishioka K, et al. Close association between cartilage destruction and cartilage intermediate lamina proteins in a subset of patients with osteoarthritis and rheumatoid arthritis. ARTHRITIS Rheum 2001;44:838-45. 10. Kerna I, Kisand K, Tam A, Tam A. Cartilage intermediate lamina protein is produced in the synovium of osteoarthritic joints and is upregulated in osteoarthritis-associated fibrosis. Bone Abstr [Internet] BioScientifica; 2013 [cited 2022 Jul 19];1. Available from: http: / / www.bone-abstracts.org / ba / 0001 / ba0001pp15

[0081] 11. Park DR, Kim J, Kim GM, Lee H, Kim M, Hwang D, et al. Blockers of osteoclast-associated receptors prevent articular cartilage destruction through the regulation of chondrocyte apoptosis. Nat Commun 2020 111 [Internet] Nature Publishing Group; 2020 [cited 2022 Jul 19];11:1-11. Available from: https: / / www.nature.com / articles / s41467-020-18208-y 12. Bleil J, Schieper J, Meier R, Schlichting U, Hempfing A, Seilb U, et al. Cartilage within the facet joints of patients with ankylosing spondylitis (AS) shows signs of cartilage destruction rather than signs of chondrocyte hypertrophy: close relevance to joint reconstruction in AS. Arthritis Res Ther [Internet] BioMed Central Ltd.; 2015 [cited 2021 May 31];17. Available from: / pmc / articles / PMC4506408 / 13. Rannow F, Francois M, Kohlvor MT, Berenbaum F. Cartilage damage in rheumatoid arthritis. 2005 [cited 2022 Jul 19]; Available from: http: / / france.elsevier.com / direct / BONSOI / 14. Mehana E-SE, Karfagha AF, El-Brehi SS. The role of matrix metalloproteinases in the pathogenesis of osteoarthritis. An updated review. 2019; 15. Kuzminski A, Przybyszewski M, Graczyk M, Barczy Z. The role of extracellular matrix metalloproteinases and their inhibitors in allergic diseases. Postep Dermatologii Alergol 2012;29:384-9.

[0082] 16. Van Lint P, Leibert C. Matrix metalloproteinase-8: cleavage can be determined. Cytokine Growth Factor Rev 2006;17:217-23. 17. Chen YE. MMP-12, a classical enzyme, plays a new role in the pathogenesis of rheumatoid arthritis? Am J Pathol [Internet] American Society for Investigative Pathology; 2004 [cited 2022 Aug 3];165:1069. Available from: / pmc / articles / PMC1618647 / 18. Wang X, Ryan J, Koike T, Seung H, Ichikawa T, Kitajima S, et al. Overexpression of human matrix metalloproteinase-12 enhances the development of inflammatory arthritis in transgenic rabbits. Am J Pathol [Internet] American Society for Investigative Pathology; 2004 [cited 2022 Aug 3];165:1375. Available from: / pmc / articles / PMC1618618 / 19. Tsuruha JI, Masuko-Hongou K, Kato T, Sakata M, Nakamura H, Nishioka K. Close association between cartilage destruction and cartilage intermediate lamina proteins in a subset of patients with osteoarthritis and rheumatoid arthritis. Arthritis Rheum 2001;44:838-45. 20. Husakova M, Bay-Jensen AC, Foraytova S, Tsegturkova K, Tomsik M, Gregova M, et al. Metabolites of collagen types I, II, III, and IV may serve as markers of disease activity in axial spondyloarthritis. Sci Rep 2019;9:1-10.

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Claims

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

2. The monoclonal antibody of claim 1, wherein the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence WSLNPDTGLWE (SEQ ID NO: 2).

3. The monoclonal antibody according to claim 1 or 2, which does not specifically bind to a peptide having the N-terminal amino acid sequence LNPDTGLWE (SEQ ID NO: 3).

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

5. 5. The monoclonal antibody of any one of claims 1 to 4, wherein the monoclonal antibody is raised against a synthetic peptide having the N-terminal amino acid sequence SLNPDTGLWE (SEQ ID NO: 1).

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

7. 10. The method of claim 6, wherein the method is for detecting and / or monitoring a disease in a subject and / or assessing the likelihood that the subject will respond to treatment with a disease therapeutic agent, further comprising the steps of: iii) Correlating the determined amount of binding with values ​​associated with normal healthy individuals and / or with values ​​associated with known disease severity and / or with values ​​obtained from the subject at previous time points and / or with predetermined cut-off values.

8. The method of claim 7, wherein the disease is a joint disease.

9. 9. The method of claim 8, wherein the joint disease is rheumatoid arthritis, ankylosing spondylitis, or osteoarthritis.

10. 10. The method of any one of claims 7 to 9, wherein said treatment with a disease therapeutic agent is an anti-TNF-α therapy.

11. The method of any one of claims 6 to 10, wherein the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence WSLNPDTGLWE (SEQ ID NO: 2).

12. The method of any one of claims 6 to 11, wherein the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence LNPDTGLWE (SEQ ID NO: 3).

13. The method of any one of claims 6 to 12, wherein the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence NPDTGLWE (SEQ ID NO: 4).

14. 14. The method of any one of claims 6 to 13, wherein the monoclonal antibody is raised against a synthetic peptide having the N-terminal amino acid sequence SLNPDTGLWE (SEQ ID NO: 1).

15. 15. The method of any one of claims 6 to 14, wherein the patient sample is selected from blood, serum, or plasma.

16. The method of any one of claims 6 to 15, wherein the immunoassay is a competitive assay or a sandwich assay.

17. 17. The method of any one of claims 6 to 16, wherein the immunoassay is a radioimmunoassay or an enzyme-linked immunosorbent assay.

18. A monoclonal antibody that specifically binds to the N-terminal amino acid sequence SLNPDTGLWE (SEQ ID NO: 1), and at least one of the following: - streptavidin-coated well plates; - biotinylated peptide SLNPDTGLWE-L-biotin (SEQ ID NO: 15), where L is an optional linking group; - secondary antibodies used in sandwich immunoassays; a calibration protein comprising the N-terminal amino acid sequence SLNPDTGLWE (SEQ ID NO: 1); - antibody biotinylation kit; - Antibody HRP labeling kit; - antibody radiolabeling kit; and, - Assay Visualization Kit 1. An immunoassay kit comprising:

19. The immunoassay kit of claim 18, wherein the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence WSLNPDTGLWE (SEQ ID NO: 2).

20. The immunoassay kit of claim 18 or 19, wherein the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence LNPDTGLWE (SEQ ID NO: 3).

21. The immunoassay kit of any one of claims 18 to 20, wherein the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence NPDTGLWE (SEQ ID NO: 4).

22. 22. The immunoassay kit of any one of claims 18 to 21, wherein the monoclonal antibody is raised against a synthetic peptide having the N-terminal amino acid sequence SLNPDTGLWE (SEQ ID NO: 1).

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