Antibody specifically recognizing ECM1, method for detecting disease having high risk of fracture using same, and detection reagent

Abstract

The present invention addresses the problem of providing a method for detecting a disease having a high risk of fracture.　Provided is an antibody specifically recognizing extracellular matrix protein 1 (ECM1), the antibody being characterized by: recognizing an epitope on ECM1 having the amino acid sequence represented by positions 20-540 of SEQ ID NO: 10, the epitope being located within positions 194-279 or 454-540 of SEQ ID NO: 10; or recognizing an epitope on a polypeptide having an amino acid sequence with at least 90% identity to the amino acid sequence represented by positions 20-540 of SEQ ID NO: 10, the epitope being located within positions corresponding to positions 194-279 or 454-540 of SEQ ID NO: 10.

Description

Antibody that specifically recognizes ECM1, method for detecting diseases associated with high risk of fracture using the same, and detection reagent

[0001] The present invention relates to an antibody that specifically recognizes extracellular matrix protein 1 (ECM1), a method for detecting diseases associated with a high risk of fracture using the antibody, and a detection reagent.

[0002] Diseases associated with a high risk of fracture include osteopenia and osteoporosis. Osteopenia refers to a condition in which bone mass or bone density is lower than normal, even if not diagnosed as osteoporosis. Osteoporosis is a disease characterized by low bone mass and abnormalities in the microstructure of bone tissue, resulting in an increased risk of fracture. Criteria for determining high risk of fracture include the World Health Organization (WHO) diagnostic categories based on bone density. These are based on the value (T-score) obtained by dividing the mean value of a young adult by the standard deviation (SD) of the young adult population, and are classified as follows: Normal: Bone density is at least one standard deviation (SD) below the mean value of a young adult (T-score of -1 or greater); Osteopenia: T-score less than -1 and greater than -2.5; Osteoporosis: T-score of -2.5 or less. It is known that an increased risk of fracture significantly increases the risk of death and decreases quality of life. In diseases with a high risk of fracture, early diagnosis and therapeutic intervention are important.

[0003] Currently, the gold standard for diagnosing osteoporosis is bone mineral density measurement using the DXA (Dual-energy X-ray Absorbtiometry) method, and the diagnosis is based on the T-score. The WHO defines a T-score of ≦−2.5 as the diagnostic criterion for primary osteoporosis. However, because it has been reported that approximately 50% of postmenopausal women who have suffered osteoporotic fractures have normal T-scores (Non-Patent Document 1), a history of fragility fractures is also included as one of the diagnostic criteria in Japan.

[0004] In addition, bone metabolic markers are used as indicators related to osteoporosis. Bone metabolic markers are recognized as indicators independent of bone mineral density, and evaluation of bone metabolic turnover is useful for selecting therapeutic drugs and assessing therapeutic effects. However, bone metabolic markers are known to vary day to day and within the day, so they are not included in diagnostic criteria.

[0005] The fracture risk assessment tool FRAX (fracture risk assessment tool) is also used clinically. FRAX predicts 10-year fracture risk by inputting age, sex, BMI, and the presence or absence of fracture risk factors. Therefore, it has been adopted as a treatment initiation criterion in Europe, the United States, and Japan. However, FRAX is not used for diagnosis due to limitations in its prediction accuracy and the limited number of items that can be selected as risk factors. Meanwhile, there have been reports of measuring ECM1 using a commercially available ELISA kit (Non-Patent Document 2). However, in practice, further detection performance is required.

[0006] Japanese Patent Application Laid-Open No. 2009-240300

[0007] Nguyen et al, J. Clin. Endocrinol. Metab., 2007; 92(3): 955-962Journal of Proteomics 300 (2024) 105166

[0008] As described above, the only quantitative index currently used to diagnose osteoporosis is the T-score, which is based on bone mineral density. However, it is difficult to accurately diagnose the fracture risk of osteoporosis patients using only the T-score. Meanwhile, early diagnosis and therapeutic intervention for diseases associated with a high risk of fracture are important challenges for preventing fractures. Therefore, an objective of the present invention is to provide a method for detecting diseases associated with a high risk of fracture.

[0009] As a result of extensive research, the present inventors have found that antibodies that recognize specific sites on ECM1 are useful for detecting diseases associated with a high risk of fracture, and have thus completed the present invention.

[0010] That is, the present invention is as follows: [1] An antibody that specifically recognizes ECM1, characterized by having an epitope at positions 194 to 279 or 454 to 540 of extracellular matrix protein 1 (ECM1) represented by positions 20 to 540 of the amino acid sequence represented by SEQ ID NO: 10, in SEQ ID NO: 10, or having an epitope at a position corresponding to positions 194 to 279 or 454 to 540 of SEQ ID NO: 10 in a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by positions 20 to 540 of SEQ ID NO: 10. [2] A method for detecting a disease associated with a high risk of fracture, comprising the step of measuring the level of ECM1 in a sample collected from a living body using the antibody according to [1]. [3] The method of [2], wherein the level of ECM1 is measured using: (1) an antibody having an epitope at positions 194 to 279 of SEQ ID NO: 10 of ECM1 represented by positions 20 to 540 of the amino acid sequence represented by SEQ ID NO: 10, or at positions corresponding to positions 194 to 279 of SEQ ID NO: 10 in a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by positions 20 to 540 of SEQ ID NO: 10; and (2) an antibody having an epitope at positions 454 to 540 of SEQ ID NO: 10 of ECM1 represented by positions 20 to 540 of the amino acid sequence represented by SEQ ID NO: 10, or at positions corresponding to positions 454 to 540 of SEQ ID NO: 10 in a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by positions 20 to 540 of SEQ ID NO: 10. [4] The method of [2] or [3], wherein the subject is determined to have a high possibility of having a disease associated with a high risk of fracture if the level of ECM1 in the sample is higher than a value obtained from a control subject not having a disease associated with a high risk of fracture or a predetermined reference value. [5] The method according to any one of [2] to [4], wherein the disease associated with a high risk of fracture is osteoporosis or osteopenia. [6] The method according to any one of [2] to [5], wherein the sample is whole blood, serum, or plasma. [7] The method according to any one of [2] to [6], wherein the measurement of the ECM1 level is performed by immunoassay. [8] The method according to [7], wherein the immunoassay is performed by ELISA or CLEIA.[9] A reagent or kit for use in the detection method of [2], characterized by containing the antibody of [1].

[10] The reagent or kit of [9], comprising: (1) an antibody having an epitope at positions 194 to 279 of SEQ ID NO: 10 of ECM1 represented by positions 20 to 540 of the amino acid sequence represented by SEQ ID NO: 10, or at positions corresponding to positions 194 to 279 of SEQ ID NO: 10 in a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by positions 20 to 540 of SEQ ID NO: 10, and (2) an antibody having an epitope at positions 454 to 540 of SEQ ID NO: 10 of ECM1 represented by positions 20 to 540 of the amino acid sequence represented by SEQ ID NO: 10, or at positions corresponding to positions 454 to 540 of SEQ ID NO: 10 in a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by positions 20 to 540 of SEQ ID NO: 10.

[0011] The present invention can provide a detection method with higher efficiency in detecting diseases associated with a high risk of fracture.

[0012] 1 is a diagram showing various recombinant antigens obtained in Example 1. FIG. 2 is a diagram showing the reactivity of antibody EMArEC04M01 to various recombinant antigens obtained in Example 4. FIG. 3 is a diagram showing the reactivity of antibody EMCrEC20M04 to various recombinant antigens obtained in Example 4. FIG. 4 is a diagram showing the ECM1 concentrations in a group of healthy subjects and a group of patients at high risk of fracture when antibodies EMArEC04M01 and EMCrEC20M04 are used in combination, obtained in Example 5. FIG. 5 is a diagram showing the reactivity of a commercially available ELISA kit to various recombinant antigens obtained in Comparative Example 1. FIG. 6 is a diagram showing the ECM1 concentrations in a group of healthy subjects and a group of patients at high risk of fracture when a commercially available ELISA kit is used, obtained in Comparative Example 2. FIG. 7 is a diagram showing the correlation between CLEIA measurement values ​​using an AIA-CL reagent obtained in Example 6 and ELISA measurement values ​​obtained in Example 7.

[0013] <Antibody that specifically recognizes a specific site in ECM1> A first embodiment of the present invention is an antibody that specifically recognizes ECM1, characterized in that it has an epitope at positions 194 to 279 or 454 to 540 of the amino acid sequence represented by SEQ ID NO: 10, of ECM1 represented by positions 20 to 540 of the amino acid sequence represented by SEQ ID NO: 10. As will be described later, by measuring ECM1 using such an antibody, diseases associated with a high risk of fracture can be efficiently detected.

[0014] Furthermore, the amino acid sequence of ECM1 is typically represented by positions 20 to 540 of SEQ ID NO: 10, but is not particularly limited as long as it allows efficient detection of diseases associated with a high risk of fracture by the method described below, and may be a polypeptide having an amino acid sequence that is 90% or more identical, preferably 95% or more identical, and more preferably 98% or more identical to the amino acid sequence represented by positions 20 to 540 of SEQ ID NO: 10. In this case, the epitope may be located at a position corresponding to positions 194 to 279 or 454 to 540 of the amino acid sequence represented by SEQ ID NO: 10.

[0015] Such antibodies may be polyclonal or monoclonal. Antibody origins include, for example, mouse, rat, rabbit, chimeric, humanized, and human antibodies. The antibodies may be complete antibodies or fragment antibodies (antigen-binding fragments).

[0016] Antisera may be used as polyclonal antibodies. In the present invention, the term "polyclonal antibody" also encompasses antisera before purification. Furthermore, antigen-binding fragments of antibodies may be used instead of antibodies. Hereinafter, unless otherwise clear from the context, the term "antibody" also encompasses antigen-binding fragments of the antibodies. Polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments can all be prepared by well-known, conventional methods. Commercially available antibodies may be used, or antibodies prepared by well-known methods may be used.

[0017] Specifically, a polyclonal antibody having an epitope at positions 194 to 279 or 454 to 540 of the amino acid sequence represented by SEQ ID NO: 10 (hereinafter sometimes referred to as the "specific site") of ECM1 represented by positions 20 to 540 of the amino acid sequence represented by SEQ ID NO: 10 can be obtained, for example, by mixing multiple monoclonal antibodies that specifically recognize the site. Alternatively, the polyclonal antibody can be obtained by immunizing a non-human animal with an immunogen containing a polypeptide containing the site of ECM1 prepared by well-known techniques such as chemical synthesis, or a polynucleotide encoding such a polypeptide, together with an appropriate adjuvant, and obtaining antisera from blood collected from the animal, and purifying the polyclonal antibody in the antisera. Immunization is usually performed multiple times over several weeks to increase the antibody titer in the immunized animal. Purification of the antibody in the antisera can be achieved, for example, by ammonium sulfate precipitation, fractionation by anion chromatography, affinity column purification, or the like.

[0018] One well-known method for producing monoclonal antibodies is the hybridoma method. Specifically, for example, antibody-producing cells such as splenocytes or lymphocytes are collected from a non-human animal immunized as described above, and these are fused with myeloma cells to prepare hybridomas. Hybridomas that produce antibodies that bind to a specific site on ECM1 are selected, and these hybridomas are grown to obtain monoclonal antibodies that specifically recognize the specific site on ECM1 from the culture supernatant.

[0019] "Antigen-binding fragment" refers to, for example, an Fab fragment or F(ab') of an immunoglobulin. ２ The term "antibody fragment" refers to an antibody fragment that maintains the ability to bind to a specific site of the corresponding antigen (antigen-antibody reactivity), such as a Fab fragment or F(ab') fragment. ２ As is well known, fragments can be obtained by treating antibodies with proteolytic enzymes such as papain and pepsin. Antigen-binding fragments include Fab fragments and F(ab') fragments. ２The term "antigen-binding fragment" is not limited to a single fragment, but may be any fragment that maintains binding to the corresponding antigen, or may be prepared by genetic engineering techniques. For example, an antibody in which a single chain fragment of variable region (scFv) is expressed in Escherichia coli by genetic engineering techniques can also be used. Methods for producing scFv are also well known. For example, mRNA from a hybridoma prepared as described above is extracted, single-stranded cDNA is prepared, and PCR is performed using primers specific to the immunoglobulin H chain and L chain to amplify the immunoglobulin H chain gene and L chain gene. These are linked with a linker, and the vector is inserted into a plasmid vector with appropriate restriction enzyme sites. Escherichia coli is transformed with the scFv, and the scFv can be recovered from the E. coli to produce the scFv. Such scFvs are also encompassed by the term "antigen-binding fragment."

[0020] <Method for detecting diseases associated with a high risk of fracture> A second embodiment of the present invention is a method for detecting diseases associated with a high risk of fracture, comprising a step of measuring ECM1 levels in a sample collected from a living body using the above-mentioned antibody.

[0021] It is preferable to use the following two antibodies: (1) an antibody having an epitope at positions 194 to 279 in SEQ ID NO: 10 of ECM1 represented by positions 20 to 540 of the amino acid sequence represented by SEQ ID NO: 10, or at positions corresponding to positions 194 to 279 in SEQ ID NO: 10 of a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by positions 20 to 540 in SEQ ID NO: 10, and (2) an antibody having an epitope at positions 454 to 540 in SEQ ID NO: 10 of ECM1 represented by positions 20 to 540 of the amino acid sequence represented by SEQ ID NO: 10, or at positions corresponding to positions 454 to 540 in SEQ ID NO: 10 of a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by positions 20 to 540 in SEQ ID NO: 10.

[0022] In the present invention, the sample may be, for example, blood (whole blood, serum, or plasma) or urine, preferably blood, and more preferably serum or plasma. The use of a blood sample enables minimally invasive quantitative testing. The detection method of the present invention is typically performed in vitro.

[0023] The animal from which the specimen is collected is not particularly limited as long as it is a mammal, but is preferably a human.

[0024] Using the above-mentioned antibody, if the ECM1 level is higher than the value obtained from a control subject who does not have a disease associated with a high risk of fracture or a predetermined reference value, it can be determined that a subject is likely to have a disease associated with a high risk of fracture. The levels of these proteins can be expressed, for example, by the concentration of the protein.

[0025] Data obtained by this detection method can be used to assist doctors and others in diagnosing diseases associated with a high risk of fracture. In other words, the method of the present invention includes the step of detecting diseases associated with a high risk of fracture, but does not include the final decision to diagnose the disease. Doctors refer to the detection results obtained by the method of the present invention to make diagnoses and determine treatment plans. Therefore, the method of the present invention can be rephrased as a method for providing information for determining whether a disease is associated with a high risk of fracture. The information for determining can also be rephrased as information that serves as a basis for making a decision.

[0026] In this embodiment, the control subject who does not have a disease that poses a high risk of fracture may be a subject whose bone mineral density and bone quality are within the normal range and who does not have any disease (hereinafter also referred to as a healthy subject), or may be a subject who has another disease as long as it does not affect bone mineral density or bone quality.

[0027] In this embodiment, the reference value can be determined, for example, by measuring the ECM1 concentration in a sample collected from the control subject using the aforementioned antibody in advance, and using the median, mean, upper limit, index, or other value calculated from the measured value. This reference value can also be determined by dividing subjects into groups based on parameters such as gender, age, history of fracture, and treatment history, and setting an optimal value for each group. The number of control subjects may be any number that allows for a statistically appropriate determination of the reference value, and may be, for example, 5 or more, 10 or more, 20 or more, or 50 or more, or 10,000 or less, 1,000 or less, or 100 or less, or a combination thereof. The reference value can be determined, for example, so that 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 100% of a sample group of humans who do not have a disease associated with a high risk of fracture are determined to be unlikely to have a disease associated with a high risk of fracture.

[0028] Examples of diseases associated with a high risk of fracture include osteoporosis, osteopenia, hypophosphatemic rickets, osteomalacia, chronic kidney disease (CKD), type 2 diabetes, and chronic obstructive pulmonary disease (COPD). Among these, osteoporosis and osteopenia are particularly preferred.

[0029] In the present invention, the method for measuring the level of ECM1 protein using the aforementioned antibody is not particularly limited, and may include immunoassay, liquid chromatography, electrophoresis, etc., as long as the antibody is used. Immunoassay itself is well known in the art. Immunoassays are classified based on the reaction format, including sandwich assays, competitive assays, agglutination assays, and Western blotting, with the sandwich assay using the aforementioned two antibodies being preferred. Furthermore, based on the label, they include enzyme immunoassays, radioimmunoassays, and fluorescent immunoassays. For example, enzyme-linked immunosorbent assays (ELISAs), immunoblots, fluorescent antibody assays (FAs), radioimmunoassays (RIAs), fluorescent enzyme immunoassays (FLEIAs), chemiluminescent enzyme immunoassays (CLEIAs), chemiluminescent immunoassays (CLIAs), electrochemiluminescent immunoassays (ECLIAs), immunochromatography (ICA), and Western blotting (WB) can be used. As the immunological measurement method, for example, the ELISA method or the CLEIA method can be preferably used, and the sandwich ELISA method or the sandwich CLEIA method, which are expected to have high detection sensitivity, can be particularly preferably used.

[0030] Immunological assays themselves are well-known techniques, but to briefly describe them, for example, in the sandwich method, one of the two antibodies mentioned above is immobilized on a solid phase (solid-phase antibody) and reacted with a sample. After washing as necessary, the other of the two antibodies is reacted with a labeled antibody, and after washing, the labeled antibody bound to the solid phase is measured.

[0031] Labeled antibodies can be measured by measuring signals from the labeling substance. The method for measuring the signal is appropriately selected depending on the type of labeling substance. For example, in the case of enzyme labeling, enzyme activity can be determined and the analyte measured by reacting a substrate such as a chromogenic, fluorescent, or luminescent substrate corresponding to the enzyme with the enzyme and measuring the resulting color or luminescent signal using an appropriate instrument such as an absorptiometer or luminometer. For example, when ALP is used as the labeling substance, a luminescent substrate such as 3-(4-methoxyspiro(1,2-dioxetane-3,2'-tricyclo[3.3.1.13,7]decane)-4-yl)phenylphosphate disodium (e.g., trade name AMPPD) can be used. Labeled antibodies may be directly bound to the labeling substance, or may be indirectly bound to the antibody by binding a specific binding molecule such as biotin or a hapten to the antibody and then reacting with a partner of the specific binding molecule bound to the labeling substance (e.g., streptavidin or a hapten antibody). Immunological measurements are performed using the aforementioned antibody on standard samples of known concentrations containing various concentrations of ECM1, and a calibration curve is created by plotting the correlation between the amount of signal from the label and the concentration of ECM1 in the standard samples.The same procedure is then performed on a sample with an unknown ECM1 concentration to measure the amount of signal from the label, and the measured value is applied to this calibration curve, thereby quantifying the concentration of ECM1 in the sample.

[0032] A third embodiment of the present invention is a reagent or kit for use in a method for detecting a disease associated with a high risk of fracture, comprising a step of measuring the ECM1 level in a sample collected from a living body, the reagent or kit comprising the above-mentioned antibody. For details, the descriptions in the above sections <Antibody that specifically recognizes a specific site of ECM1> and <Method for detecting a disease associated with a high risk of fracture> can be cited.

[0033] The reagent is an immunoassay reagent and includes the aforementioned antibody that specifically recognizes ECM1 as a detection reagent, and the antibody is preferably a labeled antibody with an arbitrary label. The reagent may also include other reagents necessary for immunoassay. It may also be a kit further including a sample diluent, a washing solution, a labeling substance, a standard sample, a blocking agent, a detection reagent, a reaction stop solution, a plate, etc. If the labeling substance used in the labeled antibody is an enzyme, the reagent may further include a substrate solution for the enzyme, etc. It may also include instructions for use, etc. The kit preferably comprises the following two antibodies: (1) an antibody having an epitope at positions 194 to 279 of SEQ ID NO: 10 of ECM1 represented by positions 20 to 540 of the amino acid sequence represented by SEQ ID NO: 10, or at positions corresponding to positions 194 to 279 of SEQ ID NO: 10 of a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by positions 20 to 540 of SEQ ID NO: 10, and (2) an antibody having an epitope at positions 454 to 540 of SEQ ID NO: 10 of ECM1 represented by positions 20 to 540 of the amino acid sequence represented by SEQ ID NO: 10, or at positions corresponding to positions 454 to 540 of SEQ ID NO: 10 of a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by positions 20 to 540 of SEQ ID NO: 10. One of the two antibodies may be a solid-phase antibody immobilized on a solid phase such as a plate or particle, and the other antibody may be a labeled antibody. Another aspect of this embodiment is the use of the aforementioned antibody in the manufacture of a reagent or kit for use in a method for detecting a disease associated with a high risk of fracture, which comprises the step of measuring ECM1 levels in a sample collected from a living body.

[0034] <Treatment Method> The method for detecting a disease associated with a high risk of fracture of the present invention can be applied to a method for treating a disease associated with a high risk of fracture. That is, as a fourth embodiment of the present invention, there is provided a method for treating a disease associated with a high risk of fracture in a subject, the method comprising: (i) identifying the subject as a subject for treatment based on a measured value of the ECM1 level in a sample collected from the subject; and (ii) administering treatment to the subject identified as a subject for treatment.

[0035] In a preferred embodiment of the method for treating a disease associated with a high risk of fracture, in step (i), preferably, if the ECM1 level in the sample is higher than the value obtained from a control subject not having a disease associated with a high risk of fracture or a predetermined reference value, the subject is identified as having a high probability of having a disease associated with a high risk of fracture and is therefore a subject to be treated. For details of step (i), the descriptions in the above sections <Antibody that specifically recognizes a specific site of ECM1> and <Method for detecting a disease associated with a high risk of fracture> can be used.

[0036] The treatment in the step (ii) includes drug therapy, exercise therapy, nutritional therapy, etc., and examples of the drug include, but are not limited to, bisphosphonate, raloxifene, active vitamin D3, calcitonin, vitamin K2, ipriflavone, calcium preparations, etc.

[0037] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0038] Example 1: Preparation of Recombinant Antigens To secure immunogens and clarify antibody recognition sites, various recombinant ECM1s listed in Table 1 were prepared. Each recombinant ECM1 had a HIS-tag attached to the N-terminus and a BNC peptide attached to the C-terminus for use in subsequent experiments. Specifically, the following procedure was followed: A HIS-tag was attached to the 5'-terminus of the nucleotide sequence encoding each recombinant ECM1, and a nucleotide sequence encoding the BNC peptide (JP 2009-240300 A), consisting of the C-terminal 7 amino acids of BNP (brain natriuretic peptide), was attached to the 3'-terminus, and the resulting mixture was co-expressed. Using the nucleotide sequence of ECM1 set forth in SEQ ID NO: 1 (the nucleotide sequence corresponding to the protein represented by GenBank Accession No.: NP_004416) as a template, amplification was performed by PCR using the forward and reverse primers shown in Table 1, and the amplified fragment was introduced into pECE Vector using In-Fusion® HD Cloning Kit (manufactured by Takara Bio Inc.). HEK293T cells were transformed with the prepared plasmid using Lipofectamine® 3000 (manufactured by Thermo Bio Inc.), and seven types of recombinant ECM1 listed in Table 1 were transiently expressed to obtain those whose N- and C-termini were modified as described above. The resulting recombinant antigens are shown in Figure 1.

[0039] As is clear from Figure 1, ECM Full corresponds to positions 20 to 540 of SEQ ID NO: 10 (corresponding to the amino acid sequence of ECM1), ECM F279 corresponds to positions 20 to 279 of SEQ ID NO: 10, ECM 107 corresponds to positions 107 to 540 of SEQ ID NO: 10, ECM 194 corresponds to positions 194 to 540 of SEQ ID NO: 10, ECM 280 corresponds to positions 280 to 540 of SEQ ID NO: 10, ECM 367 corresponds to positions 367 to 540 of SEQ ID NO: 10, and ECM 454 corresponds to positions 454 to 540 of SEQ ID NO: 10.

[0040]

[0041] Example 2: Preparation of ECM1 Monoclonal Antibody ECM1 monoclonal antibodies were prepared by a known method (rat iliac lymph node immunization method), and antibodies suitable for ELISA measurement were selected. Specifically, the procedure is as follows: (1) For immunization, the ECM Full prepared in Example 1 was used. Purification was performed using a HisTALON Gravity Column (manufactured by Takara Bio Inc.) and used as a purified immunization antigen. (2) Immunization was performed on 4-week-old female rats. An emulsion was prepared by mixing equal amounts of antigen and adjuvant, and the rats were immunized eight times at two-week intervals. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the second and subsequent immunizations. (3) Preparation of antibody-producing hybridomas Iliac lymph nodes were excised from rats (2) with sufficiently elevated antibody titers, prepared according to standard procedures, and then fused with mouse myeloma cells (SP2 / 0) by electrofusion to produce hybridomas. The fused hybridomas were suspended in E-RDF medium (Kyokuto Pharmaceuticals) containing 10% FCS (fetal calf serum) and 1x HAT (Sigma), then seeded into a 96-well plate (Thermo) and cultured for 10 days, and the culture supernatant was obtained.

[0042] (4) Hybridoma Selection Screening was performed to select antibody-producing cells. α-His antibody (Fujifilm Wako Pure Chemical Industries, Ltd.) was diluted with carbonate buffer (pH 9.8) to 200 ng / well and immobilized on a MaxiSorp 96-well plate (Nunc). After overnight incubation at 4°C, the plate was washed three times with TBS-T (Tris-Buffered Saline containing 0.05% Tween 20), and 200 μL / well of TBS solution containing 3% bovine serum albumin (BSA) was added to each well and allowed to stand at room temperature for 2 hours. After washing three times with TBS-T, purified ECM1 Full prepared in (1) above was diluted to 0.1 μg / mL in TBS-T solution containing 1% bovine serum albumin, added at 40 μL / well, and left at room temperature for 1 hour. After washing three times with TBS-T, the hybridoma culture supernatant obtained in (3) above was diluted 4-fold with TBS-T solution containing 1% bovine serum albumin, added at 40 μL / well, and left at room temperature for 1 hour. After washing three times with TBS-T, α-Rat-IgG-HRP (Funakoshi Co., Ltd.) solution diluted 20,000-fold with TBS-T solution containing 1% bovine serum albumin was added at 40 μL / well, and left at room temperature for 1 hour. After washing four times with TBS-T, TMB Microwell Peroxidase Substrate (KPL) was added, and the reaction was stopped with 1 mol / L phosphoric acid solution. The absorbance at 450 nm was measured using an absorbance plate reader. (5) Monocloning of Hybridomas Cells in screening-positive wells were monocloned by limiting dilution. The monocloned cells were cultured in large quantities using ERDF medium (Kyokuto Pharmaceuticals) supplemented with 10% FCS and purified using MonoSpin ProG (GL Sciences). Two monoclonal antibodies were selected from the obtained antibodies and designated as EMArEC04M01 and EMCrEC20M04, respectively.

[0043] Example 3: Preparation of monoclonal antibody against biotinylated ECM1 The monoclonal antibody obtained in Example 2 was labeled with Biotin Labeling Kit-NH. ２(Dojindo Laboratories) to give EMArEC04M01-Biotin and EMCrEC20M04-Biotin, respectively.

[0044] Example 4: Examination of Recognition Sites of Anti-ECM1 Antibodies The various recombinant ECM1s expressed in Example 1 were used to examine the recognition sites of the prepared monoclonal antibodies. Anti-BNC peptide antibodies were diluted with carbonate buffer (pH 9.8) to 200 ng / well and immobilized on a MaxiSorp 96-well plate (manufactured by Nunc). Specifically, the procedure was as follows: After overnight incubation at 4°C, the plate was washed three times with TBS-T (Tris-Buffered Saline containing 0.05% Tween 20), and 200 μL / well of a TBS solution containing 3% bovine serum albumin (BSA) was added to each well and allowed to stand at room temperature for 2 hours. The various recombinant antigens from Example 1 were diluted 10-fold with TBS-T containing 1% bovine serum albumin, added at 40 μL per well, and left at room temperature for 1 hour. After washing three times with TBS-T, EMArEC04M01-Biotin or EMCrEC20M04-Biotin obtained in Example 3 was diluted to 1.0 μg / mL with TBS-T containing 1% bovine serum albumin, added at 40 μL per well, and left at room temperature for 1 hour. After washing three times with TBS-T, a horseradish peroxidase (HRP)-labeled anti-rat IgG (Funakoshi Co., Ltd.) solution diluted 20,000-fold with TBS-T containing 1% bovine serum albumin was added at 40 μL per well and left at room temperature for 1 hour. After washing four times with TBS-T, TMB Microwell Peroxidase Substrate (KPL) was added, and the reaction was stopped with 1 mol / L phosphoric acid solution. The absorbance at 450 nm was measured using an absorbance plate reader. Evaluation was performed using S (with antigen) / N (without antigen).

[0045] The results for EMArEC04M01 are shown in Figure 2. EMArEC04M01 was confirmed to react with the recombinant antigens ECM Full, ECM F279, ECM 107, and ECM 194, but not with the recombinant antigens ECM 280, ECM 367, and ECM 454. These results demonstrate that antibody EMArEC04M01 recognizes an epitope located at positions 194 to 279 of the ECM1 amino acid sequence of SEQ ID NO: 10. Meanwhile, the results for antibody EMCrEC20M04 are shown in Figure 3. EMCrEC20M04 was confirmed to react with ECM Full, ECM 107, ECM 194, ECM 280, ECM 367, and the shortest ECM 454. However, it was confirmed not to react with ECM F279. These results demonstrate that the antibody EMCrEC20M04 recognizes an epitope located at positions 454 to 540 in the ECM1 amino acid sequence of SEQ ID NO: 10. These results demonstrate that a sandwich assay system using a combination of the antibodies EMArEC04M01 and EMCrEC20M04 can detect a peptide comprising positions 194 to 540 in the ECM1 amino acid sequence of SEQ ID NO: 10.

[0046] Example 5: Measurement of ECM1 Concentration in Serum Samples from Patients at High Risk for Fracture by ELISA Using Anti-ECM1 Antibody. ECM1 concentrations were measured using 50 serum samples from patients at high risk for fracture and 17 serum samples from age-matched healthy individuals (in their 50s and 60s). The rat monoclonal antibody EMArEC04M01 obtained in Example 2 was diluted with carbonate buffer (pH 9.8) to 200 ng / well and immobilized on a MaxiSorp 96-well plate (Nunc). After overnight incubation at 4°C, the plate was washed three times with TBS-T (Tris-Buffered Saline containing 0.05% Tween 20). 200 μL / well of a TBS solution containing 3% bovine serum albumin (BSA) was added to each well and allowed to stand at room temperature for 2 hours. After washing three times with TBS-T, patient serum or age-matched healthy serum was diluted 100-fold with TBS-T containing 1% bovine serum albumin, added at 40 μL / well, and left at room temperature for 1 hour. After washing three times with TBS-T, EMCrEC20M04-Biotin obtained in Example 3 was diluted to 1.0 μg / mL with TBS-T containing 1% bovine serum albumin, added at 40 μL / well, and left at room temperature for 1 hour. After washing three times with TBS-T, horseradish peroxidase (HRP)-labeled streptavidin (Funakoshi Co., Ltd.) solution diluted 50,000-fold with TBS-T containing 1% bovine serum albumin was added at 40 μL / well, and left at room temperature for 1 hour. After washing four times with TBS-T, TMB Microwell Peroxidase Substrate (KPL) was added, and the reaction was stopped with 1 mol / L phosphoric acid solution. The absorbance at 450 nm was measured using an absorbance plate reader. The measured concentration was calculated by quantifying the purified recombinant ECM Full using the commercially available ELISA kit described below.

[0047] The results are shown in Figure 4. As is clear from Figure 4, when the measurement method using the anti-ECM1 antibody of the present invention was used, the blood ECM1 levels were clearly higher in the high-risk fracture patient group compared to the healthy control group. Table 2 shows the results of ROC analysis evaluating the ability of ECM1 measured by the measurement method using the anti-ECM1 antibody of the present invention to detect high-risk fracture patients. These results demonstrated a significantly strong correlation between the independent variable, serum ECM1 concentration, and the outcome, the dichotomous variable, whether or not a patient was at high risk of fracture. The area under the ROC curve (AUC) was 0.8006. Specifically, it was revealed that the present invention, using antibodies recognizing epitopes located at positions 194 to 279 and 454 to 540 of the amino acid sequence of ECM1 of SEQ ID NO: 10, demonstrated superior diagnostic performance compared to the commercially available ELISA kit described in Comparative Example 2 below.

[0048]

[0049] Comparative Example 1: Examination of ECM1 Labeling Sites in Commercially Available Kits Similar to Example 4, various recombinant ECM1s were used to examine the recognition site of a commercially available ELISA kit, the Human ECM1 ELISA Kit (anti-ECM1 antibodies were used as both solid-phase and labeled antibodies, manufactured by ABCAM). Measurements were performed according to the attached protocol, and absorbance values ​​at 450 nm were measured using an absorbance plate reader. Evaluation was performed using an S (antigen present) / N (antigen absent) ratio. The results are shown in Figure 5. It was shown that the commercially available ELISA kit reacted with ECM Full, ECM F279, and ECM 107, but did not react with ECM 194, ECM 280, ECM 367, or ECM 454. This demonstrated that the commercially available ELISA kit recognizes an epitope located at positions 107 to 193 of the amino acid sequence of ECM1 represented by SEQ ID NO: 10.

[0050] Comparative Example 2: Measurement of ECM1 using a commercially available kit The same samples from high-risk fracture patients and healthy individuals were used as in Example 5. Serum ECM1 measurement was performed according to the protocol using the commercially available ELISA kit described in Comparative Example 1. Measurements were performed by measuring absorbance at 450 nm using an absorbance plate reader, and concentrations were calculated from a calibration curve. The results are shown in Figure 6. Compared to the healthy individual group, the high-risk fracture patient group tended to have higher blood ECM1 levels. Table 3 shows the results of an ROC analysis evaluating the detectability of high-risk fracture patients using the commercially available ELISA kit. The area under the ROC curve was 0.6353, indicating lower diagnostic performance compared to Example 5.

[0051]

[0052] Preparation Example 1: Preparation of ECM1 Measurement Reagent An ECM1 measurement reagent was prepared as follows, using the EMArEC04M01 antibody obtained in Example 2 on the solid phase side and the EMCrEC20M04 antibody obtained in Example 2 on the detection side. A measurement reagent for use in automated analysis was prepared using a cup with two cells (hereinafter, one cell of the cup will be referred to as the "microparticle-side cell" and the other cell as the "conjugate-side cell" based on their contents). A solution containing microparticles with immobilized EMArEC04M01 antibody was dispensed into the microparticle-side cell, and a solution containing alkaline phosphatase-labeled EMCrEC20M04 antibody was dispensed into the conjugate-side cell. The dispensed solution was freeze-dried, and the cup was sealed with aluminum to prepare an ECM1 measurement reagent.

[0053] Example 6 Evaluation of Clinical Samples (Sandwich CLEIA Measurement Using AIA-CL Reagent) Using the measurement reagent prepared in Preparation Example 1, 24 samples were randomly selected from the 50 serum samples from patients at high risk of fracture evaluated in Example 5 and measured. The measurement device used was the fully automated chemiluminescent enzyme immunoassay device AIA-CL2400 (manufactured by Tosoh Corporation: manufacturing and sales notification number 13B3X90002000018), and ECM1 was measured according to the following procedure. (1) 10 μL of a sample diluted 20-fold with a TBS-T solution containing 1% bovine serum albumin and 40 μL of a diluent containing a surfactant were automatically dispensed into the cell on the microparticle side of the ECM1 measurement reagent prepared in Preparation Example 1, (2) an antigen-antibody reaction was carried out for 5 minutes at a constant temperature of 37°C, (3) Bound / Free (B / F) separation was carried out to remove any material that did not bind to the microparticles, and then a buffer solution containing a surfactant was dispensed into the cell on the microparticle side for washing, and the solution was then discarded, (4) Next, an alkaline phosphatase-labeled antibody separately dissolved in a buffer solution in the conjugate cell was dispensed into the cell on the microparticle side, (5) an antigen-antibody reaction was carried out for 3 minutes at a constant temperature of 37°C, and (6) B / F separation was carried out to remove any material that did not bind to the microparticles, and then a buffer solution containing a surfactant was dispensed into the cell on the microparticle side for washing, and the solution was then discarded. (7) 3-(5-tert-butyl-4,4-dimethyl-2,6,7-trioxabicyclo[3.2.0]hept-1-yl)phenyl phosphate disodium salt (DIFURAT) was added to the cell on the particle side, and the chemiluminescence intensity per unit time obtained by decomposition of DIFURAT with alkaline phosphatase was taken as the measured value (Cps).

[0054] Example 7: Evaluation of Clinical Samples (Sandwich ELISA Measurement) ECM1 concentration was measured using the samples evaluated in Example 6. The rat monoclonal antibody EMArEC04M01 obtained in Example 2 was diluted with carbonate buffer (pH 9.8) to 200 ng / well and immobilized on a MaxiSorpBlack 96-well plate (Thermo). After overnight incubation at 4°C, the plate was washed three times with TBS-T (Tris-Buffered Saline containing 0.05% Tween 20). 200 μL / well of a TBS solution containing 3% bovine serum albumin was added to each well and allowed to stand at room temperature for two hours. After washing three times with TBS-T, patient serum was diluted 20-fold with TBS-T containing 1% bovine serum albumin, added at 40 μL / well, and allowed to stand at room temperature for one hour. After washing three times with TBS-T, the alkaline phosphatase-labeled EMCrEC20M04 antibody used in Preparation Example 1 was diluted to 1.0 μg / mL with TBS-T solution containing 1% bovine serum albumin, added at 40 μL / well, and left at room temperature for 1 hour. After washing three times with TBS-T, 3-(5-tert-butyl-4,4-dimethyl-2,6,7-trioxabicyclo[3.2.0]hept-1-yl)phenyl phosphate disodium salt (DIFURAT) was added, and the fluorescence was measured using a fluorescence plate reader.

[0055] The results are shown in Figure 7. The correlation coefficient between the measurement results obtained by sandwich ELISA in Example 7 and the results obtained by the AIA-CL2400 measurement reagent (sandwich CLEIA) in Example 6 was R ２ = 0.9229, which was good.

Claims

1. An antibody that specifically recognizes ECM1, characterized in that it has an epitope at positions 194 to 279 or 454 to 540 of SEQ ID NO: 10 in extracellular matrix protein 1 (ECM1) represented by positions 20 to 540 of the amino acid sequence represented by SEQ ID NO: 10, or characterized in that it has an epitope at positions 194 to 279 or 454 to 540 of SEQ ID NO: 10 in a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by positions 20 to 540 of SEQ ID NO:

10.

2. A method for detecting a disease associated with a high risk of fracture, comprising the step of measuring the level of ECM1 in a sample collected from a living body using the antibody described in claim 1.

3. The method of claim 2, wherein the level of ECM1 is measured using: (1) an antibody having an epitope at positions 194 to 279 in SEQ ID NO: 10 of ECM1 represented by positions 20 to 540 of the amino acid sequence represented by SEQ ID NO: 10, or at positions corresponding to positions 194 to 279 in SEQ ID NO: 10 of a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by positions 20 to 540 in SEQ ID NO: 10; and (2) an antibody having an epitope at positions 454 to 540 in SEQ ID NO: 10 of ECM1 represented by positions 20 to 540 of the amino acid sequence represented by SEQ ID NO: 10, or at positions corresponding to positions 454 to 540 in SEQ ID NO: 10 of a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by positions 20 to 540 in SEQ ID NO:

10.

4. The method described in claim 2, wherein if the level of ECM1 in the sample is higher than the value obtained from a control subject not having a disease associated with a high risk of fracture or a predetermined standard value, it is determined that the subject is likely to have a disease associated with a high risk of fracture.

5. The method according to claim 2, wherein the disease associated with a high risk of fracture is osteoporosis or osteopenia.

6. The method of claim 2, wherein the sample is whole blood, serum, or plasma.

7. The method of claim 2, wherein the measurement of the ECM1 level is an immunological assay.

8. The method according to claim 7, wherein the immunological assay is an ELISA or CLEIA method.

9. A reagent or kit for use in the detection method according to claim 2, characterized in that it contains the antibody according to claim 1.

10. The reagent or kit according to claim 9, comprising: (1) an antibody having an epitope at positions 194 to 279 in SEQ ID NO: 10 of ECM1 represented by positions 20 to 540 of the amino acid sequence represented by SEQ ID NO: 10, or at positions corresponding to positions 194 to 279 in SEQ ID NO: 10 of a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by positions 20 to 540 in SEQ ID NO: 10; and (2) an antibody having an epitope at positions 454 to 540 in SEQ ID NO: 10 of ECM1 represented by positions 20 to 540 of the amino acid sequence represented by SEQ ID NO: 10, or at positions corresponding to positions 454 to 540 in SEQ ID NO: 10 of a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by positions 20 to 540 in SEQ ID NO: 10.

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