JP7496992B2 - Method for detecting arteriosclerotic lesions, detection reagent and detection kit - Google Patents

Description

The present invention relates to a method for detecting arteriosclerotic lesions by using an antibody that recognizes a specific region of blood myosin heavy chain 11.

Due to the aging of the population and changes in lifestyle, the incidence of diseases caused by arteriosclerosis (atherosclerotic diseases), such as coronary artery disease, peripheral arterial disease, and aortic aneurysms, is increasing. In order to improve prognosis, it is necessary to diagnose atherosclerotic lesions or atherosclerotic diseases using non-invasive or relatively simple tests and to develop appropriate testing and treatment plans.

Because arteriosclerosis itself is asymptomatic, patients are unaware of its progression. Aortic aneurysms, one type of arteriosclerotic disease, are also asymptomatic, making early detection difficult. There is a demand for biomarkers that can diagnose these conditions.

Patent Document 1 describes that, as a result of studies by the present inventors using a commercially available ELISA kit for myosin heavy chain 11, arteriosclerosis can be detected by the level of myosin heavy chain 11 in blood. However, Patent Document 1 only teaches that, as a preferred example of an antibody to be used for detection, an antibody that recognizes the C-terminal region of myosin heavy chain 11 protein can be preferably used, and does not provide any detailed study on the recognition site of the antibody.

International Publication No. 2017/141980

The present invention aims to provide a means that reflects atherosclerotic lesions in the body with higher sensitivity and enables detection of atherosclerotic lesions with high diagnostic efficiency.

As a result of intensive research, the present inventors discovered that in patients with abdominal aortic aneurysm, which is an arteriosclerotic disease, and who have advanced arteriosclerotic lesions in their bodies, arteriosclerotic lesions can be detected with higher sensitivity by measuring blood myosin heavy chain 11 using an antibody that recognizes a specific region of myosin heavy chain 11 (MYH11), and thus completed the present invention described below.

(1) A method for detecting an arteriosclerotic lesion, comprising measuring a level of myosin heavy chain 11 in a blood sample from a subject by immunoassay using at least one of the following (A) to (C), wherein a value obtained by the measurement that is higher than that of a healthy subject is an indication that the subject has an arteriosclerotic lesion.
(A) an antibody or an antigen-binding fragment thereof that specifically recognizes the YDKLEKTKNRLQQELDDLV region of myosin heavy chain 11; (B) an antibody or an antigen-binding fragment thereof that specifically recognizes the ADLEKEELAEELASSLSGR region of myosin heavy chain 11; (C) a mixture of an antibody or an antigen-binding fragment thereof that specifically recognizes the YDKLEKTKNRLQQELDDLV region of myosin heavy chain 11 and an antibody or an antigen-binding fragment thereof that specifically recognizes the ADLEKEELAEELASSLSGR region of myosin heavy chain 11 (2) (A) is a polyclonal antibody, a monoclonal antibody, or an antigen-binding fragment thereof prepared using the YDKLEKTKNRLQQELDDLV region of myosin heavy chain 11 as an immunogen. (B) is a polyclonal antibody, a monoclonal antibody, or an antigen-binding fragment thereof prepared using the ADLEKEELAEELASSLSGR region of myosin heavy chain 11 as an immunogen. (C) is a polyclonal antibody, a monoclonal antibody, or an antigen-binding fragment thereof prepared using the YDKLEKTKNRLQQELDDLV region of myosin heavy chain 11 as an immunogen. The method according to (1), wherein the antibody is a mixture of a polyclonal antibody, a monoclonal antibody or an antigen-binding fragment thereof prepared using the ADLEKEELAEELASSLSGR region of myosin heavy chain 11 as an immunogen and a polyclonal antibody, a monoclonal antibody or an antigen-binding fragment thereof prepared using the YDKLEKTKNRLQQELDDLV region and the ADLEKEELAEELASSLSGR region of myosin heavy chain 11 as an immunogen.
(3) (A) is an antibody or an antigen-binding fragment thereof that is substantially free of an antibody or an antigen-binding fragment thereof that recognizes a region other than the YDKLEKTKNRLQQELDDLV region of myosin heavy chain 11,
(B) is an antibody or an antigen-binding fragment thereof that is substantially free of an antibody or an antigen-binding fragment thereof that recognizes a region other than the ADLEKEELAEELASSLSGR region of myosin heavy chain 11;
The method according to (1) or (2), wherein (C) is a mixture that is substantially free of antibodies or antigen-binding fragments thereof that recognize regions other than the YDKLEKTKNRLQQELDDLV and ADLEKEELAEELASSLSGR regions of myosin heavy chain 11.

(4) A reagent for use in detecting arteriosclerotic lesions, comprising an antibody or an antigen-binding fragment thereof that specifically recognizes the YDKLEKTKNRLQQELDDLV region of myosin heavy chain 11.
(5) The reagent described in (4), which is substantially free of an antibody or an antigen-binding fragment thereof that recognizes a region other than the YDKLEKTKNRLQQELDDLV region of myosin heavy chain 11.
(6) A reagent for use in detecting arteriosclerotic lesions, comprising an antibody or an antigen-binding fragment thereof that specifically recognizes the ADLEKEELAEELASSLSGR region of myosin heavy chain 11.
(7) The reagent described in (6), which is substantially free of an antibody or an antigen-binding fragment thereof that recognizes a region other than the ADLEKEELAEELASSLSGR region of myosin heavy chain 11.
(8) A reagent for use in detecting atherosclerotic lesions, comprising a mixture of an antibody or an antigen-binding fragment thereof that specifically recognizes the YDKLEKTKNRLQQELDDLV region of myosin heavy chain 11 and an antibody or an antigen-binding fragment thereof that specifically recognizes the ADLEKEELAEELASSLSGR region of myosin heavy chain 11.
(9) The reagent described in (8), which is substantially free of an antibody or an antigen-binding fragment thereof that recognizes regions other than the YDKLEKTKNRLQQELDDLV and ADLEKEELAEELASSLSGR regions of myosin heavy chain 11.
(10) A reagent set for use in detecting arteriosclerotic lesions, comprising at least one reagent selected from the reagent described in (4) or (5), the reagent described in (6) or (7), and the reagent described in (8) or (9).
(11) The reagent set described in (10), which is a set including one reagent selected from the reagent described in (4) or (5), the reagent described in (6) or (7), and the reagent described in (8) or (9), and a reagent containing an antibody or an antigen-binding fragment thereof that specifically recognizes a region other than the YDKLEKTKNRLQQELDDLV and ADLEKEELAEELASSLSGR regions of myosin heavy chain 11.
(12) The reagent set according to (10), which is a set containing two reagents selected from the reagent according to (4) or (5), the reagent according to (6) or (7), and the reagent according to (8) or (9).
(13) The reagent set according to any one of (10) to (12), which is a reagent set for detecting arteriosclerotic lesions by measuring the level of myosin heavy chain 11 in a blood sample by the sandwich method.
(14) A kit for use in detecting arteriosclerotic lesions, comprising the reagent according to any one of (4) to (9) or the reagent set according to any one of (10) to (13).

The present invention makes it possible to detect arteriosclerotic lesions with a higher AUC and improved diagnostic efficiency. The present invention can provide data useful for detecting the presence of arteriosclerotic lesions, which greatly assists doctors in evaluating and diagnosing arteriosclerotic lesions themselves.

The results are the results of comparing the measured levels of myosin heavy chain 11 in the blood between a normal group and a group of patients with abdominal aortic aneurysm, which are arteriosclerosis patients, using antibodies that recognize the first and second halves of the C-terminal region of MYH11. There is a significant difference between the groups at P<0.0001 (Mann Whitney test). The results are the results of comparing the measured levels of myosin heavy chain 11 in the blood between a normal group and a group of abdominal aortic aneurysm patients (arteriosclerosis patients) using a commercially available ELISA kit. P=0.1220. The blood myosin heavy chain 11 levels detected with a polyclonal antibody that recognizes the latter half of the C-terminal region and purified antiserum were compared between a normal group and a group of patients with abdominal aortic aneurysm, which are arteriosclerosis patients. P=0.0003. The levels of myosin heavy chain 11 in the blood were measured by sandwich ELISA using the same monoclonal antibody as the solid-phase antibody and the labeled antibody in normal subjects and abdominal aortic aneurysm subjects from arteriosclerosis patients, and the results were compared (P=0.7632). Using antibodies that recognize the first and second halves of the C-terminal region of MYH11, we created a measurement reagent for use in a fully automated enzyme immunoassay device (AIA-CL2400, measurement principle: CLEIA, manufactured by Tosoh Corporation), and also performed ELISA measurements using the same antibodies to examine the correlation between the results. The correlation coefficient R = 0.986. 1 is an alignment of the amino acid sequences of the four isoforms of myosin heavy chain 11. Alignment of the amino acid sequences of the four isoforms of myosin heavy chain 11 (continuation of Figure 6-1). Alignment of the amino acid sequences of the four isoforms of myosin heavy chain 11 (continued from Figure 6-2). Alignment of the amino acid sequences of the four isoforms of myosin heavy chain 11 (continued from Figure 6-3). Alignment of the amino acid sequences of the four isoforms of myosin heavy chain 11 (continued from Figure 6-4).

There are known isoforms of myosin heavy chain, such as isoforms 5, 7, 10, and 11, but the one targeted in the present invention is myosin heavy chain 11 (Gene ID 4629). Myosin heavy chain 11 is an isoform that is specifically expressed in smooth muscle, and is also called smooth muscle myosin heavy chain. When myosin heavy chain 11 is expressed in smooth muscle, two molecules of the heavy chain form a hexamer of smooth muscle myosin molecule together with two molecules each of two types of myosin light chain.

In the present invention, the level of myosin heavy chain 11 in a blood sample of a subject is measured by immunoassay using at least one of the following (A) to (C).
(A) an antibody or an antigen-binding fragment thereof that specifically recognizes the YDKLEKTKNRLQQELDDLV region of myosin heavy chain 11; (B) an antibody or an antigen-binding fragment thereof that specifically recognizes the ADLEKEELAEELASSLSGR region of myosin heavy chain 11; (C) a mixture of an antibody or an antigen-binding fragment thereof that specifically recognizes the YDKLEKTKNRLQQELDDLV region of myosin heavy chain 11 and an antibody or an antigen-binding fragment thereof that specifically recognizes the ADLEKEELAEELASSLSGR region of myosin heavy chain 11 (i.e., a mixture of (A) and (B)).

Myosin heavy chain 11 has four known isoforms, SM1A, SM1B, SM2A, and SM2B (GenBank accession numbers: NP_002465.1, NP_001035203.1, NP_074035.1, NP_001035202.1, amino acid sequences are shown in SEQ ID NOs: 1 to 4, respectively), which differ mainly in the sequence of the C-terminal portion. The region YDKLEKTKNRLQQELDDLV (SEQ ID NO: 7) recognized by antibody (A) corresponds to residues 1415 to 1433 in SM1A (SEQ ID NO: 1), residues 1422 to 1440 in SM1B (SEQ ID NO: 2), residues 1415 to 1433 in SM2A (SEQ ID NO: 3), and residues 1422 to 1440 in SM2B (SEQ ID NO: 4). The region ADLEKEELAEELASSLSGR (SEQ ID NO: 8) recognized by antibody (B) corresponds to residues 1706-1724 in SM1A (SEQ ID NO: 1), residues 1713-1731 in SM1B (SEQ ID NO: 2), residues 1706-1724 in SM2A (SEQ ID NO: 3), and residues 1713-1731 in SM2B (SEQ ID NO: 4). Therefore, the method of the present invention can be used for all four isoforms.

An antibody that specifically recognizes a specific region of myosin heavy chain 11 is an antibody that has an epitope in the specific region, recognizes the epitope, and binds to myosin heavy chain 11 protein or a fragment thereof that includes the specific region. It is preferable that such an antibody binds to the specific region of myosin heavy chain 11 and does not substantially bind to other regions. Note that "does not substantially recognize" or "does not substantially bind" means that it does not bind to other regions at a detectable level (i.e., binding to other regions is below background), or even if it binds to other regions at a detectable level due to cross-reaction, it binds only to an extent that is clearly less than the amount that binds to the specific region and that is clear to those skilled in the art that it does not specifically recognize and bind to the other regions. For example, if the amount that binds to other regions is about 1/100 or less of the amount that binds to the specific region, the antibody is an antibody that does not substantially recognize other regions or does not substantially bind to other regions. Alternatively, it is preferable that such an antibody does not substantially contain antibodies that recognize regions other than the specific region of myosin heavy chain 11. Here, "substantially free" means that even if the antibody contains an antibody that recognizes and binds to a region other than the specific region, the amount of such an antibody that binds to myosin heavy chain 11 is about 1/100 or less of the amount of the antibody that binds to the specific region. Whether the amount of binding to other regions is 1/100 or less of the amount of binding to the specific region can be examined, for example, by actually performing an immunoassay using myosin heavy chain 11 protein as a sample and a target antibody as a primary antibody, and after the detection reaction, quantifying and comparing the amount of signal derived from binding to the specific region and the amount of signal derived from binding to other regions. As described below, such an antibody can be prepared using the specific region as an immunogen. Specifically, (A) may be a polyclonal or monoclonal antibody or an antigen-binding fragment thereof prepared using the YDKLEKTKNRLQQELDDLV region of myosin heavy chain 11 as an immunogen. (B) may be a polyclonal or monoclonal antibody or an antigen-binding fragment thereof prepared using the ADLEKEELAEELASSLSGR region of myosin heavy chain 11 as an immunogen. (C) may be a mixture of a polyclonal or monoclonal antibody or an antigen-binding fragment thereof prepared using the YDKLEKTKNRLQQELDDLV region of myosin heavy chain 11 as an immunogen and a polyclonal or monoclonal antibody or an antigen-binding fragment thereof prepared using the ADLEKEELAEELASSLSGR region of myosin heavy chain 11 as an immunogen, or a polyclonal antibody or an antigen-binding fragment thereof prepared using the YDKLEKTKNRLQQELDDLV region and the ADLEKEELAEELASSLSGR region of myosin heavy chain 11 as immunogens.

The level of myosin heavy chain 11 in blood can be examined by measuring myosin heavy chain 11 protein or its fragments in a blood sample. Since myosin heavy chain 11 is not a secreted protein, it is thought to exist in blood in a fragmented form. The myosin heavy chain 11 protein or its fragments to be measured include those present in blood as monomers and those present in blood in a form bound or associated with other proteins (e.g., those present in blood in a form constituting a complete or partially degraded smooth muscle myosin molecule). Therefore, the term "myosin heavy chain 11" used as a blood biomarker for detecting arteriosclerotic lesions includes the full-length protein of myosin heavy chain 11 and its partial fragments present in blood as monomers, as well as myosin heavy chain 11 protein and its partial fragments in a form bound or associated with other proteins or protein fragments.

The blood sample may be either serum or plasma, with plasma being preferred.

The arteriosclerosis that is the subject of the present invention includes early arteriosclerosis as well as advanced, later-stage arteriosclerosis. In addition, there are three pathological types of arteriosclerosis: atherosclerosis, arteriolar sclerosis, and medial calcification, and the arteriosclerosis that is the subject of the present invention includes these three types. Atherosclerosis is a typical example of the arteriosclerosis that is the subject of the present invention.

Diseases caused by arteriosclerosis (arteriosclerotic diseases) include ischemic heart disease (angina pectoris, myocardial infarction), aortic aneurysms (thoracic and abdominal aortic aneurysms, iliac artery aneurysms, etc.), obliterative arteriosclerosis, aortic valve stenosis, and cerebrovascular diseases (cerebral infarction, stroke). By detecting arteriosclerosis itself using the present invention, it is possible to know the risk of developing such diseases before they occur, which encourages voluntary improvements in lifestyle habits and also leads to the prevention of the onset of arteriosclerotic diseases. Furthermore, in subjects in whom severe arteriosclerosis has been detected, early detection and early treatment of the disease are possible by carrying out detailed examinations and careful follow-up thereafter.

The means for measuring using an antibody that recognizes a specific site of myosin heavy chain 11 in blood is not particularly limited as long as it is a means capable of measuring a polypeptide. Examples of methods for measuring a polypeptide include immunoassays and mass spectrometry, but immunoassays do not require large-scale equipment and are easy to operate, so they can be preferably used in the present invention. There is no particular limit to the method for producing the antibody used in the present invention, and they are typically non-human animal polyclonal or monoclonal antibodies prepared in non-human animals such as mice and rabbits. As described above, the amino acid sequence of myosin heavy chain 11 and the base sequence encoding it are also publicly known, so anti-myosin heavy chain 11 antibodies that specifically recognize a specific site of myosin heavy chain 11 may be prepared and used by a conventional hybridoma method or the like.

The antibody used in the present invention may be a polyclonal antibody or a monoclonal antibody. Antiserum may be used as a polyclonal antibody. In the present invention, the term polyclonal antibody also includes antiserum before purification. Furthermore, an antigen-binding fragment of an antibody may be used instead of an antibody. Hereinafter, in this specification, the term "antibody" also includes an antigen-binding fragment of the antibody, unless otherwise clear from the context. Polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments can all be prepared by well-known conventional methods.

Specifically, a polyclonal antibody that recognizes a specific site of anti-myosin heavy chain 11 can be obtained, for example, by mixing multiple types of monoclonal antibodies that specifically recognize that site. Alternatively, the polyclonal antibody can be obtained by immunizing a non-human animal with the site of myosin heavy chain 11 prepared by a well-known method such as chemical synthesis together with an appropriate adjuvant, obtaining antiserum from blood collected from the animal, and purifying the polyclonal antibody (non-human animal anti-myosin heavy chain 11 polyclonal antibody) in the antiserum. In order to increase the antibody titer in the immunized animal, immunization is usually performed multiple times over several weeks. The antibody in the antiserum can be purified, for example, by ammonium sulfate precipitation, fractionation by anion chromatography, affinity column purification, etc.

One example of a well-known method for producing monoclonal antibodies is the hybridoma method. Specifically, for example, antibody-producing cells such as spleen cells 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 of myosin heavy chain 11 are selected, and these are grown to obtain monoclonal antibodies that specifically recognize a specific site of non-human animal anti-myosin heavy chain 11 from the culture supernatant.

The term "antigen-binding fragment" refers to an antibody fragment that maintains the binding ability (antigen-antibody reactivity) of the antibody to the corresponding antigen, such as, for example, a Fab fragment or F(ab') ₂ fragment of an immunoglobulin. It is well known that such antigen-binding fragments can also be used in immunoassays, and are as useful as the original antibody. As is well known, a Fab fragment or F(ab') ₂ fragment can be obtained by treating an antibody with a protease such as papain or pepsin. The antigen-binding fragment is not limited to a Fab fragment or F(ab') ₂ fragment, but may be any fragment that maintains the binding ability to the corresponding antigen, or may be prepared by a genetic engineering technique. For example, an antibody in which a single chain fragment of variable region (scFv) is expressed in Escherichia coli by a genetic engineering technique can also be used. Methods for producing scFv are also well known, and scFv can be produced by extracting mRNA from the hybridoma produced as described above, preparing single-stranded cDNA, amplifying immunoglobulin H-chain and L-chain genes by PCR using primers specific to the immunoglobulin H-chain and L-chain, linking them with a linker, providing appropriate restriction enzyme sites, introducing them into a plasmid vector, transforming Escherichia coli with it, and recovering scFv from Escherichia coli. Such scFvs are also encompassed in the "antigen-binding fragment".

Immunoassay itself is well known in this field. Immunoassay methods can be classified based on the reaction format, including the sandwich method, competitive method, agglutination method, and Western blot method, and can be classified based on the label, including enzyme immunoassay (ELISA, CLEIA (chemiluminescent enzyme immunoassay), etc.), radioimmunoassay, and fluorescent immunoassay. In the present invention, any immunoassay method capable of quantitative detection may be used. Although not particularly limited, sandwich methods such as sandwich ELISA and one-step or two-step sandwich CLEIA can be preferably used.

The immunoassay method itself is a well-known technique, but to briefly describe it, for example, in the sandwich method, an antibody that binds to myosin heavy chain 11 is immobilized on a solid phase (immobilized antibody), reacted with a sample, washed, and then reacted with a labeled antibody that is a labeled antibody that binds to myosin heavy chain 11 at the same or a different site as the immobilized antibody, and after washing, the labeled antibody bound to the solid phase is measured. In the present invention, any of (A) to (C) may be used for at least one of the immobilized antibody and the labeled antibody.

The measurement of a labeled antibody can be performed by measuring a signal from the labeling substance. The method of measuring the signal is appropriately selected depending on the type of labeling substance. For example, in the case of an enzyme label, a substrate such as a chromogenic substrate, fluorescent substrate, or luminescent substrate corresponding to the enzyme is reacted with the enzyme, and the resulting signal such as color development or luminescence is measured with an appropriate device such as an absorption spectrometer or luminometer, thereby determining the enzyme activity and measuring the object to be measured. 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 (for example, trade name AMPPD) can be used. The labeled antibody may be directly bound to the antibody, or the labeling substance may be indirectly bound to the antibody by binding a specific binding molecule such as biotin or hapten to the antibody and reacting with a partner of the specific binding molecule bound to the labeling substance (streptavidin or hapten antibody, etc.). For standard samples of known concentrations containing various concentrations of myosin heavy chain 11 having a specific site, an immunoassay is performed using an anti-myosin heavy chain 11 antibody or an antigen-binding fragment thereof that specifically recognizes the specific site, and a calibration curve is created by plotting the correlation between the amount of signal from the label and the concentration of myosin heavy chain 11 in the standard samples. The same procedure is then performed for a specimen with an unknown concentration of myosin heavy chain 11 to measure the amount of signal from the label, and the measured value is applied to this calibration curve, allowing the amount of myosin heavy chain 11 in the specimen to be quantified.

As described above, a smooth muscle myosin molecule contains two molecules of myosin heavy chain 11, and such a complex containing two molecules of myosin heavy chain 11 protein or a fragment thereof can be measured by the sandwich method using the same antibody that recognizes a specific site of myosin heavy chain 11. When it is desired to measure a monomer or fragment of myosin heavy chain 11 protein, or a complex containing only one molecule of myosin heavy chain 11 by the sandwich method, two types of antibodies that recognize different sites of myosin heavy chain 11 can be used as the labeled antibody and the solid-phase antibody, and at least one of them can be any one of (A) to (C). For example, when the monoclonal antibody (A) is used as one antibody, an anti-myosin heavy chain 11 antibody other than the monoclonal antibody (A) may be used as the other antibody. Specifically, any of the polyclonal antibody (A), the monoclonal or polyclonal antibody (B), the antibody mixture (C), and other anti-myosin heavy chain 11 antibodies (antibodies that specifically bind to myosin heavy chain 11 in a region other than YDKLEKTKNRLQQELDDLV and ADLEKEELAEELASSLSGR), preferably any of the monoclonal or polyclonal antibody (B), the antibody mixture (C), and other anti-myosin heavy chain 11 antibodies may be used. When the monoclonal antibody (B) is used as one of the antibodies, the other antibody may be any of the monoclonal or polyclonal antibody (A), the polyclonal antibody (B), the antibody mixture (C), and other anti-myosin heavy chain 11 antibodies, preferably any of the monoclonal or polyclonal antibody (A), the antibody mixture (C), and other anti-myosin heavy chain 11 antibodies. In the case of a polyclonal antibody, it is possible to use the same polyclonal antibody for both the labeled antibody and the solid-phase antibody to measure monomers and fragments, so it is also possible to use the polyclonal antibody (A) for both the solid-phase antibody and the labeled antibody, or to use the polyclonal antibody (B) for both the solid-phase antibody and the labeled antibody. The antibody mixture (C) can also be used as both a solid-phase antibody and a labeled antibody, and may be used in combination with the antibody (A), the antibody (B), or an antibody that specifically binds to myosin heavy chain 11 in a region different from YDKLEKTKNRLQQELDDLV and ADLEKEELAEELASSLSGR.

When both the immobilized antibody and the labeled antibody are monoclonal antibodies, and an anti-myosin heavy chain 11 monoclonal antibody that does not fall under (A) to (C) is used as the other antibody, whether the combination of monoclonal antibodies is preferable can be easily examined by actually performing an immunoassay. If desired, the recognition site of the antibody may be identified to examine whether it recognizes a different epitope. The recognition site of the antibody can be identified by a conventional method well known in the art. Briefly, for example, the corresponding antigen, myosin heavy chain 11, is partially digested with a protease such as trypsin, and the digest is bound to an affinity column to which an antibody whose recognition site is to be examined is bound by passing a partial digest solution through the column, and then the bound digest is eluted and subjected to conventional mass spectrometry to identify the recognition site of the antibody.

As mentioned above, four isoforms of myosin heavy chain 11 are known, but if an antibody that recognizes a region common to all four isoforms is used as the other antibody to be combined with at least one of antibodies (A) to (C), all four isoforms can be comprehensively measured. Furthermore, if an antibody specific to each of the four isoforms is used as the other antibody, it is possible to distinguish between isoforms or to measure only a specific isoform.

As shown in Figures 6-1 to 6-5, the regions common to the four isoforms shown in SEQ ID NOs: 1 to 4 are the regions of amino acids 1 to 211 and 219 to 1936 in SEQ ID NO: 4, and by using these regions or partial regions as immunogens in antibody preparation, antibodies that react with all four isoforms can be prepared. In addition, the region of amino acids 212 to 218 in SEQ ID NO: 4 is specific to SM1B and SM2B, the region of amino acids 1937 to 1945 in SEQ ID NO: 4 is specific to SM2A and SM2B, and the region of amino acids 1930 to 1972 in SEQ ID NO: 1 is specific to SM1A and SM1B. Therefore, by using an appropriate combination of antibodies that recognize these regions or partial regions, it is possible to distinguish between isoforms or to measure only a specific isoform.

In the present invention, the N-terminal region refers to the region on the N-terminal side including the head portion composed of the motor domain and the regulatory domain and a part of the tail portion having a coiled coil structure, and the C-terminal region refers to the region on the C-terminal side including most of the tail portion having a coiled coil structure. Specifically, for example, the region before (the N-terminal side) the 840th amino acid in the amino acid sequence of SEQ ID NO: 4 is the N-terminal region, and the region after (the C-terminal side) the 840th amino acid is the C-terminal region. An antibody that recognizes the C-terminal region is an antibody that has an epitope in the C-terminal region of the myosin heavy chain 11 protein and binds to any site in the C-terminal region. An antibody that recognizes the N-terminal region is an antibody that has an epitope in the N-terminal region of the myosin heavy chain 11 protein and binds to any site in the N-terminal region.

A higher blood myosin heavy chain 11 level than that of a healthy person is an indicator of the presence of arteriosclerotic lesions. Measuring the blood myosin heavy chain 11 level can provide data to assist in the detection of arteriosclerotic lesions. The term detection of arteriosclerotic lesions includes detection of severe arteriosclerosis. If the measured blood myosin heavy chain 11 level of a subject is higher than that of a healthy person, the subject can be determined to be at risk of having arteriosclerotic lesions. In addition, the higher the blood myosin heavy chain 11 level, the more likely it is that arteriosclerotic lesions are present in the body, or the more severe the arteriosclerosis is, the more likely it is that arteriosclerosis is. The terms "highly severe arteriosclerosis" and "severe arteriosclerosis" include a state in which there are a large number of arteriosclerotic lesions in the body, and a state in which arteriosclerotic lesions in the body are highly advanced.

Whether or not the blood myosin heavy chain 11 level is higher than that of healthy people can be determined by, for example, comparing the blood myosin heavy chain 11 level measured for a large number of healthy people with a cutoff value, which is a healthy standard value. A healthy person is a person who does not have arteriosclerosis, and people who have symptoms of arteriosclerosis are naturally excluded. In selecting healthy people, people who do not have arteriosclerosis may be selected based on self-reporting, etc., but for example, in addition to arteriosclerosis, people who do not have lifestyle-related diseases such as dyslipidemia, hypertension, diabetes, and obesity, which are known as risk factors for arteriosclerosis, may also be selected, or the selection may be made taking into consideration the absence of smoking habits, a family history of arteriosclerosis, etc. The healthy standard value may be, for example, the average value of the blood myosin heavy chain 11 level of healthy people, or a value obtained by adding a standard error to the average value. If the measured blood myosin heavy chain 11 level of a subject is higher than this cutoff value, it can be determined that the subject is likely to have arteriosclerotic lesions, and the higher the measured value, the more likely the subject is to have arteriosclerotic lesions or the more severe the arteriosclerosis. This cutoff value may be set for each age group (e.g., under 30 years old, 30s, 40s, 50s, 60s, 70 years old or older, etc.).

It is also possible to carry out the present invention for the purpose of detecting particularly severe arteriosclerosis that may have progressed to a level requiring treatment. In this case, the severe arteriosclerosis reference value may be determined from the blood myosin heavy chain 11 level measured for a group of patients with severe arteriosclerosis. The severe arteriosclerosis reference value may be the average value of the measured blood myosin heavy chain 11 level for the group of patients, or a value obtained by subtracting the standard error from the average value. For example, arteriosclerosis patients who have undergone percutaneous coronary intervention (PCI) or peripheral endovascular therapy (EVT) may be considered as "patients with severe arteriosclerosis," and the severe arteriosclerosis reference value may be determined based on the measured blood myosin heavy chain 11 level for such a patient group. Patients who have undergone PCI or EVT are patients with very advanced arteriosclerosis that requires treatment in peripheral arteries such as the arteries of the lower limbs or coronary arteries, and may be said to be patients with very severe arteriosclerosis lesions. If the subject's measured value is close to or exceeds this standard value for severe arteriosclerosis, the subject can be determined to be at risk of having particularly severe arteriosclerosis or particularly severe arteriosclerotic disease. By promptly conducting detailed examinations on such subjects and, if abnormalities are found, immediately administering treatment or carefully monitoring the condition, early detection and early treatment of arteriosclerotic disease can be achieved.

The present invention can also be used to determine the therapeutic effect of a treatment for arteriosclerotic disease, or to determine the therapeutic effect of a candidate therapeutic substance in a clinical trial, etc. In a patient who has undergone a treatment, if the blood myosin heavy chain 11 level is reduced compared to before the treatment, it can be determined that the treatment has a therapeutic effect. In a patient who has been administered a candidate therapeutic substance, if the blood myosin heavy chain 11 level is reduced compared to before the administration, it can be determined that the candidate therapeutic substance has a therapeutic effect for arteriosclerotic disease. In addition, the method of the present invention can also be implemented as a method for detecting patients who are at high risk of developing arteriosclerotic disease, including severe arteriosclerotic disease, or patients who may have arteriosclerotic disease. A blood myosin heavy chain 11 level higher than that of a healthy person is an indicator of a high risk of developing arteriosclerotic disease, or a high possibility of having arteriosclerotic disease.

The antibodies (A) to (C) used in the method of the present invention can be used as detection reagents for arteriosclerotic lesions. That is, the present invention also provides the following detection reagents.
A reagent for use in detecting arteriosclerotic lesions, comprising an antibody or an antigen-binding fragment thereof (antibody (A)) that specifically recognizes the YDKLEKTKNRLQQELDDLV region of myosin heavy chain 11.
A reagent for use in detecting arteriosclerotic lesions, comprising an antibody or an antigen-binding fragment thereof (antibody (B)) that specifically recognizes the ADLEKEELAEELASSLSGR region of myosin heavy chain 11.
A reagent for use in detecting atherosclerotic lesions, comprising a mixture (antibody mixture (C)) of an antibody or an antigen-binding fragment thereof that specifically recognizes the YDKLEKTKNRLQQELDDLV region of myosin heavy chain 11 and an antibody or an antigen-binding fragment thereof that specifically recognizes the ADLEKEELAEELASSLSGR region of myosin heavy chain 11.

These detection reagents may consist of only the antibodies or antigen-binding fragments of (A) to (C), or may further contain other components useful for stabilizing these antibodies or their antigen-binding fragments. In addition, these antibodies or their antigen-binding fragments may be in a form bound to a labeling substance or a specific binding molecule such as biotin, or may be immobilized on a solid phase such as a plate or particles.

The reagent containing (A) is preferably a reagent that does not substantially contain antibodies or antigen-binding fragments thereof that recognize regions other than the YDKLEKTKNRLQQELDDLV region of myosin heavy chain 11. As used herein, "substantially does not contain" means that even if the reagent containing (A) contains antibodies that recognize regions other than YDKLEKTKNRLQQELDDLV and bind to myosin heavy chain 11, the amount of such antibodies that bind to myosin heavy chain 11 in the reagent is approximately 1/100 or less of the amount of antibodies that bind to YDKLEKTKNRLQQELDDLV.

It is preferable that the reagent containing (B) is a reagent that does not substantially contain antibodies or antigen-binding fragments thereof that recognize regions other than the ADLEKEELAEELASSLSGR region of myosin heavy chain 11. As used herein, "substantially does not contain" means that even if the reagent containing (B) contains antibodies that recognize regions other than ADLEKEELAEELASSLSGR and bind to myosin heavy chain 11, the amount of such antibodies that bind to myosin heavy chain 11 in the reagent is approximately 1/100 or less of the amount of antibodies that bind to ADLEKEELAEELASSLSGR.

It is preferable that the reagent containing (C) is a reagent that does not substantially contain antibodies or antigen-binding fragments thereof that recognize regions other than the YDKLEKTKNRLQQELDDLV and ADLEKEELAEELASSLSGR regions of myosin heavy chain 11. The term "substantially does not contain" here means that even if the reagent containing (C) contains antibodies that recognize regions other than the YDKLEKTKNRLQQELDDLV and ADLEKEELAEELASSLSGR regions and bind to myosin heavy chain 11, the amount of such antibodies that bind to myosin heavy chain 11 in the reagent is about 1/100 or less of the amount of antibodies that bind to YDKLEKTKNRLQQELDDLV and antibodies that bind to ADLEKEELAEELASSLSGR.

The detection reagent of the present invention can be used to detect arteriosclerotic lesions in combination with a reagent containing an anti-myosin heavy chain 11 antibody or an antigen-binding fragment thereof that does not fall under (A) to (C), i.e., a reagent containing an antibody or an antigen-binding fragment thereof that specifically recognizes a region different from YDKLEKTKNRLQQELDDLV and ADLEKEELAEELASSLSGR and binds to myosin heavy chain 11, or can be used to detect arteriosclerotic lesions in combination with two or more reagents selected from a reagent containing (A), a reagent containing (B), and a reagent containing (C).

As described above, when (A) is a polyclonal antibody, a reagent containing (A) can be used for both the solid-phase antibody and the labeled antibody to perform the sandwich method, and when (B) is a polyclonal antibody, a reagent containing (B) can be used for both the solid-phase antibody and the labeled antibody to perform the sandwich method. A reagent containing (C) can be used for both the solid-phase antibody and the labeled antibody to perform the sandwich method, regardless of whether the antibodies (A) and (B) contained therein are polyclonal or monoclonal.

The present invention also provides a reagent set for use in detecting arteriosclerotic lesions, comprising at least one reagent selected from a reagent comprising (A), a reagent comprising (B), and a reagent comprising (C). The reagent set may comprise one reagent selected from a reagent comprising (A), a reagent comprising (B), and a reagent comprising (C), and a reagent containing an antibody or an antigen-binding fragment thereof that specifically recognizes a region other than the YDKLEKTKNRLQQELDDLV and ADLEKEELAEELASSLSGR regions of myosin heavy chain 11. The reagent set may also comprise two reagents selected from a reagent comprising (A), a reagent comprising (B), and a reagent comprising (C). The reagent set of the present invention may be, for example, a reagent set for detecting arteriosclerotic lesions by measuring the level of myosin heavy chain 11 in a blood sample by the sandwich method.

The present invention also provides a kit for detecting arteriosclerotic lesions (a kit for use in detecting arteriosclerotic lesions) that includes the above-described detection reagent or reagent set of the present invention. The kit is an immunoassay kit, and may also include other reagents necessary for immunoassay. Other reagents necessary for immunoassay are well known. For example, in addition to the above-described detection reagent or reagent set, the kit of the present invention may further include a sample dilution solution, a washing solution, and, when the labeling substance used in the labeled antibody is an enzyme, a substrate solution for the enzyme. The kit also typically includes instructions for use.

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

<Preparation Example 1> Preparation of a monoclonal antibody (MYSR2Re2M04) that recognizes the first half of the C-terminal region of myosin heavy chain 11 Thirty types of monoclonal antibodies that recognize the first half of the C-terminal region of myosin heavy chain 11 were prepared by a known method (rat iliac lymphatic immunization method), and one of them (MYSR2Re2M04) was selected.

For immunization, recombinant myosin heavy chain 11 using mammalian cells was used. ORF containing myosin heavy chain 11 was purchased (DNAFORM Co., Ltd.), and the first half of the C-terminal region of myosin heavy chain 11 was amplified by PCR using primers with the base sequences shown in SEQ ID NOs: 5 and 6, and introduced into pECE Vector using In-Fusion (registered trademark) HD Cloning Kit (Takara Bio Inc.). HEK293T was transformed with the prepared plasmid using Lipofectamine (registered trademark) 3000 (Thermo), and the first half of the C-terminal region of recombinant myosin heavy chain 11 (corresponding to the region of residues 844 to 1393 of SEQ ID NO: 4) was obtained by transient expression. This was used as an immunization antigen to obtain a rat monoclonal antibody (MYSR2Re2M04).

<Preparation Example 2> Preparation of purified antiserum and polyclonal antibody recognizing myosin heavy chain 11 Polyclonal antibodies recognizing myosin heavy chain 11 were prepared by a known method (Rabbit immunization method; custom antibody preparation by Sigma-Aldrich Japan). Two types of immunizing peptides were prepared by conventional chemical synthesis.

A rabbit was immunized with the region of residues 1422 to 1440 of SEQ ID NO: 4 (peptide 1, underlined portion YDKLEKTKNRLQQELDDLV in Figure 6-4), which is the amino acid sequence of the latter half of the C-terminal region of myosin heavy chain 11, and the region of residues 1713 to 1731 of SEQ ID NO: 4 (peptide 2, underlined portion ADLEKEELAEELASSLSGR in Figure 6-5), and whole blood was collected. The obtained antiserum was purified using a HiTrap Protein A Column (GE Healthcare) to obtain purified antiserum. Either peptide 1 or peptide 2 was bound to a HiTrap NHS Activated Column (GE Healthcare) using a predetermined method, and the antiserum purified with Protein A was affinity-purified to obtain two polyclonal antibodies that recognize myosin heavy chain 11. The polyclonal antibody purified using a column bound to peptide 1 (i.e., a polyclonal antibody that specifically recognizes peptide 1) was designated POS 3, and the polyclonal antibody purified using a column bound to peptide 2 (i.e., a polyclonal antibody that specifically recognizes peptide 2) was designated POS 4. Each of the obtained polyclonal antibodies was biotin-labeled using Biotin Labeling Kit-NH2 (Dojindo Laboratories). They were designated biotinylated POS 3 and biotinylated POS 4, respectively.

Measurement Example 1 (Example) Measurement of myosin heavy chain 11 by ELISA using self-made antibody Plasma samples from the arteriosclerosis group were collected from 20 patients who had developed abdominal aortic aneurysm (AAA) due to the progression of arteriosclerosis and had undergone catheter or stent treatment. Normal plasma samples as negative controls were collected from 23 Japanese healthy subjects without arteriosclerosis or its risk factors.

The rat monoclonal antibody (MYSR2Re2M04) obtained in Preparation Example 1 was diluted with carbonate buffer (pH 9.8) to 200 ng/well and immobilized on a MaxiSorp 96-well plate (Nunc). After overnight reaction 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, plasma samples from 20 AAA patients or 23 healthy Japanese subjects without arteriosclerosis or its risk factors were 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, the biotinylated POS 3 antibody obtained in Preparation Example 2 was diluted to 4 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, a horseradish peroxidase (HRP)-labeled streptavidin (Funakoshi Co., Ltd.) solution diluted 50,000-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 and adding TMB Microwell Peroxidase Substrate (KPL Co., Ltd.), the reaction was stopped with 1 mol/L phosphoric acid solution, and the absorbance value at 450 nm was measured using an absorbance measurement plate reader.

The results are shown in Figure 1. Compared to the normal group, the AAA patient group, which is an arteriosclerotic group, had clearly higher levels of myosin heavy chain 11 in the blood.

Table 1 shows the results of an evaluation of the detection ability of myosin heavy chain 11 in the arteriosclerosis group (AAA patient group) by ROC analysis. The results show that there is a significantly strong relationship between the independent variable, myosin heavy chain 11 concentration in plasma, and the outcome, the dichotomous variable, the presence or absence of arteriosclerosis. In addition, the area under the ROC curve (AUC) was 0.8391. This showed higher diagnostic performance than the commercially available ELISA kit in Measurement Example 2 described below.

Measurement Example 2 (Reference Example) Measurement of myosin heavy chain 11 using a commercially available kit Plasma samples from the arteriosclerosis group were collected from the same 20 patients as in Measurement Example 1, who had developed abdominal aortic aneurysm (AAA) due to the progression of arteriosclerosis and had undergone catheter or stent treatment. As negative control normal plasma samples, samples were collected from the same 23 Japanese healthy subjects as in Measurement Example 1, who were free of arteriosclerosis and its risk factors.

Myosin heavy chain 11 in plasma was measured by sandwich ELISA using a human myosin heavy chain 11 ELISA kit (CUSABIO, hereafter referred to as the "commercial ELISA kit") with anti-myosin heavy chain 11 antibody as the solid-phase antibody and labeled antibody, according to the protocol. The absorbance at 450 nm was measured using a spectrophotometric plate reader.

The results are shown in Figure 2. Previous studies by the inventors have shown that the antibodies contained in commercially available ELISA kits are polyclonal antibodies (WO 2017/141980). Because the antibodies contained in commercially available ELISA kits are polyclonal antibodies, the exact recognition site is unknown.

Table 2 shows the results of ROC analysis evaluating the detection ability of myosin heavy chain 11 in arteriosclerosis (AAA patient group). The AUC was a low value of 0.6391.

Measurement Example 3 (Example) Measurement of myosin heavy chain 11 by ELISA using polyclonal antibody Plasma samples from the arteriosclerosis group were collected from the same 20 patients as in Measurement Example 1, who had developed abdominal aortic aneurysm (AAA) due to the progression of arteriosclerosis and had undergone catheter treatment or stent treatment. As negative control normal plasma samples, samples were collected from the same 23 Japanese healthy subjects as in Measurement Example 1, who were free of arteriosclerosis and its risk factors.

The purified antiserum obtained in Preparation Example 2 (corresponding to a mixture of antibodies against peptide 1 and peptide 2) was diluted with carbonate buffer (pH 9.8) to 400 ng/well and immobilized on a MaxiSorp 96-well plate (Nunc). After overnight reaction 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.

After washing three times with TBS-T, samples from 20 AAA patients or 23 Japanese healthy subjects were 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, the biotinylated POS 3 antibody obtained in Preparation Example 2 was diluted to 4 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, a horseradish peroxidase (HRP)-labeled streptavidin (Funakoshi Co., Ltd.) solution diluted 50,000-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 and adding TMB Microwell Peroxidase Substrate (KPL Co., Ltd.), the reaction was stopped with 1 mol/L phosphoric acid solution, and the absorbance value at 450 nm was measured using an absorbance measurement plate reader.

The results are shown in Figure 3. Compared to the normal group, the arteriosclerosis group (AAA patients) had significantly higher levels of myosin heavy chain 11 in their blood.

Table 3 shows the results of ROC analysis evaluating the detection ability of myosin heavy chain 11 in the arteriosclerosis group (AAA patient group). The AUC was 0.8130, a high value comparable to that of the measurement method in Measurement Example 1. These results demonstrate that arteriosclerosis can be efficiently diagnosed by using antibodies that specifically recognize the region of residues 1422 to 1440 or the region of residues 1713 to 1731 of SEQ ID NO: 4 for measurement.

Measurement Example 4 (Comparative Example) Measurement of myosin heavy chain 11 by sandwich ELISA using the same monoclonal antibody Plasma samples from the arteriosclerosis group were collected from the same 20 patients as in Measurement Example 1, who had developed abdominal aortic aneurysm (AAA) due to the progression of arteriosclerosis and had undergone catheter treatment or stent treatment. As negative control normal plasma samples, samples were collected from the same 23 Japanese healthy subjects as in Measurement Example 1, who were free of arteriosclerosis and its risk factors.

The rat monoclonal antibody (MYSR2Re2M04) obtained in Preparation Example 1 was diluted with carbonate buffer (pH 9.8) to 200 ng/well and immobilized on a MaxiSorp 96-well plate (Nunc). After overnight reaction 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, plasma samples from 20 AAA patients or 23 healthy Japanese subjects without arteriosclerosis or its risk factors were 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, the same monoclonal antibody (MYSR2Re2M04) as the immobilized antibody was biotinylated and diluted to 4 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, horseradish peroxidase (HRP)-labeled streptavidin (Funakoshi Co., Ltd.) solution diluted 50,000-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, the reaction was stopped with 1 mol/L phosphoric acid solution, and the absorbance value at 450 nm was measured using an absorbance plate reader.

The results are shown in Figure 4. These are the results of measuring myosin heavy chain 11 in the blood of a total of 43 subjects, including 23 normal subjects and 20 AAA patients with arteriosclerosis. Although there were a small number of samples that could be measured by the sandwich method using the same monoclonal antibody, many of the samples that showed high values in Measurement Example 1 showed low values, indicating that arteriosclerosis was not being detected correctly.

Table 4 shows the results of ROC analysis evaluating the ability to detect arteriosclerosis (AAA patient group) by myosin heavy chain 11. The AUC was a very low value of 0.5282, indicating that the sandwich method using an antibody that does not specifically recognize peptide 1, an antibody that specifically recognizes peptide 2, or a mixture of these antibodies has low ability to detect arteriosclerosis.

As shown in Figure 4, in the sandwich assay using an antibody that does not specifically recognize peptide 1, an antibody that specifically recognizes peptide 2, or a mixture of these antibodies, many of the samples that obtained measurements using at least one of an antibody that specifically recognizes peptide 1, an antibody that specifically recognizes peptide 2, or a mixture of these antibodies gave low values, while there were a small number of samples in which myosin heavy chain 11 was measured. These results suggest that myosin heavy chain 11 exists in multiple molecular forms in the blood.

Preparation Example 3 (Example) Construction of myosin heavy chain 11 immunoreagent for use in immunoassay device An immunoreaction reagent for use in a fully automated enzyme immunoassay device (AIA-CL2400, measurement principle: CLEIA, manufactured by Tosoh Corporation) was constructed as an immunoassay device, and correlation with ELISA measurement results was obtained. The myosin heavy chain 11 immunoreaction reagent using the antibodies prepared in Preparation Examples 1 and 2 was prepared as follows.

(1) Preparation of solid-phase suspension The monoclonal antibody (MYSR2Re2M04) recognizing myosin heavy chain 11 obtained in Preparation Example 1 and magnetic microparticles (Dynabeads (registered trademark) M-280 Tosylactivated; Thermo Fisher Scientific) were suspended in a buffer solution described in the protocol for the magnetic microparticles, and the antibody was immobilized at 37°C for 17 hours. After antibody immobilization, the antibody-immobilized microparticles were suspended in 0.05 mol/L Tris-HCl buffer solution (pH 8.0) containing 10% Blockmaster CE510 (JSR), and incubated at 37°C for 5 hours for blocking, to obtain anti-MYH11 antibody-immobilized magnetic microparticles. The adjusted antibody-immobilized magnetic microparticles were diluted and mixed with MES buffer solution containing collagen peptide, sugar, salts, etc. to prepare a solid-phase suspension, which was dispensed into one of the holes of a two-hole resin container for immunoreagents.

(2) Preparation of labeled antibody solution for detection The polyclonal antibody POS 3 recognizing myosin heavy chain 11 obtained in Preparation Example 2 was alkaline phosphatase labeled using Alkaline Phosphatase Labeling Kit - NH2 (Dojindo Laboratories) to obtain an ALP-POS 3 antibody. This was diluted and mixed with MES buffer containing collagen peptide, sugar, salts, etc. to prepare a labeled antibody solution for detection with a final concentration of 4 μg/mL, which was dispensed into the other hole of the above-mentioned resin two-hole container.

(3) Preparation of immunoreagent for myosin heavy chain 11
The two-well vessels prepared as described in (1) and (2) were freeze-dried to prepare myosin heavy chain 11 immunoreactive reagents.

Measurement Example 5 (Example) Measurement of myosin heavy chain 11 in blood using myosin heavy chain 11 immunoreaction reagent The samples used in the measurement were 21 samples (hereinafter, spiked samples) prepared by spiking the recombinant antigen used in Preparation Example 1 into EDTA plasma samples.

The immunoreaction reagent prepared in Example 3 was set in a fully automated enzyme immunoassay device and measurements were performed. The reaction scheme was as follows: of the two wells containing the immunoreaction reagent, a dissolving solution was poured into the well containing the lyophilized solid phase suspension, and 20 μL of the spiked sample was dispensed and reacted at 37°C for approximately 6 minutes (first reaction). Next, a dissolving solution was poured into the well containing the lyophilized labeled antibody solution for detection, and this was dispensed into the other well and reacted at 37°C for approximately 3 minutes (second reaction). After that, unreacted substances were removed by B/F separation, and then a luminescent substrate (3-(5-tert-butyl-4,4-dimethyl-2,6,7-trioxabicyclo[3.2.0]hept-1-yl)phenyl phosphate disodium salt) was dispensed and reacted for approximately 4 minutes, and the signal was measured from the amount of luminescence from the alkaline phosphatase activity bound to the solid phase.

Measurement Example 6: Measurement of myosin heavy chain 11 by ELISA The rat monoclonal antibody (MYSR2Re2M04) obtained in Preparation Example 1 was diluted with carbonate buffer (pH 9.8) to 200 ng/well and immobilized on a MaxiSorp 96-well plate (manufactured by Nunc). After overnight reaction at 4°C, the plate was washed three times with TBS-T (Tris-Buffered Saline containing 0.05% Tween 20), and a TBS solution containing 3% bovine serum albumin (BSA) was added to each well at 200 μL/well and allowed to stand at room temperature for 2 hours.

After washing three times with TBS-T, the spiked samples were diluted four-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, the ALP-POS 3 antibody obtained in Preparation Example 3 was diluted to 4 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 four times with TBS-T, 4-Methylumbelliferyl Phosphate (Invitrogen) was added and reacted for about 20 minutes, and the signal was measured from the amount of fluorescence of alkaline phosphatase activity bound to the solid phase.

Figure 5 shows the correlation diagram between the immunoreaction reagent using antigen-spiked samples and ELISA. It was shown that there was a significant correlation between the signal of the prepared immunoreaction reagent and the signal of the ELISA measurement. It was confirmed that the prepared anti-myosin heavy chain 11 antibody can be used as an immunoreaction reagent. It was also suggested that myosin heavy chain 11 can be measured correctly even if the measurement format is different.

Claims (14)

A method for detecting arteriosclerotic lesions, comprising measuring a level of myosin heavy chain 11 in a blood sample from a subject by immunoassay using at least one of the following (A) to (C), wherein a value obtained by the measurement that is higher than that of a healthy subject is an indication that the subject has an arteriosclerotic lesion:
(A) an antibody or an antigen-binding fragment thereof that specifically recognizes the YDKLEKTKNRLQQELDDLV region of myosin heavy chain 11; (B) an antibody or an antigen-binding fragment thereof that specifically recognizes the ADLEKEELAEELASSLSGR region of myosin heavy chain 11; (C) a mixture of an antibody or an antigen-binding fragment thereof that specifically recognizes the YDKLEKTKNRLQQELDDLV region of myosin heavy chain 11 and an antibody or an antigen-binding fragment thereof that specifically recognizes the ADLEKEELAEELASSLSGR region of myosin heavy chain 11. (A) is a polyclonal antibody, a monoclonal antibody, or an antigen-binding fragment thereof prepared using the YDKLEKTKNRLQQELDDLV region of myosin heavy chain 11 as an immunogen; (B) is a polyclonal antibody, a monoclonal antibody, or an antigen-binding fragment thereof prepared using the ADLEKEELAEELASSLSGR region of myosin heavy chain 11 as an immunogen; and (C) is a polyclonal antibody, a monoclonal antibody, or an antigen-binding fragment thereof prepared using the YDKLEKTKNRLQQELDDLV region of myosin heavy chain 11 as an immunogen. The method according to claim 1, wherein the antibody is a mixture of a polyclonal antibody, a monoclonal antibody, or an antigen-binding fragment thereof prepared using the ADLEKEELAEELASSLSGR region of myosin heavy chain 11 as an immunogen, and a polyclonal antibody, a monoclonal antibody, or an antigen-binding fragment thereof, prepared using the YDKLEKTKNRLQQELDDLV region and the ADLEKEELAEELASSLSGR region of myosin heavy chain 11 as an immunogen. (A) is an antibody or an antigen-binding fragment thereof that is substantially free of an antibody or an antigen-binding fragment thereof that recognizes a region other than the YDKLEKTKNRLQQELDDLV region of myosin heavy chain 11,
(B) is an antibody or an antigen-binding fragment thereof that is substantially free of an antibody or an antigen-binding fragment thereof that recognizes a region other than the ADLEKEELAEELASSLSGR region of myosin heavy chain 11;
The method according to claim 1 or 2, wherein (C) is a mixture that is substantially free of antibodies or antigen-binding fragments thereof that recognize regions other than the YDKLEKTKNRLQQELDDLV and ADLEKEELAEELASSLSGR regions of myosin heavy chain 11. A reagent for use in detecting arteriosclerotic lesions, containing an antibody or an antigen-binding fragment thereof that specifically recognizes the YDKLEKTKNRLQQELDDLV region of myosin heavy chain 11. The reagent according to claim 4, which is substantially free of an antibody or an antigen-binding fragment thereof that recognizes a region other than the YDKLEKTKNRLQQELDDLV region of myosin heavy chain 11. A reagent for use in detecting arteriosclerotic lesions, containing an antibody or an antigen-binding fragment thereof that specifically recognizes the ADLEKEELAEELASSLSGR region of myosin heavy chain 11. The reagent according to claim 6, which is substantially free of an antibody or an antigen-binding fragment thereof that recognizes a region other than the ADLEKEELAEELASSLSGR region of myosin heavy chain 11. A reagent for use in detecting arteriosclerotic lesions, comprising a mixture of an antibody or an antigen-binding fragment thereof that specifically recognizes the YDKLEKTKNRLQQELDDLV region of myosin heavy chain 11 and an antibody or an antigen-binding fragment thereof that specifically recognizes the ADLEKEELAEELASSLSGR region of myosin heavy chain 11. The reagent according to claim 8, which is substantially free of an antibody or an antigen-binding fragment thereof that recognizes a region other than the YDKLEKTKNRLQQELDDLV and ADLEKEELAEELASSLSGR regions of myosin heavy chain 11. A reagent set for use in detecting arteriosclerotic lesions, comprising at least one reagent selected from the reagent according to claim 4 or 5, the reagent according to claim 6 or 7, and the reagent according to claim 8 or 9. The reagent set according to claim 10, which is a set including one reagent selected from the reagent according to claim 4 or 5, the reagent according to claim 6 or 7, and the reagent according to claim 8 or 9, and a reagent containing an antibody or an antigen-binding fragment thereof that specifically recognizes a region of myosin heavy chain 11 other than the regions YDKLEKTKNRLQQELDDLV and ADLEKEELAEELASSLSGR. The reagent set according to claim 10, which is a set including two reagents selected from the reagent according to claim 4 or 5, the reagent according to claim 6 or 7, and the reagent according to claim 8 or 9. The reagent set according to any one of claims 10 to 12, which is a reagent set for detecting arteriosclerotic lesions by measuring the level of myosin heavy chain 11 in a blood sample by the sandwich method. A kit for use in detecting arteriosclerotic lesions, comprising the reagent according to any one of claims 4 to 9 or the reagent set according to any one of claims 10 to 13.