JP2021092555A - Detection method, detection reagent and detection kit for arteriosclerotic lesion - Google Patents

Abstract

To provide means capable of reflecting arteriosclerotic lesions in a body with high sensitivity, to detect the arteriosclerosis lesion with high diagnostic efficiency.SOLUTION: A method for detecting arteriosclerotic lesions comprises measuring a level of a myosin heavy chain 11 in a subject's blood sample by immunoassay using at least one selected from an antibody or antigen-binding fragment thereof that specifically recognizes the region of YDKLEKTKNRLQQELDDLV of the myosin heavy chain 11, an antibody or antigen-binding fragment thereof that specifically recognizes the region of ADLEKEELAEELASSLSGR of myosin heavy chain 11, and a mixture of them.SELECTED DRAWING: Figure 1

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, diseases caused by arteriosclerosis (arteriosclerosis) such as coronary artery disease, peripheral arterial disease, and aortic aneurysm are increasing. It is necessary to improve the prognosis by diagnosing atherosclerotic lesions or atherosclerotic diseases by non-invasive or relatively simple tests and making an appropriate test / treatment plan.

Since arteriosclerosis itself is asymptomatic, the patient cannot be aware of its progression. Early detection is difficult because aortic aneurysm, which is one of the arteriosclerotic diseases, is also asymptomatic. Biomarkers capable of diagnosing these are required.

Patent Document 1 describes that arteriosclerosis can be detected by the level of blood myosin heavy chain 11 as a result of examination using a commercially available ELISA kit or the like for myosin heavy chain 11 by the inventors of the present application. However, Patent Document 1 only teaches that an antibody that recognizes the C-terminal region of the myosin heavy chain 11 protein can be preferably used as a preferable example of the antibody used for detection, and the recognition site of the antibody is described. No detailed examination has been made.

International Publication No. 2017/141980

An object of the present invention is to provide a means for reflecting atherosclerotic lesions in the body with higher sensitivity and enabling detection of atherosclerotic lesions with high diagnostic efficiency.

As a result of diligent research, the inventors of the present application recognize a specific region of myosin heavy chain 11 (MYH11) in abdominal aortic aneurysm patients who have advanced arteriosclerotic lesions in their bodies, which is one of the arteriosclerotic diseases. We have found that atherosclerotic lesions can be detected with higher sensitivity by measuring blood myosin heavy chain 11 using an antibody, and completed the following invention of the present application.

(1) A method for detecting atherosclerotic lesions, in which the level of myosin heavy chain 11 in a blood sample of a subject is determined by immunoassay using at least one of the following (A) to (C). A method, including measurement, in which a measured value higher than that of a healthy subject is an indicator that the subject has an atherosclerotic lesion.
(A) An antibody that specifically recognizes the region of YDKLEKTKNRLQQELDDLV of myosin heavy chain 11 or an antigen-binding fragment thereof (B) An antibody that specifically recognizes the region of ADLEKEELAEELASSLSGR of myosin heavy chain 11 or an antigen-binding fragment thereof (C) ) A mixture of an antibody that specifically recognizes the region of YDKLEKTKNRLQQELDDLV of myosin heavy chain 11 or an antigen-binding fragment thereof and an antibody that specifically recognizes the region of ADLEKEELAEELASSLSGR of myosin heavy chain 11 or an antigen-binding fragment thereof (2) (A) is a polyclonal antibody, a monoclonal antibody or an antigen-binding fragment thereof prepared by using the region of YDKLEKTKNRLQQELDDLV of myosin heavy chain 11 as an immunogen, and (B) immunizes the region of ADLEKEELAEELASSLSGR of myosin heavy chain 11. A polyclonal antibody, a monoclonal antibody or an antigen-binding fragment thereof prepared as a source, and (C) is a polyclonal antibody, a monoclonal antibody or an antigen-binding fragment thereof prepared using the region of YDKLEKTKNRLQQELDDLV of myosin heavy chain 11 as an immunogen. And a mixture of a polyclonal antibody, a monoclonal antibody or an antigen-binding fragment thereof prepared using the region of ADLEKEELAEELASSLSGR of myosin heavy chain 11 as an immunogen, or the region of YDKLEKTKNRLQQELDDLV and the region of ADLEKEELAEELASSLSGR of myosin heavy chain 11 as an immunogen. The method according to (1), which is a prepared polyclonal antibody.
(3) (A) is an antibody or an antigen-binding fragment thereof that substantially does not contain an antibody that recognizes a region other than the region of YDKLEKTKNRLQQELDDLV of myosin heavy chain 11 or an antigen-binding fragment thereof.
(B) is an antibody or an antigen-binding fragment thereof that substantially does not contain an antibody that recognizes a region other than the region of ADLEKEELAEELASSLSGR of myosin heavy chain 11 or an antigen-binding fragment thereof.
The method according to (1) or (2), wherein (C) is a mixture substantially free of an antibody or an antigen-binding fragment thereof that recognizes a region other than the regions of YDKLEKTKNRLQQELDDLV and ADLEKEELAEELASSLSGR of myosin heavy chain 11.

(4) A reagent containing an antibody that specifically recognizes the region of YDKLEKTKNRLQQELDDLV of myosin heavy chain 11 or an antigen-binding fragment thereof, and used for detecting atherosclerotic lesions.
(5) The reagent according to (4), which does not substantially contain an antibody or an antigen-binding fragment thereof that recognizes a region other than the region of YDKLEKTKNRLQQELDDLV of myosin heavy chain 11.
(6) A reagent containing an antibody that specifically recognizes the region of ADLEKEELAEELASSLSGR of myosin heavy chain 11 or an antigen-binding fragment thereof, and used for detecting atherosclerotic lesions.
(7) The reagent according to (6), which does not substantially contain an antibody or an antigen-binding fragment thereof that recognizes a region other than the region of ADLEKEELAEELASSLSGR of myosin heavy chain 11.
(8) A mixture of an antibody or an antigen-binding fragment thereof that specifically recognizes the region of YDKLEKTKNRLQQELDDLV of myosin heavy chain 11 and an antibody or an antigen-binding fragment thereof that specifically recognizes the region of ADLEKEELAEELASSLSGR of myosin heavy chain 11. A reagent for use in the detection of arteriosclerotic lesions.
(9) The reagent according to (8), which does not substantially contain an antibody or an antigen-binding fragment thereof that recognizes a region other than the regions of YDKLEKTKNRLQQELDDLV and ADLEKEELAEELASSLSGR of myosin heavy chain 11.
(10) Atherosclerotic lesions comprising the reagent according to (4) or (5), the reagent according to (6) or (7), and at least one reagent selected from the reagents according to (8) or (9). A set of reagents for use in the detection of.
(11) One reagent selected from the reagent according to (4) or (5), the reagent according to (6) or (7), and the reagent according to (8) or (9), and myosin heavy chain 11. The reagent set according to (10), which is a set containing an antibody that specifically recognizes a region other than the region of YDKLEKTKNRLQQELDDLV and ADLEKEELAEELASSLSGR or a reagent containing an antigen-binding fragment thereof.
(12) 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). 10) The reagent set according to the above.
(13) The reagent set according to any one of (10) to (12), which is a reagent set for detecting arteriosclerotic lesions by measuring 11 levels of myosin heavy chain in a blood sample by a sandwich method. ..
(14) For use in detecting atherosclerotic lesions, which comprises the reagent according to any one of (4) to (9) or the reagent set according to any one of (10) to (13). Kit.

According to the present invention, when detecting an atherosclerotic lesion, it is possible to perform detection with a higher AUC and improved diagnostic efficiency. According to the present invention, it is possible to provide useful data for detecting the presence of an atherosclerotic lesion, which greatly assists a doctor in evaluating and diagnosing the atherosclerotic lesion itself.

This is the result of comparing the measured values of blood myosin heavy chain 11 level between the normal group and the abdominal aortic aneurysm patient group, which is an arteriosclerosis patient, using an antibody that recognizes the first half and the second half of the C-terminal region of MYH11. There was a significant difference between the groups with P <0.0001 (Mann Whitney test). This is the result of comparing the measured values of blood myosin heavy chain 11 levels between the normal group and the abdominal aortic aneurysm patient group, which is an arteriosclerosis patient, using a commercially available ELISA kit. P = 0.1220. It is the result of comparing the blood myosin heavy chain 11 level detected by the polyclonal antibody recognizing the C-terminal latter half region and the purified antiserum between the normal group and the abdominal aortic aneurysm patient group which is an arteriosclerosis patient. P = 0.0003. Blood myosin heavy chain 11 levels were measured for normal specimens and abdominal aortic aneurysm specimens of arteriosclerosis patients by sandwich ELISA using the same monoclonal antibody as solid-phase antibody and labeled antibody, and the measurement results were compared. P = 0.7632. An assay reagent used for a fully automatic enzyme immunoassay device (AIA-CL2400, measurement principle: CLEIA, manufactured by Tosoh Corporation) was constructed using an antibody that recognizes the first half and the second half of the C-terminal region of MYH11, and an ELISA using the same antibody was constructed. This is the result of making measurements and examining their correlation. Correlation coefficient R = 0.986. Alignment of amino acid sequences of four isoforms of myosin heavy chain 11. Alignment of amino acid sequences of four isoforms of myosin heavy chain 11 (continuation of FIG. 6-1). Alignment of amino acid sequences of four isoforms of myosin heavy chain 11 (continuation of FIG. 6-2). Alignment of amino acid sequences of four isoforms of myosin heavy chain 11 (continuation of FIG. 6-3). Alignment of amino acid sequences of four isoforms of myosin heavy chain 11 (continuation of FIG. 6-4).

Isoforms such as 5, 7, 10, and 11 are known for the myosin heavy chain, and among these, the myosin heavy chain 11 (Gene ID 4629) is the target of the present invention. Myosin heavy chain 11 is an isoform specifically expressed in smooth muscle, and is also called smooth muscle myosin heavy chain. The myosin heavy chain 11 expressed in smooth muscle forms a hexameric smooth muscle myosin molecule together with two molecules of each of the two types of myosin light chains.

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

The myosin heavy chain 11 contains four types of isoforms, SM1A, SM1B, SM2A, and SM2B (GenBank accession numbers: NP_002465.1, NP_001035203.1, NP_074035.1, NP_001035202.1, amino acids, which mainly have different C-terminal sequences. The sequences are shown in SEQ ID NOs: 1 to 4, respectively). The region YDKLEKTKNRLQQELDDLV (SEQ ID NO: 7) recognized by the antibody (A) is 1415 to 1433 residues in SM1A (SEQ ID NO: 1), 1422-1440 residues in SM1B (SEQ ID NO: 2), and SM2A (SEQ ID NO: 2). In 3), it corresponds to residues 1415 to 1433, and in SM2B (SEQ ID NO: 4), it corresponds to residues 1422-1440. The region ADLEKEELAEELASSLSGR (SEQ ID NO: 8) recognized by the antibody (B) is 1706 to 1724 residues in SM1A (SEQ ID NO: 1), 1713 to 1731 residues in SM1B (SEQ ID NO: 2), and SM2A (SEQ ID NO: 1). In 3), it corresponds to residues 1706 to 1724, and in SM2B (SEQ ID NO: 4), it corresponds to residues 1713 to 1731. Therefore, the method of the present invention can handle 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 and recognizes the epitope and comprises the myosin heavy chain 11 protein or a fragment thereof containing the specific region. It is an antibody that binds. Such an antibody preferably binds to the specific region of the myosin heavy chain 11 and does not substantially bind to other regions. Note that "substantially unrecognized" or "substantially unbound" means that it does not bind to other regions at a detectable level (that is, the binding to other regions is below the background). Or, even if it binds to other regions at a level that can be detected by cross-reactivity, it is clearly less than the amount that binds to the specific region, and it specifically recognizes and binds to other regions. Not that means that they combine only to a degree that is obvious to those skilled in the art. For example, if the amount that binds to another region is less than about 1/100 of the amount that binds to a particular region, the antibody does not substantially recognize the other region or is substantially in the other region. It is an antibody that does not bind to. Alternatively, such antibody is preferably an antibody that does not substantially contain an antibody that recognizes a region other than the specific region of the myosin heavy chain 11. The term "substantially not contained" as used herein means that even if an antibody that recognizes and binds to a region other than the specific region is contained in the antibody, such an antibody is contained in the myosin heavy chain 11. It means that the amount of binding is about 1/100 or less of the amount of antibody that binds to the specific region. Whether or not the amount of binding to another region is 1/100 or less of the amount of binding to a specific region is determined by actually performing immunoassay using, for example, myosin heavy chain 11 protein as a sample and the target antibody as the primary antibody. After the detection reaction, the amount of signal derived from binding to a specific region and the amount of signal derived from binding to another region can be quantified and compared. Such antibodies can be prepared using the particular region as an immunogen, as described below. Specifically, (A) may be a polyclonal or monoclonal antibody prepared by using the region of YDKLEKTKNRLQQELDDLV of myosin heavy chain 11 as an immunogen, or an antigen-binding fragment thereof. (B) may be a polyclonal or monoclonal antibody prepared by using the region of ADLEKEELAEELASSLSGR of myosin heavy chain 11 as an immunogen, or an antigen-binding fragment thereof. In (C), a polyclonal or monoclonal antibody prepared by using the region of YDKLEKTKNRLQQELDDLV of myosin heavy chain 11 as an immunogen or an antigen-binding fragment thereof, and a polyclonal or monoclonal antibody prepared by using the region of ADLEKEELAEELASSLSGR of myosin heavy chain 11 as an immunogen. It may be an antibody or a mixture with an antigen-binding fragment thereof, or a polyclonal antibody or an antigen-binding fragment thereof prepared by using the region of YDKLEKTKNRLQQELDDLV and the region of ADLEKEELAEELASSLSGR of myosin heavy chain 11 as immunogens.

Blood myosin heavy chain 11 levels can be determined by measuring myosin heavy chain 11 protein or fragments thereof in blood samples. Since myosin heavy chain 11 is not a secretory protein, it is considered that it is also present in a fragmented form in blood. The myosin heavy chain 11 protein to be measured or a fragment thereof includes one that is present as a monomer in blood and one that is present in blood in a form bound or associated with other proteins or the like (for example, complete or partial). Those present in the blood in the form constituting the solved smooth muscle myosin molecule) are included. Therefore, the term "myosin heavy chain 11" used as a blood biomarker for detecting arteriosclerotic lesions includes a full-length protein of myosin heavy chain 11 present as a monomer in blood and a partial fragment thereof, as well as a partial fragment thereof. Includes myosin heavy chain 11 proteins and their partial fragments that are bound or associated with other proteins or protein fragments.

The blood sample may be serum or plasma, but plasma can be preferably used.

The arteriosclerosis of interest in the present invention includes from early arteriosclerosis to advanced late arteriosclerosis. There are three types of arteriosclerosis pathologically, atherosclerosis, arteriolosclerosis, and medial calcification, and these three types are included in the arteriosclerosis targeted in the present invention. Atherosclerosis can be mentioned as a typical example of the target arteriosclerosis.

Diseases caused by arteriosclerosis (arteriosclerosis) include ischemic heart disease (angina, myocardial infarction), aortic aneurysm (thoracic / abdominal aortic aneurysm, iliac aneurysm, etc.), arteriosclerosis obliterans, Examples include aortic valve stenosis and cerebrovascular disease (cerebral infarction, stroke). By detecting arteriosclerosis itself according to the present invention, it is possible to know the risk of developing such a disease before it develops, so that it promotes spontaneous improvement of lifestyle and also prevents the onset of arteriosclerosis. Connect. In addition, in the subject in which severe arteriosclerosis is detected, early detection and early treatment of the disease become possible by carrying out a detailed examination and subsequent careful follow-up.

The means for measuring using an antibody that recognizes a specific site of the blood myosin heavy chain 11 is not particularly limited as long as it is a means capable of measuring a polypeptide. Examples of the method for measuring a polypeptide include immunoassay and mass spectrometry, but the immunoassay can be preferably used in the present invention because it does not require large-scale equipment and the measurement operation is simple. The method for producing the antibody used in the present invention is not particularly limited, and is typically a non-human animal polyclonal or monoclonal antibody prepared in a non-human animal such as a mouse or a rabbit. Further, as described above, since the amino acid sequence of myosin heavy chain 11 and the base sequence encoding the same are also known, the anti-myosin heavy chain 11 specifically recognizes a specific site of myosin heavy chain 11 by a conventional hybridoma method or the like. Antibodies may be prepared and used.

The antibody used in the present invention may be a polyclonal antibody or a monoclonal antibody. Antiserum may be used as the polyclonal antibody. In the present invention, the term polyclonal antibody also includes antiserum before purification. Further, an antigen-binding fragment of the antibody can be used instead of the antibody. Hereinafter, in the present specification, unless it is clear from the context that this is not the case, the term "antibody" also includes an antigen-binding fragment of the antibody. The polyclonal antibody, the monoclonal antibody, and the antigen-binding fragment can all be prepared by a well-known conventional method.

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

As an example of a well-known method for producing a monoclonal antibody, a hybridoma method can be mentioned. Specifically, for example, antibody-producing cells such as splenocytes and lymphocytes are collected from the non-human animals immunized as described above, and these are fused with myeloma cells to prepare a hybridoma, and the myosin heavy chain 11 is prepared. A hybridoma that produces an antibody that binds to a specific site can be selected and propagated to obtain a monoclonal antibody that specifically recognizes the specific site of non-human animal anti-myosin heavy chain 11 from the culture supernatant.

The "antigen-binding fragment" means an antibody fragment that maintains the binding property (antigen-antibody reactivity) of the antibody to the corresponding antigen, such as a Fab fragment of immunoglobulin or an F (ab') _{2 fragment.} To do. It is well known that such antigen-binding fragments are also available for immunoassays and are as useful as the original antibodies. Fab fragments and F (ab') ₂ fragments can be obtained by treating the antibody with a proteolytic enzyme such as papain or pepsin, as is well known. The antigen-binding fragment is not limited to the Fab fragment and the F (ab') ₂ fragment, and may be any fragment that maintains the binding property to the corresponding antigen, and may be obtained by a genetic engineering method. It may be prepared. In addition, for example, an antibody in which a single chain fragment of variable region (scFv) is expressed in Escherichia coli by a genetic engineering method can be used. The method for producing scFv is also well known. The mRNA of the hybridoma prepared as described above is extracted, single-chain cDNA is prepared, and PCR is performed using primers specific for immunoglobulin H chain and L chain to immunize. ScFv by amplifying globulin H-chain and L-chain genes, linking them with a linker, imparting appropriate limiting enzyme sites and introducing into a plasmid vector, transforming E. coli with it, and recovering scFv from E. coli. Can be produced. Such scFv is also included in the "antigen-binding fragment".

Immunoassay itself is well known in this field. When immunoassays are classified based on the reaction type, there are sandwich method, competition method, aggregation method, western blot method, etc., and when classified based on the label, enzyme-linked immunosorbent assay (ELISA, CLEIA (chemoluminescent enzyme-linked immunosorbent assay)) ) Etc.), radioimmunoassay, fluorescent immunoassay, etc. In the present invention, any immunoassay method capable of quantitative detection may be used. Although not particularly limited, for example, a sandwich method such as sandwich ELISA or 1-step or 2-step sandwich CLEIA can be preferably used.

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

The labeled antibody can be measured by measuring the signal from the labeling substance. The method for measuring the signal is appropriately selected according to the type of labeling substance. For example, in the case of enzyme labeling, a substrate such as a color-developing substrate, a fluorescence substrate, or a luminescent substrate corresponding to the enzyme is reacted with the enzyme, and the resulting signal such as color development or luminescence is appropriately transmitted by an absorptiometer, a luminometer, or the like. By measuring with an instrument, the enzyme activity can be obtained and the measurement target can be measured. For example, when ALP is used as a labeling substance, 3- (4-methoxyspiro (1,2-dioxetane-3,2'-tricyclo [3.3.1.13,7] decane) -4-yl) phenyl phosphate A luminescent substrate such as 2 sodium (for example, trade name AMPPD) can be used. In the labeled antibody, the labeling substance may be directly bound to the antibody, or a specific binding molecule such as biotin or hapten is bound to the antibody, and a partner (streptavidin or hapten antibody, etc.) of the specific binding molecule to which the labeling substance is bound is used. ) May be indirectly bound to the labeling substance. An immunoassay was performed on a standard sample having a known concentration containing myosin heavy chain 11 having a specific site at various concentrations using an anti-myosin heavy chain 11 antibody that specifically recognizes a specific site or an antigen-binding fragment thereof, and labeled. A calibration curve was created by plotting the correlation between the amount of signal from and the concentration of myosin heavy chain 11 in the standard sample, and the same operation was performed for a sample with unknown concentration of myosin heavy chain 11 to signal from the label. By measuring the amount and applying the measured value to this calibration curve, the myosin heavy chain 11 in the sample can be quantified.

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

When both the immobilized antibody and the labeled antibody are monoclonal antibodies and the anti-myosin heavy chain 11 monoclonal antibody which does not correspond to (A) to (C) is used as the other antibody, is the combination of the monoclonal antibodies preferable? Whether or not it can be easily investigated by actually performing an immunoassay. If desired, the recognition site of the antibody may be identified to see if it recognizes a different epitope. Identification of the recognition site of the antibody can be performed by a conventional method well known in this field. Briefly, for example, the corresponding antigen, myosin heavy chain 11, is partially digested with a proteolytic enzyme such as trypsin, and the digested product is passed through a partially digested product solution to an affinity column to which an antibody whose recognition site should be examined is bound. The recognition site of the antibody can be identified by binding and then eluting the bound digest and performing conventional mass analysis.

As described above, four types of isoforms are known for myosin heavy chain 11, but a region common to all four types is recognized as the other antibody to be combined with at least one of the antibodies (A) to (C). All four isoforms can be comprehensively measured by using the antibody to be used. Further, if an antibody specific to each of the four isoforms is used as the other antibody, it is possible to measure the isoforms separately or to measure only a specific isoform.

As shown in FIGS. 6-1 to 6-5, the regions common to the four isoforms shown in SEQ ID NOs: 1 to 4 are amino acids 1 to 211 in SEQ ID NO: 4 and amino acids 219 to 1936. It is a region of amino acids, and if these regions or subregions thereof are used as immunogens at the time of antibody preparation, an antibody that reacts with all four isoforms can be prepared. Further, the region of amino acids 212 to 218 in SEQ ID NO: 4 is a region specific to SM1B and SM2B, and the region of amino acids 1937 to 1945 in SEQ ID NO: 4 is a region specific to SM2A and SM2B. Since the regions of amino acids 1930 to 1972 in SEQ ID NO: 1 are regions specific to SM1A and SM1B, isoforms can be distinguished by using an appropriate combination of antibodies recognizing these regions or their partial regions. It is possible to measure only specific isoforms.

In the present invention, the N-terminal region is an N-terminal region including a head composed of a motor domain and a regulatory domain and a part of a tail having a coiled coil structure, and the C-terminal region is defined as a C-terminal region. , A region on the C-terminal side that includes most of the tail that has a coiled coil structure. Specifically, for example, in the amino acid sequence of SEQ ID NO: 4, the region from the vicinity of amino acid No. 840 to the front (N-terminal side) is the N-terminal region, and the region from the vicinity of amino acid No. 840 to the back (C-terminal side) is the C-terminal region. is there. The 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.

Blood myosin heavy chain 11 levels higher than those of healthy subjects are indicators of having arteriosclerotic lesions. Measurement of blood myosin heavy chain 11 levels can provide data to assist in the detection of arteriosclerotic lesions. The term detection of atherosclerosis includes the detection of severe arteriosclerosis. If the blood myosin heavy chain 11 level measurement of the subject is higher than that of the healthy subject, it can be determined that the subject may have an atherosclerotic lesion. In addition, it can be determined that the higher the blood myosin heavy chain 11 level, the higher the possibility that arteriosclerotic lesions are present in the body, or the higher the severity of arteriosclerosis. The terms "severe arteriosclerosis" and "severe arteriosclerosis" refer to a condition in which there are many atherosclerotic lesions in the body and a condition in which the arteriosclerosis lesions in the body are highly advanced. Is included.

Whether or not the blood myosin heavy chain 11 level is higher than that of a healthy subject is determined by, for example, using a healthy reference value determined from the blood myosin heavy chain 11 level measured for a large number of healthy subjects as a cutoff value. It can be judged by comparing with the value. Healthy individuals are those who do not have arteriosclerosis, and those who have symptoms of arteriosclerosis are naturally excluded. In selecting healthy people, people without arteriosclerosis may be selected based on self-reports, etc. For example, in addition to arteriosclerosis, dyslipidemia, hypertension, and diabetes, which are known as risk factors for arteriosclerosis. , A person who does not have a lifestyle-related disease such as obesity may be selected, or further, a person who does not have a smoking habit and a family history of arteriosclerosis may be selected. The healthy reference value may be, for example, an average value of 11 levels of myosin heavy chain in the blood of a healthy person, or a value obtained by adding a standard error to the average value. If the measured value of the blood myosin heavy chain 11 level of the subject is higher than this cutoff value, there is a possibility of having arteriosclerotic lesions, and the higher the measured value, the higher the possibility of having arteriosclerotic lesions. Alternatively, it can be determined that the severity of arteriosclerosis may be higher. This cutoff value may be set for each age group (for example, under 30 years old, 30s, 40s, 50s, 60s, 70 years or older, etc.).

It is also possible to practice the present invention for the purpose of detecting particularly serious arteriosclerotic diseases that may have progressed to the level of treatment required. In this case, the severe arteriosclerosis reference value may be set from the blood myosin heavy chain 11 level measured for the group of patients developing serious arteriosclerosis. As the reference value for severe arteriosclerosis, the average value of the measured values of the blood myosin heavy chain 11 level of the patient group, or the value obtained by subtracting the standard error from the average value can be adopted. For example, an arteriosclerotic patient who has undergone percutaneous coronary angioplasty (PCI) or peripheral arteriosclerosis (EVT) is referred to as a "patient with severe arteriosclerotic disease" and the blood of such a group of patients. A reference value for severe arteriosclerosis can be determined based on the measured value of the medium myosin heavy chain 11 level. Patients who have undergone PCI or EVT are patients with extremely advanced arteriosclerosis that requires treatment for peripheral arteries such as lower limb arteries and coronary arteries, and patients with very serious arteriosclerosis lesions. Can be done. If the subject's measurements are close to or exceed this severe arteriosclerosis reference value, the subject has particularly severe arteriosclerosis or particularly serious arteriosclerosis. It can be judged that there is a risk. It is possible to detect and treat arteriosclerosis at an early stage by conducting a detailed examination of such a subject as soon as possible, and if any abnormality is confirmed, immediately treat it or carefully follow up. become.

Further, the present invention can also be utilized for determining the therapeutic effect of a therapeutic treatment for arteriosclerosis and determining the therapeutic effect of a therapeutic drug candidate substance in clinical trials and the like. In a patient who has undergone a therapeutic treatment, when the blood myosin heavy chain 11 level is lowered as compared with that before the therapeutic treatment, it can be determined that the therapeutic effect is obtained by the therapeutic treatment. In addition, in patients who received the therapeutic drug candidate substance, if the blood myosin heavy chain 11 level decreased as compared with before administration, it is judged that the therapeutic drug candidate substance has a therapeutic effect on arteriosclerosis. be able to. In addition, the method of the present invention can also be carried out as a method for detecting patients at high risk of developing arteriosclerosis, including serious arteriosclerosis, or patients who may have arteriosclerosis. Is. Blood myosin heavy chain 11 levels, which are higher than those of healthy subjects, are indicators of a high risk of developing arteriosclerosis or a high probability of having arteriosclerotic disease.

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

These detection reagents may consist only of 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. It may be included. Further, these antibodies or antigen-binding fragments thereof may be in a form in which a specific binding molecule such as a labeling substance or biotin is bound, or in a form immobilized on a solid phase such as a plate or particles.

The reagent containing (A) is preferably a reagent that does not substantially contain an antibody or an antigen-binding fragment thereof that recognizes a region other than the region of YDKLEKTKNRLQQELDDLV of myosin heavy chain 11. The term "substantially free" as used herein means that an antibody that recognizes a region other than YDKLEKTKNRLQQELDDLV and binds to myosin heavy chain 11 may be contained in the reagent containing (A). This means that the amount of such antibody binding to myosin heavy chain 11 is about 1/100 or less of the amount of antibody binding to YDKLEKTKNRLQQELDDLV.

The reagent containing (B) is preferably a reagent that does not substantially contain an antibody that recognizes a region other than the region of ADLEKEELAEELASSLSGR of myosin heavy chain 11 or an antigen-binding fragment thereof. The term "substantially free" as used herein means that an antibody that recognizes a region other than ADLEKEELAEELASSLSGR and binds to myosin heavy chain 11 may be contained in the reagent containing (B). This means that the amount of such antibody binding to myosin heavy chain 11 is about 1/100 or less of the amount of antibody binding to ADLEKEELAEEL ASSLSGR.

The reagent containing (C) is preferably a reagent that does not substantially contain an antibody or an antigen-binding fragment thereof that recognizes a region other than the regions of YDKLEKTKNRLQQELDDLV and ADLEKEELAEELASSLSGR of myosin heavy chain 11. The term "substantially free" here may mean that an antibody that recognizes a region other than the regions of YDKLEKTKNRLQQELDDLV and ADLEKEELAEELASSLSGR and binds to myosin heavy chain 11 is contained in the reagent containing (C). Also means that in the reagent, the amount of such antibody binding to myosin heavy chain 11 is about 1/100 or less of the amount of antibody binding to YDKLEKTKNRLQQELDDLV and ADLEKEELAEELASSLSGR.

The detection reagent of the present invention specifically recognizes a region different from YDKLEKTKNRLQQELDDLV and ADLEKEELAEELASSLSGR, that is, a reagent containing an anti-myosin heavy chain 11 antibody or an antigen-binding fragment thereof, which does not correspond to (A) to (C). It can also be used in the detection of arteriosclerotic lesions in combination with a reagent containing an antibody that binds to myosin heavy chain 11 or an antigen-binding fragment thereof, a reagent containing (A), a reagent containing (B), and (C). Two or more reagents selected from the reagents containing the above can also be used in combination for the detection of arteriosclerotic lesions.

Further, as described above, when (A) is a polyclonal antibody, the sandwich method can be carried out by using the reagent containing (A) for both the solid phase antibody and the labeled antibody, and (B) is the polyclonal antibody. In the case, the sandwich method can be carried out by using the reagent containing (B) for both the solid-phase antibody and the labeled antibody. The reagent containing (C) is a solid-phase antibody and a labeled antibody containing (C) regardless of whether the antibody (A) and the antibody (B) contained therein are polyclonal or monoclonal antibody. The sandwich method can be carried out using both of these.

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

The present invention also provides a kit for detecting atherosclerotic lesions (a kit for use in detecting atherosclerotic lesions), which comprises the above-mentioned detection reagent or reagent set of the present invention. The kit is an immunoassay kit and may include other reagents and the like necessary for immunoassay. Other reagents required for immunoassay are well known. For example, in the kit of the present invention, in addition to the above-mentioned detection reagent or reagent set, a sample diluent, a washing solution, and a substrate solution of the enzyme when the labeling substance used in the labeling antibody is an enzyme are further included. Can be included. Also, kits usually include instructions for use.

Hereinafter, the present invention will be described in more detail based on Examples. However, the present invention is not limited to the following examples.

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

Recombinant myosin heavy chain 11 using mammalian cells was used for immunization. An ORF containing myosin heavy chain 11 was purchased (Danaform Co., Ltd.), and the first half of the myosin heavy chain 11 C-terminal region was amplified by the PCR method using the primers of the nucleotide sequences shown in SEQ ID NOs: 5 and 6. Introduced into pECE Vector using In-Fusion® HD Cloning Kit (Takara Bio Inc.). HEK293T was transformed with Lipofectamine® 3000 (Thermo) using the prepared plasmid, and the first half of the recombinant myosin heavy chain 11 C-terminal region (SEQ ID NOs: 4 844 to 1393) was transiently expressed. (Corresponding to the region of the residue) was obtained. Using this as an immune antigen, a Rat monoclonal antibody (MYSR2Re2M04) was obtained.

<Production Example 2> Preparation of purified antiserum and polyclonal antibody that recognizes myosin heavy chain 11 A polyclonal antibody that recognizes myosin heavy chain 11 was produced by a known method (Rabbit immunization method; Sigma Aldrich Japan custom antibody production). As the two types of immunopeptides, those prepared by conventional chemical synthesis were used.

The region of residues 1422 to 1440 of SEQ ID NO: 4 (underlined portion of FIG. 6-4, peptide 1) and 1713 to 1731 of SEQ ID NO: 4, which are the amino acid sequences of the latter half of the myosin heavy chain 11 C-terminal region. The residue region (underlined part ADLEKEELAEELASSLSGR in FIG. 6-5, peptide 2) was mixed-immunized to rabbits, and total blood was collected. The obtained antiserum was purified by HiTrap Protein A Column (GE Healthcare) to obtain purified antiserum. Myosin heavy chain 11 is recognized by binding either peptide 1 or peptide 2 to the HiTrap NHS Activated Column (GE Healthcare) by a predetermined method and affinity-purifying the antiserum purified with Protein A. Two types of polyclonal antibodies were obtained. Polyclonal antibody purified on a column to which peptide 1 is bound (that is, a polyclonal antibody that specifically recognizes peptide 1) is POS 3, and a polyclonal antibody purified on a column to which peptide 2 is bound (that is, peptide 2 is specific). The polyclonal antibody) recognized in the above was designated as POS 4. Each of the obtained polyclonal antibodies was biotin-labeled using Biotin Labeling Kit-NH2 (Dojin Chemical Laboratory). Biotinylated POS 3 and biotinylated POS 4 were used, respectively.

<Measurement Example 1 (Example)> Measurement of myosin heavy chain 11 by ELISA using self-made antibody A plasma sample of the atherosclerosis group develops abdominal aortic aneurysm (AAA) due to the progress of arteriosclerosis, and catheter treatment or stent treatment is performed. It was collected from 20 patients who received it. Negative control normal plasma samples were collected from 23 healthy Japanese subjects without 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 (manufactured by Nunc). After reacting overnight at 4 ° C., the cells were washed 3 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 200 μL / well. Was added to each well and left at room temperature for 2 hours.

After washing 3 times with TBS-T, samples of 20 AAA patient plasmas or 23 healthy Japanese subjects without arteriosclerosis and its risk factors were mixed with TBS-T solution containing 1% bovine serum albumin 4 It was diluted 2-fold, added at 40 μL / well, and left at room temperature for 1 hour.

After washing with TBS-T three times, the biotinylated POS 3 antibody obtained in Production Example 2 was diluted with a TBS-T solution containing 1% bovine serum albumin to 4 μg / mL, and at 40 μL / well. It was added and left at room temperature for 1 hour. After washing with TBS-T three times, a horseradish peroxidase (HRP) -labeled Streptavidin (Funakoshi Co., Ltd.) solution diluted 50,000 times with a TBS-T solution containing 1% bovine serum albumin was added at 40 μL / well. , Left at room temperature for 1 hour. Washing was performed 4 times with TBS-T, TMB Microwell Peroxidase Substrate (manufactured by KPL) was added, the reaction was stopped with a 1 mol / L phosphoric acid solution, and the absorption value at 450 nm was measured with an absorption measurement plate reader.

The results are shown in FIG. Compared with the normal group, the blood myosin heavy chain 11 was clearly higher in the AAA patient group, which is the arteriosclerosis group.

Table 1 shows the results of evaluating the detectability of the arteriosclerosis group (AAA patient group) by myosin heavy chain 11 by ROC analysis. From this result, it was shown that the relationship between the concentration of myosin heavy chain 11 in plasma, which is an independent variable, and the outcome of the presence or absence of arteriosclerosis, which is a dichotomous variable, is significantly strong. The area under the curve (AUC) was 0.8391. It showed higher diagnostic performance as compared with the commercially available ELISA kit of Measurement Example 2 described later.

<Measurement Example 2 (Reference Example)> Measurement of Myosin Heavy Chain 11 Using a Commercially Available Kit The plasma sample of the atherosclerosis group developed abdominal aortic aneurysm (AAA) due to the progress of arteriosclerosis and received catheter treatment or stent treatment. , Specimens collected from the same 20 patients as in Measurement Example 1 were used. As the negative control normal plasma sample, a sample collected from 23 healthy Japanese subjects, the same as in Measurement Example 1, without arteriosclerosis and its risk factors was used.

For the measurement of myosin heavy chain 11 in plasma, a human myosin heavy chain 11 ELISA kit (CUSABIO, hereinafter referred to as "commercial ELISA kit") using an anti-myosin heavy chain 11 antibody as a solid phase antibody and a labeled antibody is used. It was performed according to the protocol by the sandwich ELISA that was used. The measurement was performed by measuring the absorption value at 450 nm with an absorption measurement plate reader.

The results are shown in FIG. Past studies by the inventors have shown that the antibodies contained in commercial ELISA kits use polyclonal antibodies (WO 2017/141980). Since the antibody contained in the commercially available ELISA kit is a polyclonal antibody, the detailed recognition site is unknown.

Table 2 shows the results of evaluating the detectability of the arteriosclerosis group (AAA patient group) by myosin heavy chain 11 by ROC analysis. The AUC was as low as 0.6391.

<Measurement Example 3 (Example)> Measurement of myosin heavy chain 11 by ELISA using polyclonal antibody A plasma sample of the atherosclerosis group develops abdominal aortic aneurysm (AAA) due to the progress of arteriosclerosis, and catheter treatment or stent treatment is performed. Specimens collected from the same 20 patients as in Measurement Example 1 received were used. As the negative control normal plasma sample, a sample collected from 23 healthy Japanese subjects, the same as in Measurement Example 1, without arteriosclerosis and its risk factors was used.

The purified antiserum (corresponding to a mixture of antibody against peptide 1 and antibody against peptide 2) obtained in Preparation Example 2 was diluted with carbonate buffer (pH 9.8) to 400 ng / well, and MaxiSorp 96-well plate (Nunc). It was immobilized on (manufactured by the company). After reacting overnight at 4 ° C., the cells were washed 3 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 200 μL / well. Was added to each well and left at room temperature for 2 hours.

After washing 3 times with TBS-T, 20 AAA patients or 23 healthy Japanese samples were diluted 4-fold with TBS-T solution containing 1% bovine serum albumin to 40 μL / well. And left at room temperature for 1 hour.

After washing with TBS-T three times, the biotinylated POS 3 antibody obtained in Production Example 2 was diluted with a TBS-T solution containing 1% bovine serum albumin to 4 μg / mL, and at 40 μL / well. It was added and left at room temperature for 1 hour. After washing with TBS-T three times, a horseradish peroxidase (HRP) -labeled Streptavidin (Funakoshi Co., Ltd.) solution diluted 50,000 times with a TBS-T solution containing 1% bovine serum albumin was added at 40 μL / well. , Left at room temperature for 1 hour. Washing was performed 4 times with TBS-T, TMB Microwell Peroxidase Substrate (manufactured by KPL) was added, the reaction was stopped with a 1 mol / L phosphoric acid solution, and the absorption value at 450 nm was measured with an absorption measurement plate reader.

The results are shown in FIG. Compared with the normal group, the blood myosin heavy chain 11 was clearly higher in the AAA patient group, which is the arteriosclerosis group.

Table 3 shows the results of evaluating the detectability of the arteriosclerosis group (AAA patient group) by myosin heavy chain 11 by ROC analysis. The AUC was 0.8130, which was a high value comparable to the measurement method of Measurement Example 1. From this result, it was shown that arteriosclerosis can be efficiently diagnosed by using an antibody that specifically recognizes 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 A plasma sample in the atherosclerosis group develops abdominal aortic aneurysm (AAA) as arteriosclerosis progresses, and catheter treatment or stent Specimens collected from the same 20 patients as those in Measurement Example 1 who received treatment were used. As the negative control normal plasma sample, a sample collected from 23 healthy Japanese subjects, the same as in Measurement Example 1, without arteriosclerosis and its risk factors was used.

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 reacting overnight at 4 ° C., the cells were washed 3 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 200 μL / well. Was added to each well and left at room temperature for 2 hours.

After washing 3 times with TBS-T, samples of 20 AAA patient plasmas or 23 healthy Japanese subjects without arteriosclerosis and its risk factors were mixed with TBS-T solution containing 1% bovine serum albumin 4 It was diluted 2-fold, added at 40 μL / well, and left at room temperature for 1 hour.

After washing with TBS-T three times, the same monoclonal antibody (MYSR2Re2M04) as the immobilized antibody was biotinylated and diluted with a TBS-T solution containing 1% bovine serum albumin to 4 μg / mL. It was added at 40 μL / well and left at room temperature for 1 hour. After washing with TBS-T three times, a horseradish peroxidase (HRP) -labeled Streptavidin (Funakoshi Co., Ltd.) solution diluted 50,000 times with a TBS-T solution containing 1% bovine serum albumin was added at 40 μL / well. , Left at room temperature for 1 hour. Washing was performed 4 times with TBS-T, TMB Microwell Peroxidase Substrate (manufactured by KPL) was added, the reaction was stopped with a 1 mol / L phosphoric acid solution, and the absorption value at 450 nm was measured with an absorption measurement plate reader.

The results are shown in FIG. It is the measurement result of the blood myosin heavy chain 11 of 23 normal patients and 20 AAA patients who are arteriosclerosis patients, for a total of 43 patients. Although there were a small number of samples that could be measured by the sandwich method using the same monoclonal antibody, most of the samples that showed high values in Measurement Example 1 had low values, indicating that arteriosclerosis could not be detected correctly.

Table 4 shows the results of evaluating the detectability of the arteriosclerosis group (AAA patient group) by myosin heavy chain 11 by ROC analysis. The AUC is a very low value of 0.5282, and the sandwich method using an antibody that specifically recognizes peptide 1, an antibody that specifically recognizes peptide 2, or an antibody that does not correspond to a mixture of these antibodies causes arteriosclerosis. It was shown that the detection performance was low.

Further, as shown in FIG. 4, in the sandwich method using an antibody that specifically recognizes peptide 1, an antibody that specifically recognizes peptide 2, and an antibody that does not correspond to any mixture of these antibodies, peptide 1 is specific. While many of the samples obtained by the sandwich method using at least one of an antibody that specifically recognizes the antibody, an antibody that specifically recognizes peptide 2, and a mixture of these antibodies had low values, while There were also a small number of samples in which myosin heavy chain 11 was measured. These results suggest that myosin heavy chain 11 is present in the blood in multiple molecular modes.

<Production Example 3 (Example)> Construction of an immunoreagent for myosin heavy chain 11 used in an immunoassay device Immunity used in a fully automatic enzyme-linked immunosorbent assay device (AIA-CL2400, measurement principle: CLEIA, manufactured by Tosoh Corporation) as an immunoassay device. A reaction reagent was constructed, and the correlation with the ELISA measurement results was determined. The myosin heavy chain 11 immunoreactivity 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) for recognizing the myosin heavy chain 11 obtained in Production Example 1 and the magnetic fine particles (Dynabeads (registered trademark) M-280 Tosylactivated; Thermo Fisher Scientific) were used. The antibody was immobilized at 37 ° C. for 17 hours by suspending in the buffer solution described in the magnetic fine particle protocol. After antibody immobilization, the antibody-immobilized fine particles were suspended in 0.05 mol / L Tris-HCl buffer (pH 8.0) containing 10% Blockmaster CE510 (JSR) and incubated at 37 ° C. for 5 hours for blocking. , Anti-MYH11 antibody-immobilized magnetic fine particles were obtained. The prepared antibody-immobilized magnetic fine particles are diluted and mixed with a MES buffer solution containing collagen peptide, sugar, salts, etc. to prepare a solid phase suspension, which is divided into one hole of a resin 2-hole container for immunoreagents. Noted.

(2) Preparation of Labeled Antibody Solution for Detection The polyclonal antibody POS 3 that recognizes the myosin heavy chain 11 obtained in Production Example 2 was labeled with alkaline phosphatase using the Alkaline Phosphatase Labeling Kit --NH2 (Dojin Chemical Laboratory). This was performed to prepare an ALP-modified POS 3 antibody. It was diluted and mixed with a MES buffer solution 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 2-hole container. ..

(3) Preparation of immunoreagent for myosin heavy chain 11
The two-hole container prepared as described in (1) and (2) was freeze-dried to prepare a myosin heavy chain 11 immunoreactivity reagent.

<Measurement Example 5 (Example)> Measurement of Blood Myosin Heavy Chain 11 Using the Myosin Heavy Chain 11 Immunoreaction Reagent The sample used for the measurement was an EDTA plasma sample in which the recombinant antigen used in Preparation Example 1 was spiked. A study was conducted using 21 samples (hereinafter referred to as spike samples).

The immune reaction reagent prepared in Preparation Example 3 was set in a fully automatic enzyme immunoassay device, and measurement was performed. The reaction scheme is as follows: Of the two holes containing the immunoreaction reagent, the solution is placed in the hole containing the freeze-dried product of the solid phase suspension to dissolve it, and 20 μL of the spike sample is dispensed at about 37 ° C. The reaction was carried out for 6 minutes (first reaction). Next, the lysate was placed in the hole containing the lyophilized detection-labeled antibody solution to dissolve it, and the solution was dispensed into the other hole and reacted at 37 ° C. for about 3 minutes (second reaction). ). Then, after B / F separation to remove the unreacted substance, the luminescent substrate (3- (5-tert-butyl-4,4-dimethyl-2,6,7-trioxabicyclo [3.2.0] hepto-1) -Il) phenyl phosphate ester disodium salt) was dispensed and reacted for about 4 minutes, and the signal was measured from the amount of luminescence of 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 Production Example 1 was diluted with carbonate buffer (pH 9.8) to 200 ng / well, and MaxiSorp 96-well plate (Nunc). It was immobilized on (manufactured by the company). After reacting overnight at 4 ° C., the cells were washed 3 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 200 μL / well. Was added to each well and left at room temperature for 2 hours.

After washing with TBS-T three times, the spiked sample was diluted 4-fold with a TBS-T solution containing 1% bovine serum albumin, added at 40 μL / well, and left at room temperature for 1 hour.

After washing with TBS-T three times, the ALP-modified POS 3 antibody obtained in Production Example 3 was diluted with a TBS-T solution containing 1% bovine serum albumin to 4 μg / mL, and at 40 μL / well. It was added and left at room temperature for 1 hour. After washing 4 times with TBS-T, 4-Methylumbelliferyl Phosphate (Invitrogen) was added and reacted for about 20 minutes, and the signal was measured from the fluorescence amount of alkaline phosphatase activity bound to the solid phase.

FIG. 5 shows a correlation diagram between the immune reaction reagent and ELISA using the antigen spike sample. It was shown that the signal of the prepared immunoreactive reagent and the signal of ELISA measurement were significantly correlated. It was confirmed that the prepared anti-myosin heavy chain 11 antibody is an antibody that can be used as an immune reaction reagent. It was also suggested that the myosin heavy chain 11 can be measured correctly even if the measurement format is different.

Claims (14)

A method for detecting atherosclerotic lesions, in which 11 levels of myosin heavy chain in a blood sample of a subject are measured by immunoassay using at least one of the following (A) to (C). A method in which the value obtained by measurement including the above is higher than that of a healthy subject, which is an index of the subject having an atherosclerotic lesion.
(A) An antibody that specifically recognizes the region of YDKLEKTKNRLQQELDDLV of myosin heavy chain 11 or an antigen-binding fragment thereof (B) An antibody that specifically recognizes the region of ADLEKEELAEELASSLSGR of myosin heavy chain 11 or an antigen-binding fragment thereof (C) ) A mixture of an antibody that specifically recognizes the region of YDKLEKTKNRLQQELDDLV of myosin heavy chain 11 or an antigen-binding fragment thereof, and an antibody that specifically recognizes the region of ADLEKEELAEELASSLSGR of myosin heavy chain 11 or an antigen-binding fragment thereof. (A) is a polyclonal antibody, a monoclonal antibody or an antigen-binding fragment thereof prepared by using the region of YDKLEKTKNRLQQELDDLV of myosin heavy chain 11 as an immunogen, and (B) is an immunogen of the region of ADLEKEELAEELASSLSGR of myosin heavy chain 11. (C) is a polyclonal antibody, a monoclonal antibody or an antigen-binding fragment thereof prepared as an immunogen, and (C) is a polyclonal antibody, a monoclonal antibody or an antigen-binding fragment thereof prepared using the region of YDKLEKTKNRLQQELDDLV of myosin heavy chain 11 as an immunogen. , A polyclonal antibody prepared by using the region of ADLEKEELAEELASSLSGR of myosin heavy chain 11 as an immunogen, a mixture with a monoclonal antibody or an antigen-binding fragment thereof, or the region of YDKLEKTKNRLQQELDDLV and the region of ADLEKEELAEELASSLSGR of myosin heavy chain 11 as an immunogen. The method according to claim 1, wherein the polyclonal antibody is obtained. (A) is an antibody or an antigen-binding fragment thereof that substantially does not contain an antibody that recognizes a region other than the region of YDKLEKTKNRLQQELDDLV of myosin heavy chain 11 or an antigen-binding fragment thereof.
(B) is an antibody or an antigen-binding fragment thereof that substantially does not contain an antibody that recognizes a region other than the region of ADLEKEELAEELASSLSGR of myosin heavy chain 11 or an antigen-binding fragment thereof.
The method according to claim 1 or 2, wherein (C) is a mixture substantially free of an antibody or an antigen-binding fragment thereof that recognizes a region other than the regions of YDKLEKTKNRLQQELDDLV and ADLEKEELAEELASSLSGR of myosin heavy chain 11. A reagent for use in the detection of atherosclerotic lesions, which contains an antibody that specifically recognizes the region of YDKLEKTKNRLQQELDDLV of myosin heavy chain 11 or an antigen-binding fragment thereof. The reagent according to claim 4, which does not substantially contain an antibody or an antigen-binding fragment thereof that recognizes a region other than the region of YDKLEKTKNRLQQELDDLV of myosin heavy chain 11. A reagent for use in the detection of atherosclerotic lesions, which contains an antibody that specifically recognizes the region of ADLEKEELAEELASSLSGR of myosin heavy chain 11 or an antigen-binding fragment thereof. The reagent according to claim 6, which does not substantially contain an antibody or an antigen-binding fragment thereof that recognizes a region other than the region of ADLEKEELAEELASS LSGR of myosin heavy chain 11. Contains a mixture of an antibody that specifically recognizes the region of YDKLEKTKNRLQQELDDLV of myosin heavy chain 11 or an antigen-binding fragment thereof, and an antibody that specifically recognizes the region of ADLEKEELAEELASS LSGR of myosin heavy chain 11 or an antigen-binding fragment thereof. , A reagent for use in the detection of arteriosclerotic lesions. The reagent according to claim 8, which does not substantially contain an antibody or an antigen-binding fragment thereof that recognizes a region other than the regions of YDKLEKTKNRLQQELDDLV and ADLEKEELAEELASSLSGR of myosin heavy chain 11. A set of reagents for use in the detection of arteriosclerotic lesions, comprising the reagent according to claim 4 or 5, the reagent according to claim 6 or 7, and at least one reagent selected from the reagents according to claim 8 or 9. .. 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 region other than the regions of YDKLEKTKNRLQQELDDLV and ADLEKEELAEELASSLSGR of myosin heavy chain 11. The reagent set according to claim 10, which is a set including a reagent containing a specifically recognized antibody or an antigen-binding fragment thereof. 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 an arteriosclerotic lesion by measuring the 11th level of myosin heavy chain in a blood sample by a sandwich method. A kit for use in the detection of atherosclerotic lesions, which comprises the reagent according to any one of claims 4 to 9 or the reagent set according to any one of claims 10 to 13.

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