Body fluid antibody biomarkers for detecting arterial lesions and for detecting cerebrovascular disease, diabetes, chronic kidney disease, or solid cancer

Abstract

To provide a comprehensive biomarker for diabetes, heart disease, cerebrovascular disorder, cancer, kidney disease, etc., which can suppress artery plaque lesion and the occurrence and progress thereof through a health care such as an early diagnosis.SOLUTION: It has been found that the above problem can be solved by measuring an antibody level in a body fluid sample, separated from a living organism, against KIAA0513 protein or a portion thereof and acquiring data on an increase in the antibody level as presence data of artery plaque lesion or as clear or presence data of brain and cardiovascular disorder, diabetes, chronic kidney disease, or solid cancer.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a means for acquiring or detecting data on a disease, and more specifically, to a means for acquiring or detecting data on arterial lesions by measuring bodily fluid antibodies against the KIAA0513 protein or a peptide having a partial sequence thereof as a biomarker, as well as a means for acquiring or detecting data on cerebrovascular disorders, diabetes, chronic kidney disease, solid cancers, etc. [Background technology]

[0002] In recent years, various disease biomarkers have been discovered, and their development is underway for the purpose of understanding the condition, predicting onset and prognosis, and identifying prevention / treatment targets through simple blood tests, etc. Biomarkers include enzyme markers and antigen markers in the past, and more recently DNA / RNA markers. On the other hand, there are still few reports of body fluid antibody markers, and few examples of their practical application. Antibody markers have also been identified in cerebrovascular and cardiovascular disorders. For example, antibody markers for acute ischemic stroke (AIS) include Hsp60 (Non-Patent Document 1), RPA2 (Non-Patent Document 2), SOSTDC1 (Non-Patent Document 3), PDCD11 (Non-Patent Document 4), MMP1, CBX1, and CBX5 (Non-Patent Document 5); markers for atherosclerosis include ATP2B4 (Non-Patent Document 6), BMP-1 (Non-Patent Documents 2 and 6), DHPS (Non-Patent Document 7), SH3BP5 (Non-Patent Document 8), and prolylcarboxypeptidase (Non-Patent Document 9); markers for acute coronary syndrome include nardilysin (NRD1) (Non-Patent Document 10); and markers for diabetes mellitus (DM), such as TUBB2C (Non-Patent Document 11), insulin (Non-Patent Document 12), glutamic acid decarboxylase (Non-Patent Document 13), adiponectin (Non-Patent Document 14), and GADD34 (Non-Patent Document 15).

[0003] As an antibody biomarker for cancer, anti-p53 antibody has been put to practical use as a marker useful for diagnosis, monitoring, and prognosis prediction of esophageal squamous cell carcinoma (ESCC) and head and neck cancer (Non-Patent Documents 16, 17). The present inventors' group screened using the serological identification of antigens by recombinant cDNA expression cloning (SEREX) method and discovered and reported the following antibody markers for ESCC: Trop2 / TACSTD2 (Non-Patent Document 18), SLC2A1 (Non-Patent Document 19), TRIM21 (Non-Patent Document 20), myomegalin (Non-Patent Document 21), makorin 1 (Non-Patent Document 22), ECSA (Non-Patent Document 23), and cyclin L2 (CCNL2) (Non-Patent Document 24); FIR / PUF60 for colorectal cancer (Non-Patent Document 25); and SH3GL1 (Non-Patent Document 26) and anti-filamin C (Non-Patent Document 27) for glioma. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Kramer J, Harcos P, Prohaszka Z, et al. Frequencies of certain complement protein alleles and serum levels of anti-heat-shock protein antibodies in cerebrovascular diseases. Stroke. 2000;31:2648-2652. PMID: 11062289 [Non-Patent Document 2] Machida T, Kubota M, Kobayashi E, et al. Identification of stroke-associated-antigens via screening of recombinant proteins from the human expression cDNA library (SEREX). J Translat Med. 2015;13:71. PMID: 25890248 [Non-Patent Document 3] Goto K, Sugiyama T, Matsumura R, et al. Identification of cerebral infarction-specific antibody markers from autoantibodies detected in patients with systemic lupus erythematosus. J Mol Biomark Diagnos. 2015;6:2. doi.org / 10.4172 / 2155-9929.1000219 [Non-Patent Document 4] Yoshida Y, Wang H, Hiwasa T, et al. Elevation of autoantibody level against PDCD11 in patients with transient ischemic attack. Oncotarget. 2018; 9, 8836-8848. PMID: 29507658 [Non-Patent Document 5] Wang H, Zhang XM, Tomiyoshi G, et al. Association of serum levels of antibodies against MMP1, CBX1, and CBX5 with transient ischemic attack and cerebral infarction. Oncotarget. 2018;9:5600-5613. PMID: 29464021 [Non-Patent Document 6] Hiwasa T, Machida T, Zhang XM, et al. Elevated levels of autoantibodies against ATP2B4 and BMP-1 in sera of patients with atherosclerosis-related diseases. Immunome Res. 2015;11:097. doi.org / 10.4172 / 1745–7580.1000097

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[0005] Cancer, heart disease, cerebrovascular disease, and renal failure are the first, second, fourth, and eighth causes of death among Japanese people, respectively (Ministry of Health, Labor, and Welfare 2018 Vital Statistics). The third, fifth, sixth, seventh, ninth, and tenth causes of death are senility, pneumonia, accidental accidents, aspiration pneumonia, dementia, and suicide, respectively. In other words, among the above causes of death, cancer, heart disease, cerebrovascular disease, and renal failure, as well as diabetes, which is a risk factor for these organic diseases, are diseases whose onset and progression can be suppressed by health management such as early diagnosis. Therefore, if disease markers can be used to provide appropriate treatment, therapy, lifestyle guidance, etc. for these diseases, it can greatly contribute to promoting human health.

[0006] Furthermore, as of 2020, the novel coronavirus (COVID-19) has become a global epidemic, and factors that can worsen the severity of the novel coronavirus include "the presence or absence of underlying diseases such as diabetes, heart disease, cerebrovascular disease, cancer, and kidney disease." It can be said that the presence or absence of these underlying diseases is a general factor in the severity of infectious diseases, not just the novel coronavirus. Therefore, there is an urgent need to provide a biomarker that can easily determine the presence or absence of an underlying disease, which is a factor in the severity of such infectious diseases.

[0007] Taking this into consideration, further examination of the background art reveals that the above-mentioned atherosclerosis markers, ATP2B4, BMP-1, and DHPS, are also elevated in the serum of patients with esophageal cancer (Non-Patent Documents 6 and 7). This indicates that "arterial lesions", of which arteriosclerosis such as atherosclerosis is a representative example, promote carcinogenesis. In fact, "angiogenesis", which is an example of "arterial lesions", is essential for the progression of solid cancer (Non-Patent Document 28), and diabetes and obesity, which induce arteriosclerosis, are risk factors for colon cancer and esophageal cancer (Non-Patent Documents 29, 30, and 31). Since internal organs and organs affect each other to a greater or lesser extent (Non-Patent Document 32), if "arterial lesions" such as arteriosclerosis and angiogenesis can be detected, it will be possible to capture the signs of cerebrovascular disorders, diabetes, kidney disease, solid cancer, and the like, and it is also thought that this will lead to the early detection of these diseases. These diseases are serious and life-threatening, and it would be of great significance if a single biomarker could detect their symptoms or find them early.

[0008] It would also be extremely significant to provide a means to easily determine the presence or absence of underlying diseases, which are factors that can lead to the severity of infectious diseases, using a single biomarker. [Means for solving the problem]

[0009] The present inventors have found that the presence of arterial lesions can be detected by measuring an increase in antibody levels against KIAA0513 protein in body fluids, and that this can be obtained as data on the presence or symptom of a disease accompanied by arterial lesions. Furthermore, they have found that the increase in the antibody levels in the body fluids can be obtained as latent or overt data on cerebrovascular disorders, diabetes, chronic kidney disease, or solid cancer. Furthermore, they have found that the latent or overt data on cerebrovascular disorders, diabetes, chronic kidney disease, or solid cancer can also be used as data on arterial lesions associated with these diseases. Furthermore, they have found that the presence of arterial lesions can be detected using an increase in antibody levels against KIAA0513 protein in body fluids as an index, and that this can be used as data on the presence or symptom of a disease accompanied by arterial lesions, or as latent or overt data on cerebrovascular disorders, diabetes, chronic kidney disease, or solid cancer, to determine the presence or absence of an underlying disease that is a factor in the aggravation of an infectious disease.

[0010] That is, firstly, the present invention provides a data acquisition method (hereinafter also referred to as "data acquisition method 1 of the present invention") which measures antibody levels in a body fluid sample isolated from a living body against a protein having the amino acid sequence of SEQ ID NO: 1 or 2 constituting KIAA0513 protein, or a part thereof, or a protein or a part thereof in which 10% or less (rounded down) of the amino acid sequence has been deleted, substituted or added, and acquires data on an increase in the antibody level as data on the presence of arterial lesions.

[0011] Secondly, the present invention provides a data acquisition method (hereinafter also referred to as "data acquisition method 2 of the present invention") for measuring antibody levels in a body fluid sample isolated from a living body against a protein having the amino acid sequence of SEQ ID NO: 1 or 2 constituting the KIAA0513 protein, or a part thereof, or a protein or a part thereof in which 10% or less (rounded down) of the amino acid sequence has been deleted, substituted or added, and acquiring data on an increase in the antibody levels as latent or manifest data on cerebrovascular disorders, diabetes, chronic kidney disease, or solid cancer.

[0012] Disease data obtained by data acquisition method 2 of the present invention Latent or overt The data can be obtained as data on arterial lesions associated with these diseases.

[0013] In this specification, when the term "data acquisition method of the present invention" is mentioned, it collectively refers to data acquisition methods 1 and 2 of the present invention, unless otherwise specified.

[0014] There are three known splicing variants of the KIAA0513 protein: isoform c (301 amino acids, NP_001284695.1: SEQ ID NO: 1), isoform b (301 amino acids, NP_001273495.1), and isoform a (411 amino acids, NP_055547.1: SEQ ID NO: 2).

[0015] The full length 301 amino acids of KIAA0513 isoforms c and b are completely the same as the first 301 amino acids of KIAA0513 isoform a. Although the base sequences of the mRNA precursors encoded by KIAA0513 isoforms c and b are different, the amino acid sequences of the KIAA0513 proteins expressed using spliced ​​mature mRNA as a template are the same. Thus, in this specification, when "KIAA0513 protein" is described without any particular mention, it means KIAA0513 protein isoforms b and / or c (SEQ ID NO: 1), and the case of isoform a will be specified separately.

[0016] The present inventors also purified GST-KIAA0513 isoforms c and a, and examined serum antibodies against KIAA0513 protein in the sera of healthy subjects (HD), patients with acute cerebral infarction (AIS), and patients with cardiovascular disease (CVD) (Example 6).

[0017] As a result, KIAA0513 isoforms c and a showed higher antibody levels in the sera of AIS and CVD patients compared to HD patients (Figures 7a, b), and the reactivity of KIAA0513 isoform c was higher than that of KIAA0513 isoform a, but relatively the two were closely correlated (Figure 7c).

[0018] This indicates that the major epitope sites of humoral antibodies against KIAA0513 protein are KIAA0513 isoform c and a This means that it is located in the first 301 amino acids of the KIAA0513 protein. This shows that a "part of the KIAA0513 protein" used for detecting a body fluid antibody against the KIAA0513 protein using an antigen-antibody reaction preferably includes the above-mentioned overlapping amino acid sequence region of the KIAA0513 isoforms c, b, and a.

[0019] Other examples of the data acquisition method of the present invention include "a method for detecting an arterial lesion by measuring an antibody level in a body fluid sample isolated from a living body against a protein having the amino acid sequence of SEQ ID NO: 1 or 2 constituting the KIAA0513 protein, or a part thereof, or a protein having 10% or less (rounded down to the nearest whole number) of amino acid residues in the amino acid sequence deleted, substituted or added, and using an increase in the antibody level as an indicator" and "a method for diagnosing or detecting cerebrovascular disorders, diabetes, chronic kidney disease, or solid cancer by measuring an antibody level in a body fluid sample isolated from a living body against a protein having the amino acid sequence of SEQ ID NO: 1 or 2 constituting the KIAA0513 protein, or a part thereof, or a protein having 10% or less (rounded down to the nearest whole number) of amino acid residues in the amino acid sequence deleted, substituted or added, and using an increase in the antibody level as an indicator."

[0020] Furthermore, the present invention provides a data acquisition kit (hereinafter also referred to as the kit of the present invention) for carrying out the data acquisition method 1 or 2 of the present invention.

[0021] The data acquisition kit of the present invention can be expressed in another way as, for example, the above-mentioned "kit for performing a method for detecting an arterial lesion by measuring an antibody level in a body fluid sample isolated from a living body against a protein having the amino acid sequence of SEQ ID NO: 1 or 2 constituting the KIAA0513 protein, or a part thereof, or a protein having 10% or less (rounded down to the nearest whole number) of the amino acid residues of the amino acid sequence deleted, substituted or added, or a part thereof, and using an increase in the antibody level as an indicator" and a "method for diagnosing or detecting cerebrovascular disorder, diabetes, chronic kidney disease, or solid cancer by measuring an antibody level in a body fluid sample isolated from a living body against a protein having the amino acid sequence of SEQ ID NO: 1 or 2 constituting the KIAA0513 protein, or a part thereof, or a protein having 10% or less (rounded down to the nearest whole number) of the amino acid sequence deleted, substituted or added, or a part thereof, and using an increase in the antibody level as an indicator." Effect of the Invention

[0022] First, the present invention makes it possible to obtain data on the presence of arterial lesions. This makes it possible to grasp the signs and presence of diseases accompanied by arterial lesions, and provide a way to take measures such as lifestyle advice and medication based on the data, thereby contributing to health promotion and the prevention and treatment of diseases. Second, it makes it possible to obtain latent or manifest data on cerebrovascular disorders, diabetes, chronic kidney disease, or solid cancer. This makes it possible to predict the onset and early detection of the above serious diseases, and provide a way to take measures such as lifestyle advice and medication based on the data, thereby contributing to the prevention and treatment of diseases.

[0023] Furthermore, the present invention provides basic data for determining the presence or absence of underlying diseases that may contribute to the aggravation of infectious diseases. [Brief description of the drawings]

[0024] [Figure 1]The results of quantifying KIAA0513 serum antibody levels in healthy subjects (HD), patients with acute cerebral infarction (AIS), and patients with transient ischemic attack (TIA) using the AlphaLISA method are shown. The results when KIAA0513 protein antigen was used are shown in the distribution diagram in (a). The vertical axis of these distribution diagrams is alpha counts indicating antibody levels, and the bars in each distribution diagram indicate the mean value and the mean ± SD value. The results of ROC analysis are shown in (b) and (c). The values ​​in the ROC diagrams are the AUC value, cutoff value, sensitivity, specificity, and 95% confidence interval. [Diagram 2] The results of measuring KIAA0513 serum antibody levels in HD and diabetes mellitus (DM) patients using the AlphaLISA method are shown in (a). The results when using the KIAA0513 protein antigen are shown in (b). The distribution diagram and ROC diagram are explained as in Fig. 1. [Diagram 3] The results of measuring KIAA0513 serum antibody levels in patients with HD, cardiovascular disease (CVD), and obstructive sleep apnea syndrome (OSAS) using the AlphaLISA method are shown. The distribution diagram is shown in a, and the results of ROC analysis are shown in b and c. The explanation of the distribution diagram and ROC analysis diagram is as described in Figure 1. [Figure 4] The results of measuring KIAA0513 serum antibody levels in HD and chronic kidney disease (CKD) patients using the AlphaLISA method are shown. The results are shown when the KIAA0513 protein antigen was used. The distribution diagram is shown in a, and the results of ROC analysis are shown in b, c, and d. The explanation of the distribution diagram and ROC analysis diagram is as described in Figure 1. [Diagram 5] The results of measuring KIAA0513 serum antibody levels in HD and cancer patients using the AlphaLISA method are shown. The serum antibody levels in HD, esophageal cancer (EC), gastric cancer (GC), colon cancer (CC), lung cancer (LC), and breast cancer (MC) are shown when the KIAA0513 protein antigen was used. The distribution diagram is shown in a, and the results of ROC analysis are shown in bf. The explanation of the distribution diagram and ROC analysis diagram is as described in Figure 1. [Figure 6]Comparison of survival rates between KIAA0513 antibody-positive and -negative patients in postoperative cases of esophageal cancer (EC) and gastric cancer (GC). Survival rates after surgery were compared using Kaplan-Meyer plots. [Figure 7] Comparison of serum antibody levels when KIAA0513 isoform c and isoform a were used as antigens. Scatter plots show serum antibody levels against KIAA0513 isoform c (a) and KIAA0513 isoform a (b) in HD, AIS, and CVD patients. (c) is a correlation diagram of antibody levels (alpha value) when KIAA0513 isoform c and isoform a were used as antigens in each sample. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] (1) Data Acquisition Method of the Present Invention (a) Acquired Data The data acquired in the data acquisition method 1 of the present invention is "data on the presence of arterial lesions." Arterial lesions are abnormalities in the structure or constituent factors of an artery, and can take the form of occlusion, dilation, fistula formation, etc. Arteriosclerosis (atherosclerosis, arteriolosclerosis, medial sclerosis), chronic inflammation, abnormal angiogenesis in solid cancer, etc., become apparent as arteriosclerosis progresses. It is also known that arteriosclerosis is often caused by the progression of diabetes and obesity, and that cerebrovascular disorders, chronic kidney disease, aortic aneurysm, retinopathy, foot gangrene, etc. are known to be caused by the progression of arteriosclerosis, particularly atherosclerosis. Diabetes and obesity are also risk factors for solid cancers (colon cancer, esophageal cancer, etc.). Therefore, the "data on the presence of arterial lesions" obtained by the data acquisition method 1 of the present invention is existence The "data" can be used as data on the possibility of the occurrence or progression of diabetes, arteriosclerosis, solid cancer, etc., and further, can be used as data on the presence or absence of serious circulatory system diseases, such as cerebrovascular disorders, chronic kidney disease, aortic aneurysm, retinopathy, and foot gangrene.

[0026] The data acquired in the data acquisition method 2 of the present invention is "data on latent or overt diabetes, cerebrovascular disease, chronic kidney disease, or solid cancer." Examples of cerebrovascular disease include cerebral infarction, stroke, myocardial infarction, and angina pectoris. Examples of solid cancer include esophageal cancer, gastric cancer, colon cancer, lung cancer, and breast cancer. An increase in the level of body fluid antibodies against the KIAA0513 protein is highly likely to be latent or overt data on esophageal cancer, colon cancer, gastric cancer, or lung cancer, and is particularly likely to be data on esophageal cancer. The data acquisition method 2 of the present invention often overlaps with the data acquisition method 1.

[0027] For example, by periodically performing the data acquisition method 1 or 2 of the present invention on a subject and monitoring the level of body fluid antibodies against the "KIAA0513 protein", it is possible to predict the onset of the above-mentioned diseases with high accuracy. In particular, once serious diseases of the circulatory system such as cerebral infarction and myocardial infarction and solid cancers occur, they are life-threatening, and even if they do not lead to death, the patient suffers from severe aftereffects. Therefore, it is best to predict the onset of these diseases, and the anti-KIAA0513 body fluid antibody marker can be used as a biomarker for obtaining data for such onset prediction.

[0028] Furthermore, when an epidemic of a particular infectious disease is recognized, the presence or absence of an underlying disease that is a factor in the aggravation of the disease can be determined based on the data obtained by the data acquisition method 1 or 2 of the present invention, thereby making it possible to more appropriately treat the subject suffering from the infectious disease. If the obtained data suggests, for example, the presence of an arterial lesion, it is possible to take measures to preferentially hospitalize the subject suffering from the infectious disease in preparation for the aggravation of the disease. If no such suggestion is found, the subject can be placed on home standby, and the hospital's medical personnel and beds can be efficiently allocated, allowing treatment to be given according to the risk of aggravation of each of the subjects suffering from the infectious disease, thereby reducing the risk of the collapse of the medical system due to a shortage of medical personnel and beds caused by the epidemic.

[0029] The above infectious diseases are not particularly limited as long as they are various viral diseases, bacterial diseases, etc., and the presence or absence of an underlying disease is a factor in the severity of the disease, but in particular, for infectious diseases of the respiratory tract system, which are prevalent on a large scale, it is desirable to estimate the risk of the disease becoming severe through mass screening, and the present invention in a simple form such as immunochromatography is particularly suitable as a target for application. The above infectious diseases include, but are not limited to, infectious diseases of the respiratory tract system, such as infections caused by betacoronaviruses such as the new coronavirus (COVID-19), SARS, and MARS; infections caused by influenza viruses; and infections caused by respiratory syncytial virus, adenovirus, rhinovirus, etc.

[0030] (b) A body fluid sample isolated from a living organism The body fluid in the "body fluid sample separated from a living body" used as the subject of data acquisition in the data acquisition method of the present invention is blood, lymph, etc., and the "sample" is the body fluid itself in a state separated from the body, or a processed product. Among the body fluid samples, a blood sample is preferred. Examples of blood samples include whole blood, serum, plasma, etc., with serum samples or plasma samples being preferred, and serum samples being particularly preferred. The blood sample may be subjected to an anticoagulation treatment such as heparin treatment.

[0031] (c) Body fluid antibody capture antigen (KIAA0513 antigen) The KIAA0513 protein having the amino acid sequence shown in SEQ ID NO: 1 or 2 is the basis for capturing body fluid antibodies in the data acquisition method of the present invention. The KIAA0513 protein can be used in its entirety as a body fluid antibody capture antigen, or a portion of it can be used. Furthermore, to establish a more efficient detection system, it is also possible to select an amino acid sequence (peptide) with a short chain length, and in this case, the number of amino acids in the sequence is preferably about 7-30, and more preferably about 10-20.

[0032] Furthermore, 10% or less of the amino acid residues (decimals are discarded) of all or part of these amino acid sequences may be modified (deleted, substituted, or added). However, it is required that the target humoral antibody (humoral antibody against KIAA0513 protein or a peptide having a partial sequence thereof) can be captured, that is, when a humoral antibody capture antigen candidate with modified amino acid residues is subjected to capture of a humoral antibody by, for example, a method similar to that of the Examples of this specification, data on the target arterial lesion or disease can be obtained from the level of the captured antibody.

[0033] "Deletion" refers to a state in which any amino acid residue in the target amino acid sequence is deleted, and the amino acid residues on the N-terminus (before) and C-terminus (after) of the deleted amino acid residue are linked by peptide bonds (in the case of deletion of the N-terminus and C-terminus amino acid residues, the amino acid residue is simply deleted), and the number of deleted residues is counted as the "number of amino acid deletions." "Substitution" refers to a state in which any amino acid residue in the target amino acid sequence is replaced with "another amino acid residue," and the replaced amino acid residue is linked to each amino acid residue on the N-terminus (before) and C-terminus (after) by peptide bonds (in the case of substitution of the N-terminus amino acid residue, only the peptide bond with the amino acid residue on the C-terminus, and in the case of substitution of the C-terminus amino acid residue, only the peptide bond with the amino acid residue on the N-terminus), and the number of replaced residues is counted as the "number of amino acid substitutions." "Addition" refers to a state in which one or more new amino acid residues are inserted into each of one or more peptide bond positions in the target amino acid sequence, forming new peptide bonds. The concept of "addition" also includes the addition of one or more new amino acid residues at the N-terminus and C-terminus. The number of these "added" amino acid residues is counted as the "number of added amino acids." Hereinafter, "the protein having the amino acid sequence of SEQ ID NO: 1 or 2 constituting the KIAA0513 protein, or a part thereof, or a protein having 10% or less (rounded down) of the amino acid sequence deleted, substituted or added, or a part thereof" is also referred to as "KIAA0513 antigen."

[0034] A KIAA0513 antigen having a predetermined amino acid sequence can be obtained by a conventional method. Based on amino acid sequence Based on the base sequence of the KIAA0513 gene, nucleic acid amplification primers are designed for amplifying double-stranded DNA having all or part of this base sequence, and a gene amplification product is obtained as all or part of the KIAA0513 gene by PCR or other methods using the amplification primers. This is then used to incorporate into a prokaryotic cell expression vector such as pGEX-4T, and transformants transformed with the vector are selected. A drug such as IPTG (isopropyl-β-D-thiogalactoside) is then added to induce expression of all or part of the specified KIAA0513 gene, and all or part of the expressed KIAA0513 protein can be purified by affinity chromatography using, for example, glutathione-Sepharose (GE Healthcare Life Sciences).

[0035] Furthermore, the proteins or peptides used as the KIAA0513 antigens can be modified as necessary, for example, into GST (glutathione S-transferase)-fused proteins or peptides or biotin-fused proteins or peptides used in the AlphaLISA (Amplified Luminescence Proximity Homogeneous Assay-Linked ImmunoSorbent Assay) method, as described below.

[0036] (d) Data Acquisition In the data acquisition method of the present invention, the antibody level against the KIAA0513 antigen in a body fluid sample isolated from a living body, such as a blood sample, is quantified, and if the quantified value is greater than a standard value (cutoff value), an increase in the KIAA0513 antigen in the body fluid sample donor is recognized. This can be used as an indicator to provide data on the presence of arterial lesions in the test sample donor, or latent or overt data on cerebrovascular disorders, diabetes, chronic kidney disease, or solid cancer.

[0037] The standard value for the body fluid antibody level against the KIAA0513 antigen in a test sample can be derived, including the cutoff value, for example, by setting up a control sample population consisting of individuals (healthy individuals) who are found to have no abnormalities such as diabetes, circulatory system disease, cancer, etc. in a health check, determining the quantitative value of the above-mentioned body fluid antibody against the KIAA0513 antigen in the test sample in the sample population, and subjecting this to statistical processing to determine the average, standard deviation, etc.

[0038] The measurement of the body fluid antibody level can be carried out, for example, by contacting a body fluid sample separated from a living body with an immobilized product of KIAA0513 antigen, and detecting the binding of an antibody against the KIAA0513 antigen in the body fluid sample separated from the living body with the body fluid antibody capture antigen as a signal based on an antigen-antibody reaction. Specifically, quantitative measurement can be carried out using means such as immunochromatography, AlphaLISA, ELISA, indirect fluorescent antibody method, Western blot method (immunoblot method), turbidimetric method, nephelometric method, latex agglutination turbidimetric method, and CLEIA method. All of the quantitative measurement means exemplified here are established quantitative measurement means when the quantitative target substance is an antibody in a body fluid sample separated from a living body.

[0039] For example, in the immunochromatography method, a buffer solution containing a mixture of body fluid and gold colloid-bound anti-human IgG antibody is added to a cellulose membrane coated with the KIAA0513 antigen and slowly moved, and if an antibody against the KIAA0513 antigen is present in the body fluid, a line is formed at the coated position, which can be visually confirmed. The immunochromatography method is particularly suitable for mass screening because it is easier to simplify than other quantitative measurement means. In other words, by applying the data acquisition method of the present invention to the immunochromatography method, it becomes possible to acquire desired disease-related data in a short time (about 10 minutes) even in small facilities such as clinics and public health centers.

[0040] In the AlphaLISA method, when the GST-fused KIAA0513 antigen is used as the body fluid antibody capture antigen, glutathione-conjugated donor beads are used, and the body fluid antibody capture antigen, blood sample, and acceptor beads bound to anti-human IgG antibodies are mixed and incubated at room temperature for several hours to several days, and the formed antigen-antibody complex is irradiated with 680 nm light, and the generated light of 520-620 nm is detected, thereby allowing the desired body fluid antibody to be quantified. In the indirect fluorescent antibody method, a body fluid sample isolated from a living body is contacted with a protein array on which the KIAA0513 antigen is immobilized, and the formed KIAA0513 antigen-anti-KIAA0513 body fluid antibody complex is further contacted with a fluorescently labeled secondary antibody, allowing the body fluid antibody against the KIAA0513 protein to be quantified. In the ELISA method, the secondary antibody used in the indirect fluorescent antibody method is labeled with an enzyme to perform quantification. Various types of secondary antibody labels can be selected. In the Western blot method, the KIAA0513 antigen is electrophoresed on an SDS-polyacrylamide gel, then transferred from the gel to a carrier such as a nitrocellulose membrane, and a body fluid sample isolated from a living organism is brought into contact with the KIAA0513 antigen-anti-KIAA0513 body fluid antibody complex formed thereon is detected using a secondary antibody, thereby enabling quantitative measurement. In the turbidimetric method and nephelometric method, the KIAA0513 antigen-anti-KIAA0513 body fluid antibody complex formed by contacting a body fluid sample isolated from a living organism with the KIAA0513 antigen is detected by turbidity (nephelometric method) or change in scattered light intensity (nephelometric method), thereby enabling quantitative measurement. In the latex agglutination nephelometric method, the KIAA0513 antigen-bound latex particles are brought into contact with a body fluid sample isolated from a living organism, whereby aggregates of the latex particles are formed by interaction between the anti-KIAA0513 antibodies bound to the latex particles in the body fluid, and these aggregates are detected, thereby enabling quantitative measurement. In the CLEIA method, KIAA0513 antigen-bound magnetic particles are brought into contact with a body fluid sample isolated from a living organism, forming a KIAA0513 antigen-antibody complex on the magnetic particles, which is then magnetically collected. Unreacted material is then removed, and the complex is detected by an appropriate fluorescent treatment, etc., to perform a quantitative measurement.

[0041] (2) About the kit of the present invention In the kit of the present invention, for example, a set of GST-fused KIAA0513 antigen, glutathione-bound donor beads, and anti-human IgG-bound acceptor beads can be mentioned as a configuration for carrying out the AlphaLISA method as a quantitative measurement means.

[0042] Furthermore, the kit of the present invention for performing ELISA as a quantitative measurement means can include a plate on which the above-mentioned KIAA0513 antigen is immobilized, a labeled secondary antibody against the anti-KIAA0513 antibody, and a reagent for revealing the label of the secondary antibody.

[0043] Furthermore, the above-mentioned latex particles to which the KIAA0513 antigen is bound can be included as a component of a kit for carrying out latex agglutination turbidimetry.

[0044] Furthermore, a kit for performing the CLEIA method can include magnetic particles bound to the above-mentioned KIAA0513 antigen, a secondary antibody labeled against the anti-KIAA0513 antibody, and a reagent for revealing the label of the secondary antibody.

[0045] The above-mentioned kit configurations are merely examples, and kits for carrying out other quantitative measurement means are also included in the scope of the kit of the present invention. Furthermore, it is possible to further reduce the above-mentioned components exemplified here to increase the degree of outsourcing or self-procurement of tests, or conversely, it is possible to increase the components such as dilution solutions and tubes for reagents to set up immediate use of the kit of the present invention. It is also possible to add other elements according to the specific tests. EXAMPLES

[0046] Examples of the present invention will be disclosed below.

[0047] [Manufacturing examples and analysis methods] <Antigen Proteins Used in the Present Example> (1) Identification of antigenic proteins In this example, a human protein microarray (ProtoArray (registered trademark): manufactured by Thermo Fisher Scientific) was used to identify the KIAA0513 protein as an antigen recognized by serum antibodies from patients with atherosclerosis.

[0048] The full-length 411 amino acid region of "KIAA0513 isoform a" (SEQ ID NO: 2) with the N-terminus modified by GST, or the region of 301 amino acids from the N-terminus was used. The latter "region of 301 amino acids from the N-terminus" is KIAA0513 isoform c (SEQ ID NO: 1).

[0049] (2) Preparation of antigen protein (expression and purification of KIAA0513 protein) Human KIAA0513 protein was prepared as follows.

[0050] The cDNAs encoding human KIAA0513 isoform a and isoform c were separately inserted into the EcoRI / SalI site of the plasmid vector pGEX-4T-1 (GE Healthcare Life Sciences, Pittsburgh, PA) where the Glutathione S-transferase (GST) gene site is provided upstream of the cloning site, to prepare the recombinant plasmids for gene expression, pGEX-4T-1-KIAA0513 (isoform a) and pGEX-4T-1-KIAA0513 (isoform c), respectively. These were introduced into Escherichia coli BL-21 for transformation, and the expression of each KIAA0513 cDNA was induced by treatment at 25°C for 4 hours with 0.1 mM IPTG (isopropyl-β-D-thiogalactoside). After induction, the Escherichia coli was collected, dissolved by adding BugBuster Master Mix (Merck Millipore, Darmstadt, Germany), and a protein extract was obtained. The GST-KIAA0513 protein isoforms a and c in the protein extract were purified by glutathione-Sepharose (GE Healthcare Life Sciences) column chromatography, and the buffer was exchanged with PBS, which was designated as the above-mentioned "KIAA0513 protein". GST was purified in the same manner as a control.

[0051] <Analysis by AlphaLISA method> Prior to the disclosure of individual examples, the outline of the analysis by the AlphaLISA method used in this example will be described below.

[0052] The AlphaLISA method (Amplified Luminescence Proximity Homogeneous Assay) specifically uses a 384-well microtiter plate (white opaque OptiPlate TMThe assay was performed using a 100-fold diluted serum with AlphaLISA dedicated buffer (25 mM HEPES, pH 7.4, 0.1% casein, 0.5% Triton X-100, 1 mg / mL dextran-500, 0.05% Proclin-300) and 2.5 μL of the serum was added to each well, and the antigen GST or KIAA0513 protein (KIAA0513 isoform a and isoform c, respectively) (10 μg / mL) was diluted in AlphaLISA dedicated buffer and mixed. After incubation at room temperature for 6-8 hours, anti-human IgG-conjugated acceptor beads (2.5 μL at 40 μg / mL) and glutathione- or streptavidin-conjugated donor beads (2.5 μL at 40 μg / mL) were diluted in AlphaLISA dedicated buffer and mixed. After leaving the plates at room temperature for 1-14 days, the number of photons generated was measured using an EnSpire Alpha microplate reader (PerkinElmer) and defined as the "Alpha Count." The levels of antibodies specific to the KIAA0513 antigen were calculated by subtracting the values ​​of the control GST for the KIAA0513 protein and the buffer control for the biotinylated peptide.

[0053] [Example 1] Study of Transient Ischemic Attack (TIA) and Acute Infarction (AIS) Patients Using AlphaLISA AIS is a condition in which cerebral blood flow is blocked, causing sudden and severe damage to motor and sensory functions. Transient ischemic attack (TIA) is a disease in which brain cells are damaged for the same reasons as cerebral infarction, but recovery occurs within 24 hours, and often within a few minutes. Each serum sample was collected within two weeks of onset.

[0054] The levels of serum antibodies (s-KIAA0513-Abs: serum KIAA0513 antibodies) against KIAA0513 isoform c (SEQ ID NO: 1) were examined in patients with AIS and TIA. Sera from healthy subjects (HD), AIS and TIA patients were obtained from Chiba Prefectural Sahara Hospital. The results of AlphaLISA showed that the levels of s-KIAA0513-Ab were significantly higher in patients with TIA and AIS than in HD (Figure 1).

[0055] Using a cutoff value of the mean HD value plus 2 × standard deviation (SD), the s-KIAA0513-Ab positivity rates in HD, AIS patients, and TIA patients were 0.0%, 7.6%, and 15.6%, respectively (Table 1 ).

[0056] ROC analysis showed that the area under the ROC curve (AUC) of s-KIAA0513-Abs was 0.6439 [95% confidence interval (CI) = 0.5873-0.7004] for AIS (Fig. 1b) and 0.6604 (95% CI = 0.5633-0.7575) for TIA (Fig. 1c).

[0057] Therefore, TIA, a precursor of AIS, was also positively associated with serum KIAA0513 antibody levels. These results suggest that s-KIAA0513-Ab is useful for predicting the onset of AIS.

[0058] [Table 1]

[0059] [Example 2] Study on diabetes mellitus (DM) patients using AlphaLISA method Next, we examined the levels of s-KIAA0513-Abs in DM patients.

[0060] Serum samples from HD and DM patients were obtained from Chiba University Hospital.

[0061] The levels of s-KIAA0513-Abs were significantly higher in samples from patients with DM than in samples from HD (Fig. 2a).

[0062] When the mean plus 2 × SD of the values ​​in the HD samples was used as the cutoff value, the positivity rates of s-KIAA0513-Ab in HD and DM patients were 2.5% and 26.5%, respectively (Table 2).

[0063] Conducting ROC analysis, this When the ability of antibody markers to indicate the presence of DM was evaluated, the AUC value of s-KIAA0513-Abs was 0.7361, with sensitivity and specificity values ​​of 50.55% and 87.65%, respectively (Fig. 2b).

[0064] [Table 2]

[0065] [Example 3] Study on patients with cardiovascular disease (CVD) and obstructive sleep apnea syndrome (OSAS) using AlphaLISA method Next, we investigated the levels of s-KIAA0513-Abs in patients with CVD and OSAS.

[0066] CVD included acute myocardial infarction and unstable angina obtained from Chiba University Hospital. Because OSAS is related to atherosclerosis and is associated with a high risk of acute ischemic stroke (AIS) and CVD, serum from OSAS patients obtained from Chiba University Hospital was also examined.

[0067] As a result, the levels of s-KIAA0513-Abs were higher in both CVD and OSAS patients compared with HD patients (Figure 3a). The positive rates of s-KIAA0513-Abs in HD patients were 5.3%, those in CVD patients were 10.3%, and those in OSAS patients were 11.6% (Table 3).

[0068] ROC analysis revealed that the AUC of s-KIAA0513-Abs for CVD was 0.6492 (95% CI: 0.5821-0.7163) (Fig. 3b), and the AUC of s-KIAA0513-Abs for OSAS was 0.6463 (95% CI: 0.5616-0.7309) (Fig. 3e).

[0069] The low P value of s-KIAA0513-Ab in CVD ( 0.001 The P value for OSAS was 0.0013 when compared with the mean of s-KIAA0513-Ab (< 0.0013). This suggests that the association between the s-KIAA0513-Ab marker and OSAS is lower than that of CVD (Table 3). In other words, it was presumed that OSAS does not directly correspond to the s-KIAA0513-Ab marker, but indirectly through CVD, etc., which occurs concomitantly with OSAS.

[0070] [Table 3]

[0071] [Example 4] Study on chronic kidney disease (CKD) patients using AlphaLISA method Next, we investigated the levels of s-KIAA0513-Abs in CKD patients, which are closely related to atherosclerosis.

[0072] CKD was divided into three groups as follows:

[0073] The types were classified as follows: type 1, diabetic nephropathy; type 2, nephrosclerosis; and type 3, glomerular nephropathy.

[0074] Serum samples from CKD patients were obtained from the Kumamoto cohort, and serum samples from HD patients were obtained from Chiba University.

[0075] Serum levels of s-KIAA0513-Abs were significantly higher in all three CKD groups than in HD (Fig. 4a). The s-KIAA0513-Ab positivity rates in HD, CKD type 1, CKD type 2, and CKD type 3 patients were 6.1%, 29.0%, 37.5%, and 20.3%, respectively (Table 4), with the highest positivity observed in serum samples from CKD type 2 patients.

[0076] ROC analysis revealed that the AUC of s-KIAA0513-Abs was 0.7434 (95% CI: 0.6783-0.8085) in type 1 CKD (Fig. 4b), 0.8080 (95% CI: 0.7264-0.8897) in type 2 CKD (Fig. 4c), and 0.6906 (95% CI: 0.6177-0.7635) in type 3 CKD (Fig. 4d).

[0077] [Table 4]

[0078] [Example 5] Study on cancer patients using AlphaLISA method Because atherosclerosis is often associated with cancer, we investigated the levels of s-KIAA0513-Abs in serum samples from patients with esophageal cancer (EC), gastric cancer (GC), colon cancer (CC), lung cancer (LC), and breast cancer (MC) obtained from Toho University Hospital.

[0079] The results showed that the levels of s-KIAA0513-Abs were significantly higher in serum samples from all cancer patients compared to HD patients, and the highest mean s-KIAA0513-Ab levels were observed in esophageal cancer patients (Fig. 5a, Table 5). The ROC analysis also showed a high AUC value of 0.8300 for esophageal cancer (Fig. 5b-f).

[0080] We next investigated whether s-KIAA0513-Ab levels were associated with the survival of esophageal cancer (EC) and gastric cancer (GC) patients after surgery. Using the cutoff value obtained by ROC analysis, s-KIAA0513-Ab levels were divided into positive and negative (Fig. 5b, c). As a result, there was no significant difference in overall survival between the s-KIAA0513-Ab positive and negative groups of esophageal cancer (P = 0.3509), but the antibody-positive group showed a tendency toward poor prognosis in the late postoperative period (40-60 weeks) (Fig. 6a). On the other hand, no obvious difference in survival rate was observed between the s-KIAA0513-Ab positive and negative groups of gastric cancer (Fig. 6b).

[0081] [Table 5]

[0082] [Example 6] Comparison of serum antibody levels when KIAA0513 isoform c and isoform a were used as antigens The antibody levels against KIAA0513 isoform c (sequence number 1) and KIAA0513 isoform a (sequence number 2) in the sera of healthy subjects (HD), patients with acute cerebral infarction (AIS), and patients with cardiovascular disease (CVD) were examined using the AlphaLISA method.

[0083] As a result, KIAA0513 isoforms c and a similarly showed higher antibody levels in the sera of AIS and CVD patients compared to HD patients (Fig. 7a, b), and the reactivity of KIAA0513 isoform c to serum antibodies was higher than that of KIAA0513 isoform a, but relatively the two were closely correlated (Fig. 7c).

[0084] This means that the major epitope site of serum antibodies against the KIAA0513 protein is located at amino acid 301 of KIAA0513 isoform c.

[0085] [Example 7] Correlation analysis between serum antibody levels and subject data (1) Mann-Whitney U analysis Correlation analysis between s-KIAA0513-Ab levels and subject data was performed using 665 samples from Chiba Prefectural Sawara Hospital, including 139 HD samples, 225 AIS patients, 44 TIA patients, 17 silent ischemic stroke patients, 122 deep and subcortical leukomalacia (DSWMH) patients, 59 chronic ischemic stroke (CIS) patients, and 41 control samples.

[0086] In this analysis, the Mann-Whitney U test was used to compare s-KIAA0513-Ab levels between men / women, those with or without DM, hypertension (HT), CVD, dyslipidemia, and obesity [body mass index (BMI) <25, “normal or underweight”; BMI ≥25, “obese”], and those with or without smoking and alcohol consumption habits. As a result, significant differences were observed only in those with or without HT (Table 6).

[0087] [Table 6]

[0088] [Table 6 (continued)]

[0089] (2) Spearman correlation analysis Spearman correlation analysis was used to examine correlations between s-KIAA0513-Ab levels and subject parameters, including general information such as age, height, weight, and BMI, degree of arterial stenosis including maximum intima-media thickness (max-IMT), lifestyle factors such as smoking duration (years) and frequency of alcohol intake (times / week), and continuous variables of blood test data. 0513 Significant correlations of s-KIAA0513-Ab were observed with age, max-IMT, ALP, potassium, C-reactive protein (CRP), body glucose (BS), and smoking duration (Table 7). Inverse relationships were observed between antibody levels and height and weight. Correlation with max-IMT indicates that s-KIAA0513-Ab levels are related to stenosis and atherosclerosis.

[0090] Similar results were confirmed using other cohorts. Spearman correlation analysis of the CKD cohort (300 cases) showed significant correlations with plaque score, max-IMT, and CAVI (right), all of which are indicators of atherosclerosis (Table 8). CRP was also associated with s-KIAA0513-Abs in the CKD cohort, indicating the involvement of inflammation. On the other hand, age, height, weight, BMI, and potassium did not show significant correlations with s-KIAA0513-Abs in the CKD cohort. Since AIS is closely related to age and s-KIAA0513-Abs is positively correlated with AIS, s-KIAA0513-Abs and aging may be indirectly related.

[0091] [Table 7]

[0092] [Table 7 (continued)]

[0093] [Table 8]

[0094] [Table 8 (continued)]

[0095] [Summary of the Examples] Primary screening using human protein microarrays identified KIAA0513 protein as an antigen recognized by serum antibodies in patients with atherosclerosis. Recombinant GST-tagged KIAA0513 protein was then purified. Using this purified protein as an antigen, serum antibody levels were examined by AlphaLISA. The results showed significantly higher levels of s-KIAA0513-Abs in patients with AIS, TIA, DM, CVD, OSA, CKD, EC, GC, CC, LC, and MC than in patients with HD (Figures 1-5, Tables 1-5). Among these diseases, particularly high AUC values ​​were observed in DM, type 2 CKD, and EC (Figures 2, 4, 5). Furthermore, correlation analysis showed that serum antibody levels against KIAA0513 protein were well positively correlated with max-IMT, plaque score, and CAVI, all of which are indicators of atherosclerosis-related lesions (Tables 7, 8). On the other hand, serum antibody levels against KIAA0513 protein were weakly correlated with BS (P = 0.0229) (Table 7), but no correlation was observed with HbA1c, a typical DM marker (Table 7). Therefore, although DM patients showed higher serum antibody levels against KIAA0513 protein than HD patients, this antibody marker is not thought to mainly reflect the increase in blood glucose levels in DM, but rather corresponds to atherosclerotic lesions induced by DM. In addition, since angiogenesis is essential for the progression of solid cancers including EC, it is thought that abnormal formation of blood vessels in solid cancers promotes the expression of KIAA0513 protein, just as in atherosclerosis. In fact, DM and atherosclerosis are known to be risk factors for cancer.

[0096] At present, little is known about the function of the KIAA0513 protein; however, since it has been reported to bind to proteins involved in synaptic transmission and apoptosis induction, it is presumed to be involved at least in these signal transduction pathways.

[0097] In general, humoral antibody markers have higher sensitivity for detecting disease-related data than humoral antigen markers. Among them, the KIAA0513 antigen has particularly high antigenicity, so this anti-KIAA0513 humoral antibody marker is ultrasensitive. Moreover, it has been found that it is possible to detect not only latent or manifest disease data related to cancer, cerebral infarction, myocardial infarction, diabetes, chronic kidney disease, etc., which are fatal to humans, but also data indicating the presence of arterial lesions such as arteriosclerosis and abnormal angiogenesis in solid cancers. [Industrial Applicability]

[0098] Even for serious, life-threatening diseases such as cancer, cerebral infarction, myocardial infarction, diabetes, and chronic kidney disease, there are few easy-to-use markers, and it has been necessary to use individual markers for each disease. The data acquisition method of the present invention makes it possible to acquire disease data with ultra-high sensitivity, and to comprehensively detect data related to these serious diseases. In addition, by detecting data indicating the presence of arterial lesions such as arteriosclerosis and abnormal angiogenesis in solid cancer, it is possible to use the data as an indicator for disease prevention, making it ideal for mass medical examinations, etc.

[0099] As described above, the present invention makes it possible to comprehensively detect the presence or absence of underlying diseases that are factors that aggravate infectious diseases using a single biomarker. For example, by performing the data acquisition method of the present invention in mass screening in response to the spread of respiratory tract infections caused by the novel coronavirus (COVID-19) or influenza viruses, it becomes possible to efficiently allocate medical personnel and hospital beds, and provide more appropriate treatment.

Claims

1. A data acquisition method comprising measuring antibody levels in a body fluid sample isolated from a living body against a protein having the amino acid sequence of SEQ ID NO: 1 or 2 constituting KIAA0513 protein, or a protein in which 10% or less (rounded down) of the amino acid sequence has been deleted, substituted or added, and acquiring data on an increase in the antibody levels as data on the presence of arterial lesions.

2. A data acquisition method comprising measuring antibody levels in a body fluid sample isolated from a living body against a protein having the amino acid sequence of SEQ ID NO: 1 or 2 constituting KIAA0513 protein, or a protein in which 10% or less (rounded down) of the amino acid sequence has been deleted, substituted or added, and acquiring data on increases in the antibody levels as latent or manifest data on cerebrovascular disorders, diabetes, chronic kidney disease, or solid cancer.

3. 3. The data acquisition method according to claim 2, wherein latent or manifest data on cerebrovascular disease, diabetes, chronic kidney disease, or solid cancer is acquired as data on arterial lesions associated with these diseases.

4. The data acquisition method according to any one of claims 1 to 3, wherein the body fluid sample separated from the living body is a blood sample.

5. 5. The data acquisition method according to claim 4, wherein the blood sample is serum or plasma.

6. The data acquisition method according to any one of claims 1 to 5, characterized in that the antibody level measurement method is an immunochromatography method, an ELISA method, an AlphaLISA method, an indirect fluorescent antibody method, a Western blot method (immunoblot method), a turbidimetric method, a nephelometric method, a latex agglutination turbidimetric method, or a CLEIA method.

7. A data acquisition kit for carrying out the data acquisition method according to any one of claims 1 to 6.

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