Patent Application
20240118288
The present invention relates to means for acquiring data concerning a disease or disorder (hereinafter also referred to simply as “disease”) or for detecting a disease. More particularly, the invention relates to means for acquiring data concerning an artery lesion or for detecting an artery lesion, and to means for acquiring data concerning cerebrovascular or cardiovascular disorder, diabetes mellitus, chronic kidney disease, solid cancer, etc. or detecting such diseases, these means being based on measuring the level of a biomarker which is a humoral antibody with respect to protein KIAA0513 or a peptide having a partial sequence of the protein.
A variety of disease-specific biomarkers have been found in recent years, and these biomarkers are further refined from many aspects including assessment of the disease condition, prediction of the onset and prognosis of the disease, and determination of a prophylactic/therapeutic target, through a simple blood test or the like. Conventional biomarkers include an enzyme marker and an antigen marker, and a DNA/RNA marker is reported in recent years. In contrast, a limited number of humoral antibody markers have been reported or implemented. Meanwhile, many antibody markers have been identified with respect to cerebrovascular and cardiovascular disorders. For example, 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) are reported as antibody markers with respect to acute ischemic stroke (AIS); ATP2B4 (Non-Patent Document 6), BMP-1 (Non-Patent Document 2, 6), DHPS (Non-Patent Document 7), SH3BP5 (Non-Patent Document 8), and prolylcarboxypeptidase (Non-Patent Document 9) are reported as markers with respect to atherosclerosis; nardilysin (NRD1) is reported as a marker with respect to acute coronary syndrome (Non-Patent Document 10); and 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) are reported as markers with respect to diabetes mellitus (DM).
Regarding antibody biomarkers with respect to cancers, an anti-p53 antibody has been implemented as a marker which can be effectively employed for the diagnosis, monitoring, and prediction of prognosis of esophageal squamous cell carcinoma (ESCC) or head and neck cancer (Non-Patent Documents 16 and 17). The group to which the present inventors belong previously conducted screening through serological identification of antigens by recombinant cDNA expression cloning (SEREX), and found and reported 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) as antibody markers with respect to ESCC; FIR/PUF60 (Non-Patent Document 25) as an antibody marker with respect to colorectal cancer; and SH3GL1 (Non-Patent Document 26) and anti-filamin C (Non-Patent Document 27) as antibody markers with respect to glioma.
According to the Vital Statistics (2018) of the Ministry of Health, Labour and Welfare of Japan, cancers, heart diseases, cerebrovascular diseases, and renal failure are ranked as the first, second, fourth, and eighth leading causes of death of Japanese, respectively. Also, senescence, pneumonia, unexpected accidents, aspiration pneumonitis, dementia, and suicide are ranked as the third, fifth, sixth, seventh, ninth, and tenth causes of death of Japanese, respectively. According to the WHO, ischemic heart disease, cerebral stroke, trachea/bronchus/lung cancers, diabetes mellitus, and kidney diseases are ranked as the first, second, fifth, eighth, and ninth leading causes of death in the world (2019). Among these causes of death, cancers, heart diseases, cerebrovascular diseases, and renal failure, and diabetes mellitus which is a risk factor for such organic diseases, are conditions whose onset and progress can be prevented by health administration such as early diagnosis. Thus, if appropriate treatment, therapy, daily life guidance, etc. with respect to these conditions can be conducted by use of disease-specific markers, it can make a significant contribution to promotion of human health.
In the coronavirus (COVID-19) pandemic of 2020, the presence of pre-existing condition (e.g., diabetes mellitus, heart diseases, cerebrovascular diseases, cancers, and kidney diseases) is thought to have been one key factor for aggravation of COVID-19. Needless to say, such pre-existing conditions are also general factors for aggravation of infections other than COVID-19. Thus, there is keen demand for provision of a biomarker which can easily detect the presence of such a pre-existing condition, which may cause aggravation of such infections.
Under such circumstances, the related art will further be reviewed. The serum levels of the aforementioned humoral antibodies (i.e., ATP2B4, BMP-1, and DHPS), serving as markers with respect to atherosclerosis, were found to rise also in samples of esophageal cancer patients (see Non-Patent Documents 6 and 7). That is, “artery lesions” typically including arteriosclerosis (e.g., atherosclerosis) can conceivably promote carcinogenesis. Actually, “angiogenesis,” which is one example of “artery lesions,” is an essential factor for the progress of solid cancer (see Non-Patent Document 28), and diabetes mellitus and obesity, which may induce arteriosclerosis, are risk factors for colorectal cancer and esophageal cancer (see Non-Patent Documents 29, 30, and 31). Meanwhile, since internal organs interact with one another to a greater or lesser extent (see Non-Patent Document 32), detection of “artery lesions” such as arteriosclerosis and angiogenesis may capture symptoms of cerebrovascular and cardiovascular disorders, diabetes mellitus, kidney diseases, solid cancers, etc., thereby possibly leading to early detection of such diseases. If these life-threatening diseases can be detected in an early stage, or symptoms thereof can be detected, by use of a single biomarker, the value of the biomarker would be highly significant.
In addition, it would be remarkably significant to provide means for easily determining, by use of a single biomarker, the presence of a pre-existing condition, which may cause aggravation of infections.
The present inventors have found that the presence of an artery lesion is detected by determining a rise in the level of an antibody to protein KIAA0513 in body fluid, whereby data showing the symptoms or presence of a disease involving the artery lesion can be acquired. The inventors have also found that data showing the potential or actual presence of cerebrovascular or cardiovascular disorder, diabetes mellitus, chronic kidney disease, or a solid cancer can also be acquired by determining a rise in the aforementioned humoral antibody level. The inventors have further found that the data showing the potential or actual presence of cerebrovascular or cardiovascular disorder, diabetes mellitus, chronic kidney disease, or a solid cancer may also be employed as data showing the presence of an artery lesion involved in the diseases. The inventors have yet further found that data showing the symptoms or presence of a disease involving an artery lesion, or data showing the potential or actual presence of cerebrovascular or cardiovascular disorder, diabetes mellitus, chronic kidney disease, or a solid cancer are acquired by detecting the presence of an artery lesion based on a rise in the level of an antibody to protein KIAA0513 in body fluid as an index, whereby the presence of a pre-existing condition which is a factor for aggravation of infections can be determined.
Accordingly, in a first aspect of the present invention, there is provided a method for acquiring data, the method comprising: determining a level of an antibody to a protein having an amino acid sequence represented by SEQ ID NO: 1 or 2 which forms protein KIAA0513, or a portion of the protein, or to a protein having an amino acid sequence represented by SEQ ID NO: 1 or 2 in which 10% or less of the amino acid residues (the decimal point being suppressed) are deleted, substituted, or added, or a portion of the protein, the antibody being potentially present in a body fluid sample collected from a living body; and acquiring data showing a rise in the antibody level as data showing the presence of an artery lesion (hereinafter, the method may also be referred to as the “data acquisition method of the present invention 1”).
In a second aspect of the present invention, there is provided a method for acquiring data, the method comprising: determining a level of an antibody to a protein having an amino acid sequence represented by SEQ ID NO: 1 or 2 which forms protein KIAA0513, or a portion of the protein, or to a protein having an amino acid sequence represented by SEQ ID NO: 1 or 2 in which 10% or less of the amino acid residues (the decimal point being suppressed) are deleted, substituted, or added, or a portion of the protein, the antibody being potentially present in a body fluid sample collected from a living body; and acquiring data showing a rise in the antibody level as data showing the potential or actual presence of cerebrovascular or cardiovascular disorder, diabetes mellitus, chronic kidney disease, or a solid cancer (hereinafter, the method may also be referred to as the “data acquisition method of the present invention 2”).
The data showing the onset of the diseases which data are obtained through the data acquisition method of the present invention 2 may also be employed as data showing the presence of an artery lesion involved in the diseases.
Unless otherwise specified, the expression “the data acquisition method of the present invention” in the present specification collectively refers to the data acquisition methods of the present invention 1 and 2.
Meanwhile, protein KIAA0513 is known to include three splicing variants: 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).
The 301 full-length amino acids of KIAA0513 isoforms c or b completely coincide with the initial 301 amino acids of KIAA0513 isoform a. The nucleotide sequence of an mRNA precursor encoding KIAA0513 isoform c differs from that encoding isoform b. However, the amino acid sequence of protein KIAA0513 formed via expression of mature mRNA formed through splicing as a template is common. Thus, in the present specification, without being particularly specified, the expression “protein KIAA0513” refers to protein KIAA0513 isoforms b and/or c (SEQ ID NO: 1). A particular specification is given, in the case where “protein KIAA0513” is intended to refer to isoform a.
Additionally, the present inventors have obtained purified GST-KIAA0513 isoforms c and a, and have investigated the serum level of an antibody to protein KIAA0513 in serum samples of healthy donors (HD), acute ischemic stroke (AIS) patients, and cardiovascular disorder (CVD) patients (Example 6).
As a result, they have found that both KIAA0513 isoforms c and a exhibit a higher serum antibody level in AIS patients and CVD patients, as compared with in HDs (
The test results have revealed that the main epitope site of a humoral antibody to protein KIAA0513 is present in KIAA0513 isoform c and the initial 301 amino acids of KIAA0513 isoform b. Therefore, “a portion of protein KIAA0513” for use in detection of the humoral antibody to protein KIAA0513 based on antigen-antibody reaction preferably includes the aforementioned overlapping (common) amino acid sequence of the KIAA0513 isoforms c, b, and a.
The data acquisition method of the present invention may also be expressed as, for example, “a method for detecting an artery lesion, the method comprising: determining a level of an antibody to a protein having an amino acid sequence represented by SEQ ID NO: 1 or 2 which forms protein KIAA0513, or a portion of the protein, or to a protein having an amino acid sequence represented by SEQ ID NO: 1 or 2 in which 10% or less of the amino acid residues (the decimal point being suppressed) are deleted, substituted, or added, or a portion of the protein, the antibody being potentially present in a body fluid sample collected from a living body; and detecting an artery lesion based on a rise in the antibody level as an index,” and a method for diagnosing or detecting cerebrovascular or cardiovascular disorder, diabetes mellitus, chronic kidney disease, or a solid cancer, the method comprising: determining a level of an antibody to a protein having an amino acid sequence represented by SEQ ID NO: 1 or 2 which forms protein KIAA0513, or a portion of the protein, or to a protein having an amino acid sequence represented by SEQ ID NO: 1 or 2 in which 10% or less of the amino acid residues (the decimal point being suppressed) are deleted, substituted, or added, or a portion of the protein, the antibody being potentially present in a body fluid sample collected from a living body; and diagnosing or detecting cerebrovascular or cardiovascular disorder, diabetes mellitus, chronic kidney disease, or a solid cancer based on a rise in the antibody level as an index.”
The present invention also provides a kit for acquiring data, the kit being employed in carrying out the data acquisition method 1 or 2 of the present invention (hereinafter, the kit may also be referred to simply as the “kit of the present invention”).
The data acquiring kit of the present invention may also be expressed as, for example, “a kit for carrying out a method for detecting an artery lesion, the method comprising: determining a level of an antibody to a protein having an amino acid sequence represented by SEQ ID NO: 1 or 2 which forms protein KIAA0513, or a portion of the protein, or to a protein having an amino acid sequence represented by SEQ ID NO: 1 or 2 in which 10% or less of the amino acid residues (the decimal point being suppressed) are deleted, substituted, or added, or a portion of the protein, the antibody being potentially present in a body fluid sample collected from a living body; and detecting an artery lesion based on a rise in the antibody level as an index,” and “a kit for carrying out a method for diagnosing or detecting cerebrovascular or cardiovascular disorder, diabetes mellitus, chronic kidney disease, or a solid cancer, the method comprising: determining a level of an antibody to a protein having an amino acid sequence represented by SEQ ID NO: 1 or 2 which forms protein KIAA0513, or a portion of the protein, or to a protein having an amino acid sequence represented by SEQ ID NO: 1 or 2 in which 10% or less of the amino acid residues (the decimal point being suppressed) are deleted, substituted, or added, or a portion of the protein, the antibody being potentially present in a body fluid sample collected from a living body; and diagnosing or detecting cerebrovascular or cardiovascular disorder, diabetes mellitus, chronic kidney disease, or a solid cancer based on a rise in the antibody level as an index.”
According to the present invention, firstly, data showing the presence of an artery lesion can be acquired. Based on the data, the symptoms and presence of a disease involving the artery lesion can be grasped. Through taking measures including daily-life guidance and medication on the basis of the data, promotion of health as well as prevention/treatment of diseases can be successfully achieved. Secondarily, there can be acquired data showing the potential or actual presence of cerebrovascular or cardiovascular disorder, diabetes mellitus, chronic kidney disease, or a solid cancer. Based on the data, the onset of the aforementioned grave diseases can be predicted, and these diseases can be detected in an early stage. Through taking measures including daily-life guidance and medication on the basis of the data, prevention/treatment of diseases can be successfully achieved.
The present invention further provides basic data for determining the presence of a pre-existing condition, which is a factor for aggravation of infections.
The data acquired through the data acquisition method of the present invention 1 are “data showing the presence of an artery lesion.” The artery lesion corresponds to abnormality in the structure and components of an artery, and modes thereof include occlusion, expansion, and fistulation. The progress of artery lesion involves arteriosclerosis (atherosclerosis, arteriolosclerosis, or medial sclerosis), anomalous angiogenesis in chronic inflammation or solid cancer, and other conditions. In many cases, arteriosclerosis is known to be triggered by progress of diabetes mellitus and obesity. Particularly, cerebrovascular and cardiovascular disorders, chronic kidney disease, aortic aneurysm, retinopathy, feet gangrene, etc. are known to be triggered by progress of arteriosclerosis, in particular, atherosclerosis. Diabetes mellitus and obesity are also risk factors for solid cancers (colorectal cancer, esophageal cancer, etc.). Thus, the “data showing the presence of an artery lesion” obtained by the data acquisition method of the present invention 1 may be employed as data showing the possibility of onset or progress of diabetes mellitus, arteriosclerosis, solid cancer, etc. In addition, the data may also be employed as data showing the potential or actual presence of severe circulatory diseases such as cerebrovascular and cardiovascular disorders, chronic kidney disease, aortic aneurysm, retinopathy, and feet gangrene.
The data acquired through the data acquisition method of the present invention 2 are “data showing the potential or actual presence of diabetes mellitus, cerebrovascular or cardiovascular disorder, chronic kidney disease, or solid cancers.” Examples of the cerebrovascular or cardiovascular disorder include cerebral infarction, cerebral stroke, myocardial infarction, and angina pectoris. Examples of the solid cancer include esophageal cancer, gastric cancer, colorectal cancer, lung cancer, and mammary cancer. The rise in the level of the humoral antibody to protein KIAA0513 can be employed as reliable data showing the potential or actual presence of esophageal cancer, colorectal cancer, gastric cancer, or lung cancer, particularly esophageal cancer. The data acquisition method of the present invention 2 often overlaps with the data acquisition method of the present invention 1.
For example, the onset of any of the aforementioned diseases in a subject can be accurately predicted by periodically conducting the data acquisition method of the present invention 1 or 2 on the patient, including monitoring of the level of the humoral antibody to “protein KIAA0513.” Particularly, once a subject has developed a severe circulatory disease such as cerebral infarction or myocardial infarction or a solid cancer, the life of the subject is endangered. Even if death is avoided, the subject may suffer a severe sequela. Therefore, prediction of the onset of such diseases is the best solution, and a humoral anti-KIAA0513 antibody marker may be employed as a biomarker for acquiring data concerning prediction of the onset.
When epidemic of a specific infection is recognized, the presence of a pre-existing condition, which is a factor for aggravation of the infection, is determined on the basis of the data acquired through the data acquisition method of the present invention 1 or 2. As a result, the patient suffering the infection can be more suitably treated. In the case where the obtained data indicate the presence of, for example, an artery lesion, the patient contracting the infection is preferentially hospitalized so as to prevent aggravation. In the case where the data indicate no artery lesions, the subject is made to stay at home, whereby hospital capacity including medical staff, beds, etc. can be effectively furnished. In this case, the infected subjects can be appropriately treated in accordance with their degree of aggravation risk. Thus, the risk of medical care breakdown, which would otherwise be caused by shortage of medical staff and beds due to epidemic of infection, can be reduced.
The aforementioned infection includes various viral diseases, bacterial diseases, etc. No particular limitation is imposed on the infection, so long as the presence of a pre-existing condition is an aggravation factor of the infection. Particularly in the case of infections in the respiratory system, which occasionally cause outbreak, the aggravation risk of the infections is desirably estimated by mass examination (screening). The present invention, which can be implemented in a simple mode (e.g., immunochromatography), is particularly suited for such mass examination. Examples of the aforementioned infections include, but are not limited to, respiratory system infections such as infections with beta-coronaviruses (for example, COVID-19, SARS, and MARS); infections with influenza viruses; and infections with RS virus, adenovirus, rhinovirus, and the like.
(b) Body Fluid Sample Collected from Living Body
The term “body fluid” of “a body fluid sample collected from a living body” which is a target for data acquisition in the data acquisition method of the present invention, includes blood, lymph, or the like. The term “sample” refers to body fluid itself as collected from a body, or a processed product of the fluid. Among such body fluid samples, a blood sample is preferred. Examples of the blood sample include whole blood samples, serum samples, and plasma samples. Of these, serum samples and plasma samples are preferred, with serum samples being particularly preferred. The blood samples may undergo anti-clotting treatment (e.g., treatment with heparin) in advance.
In the data acquisition method of the present invention, protein KIAA0513 having an amino acid sequence represented by SEQ ID NO: 1 or 2 is the basis for capturing a humoral antibody. The entirety or a portion of protein KIAA0513 may be used as a humoral antibody-capturing antigen. Furthermore, in order to establish a more efficient detection system, a shorter chain amino acid sequence (peptide) may be selected. In this case, the number of amino acids of the amino acid sequence is about 7 to about 30, more preferably about 10 to 20.
In the entirety or a portion of any of the amino acid sequences, 10% or less of the amino acid residues (the decimal point being suppressed) may undergo modification (deletion, substitution, or addition), so long as the target humoral antibody (i.e., a humoral antibody to protein KIAA0513 or to a peptide having a partial sequence thereof) can be captured. In other words, when the amino acid-modified antigen candidate for capturing a target humoral antibody is used for capturing the humoral antibody through, for example, a technique similar to the technique described in the Examples of the present specification, data concerning the target artery lesion or disease must be acquired on the basis of the level of the captured antibody.
The term “deleting” refers to deletion of any amino acid residue in the relevant amino acid sequence. The amino acid residue at the N-terminal side of the deleted amino acid residue and that at the C-terminal side of the deleted amino acid residue are linked via a peptide bond. In the case of deletion of the N-terminal amino acid residue or the C-terminal amino acid residue, no linkage is present. The number of deleted residues is counted as “the number of amino acid deletions.” The term “substituting” refers to substitution of any amino acid residue in the relevant amino acid sequence, “with another amino acid residue.” The new amino acid residue is linked to the amino acid residue at the N-terminal side thereof and that at the C-terminal side thereof via a peptide bond. In the case of substitution of the N-terminal side or C-terminal side amino acid residue, the amino acid residue is linked via a peptide bond to another C-terminal side or N-terminal side amino acid residue. The number of substituted residues is counted as “the number of amino acid substitutions.” The term “adding” refers to addition of one or more new amino acid residues to one or more peptide bond sites in the relevant amino acid sequence, to thereby form a new peptide bond(s). Addition of an amino acid residue to the C terminal or the N terminal is also encompassed in the concept “addition.” The number of added substituted amino acid residues is counted as “the number of amino acid additions.” Hereinafter, “a protein having an amino acid sequence represented by SEQ ID NO: 1 or 2 which forms protein KIAA0513, or a portion of the protein, or a protein having an amino acid sequence represented by SEQ ID NO: 1 or 2 in which 10% or less of the amino acid residues (the decimal point being suppressed) are deleted, substituted, or added, or a portion of the protein” is also referred to as a “KIAA0513 antigen.”
The KIAA0513 antigen having a specific amino acid sequence may be obtained through a customary method. In one specific procedure, based on the nucleotide sequence of the KIAA0513 gene represented by SEQ ID NO: 1 or 2, nucleic acid amplification primers for amplifying a double-strand DNA including the entirety or a portion of nucleotide sequence are designed. Through PCR or a similar technique employing the amplification primers, a gene amplification product is yielded as the entirety or a portion of the KIAA0513 gene. The amplification product is incorporated into a prokaryotic cell expression vector (e.g., pGEX-4T), and a transformant including the vector is selected. Expression of the entirety or a portion of the KIAA0513 gene is induced by adding a chemical agent such as IPTG (isopropyl-β-
If needed, a modification structure may be appropriately added to the protein or the peptide used as such a KIAA0513 antigen. For example, as mentioned hereinbelow, there may be employed a GST (glutathione S-transferase)-fused protein or peptide, a biotin-fused protein or peptide, etc., which are used in AlphaLISA (Amplified Luminescence Proximity Homogeneous Assay-Linked ImmunoSorbent Assay).
According to the data acquisition method of the present invention, the level of an antibody to the KIAA0513 antigen in a body fluid sample (e.g., a blood sample) collected from a living body is determined. When the determined value is greater than a standard value (cut-off value), the increase in KIAA0513 antigen level in the body fluid sample donor is confirmed. The data can be employed as an index for showing the presence of an artery lesion in the body fluid sample donor, or for showing the potential or actual presence of cerebrovascular or cardiovascular disorder, diabetes mellitus, chronic kidney disease, or a solid cancer in the body fluid sample donor.
The standard value of the humoral antibody level with respect to the KIAA0513 antigen in a test sample may be derived through the following procedure. First, a control sample population is established from subjects exhibiting no abnormality (e.g., diabetes, a respiratory disease, or a cancer) in a medical examination (i.e., healthy donors). The level of a humoral antibody to the KIAA0513 antigen is determined, with respect to the test samples obtained from the control sample population. The determination data are statistically processed, to thereby obtain an average, a standard deviation, etc., and derive the standard value including the cut-off value, on the basis of the data.
In one possible embodiment of the humoral antibody level determination, the KIAA0513 antigen which is immobilized on a substrate is brought into contact with a body fluid sample collected from a living body. Bonding (based on antigen-antibody reaction) of a humoral antibody to the KIAA0513 antigen in the body fluid sample collected from the living body is detected as a signal. More specifically, the determination can be performed through immuno-chromatography, AlphaLISA, ELISA, indirect immunofluorescence, Western blotting (immunoblotting), turbidimetry, nephelometry, latex-coagulation turbidimetry, CLEIA, or the like. All the exemplified determination techniques have been established for determining the level of a target substance (antibody) in a body fluid sample collected from a living body.
In one mode of the immuno-chromatographic method, a body fluid sample and a buffer liquid containing a gold colloid-bound anti-human IgG antibody are added to a cellulose membrane onto which the KIAA0513 antigen has been applied, and the liquid mixture is slowly moved on the membrane. If an antibody to the KIAA0513 antigen is present in the body fluid sample, a line is formed at a position where the antigen is applied on the surface of the membrane. The line can be visually observed. Since the immuno-chromatographic method can be conducted in a simpler manner, as compared with other assay means, the method is particularly suited for mass examination. In other words, when the data acquisition method of the present invention is carried out through the immuno-chromatographic method, desired disease-related data can be acquired in a short period of time (about 10 minutes) even in small facilities (for example, a clinic), a public health center, and other facilities.
In the case of AlphaLISA, when a GST-fused KIAA0513 antigen is used as an antigen for capturing a humoral antibody, glutathione-conjugated donor beads are used. The antigen for capturing a humoral antibody, a blood sample, and acceptor beads to which an anti-human IgG antibody has been bound are mixed together, and the mixture is incubated at room temperature for several hours to several days, to thereby form an antigen-antibody complex. The complex is irradiated with light (680 nm), and the resultant light having a wavelength of 520 to 620 nm is detected, whereby the target humoral antibody can be quantitated. In indirect immunofluorescence assay, a body fluid sample collected from a living body is brought into contact with a protein array in which the KIAA0513 antigen has been immobilized on a substrate, to thereby form a complex of the KIAA0513 antigen with an anti-KIAA0513 humoral antibody. Then, a fluorescent-labeled secondary antibody is brought into contact with the humoral antibody complex, whereby a humoral antibody to protein KIAA0513 can be quantitated. In ELISA, the same secondary antibody as employed in indirect immunofluorescence assay is used, except that the label is changed to an enzyme, to thereby conduct quantitation. The label for the secondary antibody can be selected from various enzymes. In Western blotting, a KIAA0513 antigen is subjected to electrophoresis by use of SDS-polyacrylamide gel, and the resultant band is transferred to a carrier such as a nitrocellulose membrane. Then, a body fluid sample collected from a living body is brought into contact with the carrier, to thereby form a (KIAA0513 antigen)-(anti-KIAA0513 humoral antibody) complex. Detection of the complex is performed by use of a secondary antibody, to thereby achieve quantitation. In turbidimetry or nephelometry, a body fluid sample collected from a living body is brought into contact with the KIAA0513 antigen, to thereby form a (KIAA0513 antigen)-(anti-KIAA0513 humoral antibody) complex. Detection of the complex is performed on the basis of change in turbidity (in the case of turbidimetry) or scattered light (in the case of nephelometry), to thereby achieve quantitation. In latex agglutination turbidimetry, a body fluid sample collected from a living body is brought into contact with latex particles to which the KIAA0513 antigen has been bound, to thereby form agglutinated latex particles via interaction between the humoral anti-KIAA0513 antibodies bounded to latex particles, and the thus-formed agglutinated latex particles are quantitated. In one possible mode of CLEIA assay, a body fluid sample collected from a living body is brought into contact with magnetic particles to which the KIAA0513 antigen has been bound, to thereby form, on the surfaces of the magnetic particles, a (KIAA0513 antigen)-(anti-KIAA0513 antibody) complex. Unreacted matter is removed through collecting magnetism, and the product is subjected to an appropriate fluorescent chemical treatment or the like. Thus thus-treated complex is quantitated.
An embodiment of the kit of the present invention employs AlphaLISA as an assay means and has a set including a GST-fused KIAA0513 antigen, glutathione-conjugated donor beads, and anti-human IgG-conjugated acceptor beads.
Another embodiment of the kit of the present invention employs ELISA as an assay means and has a set including a plate on which the aforementioned KIAA0513 antigen has been immobilized, a labeled secondary antibody to the anti-KIAA0513 antibody, and a reagent for developing the label of the secondary antibody.
Still another embodiment of the kit of the present invention employs latex agglutination turbidimetry and includes latex particles on which the aforementioned KIAA0513 antigen has been immobilized.
Yet another embodiment of the kit of the present invention employs CLEIA and includes magnetic particles on which the aforementioned KIAA0513 antigen has been immobilized, a labeled secondary antibody with respect to the anti-KIAA0513 antibody, and a reagent for developing the label of the secondary antibody.
Notably, the aforementioned embodiments of the kit are illustrated merely as examples, and kits employing other assay means are also encompassed in the scope of the kit of the present invention. Furthermore, the number of the aforementioned kit elements may be further reduced so as to encourage self-supply or out-sourcing of medical tests. In contrast, elements such as diluent and reagent tubes are added to the kit so as to realize immediate use of the kit of the present invention. Other additional elements suited for the mode of tests may also be added.
Examples of the present invention will next be described.
In the Examples, by means of a human protein microarray (ProtoArray (registered trademark), product of Thermo Fisher Scientific), proteins KIAA0513 were identified as antigens which are recognized by a serum antibody of atherosclerosis patients.
In a specific procedure, the N-terminal of “KIAA0513 isoform a” (SEQ ID NO: 2) was modified with GST. The full-length region of 411 amino acids, or a portion of 301 amino acids from the N-terminal was used. Notably, the latter “portion of 301 amino acids from the N-terminal” corresponds to KIAA0513 isoform c (SEQ ID NO: 1).
Human proteins KIAA0513 were prepared through the following procedure.
Each of the cDNA fragments encoding human KIAA0513 isoforms a and c was inserted into an EcoRI/SalI site of a plasmid vector pGEX-4T-1 (GE Healthcare Life Sciences, Pittsburgh, PA), in which a glutathione S-transferase (GST) gene site is provided on the upstream side of the cloning site, to thereby produce recombinant plasmids pGEX-4T-1-KIAA0513 (isoform a) and pGEX-4T-1-KIAA0513 (isoform c) for gene expression, respectively. Each plasmid was incorporated into E. coli BL-21 for transformation, and expression of the corresponding cDNA encoding protein KIAA0513 was induced through a treatment with 0.1 mM IPTG (isopropyl-β-
Prior to disclosure of specific working examples, the analysis through AlphaLISA employed in the Examples will next be outlined.
Specifically, the AlphaLISA (Amplified Luminescence Proximity Homogeneous Assay) was performed by use of a 384-well microtiter plate (white opaque OptiPlate™, Perkin Elmer, Waltham, MA). To each well, a serum (2.5 μL) 100-fold diluted with AlphaLISA buffer (25 mM HEPES, pH: 7.4, 0.1% casein, 0.5% Triton X-100, 1-mg/mL dextran-500, 0.05% Proclin-300) was added. GST or protein KIAA0513 (i.e., KIAA0513 isoform a or c) (10 μg/mL) (antigen), diluted with AlphaLISA buffer, was mixed with the serum in each well. The mixture was incubated at room temperature for 6 to 8 hours. Separately, 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 with AlphaLISA buffer, and the thus-diluted product was mixed with the incubated product. The resultant mixture was allowed to stand at room temperature for 1 to 14 days. Thereafter, generated photons were counted by means of EnSpire Alpha microplate reader (Perkin Elmer), to thereby determine “Alpha Count.” In the case of protein KIAA0513, a control GST value was subtracted, whereas in the case of the biotinylated peptide, a control buffer value was subtracted, whereby the antibody level specific to each KIAA0513 antigen was determined.
Studies of biomarker in transient ischemic attack (TIA) patients and acute ischemic stroke (AIS) patients through AlphaLISA
Acute ischemic stroke (AIS) refers to a pathological condition in which a patient receives sudden damage in motor function or sensory function due to stagnation of blood flow in the brain. Transient ischemic attack (TIA) refers to a disease in which cerebral nerve cells are damaged through the same mechanism as that of cerebral infarction, but the damage is restored within 24 hours, generally several minutes. In both cases, blood serum samples are collected from relevant patients ≤2 weeks after the onset thereof.
The serum antibody (s-KIAA0513-Abs: serum KIAA0513 antibody) level with respect to KIAA0513 isoform c (SEQ ID NO: 1) was determined in AIS patients and TIA patients. Serum samples of healthy donors (HDs), AIS patients, and TIA patients were collected from Chiba Prefectural Sawara Hospital. In AlphaLISA, the s-KIAA0513-Ab level was found to be significantly higher in TIA patients and AIS patients than in HDs (see
When the cut-off value was set to “the average+2SD for HD samples,” the s-KIAA0513-Ab positive ratios of HDs, AIS patients, and TIA patients were 0.0%, 7.6%, and 15.6%, respectively (see Table 1).
Through ROC analysis, area under the curve (AUC) values of s-KIAA0513-Abs were found to be 0.6439 [95% confidence interval (CI)=0.5873-0.7004] with respect to AIS (see
Thus, TIA, which is a prodromal stage of AIS, was also found to positively correlate with the serum KIAA0513 antibody level. Therefore, the s-KIAA0513-Ab level served as a marker effectively predicting the onset of AIS.
Next, the s-KIAA0513-Abs level of DM patients was investigated.
Serum samples of HDs and DM patients were obtained from Chiba University Hospital.
The s-KIAA0513-Abs level was found to be significantly higher in DM patients than in HDs (see
When the cut-off value was set to “the average+2SD for HD samples,” the s-KIAA0513-Ab positive ratios of HDs and DM patients were 2.5% and 26.5%, respectively (see Table 2).
The ability of the antibody marker to indicate the presence of DM was evaluated through ROC analysis. The area under the curve (AUC) value of s-KIAA0513-Abs was 0.7361, and the values of sensitivity and specificity were 50.55% and 87.65%, respectively (see
Studies of biomarker in cardiovascular disorder (CVD) patients and obstructive sleep apnea syndrome (OSAS) patients through AlphaLISA
Next, the s-KIAA0513-Abs levels of CVD patients and OSAS patients were investigated.
The CVD patients in Chiba University Hospital included acute myocardial infarction patients and unstable angina patients. Generally, OSAS is known to relate to atherosclerosis and is a high risk factor for acute ischemic stroke (AIS) and CVD. Thus, serum samples of OSAS patients obtained from Chiba University Hospital were also analyzed.
As a result, the s-KIAA0513-Abs level was found to be higher in CVD patients and OSAS patients than in HDs (
Through ROC analysis, area under the curve (AUC) values of s-KIAA0513-Abs were found to be 0.6492 (95% CI: 0.5821-0.7163) with respect to CVD (see
The P value of s-KIAA0513-Ab with respect to CVD was small (<0.0001), but the P value with respect to OSAS was 0.0013. The data suggest that the correlation between the s-KIAA0513-Ab marker and OSAS is less significant than the correlation between the s-KIAA0513-Ab marker and CVD (see Table 3). Thus, conceivably, OSAS does not correlate directly with the s-KIAA0513-Ab marker, but does indirectly correlate with the marker by the mediation of CVD or the like, which is induced by OSAS.
Next, the s-KIAA0513-Abs level of CKD patients was investigated. CKD is closely related to atherosclerosis.
The studied CKD patients were divided into three groups.
Specifically, the groups were the diabetic nephropathy group (Type 1), the nephrosclerosis group (Type 2), and the glomerulonephropathy group (Type 3).
Serum samples of CKD patients were obtained from a cohort of Kumamoto Prefecture, and those of HDs were obtained from Chiba University.
The serum s-KIAA0513-Abs level was found to be significantly higher in all the three CKD groups than in HD samples (
Through ROC analysis, area under the curve (AUC) values of s-KIAA0513-Abs were found to be 0.7434 (95% CI: 0.6783-0.8085) with respect to Type 1 CKD (see
Atherosclerosis is known to be often related to cancers. Thus, the s-KIAA0513-Abs level in serum samples of esophageal cancer (EC), gastric cancer (GC), colon cancer (CC), lung cancer (LC), and mammary cancer (MC) patients was investigated. The serum samples were obtained from Toho University Medical Center.
As a result, the s-KIAA0513-Abs level was significantly higher in serum samples of all cancer patients, as compared with those of HDs. The highest average value of s-KIAA0513-Ab level was identified in the case of esophageal cancer (see
Next, correlation of the s-KIAA0513-Ab level with survival rates of esophageal cancer (EC) patients and gastric cancer (GC) patients after surgery was investigated. On the basis of the cut-off value obtained through ROC analysis, analytical samples were divided into an s-KIAA0513-Ab positive group and an s-KIAA0513-Ab negative group (see
The serum antibody levels with respect to KIAA0513 isoform c (SEQ ID NO: 1) and KIAA0513 isoform a (SEQ ID NO: 2) were determined in healthy donors (HDs), acute ischemic stroke (AIS) patients, and cardiovascular disorder (CVD) patients through AlphaLISA.
As a result, with respect to both KIAA0513 isoforms c and a, the serum antibody levels were found to be higher in AIS patients and CVD patients than in HDs (see
The test results have revealed that the main epitope site of a serum antibody to protein KIAA0513 is present in 301 amino acids of KIAA0513 isoform c.
Correlation analysis between s-KIAA0513-Ab levels and test subject data was carried out by use of 665 serum samples collected from subjects in Chiba Prefectural Sawara Hospital. The samples included 139 HD samples, 225 samples of AIS patients, 44 samples of TIA patients, 17 samples of asymptomatic ischemic cerebral stroke patients, 122 samples of deep and subcortical white matter hyperintensity (DSWMH) patients, 59 samples of chronic ischemic stroke (CIS) patients, and 41 control samples.
In the correlation analysis, Mann-Whitney U analysis was employed to compare s-KIAA0513-Ab levels with the parameters: sex (male/female), DM, hypertension (HT), CVD, lipidemia, and obesity [“normal or thin” when body mass index (BMI) is <25; and “obese” when BMI is ≥25], smoking habit, and drinking habit. As a result, a significant difference was found only in the case of presence/absence of HT (Table 6).
Through Spearman's correlation analysis, correlations of s-KIAA0513-Ab level with continuous variables, specifically, general information parameters of subjects including age, height, weight, and BMI; degree of arteriostenosis including maximum intima-media thickening (max-IMT); life-style factors such as period of smoking (years) and alcohol frequency (/week); and blood test data, were investigated. Significant correlations were identified between s-KIAA0513-Abs level and age, max-IMT, ALP, potassium, C-reactive protein (CRP), body sugar (BS), and smoking period (see Table 7). Inverse correlations were observed between the antibody level and height and weight. Correlation between the antibody level and max-IMT indicates that the s-KIAA0513-Ab level is associated with stenosis and atherosclerosis.
Similar results were obtained using other cohorts. In Spearman's correlation analysis of a CKD cohort (300 cases), significant correlations of the antibody level were observed with plaque score, max-IMT, and CAVI (right), which are indices for atherosclerosis (see Table 8). In the CKD cohort, correlation was observed between CRP and the s-KIAA0513-Abs level, indicating certain association of inflammation. Meanwhile, in the CKD cohort, no significant correlations of the s-KIAA0513-Abs level were observed with age, height, weight, BMI, and potassium. AIS is closely related with age, and positive correlation was observed between the s-KIAA0513-Abs level and AIS. Therefore, the s-KIAA0513-Abs may be indirectly associated with aging.
<0.0001
0.0007
0.0049
0.0002
0.0295
0.0228
0.0453
0.0229
0.0009
<0.0001
0.0468
0.0371
0.0273
0.0040
0.0012
0.0205
0.0240
0.0078
0.0016
Through primary screening by use of a human protein microarray, protein KIAA0513 was identified as an antigen recognized by a serum antibody of atherosclerosis patients. Then, a recombinant GST-tagged protein KIAA0513 was purified. By use of the thus-purified protein as an antigen, the serum antibody level was determined through AlphaLISA. As a result, the s-KIAA0513-Abs level was found to be significantly higher in patients of AIS, TIA, DM, CVD, OSA, CKD, EC, GC, CC, LC, and MC than in HDs (see
Currently, no precise function of protein KIAA0513 has been elucidated. However, protein KIAA0513 has been reported to bind to a protein involved in synaptic transmission and apoptosis induction. Thus, conceivably, protein KIAA0513 may be involved in at least such signal transmission.
Generally, humoral antibody markers exhibit a higher sensitivity of detecting data concerning diseases, as compared with humoral antigen markers. Among humoral antigens, a KIAA0513 antigen has particularly high antigenicity. Thus, the anti-KIAA0513 humoral antibody marker exhibits remarkably high sensitivity. The above experiments have revealed that the present invention can acquire data concerning the potential or actual presence of diseases (e.g., cancers, cerebral infarction, myocardial infarction, diabetes mellitus, and chronic kidney disease) which are fatal to humans, as well as data showing the presence of an artery lesion (e.g., arteriosclerosis, or anomalous angiogenesis in solid cancer).
Hitherto, there have been known only a small number of easily employable markers for life-threatening grave diseases including cancers, cerebral infarction, myocardial infarction, diabetes mellitus, chronic kidney disease, etc., and individual disease-specific markers must be used. However, the data acquisition method of the present invention enables to acquire data concerning diseases at remarkably high sensitivity and to acquire comprehensive data concerning grave diseases. In addition, by acquiring data showing the presence of an artery lesion (e.g., arteriosclerosis, or anomalous angiogenesis in solid cancer), it is possible to use the acquired data as an index for disease prevention. Thus, the method of the present invention is suited for mass examination or the like.
As described above, according to the present invention, the presence of pre-existing conditions, which are factors for aggravation of an infection, can be comprehensively detected by use of a single biomarker. If the data acquisition method of the present invention is carried out in mass examination in response to epidemic of respiratory tract infection caused by novel coronavirus (COVID-19) or an influenza virus, hospital capacity including medical staff, beds, etc. can be effectively furnished, and better treatment for the infection can be attained.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2020-170489 | Oct 2020 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2021/035474 | 9/27/2021 | WO |
