JP6312302B2 - Diagnostic marker for cerebral infarction - Google Patents

Description

The present invention also relates to a diagnostic marker of cerebral infarction.

  Cerebral infarction is a state in which cerebral blood vessels are blocked or narrowed, resulting in cerebral ischemia and necrosis of brain tissue. Causes of cerebral infarction are mainly divided into thrombotic, embolic and hemodynamic, and specifically, atherothrombotic cerebral infarction, cardiogenic cerebral infarction, lacunar infarction and others.

  In either case, the cells downstream of the blocked or constricted cerebral blood vessels are necrotic, resulting in paralysis, speech impairment, blindness, dizziness, ataxia, impaired consciousness, and higher brain dysfunction, often leading to death.

  At the cerebral infarction site, nerve cells and glial cells are damaged by inflammation-related substances, oxidative stress, endoplasmic reticulum stress, etc., and the cerebral infarction lesion extends widely. Cerebral infarction is a neurodegenerative disease mainly characterized by central nervous system dysfunction. It is an acute stage of cerebral infarction that leads to infarct formation following degeneration / necrosis of nerve cells, and sub-stages from infarct formation to fixation of central neuropathy. Pathological and histological classification into chronic and chronic cerebral infarction.

  Cerebral infarction is a major illness of stroke, which is the third leading cause of death in Japan, and has a high mortality rate. It is extremely beneficial to society if a diagnosis of cerebral infarction can be made accurately.

  Patent Document 1 describes a method for diagnosing cerebral infarction by measuring the concentration of nick β2 glycoprotein I in a body fluid sample. Patent Document 2 describes a method for diagnosing cerebral infarction by determining a predetermined SNP.

JP 2003-121444 A JP 2010-166844 A

  However, the technique described above cannot properly diagnose cerebral infarction.

This invention is made | formed in view of this problem, Comprising: It aims at providing the diagnostic marker which can perform the diagnosis of cerebral infarction appropriately.

Diagnostic marker of cerebral infarction of the present invention is characterized in that it is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 113.

  According to the present invention, it is possible to appropriately diagnose cerebral infarction.

  Systemic lupus erythematosus (SLE) is a typical autoimmune disease, a chronic inflammatory disease that presents multiple organ damage due to the appearance of various autoantibodies and immune complex deposition.

  In SLE, it is known that a wide variety of autoantibodies appear due to abnormal function of T lymphocytes and increased activation of B lymphocytes. Anti-nuclear antibodies are detected in more than 95% of patients with SLE. Examples of autoantibodies specific to SLE include anti-dsDNA antibodies and anti-Sm antibodies, which are strongly associated with lupus nephritis, and their presence is one of the diagnostic criteria. Other anti-phospholipid antibodies (thrombosis, stillbirth), anti-U1RNP antibodies (Raynaud phenomenon, pulmonary hypertension), anti-SS-A antibodies (discoid rash, dry symptoms), anti-PCNA antibodies / anti-ribosome P antibodies (central nervous symptoms) ) Many autoantibodies related to clinical symptoms of SLE appear.

  30-40% of SLE patients have anti-phospholipid antibodies such as anti-cardiolipin antibodies and lupus anticoagulation factors, and the incidence of cerebral infarction and myocardial infarction due to thrombus formation is high. The present inventors anticipate that among autoantibodies appearing in SLE patients, there are proteins useful for the diagnosis of cerebral infarction and myocardial infarction, particularly proteins useful for the diagnosis of cerebral infarction. The existence was found as a new finding, and the present invention was completed based on this fact.

  That is, the present inventors performed screening by protoarray method using systemic lupus erythematosus patient sera, identified a plurality of proteins positively reacting with the sera as antigen proteins, and synthesized peptides derived from these antigen proteins. The inventors succeeded in selecting four types of peptides that react specifically with serum antibodies from patients with cerebral infarction or myocardial infarction, and completed the present invention.

  In the present invention, antigenic peptides that bind to autoantibodies in cerebral infarction or myocardial infarction patient body fluid and can be used for diagnosis of cerebral infarction and myocardial infarction are derived from the following proteins, respectively.

SEQ ID NO: 113 SOSTDC1-156 (biotin-KITVVTACKCKRYTR)
SEQ ID NO: 79 CTNND1-211 (biotin-LRNVSSERSEARRKL)
SEQ ID NO: 57 CLDND1-69 (biotin-FRYNGTVGLWRRCIT)
SEQ ID NO: 63 CCNG2-231 (biotin-KKHSKINDTEFFYWR)
That is, SOSTDC1 (SEQ ID NO: 113), CTNND1 (SEQ ID NO: 79), CLDND1 (SEQ ID NO: 57), and CCNG2 (SEQ ID NO: 63) are peptides that react well with cerebral infarction.

  From the viewpoint of multiple diseases, if the above four peptides are examined, SOSTDC1 (SEQ ID NO: 113) reacts well with cerebral infarction, CTNND1 (SEQ ID NO: 79) reacts well with myocardial infarction, and CLDND1 (SEQ ID NO: 57). ) Responds well to cerebral and myocardial infarction, and CCNG2 (SEQ ID NO: 63) responds well to diabetes.

  The following functions are known for each protein.

  SOSTDC1: Abbreviation for sclerostin domain containing 1 (Accession No. NM_015464). The N-terminus is glycated and the C-terminus has a cystine knot-like domain that is secreted. It binds directly to bone morphogenetic protein (BMP), inhibits the binding of BMP and receptor, and suppresses cell growth, differentiation and cell death by BMP. That is, SOSTDC1 acts as an antagonist of BMP.

  CTNND1: Abbreviation for catenin (cadherin-associated protein), delta 1 (Accession No. NM_001085458). A member of the Armadillo protein family. It works to transmit the signal from cadherin, a cell adhesion protein, into the cell.

  CLDND1: Abbreviation for claudin domain containing 1 (Accession No. NM_001040181). Protein with claudin domain involved in tight junctions. The function of CLDND1 itself is unknown.

  CCNG2: Abbreviation for cyclin G2 (Accession No. NM_004354). A member of the cyclin family. Activates cdk and regulates cell cycle. In particular, it is involved in cell cycle arrest at the G2 / M checkpoint after DNA damage.

  The antigen peptide of the present invention comprises an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequences shown in SEQ ID NOs: 113, 79, 57 and 63, and cerebral infarction Or the peptide or protein which has a binding activity with the autoantibody in the bodily fluid of a myocardial infarction patient is contained.

  The above “autoantibodies in cerebral infarction or myocardial infarction patient body fluid” means antibody IgG that is present in the body fluid of cerebral infarction or myocardial infarction patient and binds to a cerebral infarction or myocardial infarction specific antigen peptide. In addition, “binding activity with autoantibodies in cerebral infarction or myocardial infarction patient body fluid” means, for example, an antigen capable of forming an immune complex by an antigen-antibody reaction when reacted with serum of a cerebral infarction or myocardial infarction patient This activity can be confirmed, for example, by ELISA. In addition, “having binding activity with autoantibodies in body fluids of patients with cerebral infarction or myocardial infarction” is substantially equivalent to the activity retained by the peptide having the amino acid sequence described in each of the above SEQ ID NOs. Say.

  The antigenic peptide of the present invention used in a diagnostic kit for cerebral infarction has a function of recognizing and binding to an autoantibody in a cerebral infarction patient body fluid (eg, serum).

  When an autoantibody against a cerebral infarction specific antigen is used as a marker, diagnosis of cerebral infarction can be performed by contacting the antigenic peptide of the present invention with a test sample and detecting and quantifying the antibody present in the test sample.

  Also, from the viewpoint of multiple diseases, SOSTDC1 (SEQ ID NO: 113) responds well to cerebral infarction, CTNND1 (SEQ ID NO: 79) responds well to myocardial infarction, and CLDND1 (SEQ ID NO: 57) corresponds to cerebral infarction and myocardial infarction. Since CCNG2 (SEQ ID NO: 63) reacts well with diabetes, it can be diagnosed using a combination of four peptides consisting of the amino acid sequences shown by each SEQ ID NO.

  In the case of a diagnostic kit for ELISA for antibody detection, the constituent reagents include, for example, the antigen protein of the present invention, a secondary antibody against enzyme-labeled human immunoglobulin, a substrate solution, and the like. The antigen peptide may be immobilized on a solid phase in advance, or may be in a form immobilized on a solid phase at the time of use. In this case, the diagnostic kit may include a solid phase for immobilizing the antigen peptide. In the case of a diagnostic kit for agglutination reaction, it contains carrier particles on which an antigen peptide is immobilized. Further, the antigenic peptide of the present invention may be a peptide array in which a plurality of these types are affixed at a high density, and such a peptide array is also included in the diagnostic kit of the present invention. In this case, the kit may include a mass spectrometer, software necessary for measurement / analysis, a computer in which the software is installed, and the like.

  The test sample may be any sample as long as it can detect an autoantibody against cerebral infarction or myocardial infarction-specific antigen, for example, blood, plasma, serum, synovial fluid, lymph fluid, joint fluid, ascites, saliva, Examples thereof include cerebrospinal fluid and fractional components obtained therefrom.

  “Diagnosis” means determining whether a subject has developed cerebral infarction or myocardial infarction, determining whether there is a risk of developing cerebral infarction or myocardial infarction in the future, and determining the effect of treatment Or the determination of whether there is a risk of recurrence of cerebral infarction or myocardial infarction after treatment.

  A positive antibody marker for cerebral infarction or myocardial infarction-specific antigen means that the amount or concentration of the antibody specifically present in the body fluid of cerebral infarction or myocardial infarction is significant compared to healthy subjects or other autoimmune disease patients High antibody.

  The measurement of the expression level of SOSTDC1 (SEQ ID NO: 113), CTNND1 (SEQ ID NO: 79), CLDND1 (SEQ ID NO: 57), and CCNG2 (SEQ ID NO: 63) is not particularly limited. The expression level of the peptide may be determined relative or absolute. Peptide expression levels are preferably measured by immunological techniques. For example, a combination of separation by electrophoresis and detection or quantification by fluorescence, enzymes, radioisotopes, etc. (Western blotting, two-dimensional fluorescence) (Including electrophoresis), immunostaining (including fluorescent antibody method, enzyme antibody method, heavy metal labeled antibody method, radioisotope labeled antibody method), enzyme immunoassay adsorption method (ELISA), dot blotting method, etc. be able to.

  In the present invention, the physical quantity based on the antigen-antibody reaction measured as described above is used as a clinical index of cerebral infarction or myocardial infarction. The amount of the antigen-antibody complex in patients with cerebral infarction or myocardial infarction increases significantly as compared with that in healthy individuals, and decreases as the treatment of the disease progresses. Therefore, the onset probability of cerebral infarction or myocardial infarction can be estimated by using this antigen-antibody complex amount and its change, in particular, the change with time of the antigen-antibody complex as a clinical index.

  A method for diagnosing cerebral infarction and myocardial infarction using the antigenic peptide of the present invention will be specifically described by way of examples of enzyme immunoassay, agglutination reaction, immunochromatography, and AlphaLISA as preferred embodiments.

(1) Enzyme immunoassay method In the enzyme immunoassay method, an autoantibody against a synthetic peptide antigen in a test sample is reacted with an antigen protein immobilized on a solid phase, a labeled secondary antibody against human immunoglobulin is added, and the autoantibody By measuring the labeled secondary antibody bound to.

  As the solid phase on which the antigen peptide is immobilized, for example, avidinized polyvinyl chloride, polystyrene, polyethylene, polypropylene, polyester, fluororesin, glass, metal or the like can be used. Examples of the shape of the carrier include plates, beads, cells, test tubes and the like. Preferably, a commercially available solid phase such as a microtiter plate for enzyme immunoassay (96-well microtiter plate) can be used. The antigenic peptide is biotinylated in advance, brought into contact with the avidinized solid phase, and left at 4 to 37 ° C. for 1 to 24 hours to immobilize the antigenic peptide. At this time, in order to prevent non-specific adsorption, a buffer solution such as phosphate buffered saline (PBS) containing 1-10% albumin, gelatin, milk powder or fetal bovine serum, or Tris buffer (TBS) is used. It is preferable to use as a blocking solution to block unadsorbed portions. After immobilization, the solid phase is washed with an appropriate buffer.

  The test sample is diluted 10 to 5000 times before incubation. A diluted test sample is added to the solid phase prepared as described above and incubated, whereby the cerebral infarction or myocardial infarction specific antibody (primary antibody) present in the test sample is reacted with the antigen. Incubation is performed at 4 to 37 ° C. for 1 to 24 hours, preferably about 16 hours. After the reaction, the reaction solution is removed and the solid phase is washed.

  Next, a secondary antibody against human immunoglobulin is added. The secondary antibody is an antibody against human immunoglobulin and can be labeled with an enzyme, a radioisotope, a fluorescent substance, a luminescent substance, or the like. Among these, enzyme-labeled secondary antibodies are preferable. For example, goat anti-human immunoglobulin labeled with enzymes such as peroxidase, alkaline phosphatase, β-galactosidase, rabbit anti-human immunoglobulin, and sheep anti-human immunoglobulin are used. Can do. The secondary antibody is diluted about 1000 to 10,000 times before incubation. The reaction is carried out at 4 to 37 ° C. for about 1 to 2 hours. After completion of the reaction, the reaction solution is removed and the solid phase is washed.

  The measurement of the secondary antibody can be performed according to a known method according to the type of the labeling substance of the secondary antibody used. When an enzyme-labeled secondary antibody is used, a color development reaction is performed by adding a known color former in accordance with the selected enzyme. For example, when peroxidase is used, alkaline phosphatase is used with a substrate such as o-phenylenediamine, ABTS [2,2′-azino-bis- (3′-ethylbenzodiazolinesulfonic acid)] and hydrogen peroxide. In the case of using a substrate such as p-nitrophenyl phosphate or 4-methylumbeferyl phosphate, in the case of using β-galactosidase, o-nitrophenyl-β-D-galactoside, 4-methylumbelliferyl-β- A substrate such as D-galactoside may be used as the color former. The autoantibodies in the test sample can be quantified by measuring the absorbance of the reaction solution using a spectrophotometer, a fluorimeter, or the like for the color development that occurs depending on the amount of the autoantibody in the test sample.

(2) Aggregation reaction method In the aggregation reaction method, an autoantibody against a cerebral infarction or myocardial infarction specific antigen in a test sample is reacted with the antigen peptide of the present invention immobilized on carrier particles, and the antigen peptide and the autoantibody are reacted. By measuring the aggregation caused by the reaction with Examples of carrier particles that can be used include erythrocytes such as polystyrene latex, kaolin, bentonite, carbon powder, sheep and chicken. A known method can be applied as a method of immobilizing the antigen peptide on the carrier particles. For example, it can be carried out by a method using a coupling agent such as tannic acid, glutaraldehyde, bisazobenzidine, carbodiimides, quinones, chromium chlorides, a method by physical adsorption, or the like.

The agglutination reaction may be performed according to a method usually used in latex agglutination reaction and passive agglutination reaction. For example, a dilution series of a test sample is prepared in a well of a microplate, carrier particles on which the protein of the present invention is immobilized are added to each well, mixed, allowed to stand for 1 to 2 hours, and an agglutination reaction is observed. The autoantibodies in the test sample can be quantified by comparing with a standard sample having a known antibody titer.

(3) Immunochromatography The immunochromatography method is an immunoassay method utilizing capillary action. For example, in the immunochromatography method using the sandwich method, an insoluble thin film in which a first antibody that specifically binds to the target antigen is immobilized in a specific region. In a support (for example, a glass fiber membrane, nylon membrane, or cellulose membrane), a labeled second antibody that specifically binds to the target antigen and a sample solution that may contain the target antigen are developed. Then, an immunocomplex with the target antigen is formed on the area where the first antibody of the insoluble thin film support is fixed, and a signal such as coloring or coloration of the label is detected to measure the target antigen. In addition, as a label | marker, the protein containing an enzyme, a colored latex particle, a metal colloid, or a carbon particle can be used, for example.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but these examples do not limit the present invention.

(Example 1) Identification of SLE-specific antigen
(1) Primary screening using protoarray
SLE patient serum was collected at the National Hospital Organization Shishizu Hospital with the consent of the person or family (informed consent). Cerebral infarction patient serum, myocardial infarction patient serum, diabetic patient serum, and healthy subject samples were collected in the same manner at Chiba University Hospital and Shimoshizu Hospital.

  As primary screening, SLE patient serum (6 samples) and healthy subject serum (5 samples) were used to screen for antigenic proteins to which the immunoglobulin G antibodies in the serum bind. 67 types of antigen proteins such as SOSTDC1 and CTNND1 were identified as antigen proteins that reacted with 5 or more samples of patient serum by the protoarray method and did not react with 5 samples or more of normal serum (Table 1).

  Antigen sites (epitope) were predicted for the selected antigen proteins, and 171 types of peptides having the sequences were artificially synthesized (Tables 2 to 5). A crude product in which the amino terminus of the peptide was biotinylated was used.

  Serum antibody levels were measured by AlphaLISA method using these synthetic peptides. As a result, antibody levels against the following synthetic peptides were significantly higher in the serum of SLE patients than in the serum of healthy subjects.

SEQ ID NO: 113 SOSTDC1-156 (biotin-KITVVTACKCKRYTR)
SEQ ID NO: 79 CTNND1-211 (biotin-LRNVSSERSEARRKL)
SEQ ID NO: 57 CLDND1-69 (biotin-FRYNGTVGLWRRCIT)
SEQ ID NO: 63 CCNG2-231 (biotin-KKHSKINDTEFFYWR)
SEQ ID NO: 171 FOXJ2-426 (biotin-KMVNRLNWSSIEQSQ)
SEQ ID NO: 55 TFAM-231 (biotin-LRRTIKKQRKYGAEE)
SEQ ID NO: 128 TOP3B-628 (biotin-HRFMKYIQAKPSRLH)
SEQ ID NO: 87 MYBBP1A-1134 (biotin-LYWQAMKTLGVQRPK)
SEQ ID NO: 88 MYBBP1A-1306 (biotin-IRSPSLLQSGAKKKA)
SEQ ID NO: 76 KIF12-203 (biotin-CVSPSAQCLPETLST)
The results are shown in Tables 6 and 7. Tables 6 and 7 show serum antibody levels examined by the AlphaLISA method, and show comparison between SLE patients and healthy subjects. In the table, the average value, SD, average value + 2SD (cutoff value), total number, positive number, and positive rate of each group are shown. The number of positives (P Positive No.) and the positive rate (P Positive (%)) of each group when the average value of healthy subject samples + 2SD is set as a cutoff value are also shown. P (SLE vs HD) indicates the P value of the t test for healthy subjects and each group.

(2) Identification of cerebral infarction and myocardial infarction specific antigen Next, peptides of high purity (90% or more) were synthesized for the peptide antigens selected above. Antibody levels in the serum of patients with cerebral or myocardial infarction against them were measured by the AlphaLISA method.

  As a result, SOSTDC1-156 had a high positive rate in cerebral infarction samples, while CTNND1-211 had a high positive rate in myocardial infarction samples, and CLDND1-69 responded well to both cerebral and myocardial infarction ( Table 8 and Table 9). Tables 8 and 9 show serum antibody levels examined by the AlphaLISA method, and show a comparison between cerebral infarction patients, myocardial infarction patients, and healthy subjects.

  CCNG2-231 was found to correlate well with diabetes (Tables 10 and 11). Tables 10 and 11 show the serum antibody levels examined by the AlphaLISA method, and show a comparison between cerebral infarction patients, myocardial infarction patients, and healthy subjects.

  From these results, SOSTDC1-156 is A, CTNND1-211 is B, CLDND1-69 is C, and CCNG2-231 is D, which is an example of diagnosis, but if A and C are positive, cerebral infarction, B and C If is positive, it is diagnosed that the possibility of developing myocardial infarction is high. If D is positive, the cause may be diabetes and the progression of arteriosclerosis. Table 12 shows an example of diagnosis.

  Useful for diagnosis of cerebral infarction and myocardial infarction.

  SEQ ID NOs: 1-171: synthetic peptides

Claims (2)

A diagnostic marker for cerebral infarction, which is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 113. Oite the amino acid sequence represented by SEQ ID NO: 113, 1 or diagnostic marker of cerebral infarction according to claim 1 more amino acids is a peptide consisting of deletion, substitution or addition of amino acid sequence.