JP6153239B2 - Test method and kit for Kawasaki disease - Google Patents

Description

  The present invention relates to a test method and kit for Kawasaki disease.

  Kawasaki disease is an acute febrile rash disease mainly seen in infants under 4 years of age, and the main condition is systemic vasculitis. The diagnosis of Kawasaki disease consists of several major symptoms (fever that lasts for more than 1.5 days, 2. hyperemia of the bilateral conjunctiva, 3. redness of the lips, tongue, 4. irregular rash, 5. stiffness of the fingers in the acute phase, palms And erythema of the sole of the foot, membrane-like desquamation after antipyretic, 6. non-suppurative lymphadenopathy of the cervix) (guideline for diagnosis of Kawasaki disease). In the blood test, the white blood cell count, C-reactive protein, hepatocyte escape enzyme, increased red sediment, leukocyte fraction (neutrophil ratio), etc. are examined, and coronary artery lesions detected by tomographic echocardiography or cardiovascular angiography Confirmation is also performed.

  Kawasaki disease is a disease that resolves spontaneously, but when not treated, 25-30% of patients develop cardiac complications, typically coronary artery lesions. Therefore, in Kawasaki disease, it is important to start treatment early in the onset to calm the inflammation, and it is necessary to shorten the fever period even one day and prevent the occurrence of cardiac complications. However, the etiology and onset mechanism of Kawasaki disease are still unknown, there are no specific diagnostic tests, there are individual differences in major symptoms, and there are many cases that do not meet the diagnostic criteria. Therefore, a rapid definitive diagnosis of Kawasaki disease is difficult.

  Patents relating to the diagnosis of Kawasaki disease include a method of measuring blood VEGF (vascular endothelial growth factor) concentration (Patent Document 1: Japanese Patent Laid-Open No. 11-6832), one or more superantigens There are methods for measuring IgM (Patent Document 2: Japanese Patent Laid-Open No. 3-139294) and other gene polymorphism investigations (Patent Document 3: Japanese Patent Laid-Open No. 2009-72193), which are actually used in clinical practice. There is nothing yet.

Japanese Patent Laid-Open No. 11-6832 Japanese Unexamined Patent Publication No. 3-139294 JP 2009-72193 A

  An object of this invention is to provide the method and kit which test | inspect a Kawasaki disease rapidly and simply.

  As a result of diligent efforts, the present inventors have found that lipopolysaccharide binding protein (LBP), leucine-rich alpha-2-glycoprotein 1 (LRG1), angiotensinogen ( Angiotensinogen (AGT)) and retinol binding protein 4 (RBP4) expression levels in patients' sera are statistically significant between acute (fever) and recovery (after fever) ( p <0.0001). For LBP, LRG1, AGT, and RBP4, specific antibodies already exist, and it is possible to measure the amount of these proteins in serum or blood with high sensitivity and ease using antigen-antibody reaction. In this study, immunoblotting analysis using a very small amount of serum (or whole blood) revealed that LBP, LRG1, and AGT were highly expressed and RBP4 was low in the acute phase of Kawasaki disease. The present invention has been completed based on these findings.

The gist of the present invention is as follows.
(1) measuring at least one component selected from the group consisting of lipopolysaccharide binding protein, leucine-rich α2-glycoprotein, angiotensinogen and retinol binding protein 4 in a subject-derived specimen, Test method for Kawasaki disease.
(2) If at least one component selected from the group consisting of lipopolysaccharide-binding protein, leucine-rich α2-glycoprotein, and angiotensinogen has a high level in the subject-derived specimen, the patient is suffering from Kawasaki disease The method according to (1), wherein it is determined that the possibility is high, and when the level is low, it is determined that the possibility of suffering from Kawasaki disease is low.
(3) Retinol-binding protein 4 is determined to have a high possibility of suffering from Kawasaki disease when the level in the subject-derived specimen is low, and is suffering from Kawasaki disease when the level is high The method according to (1), wherein the possibility is determined to be low.
(4) The subject is a patient undergoing treatment for Kawasaki disease, and at least one component selected from the group consisting of lipopolysaccharide binding protein, leucine-rich α2-glycoprotein, and angiotensinogen is present in the subject-derived specimen Is measured once or multiple times at different times, and when the level is low or decreased, it is determined that treatment has recovered from Kawasaki disease. The method according to (1), wherein it is determined that recovery has not been performed or recovery has been insufficient.
(5) When the subject is a patient undergoing treatment for Kawasaki disease, and the level of retinol binding protein 4 in the subject-derived specimen is measured once or multiple times at different times, and the level is high or increased The method according to (1), wherein it is determined that treatment has recovered from Kawasaki disease, and when the level is low or does not increase, it is determined that treatment has not recovered from Kawasaki disease or recovery is insufficient. .
(6) The method according to any one of (1) to (5), wherein the subject-derived specimen is serum or whole blood.
(7) Reagent capable of specifically detecting lipopolysaccharide-binding protein, reagent capable of specifically detecting leucine-rich α2-glycoprotein, reagent capable of specifically detecting angiotensinogen and retinol-binding protein 4 can be specifically detected A test kit for Kawasaki disease comprising at least one reagent selected from the group consisting of reagents.
(8) The kit according to (7), wherein the reagent is an antibody.

  About 10,000 patients are diagnosed with Kawasaki disease annually. The number of other children with febrile illnesses of unknown cause is also very large, and the market scale will be large if Kawasaki disease is diagnosed as an initial screening test for these patients. In addition, if it can be applied to the determination of severity, it is possible to avoid the use of expensive gamma globulin preparations as a therapeutic agent, leading to savings in medical costs.

According to the present invention, Kawasaki disease can be diagnosed promptly with a very high probability by an inspection method with a small patient burden in addition to diagnosis by main symptoms. In addition, the therapeutic effect of Kawasaki disease can be confirmed.
This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2015-024506, which is the basis of the priority of the present application.

Western blot image using Anti-LBP antibody. The same number is the serum of the same patient and corresponds to the acute phase and the recovery phase. Western blot image using Anti-LRG1 [EPR 12362] antibody. The same number is the serum of the same patient and corresponds to the acute phase and the recovery phase. The graph which plotted by digitizing the intensity | strength of the band detected by Western blot using Anti-LBP antibody. The relative amounts were plotted when the standard protein (2.5 ng) was taken as 100. The graph which plotted and digitized the intensity | strength of the band detected by Western blot using Anti-LRG1 [EPR 12362] antibody. Each relative amount when the standard protein (10 ng) was set to 100 was plotted. Western blot image using Anti-ATG antibody. Serum from the same patient corresponds to the acute phase and the recovery phase. The graph which plotted and digitized the intensity | strength of the band detected by Western blot using Anti-ATG antibody. The detected band intensity was used as it was for graphing. Western blot (left) using Anti-LBP, LRG1, AGT, RBP4 antibodies. Serum of 10 Kawasaki disease patients (No. 1 to 10) (acute and convalescent in the same patient) was used. A graph in which the intensity of the band detected by Western blot is digitized and the intensity is plotted as it is (right). NS: non-significant ELISA results for LBP, LRG1, AGT and RBP4. Comparison of expression levels between Kawasaki disease acute phase, recovery phase, and healthy children (during allergy testing). The vertical axis indicates the serum protein concentration. ***: p <0.001, **: p <0.01, *: p <0.1, NS: non-significant Changes in LBP, LRG1, AGT, and RBP4 based on ELISA results. Fluctuation in the expression level between the acute and convalescent stages of Kawasaki disease and other healthy children (during allergy testing) in the same 42 patients. The vertical axis indicates the serum protein concentration, and the acute phase and the recovery phase are connected by a line. ***: p <0.001, **: p <0.01, *: p <0.1, NS: non-significant ELISA results for LBP, LRG1, AGT and RBP4. Comparison of the expression level between Kawasaki disease acute stage and other pediatric patients. The vertical axis indicates the serum protein concentration. ***: p <0.001, **: p <0.01, *: p <0.1, NS: non-significant Results of LPC, LRG1, AGT, RBP4 ROC (Receiver Operator Characteristic Curve) analysis. Sensitivity% on the vertical axis (percentage that is positive when testing a person who has truly Kawasaki disease), and 100% -specificity% (percentage of misdiagnosis of diseases other than Kawasaki disease as Kawasaki disease) on the horizontal axis I took it.

  Hereinafter, the present invention will be described in detail.

  The present invention includes measuring a level in a subject-derived specimen for at least one component selected from the group consisting of lipopolysaccharide binding protein, leucine-rich α2-glycoprotein, angiotensinogen and retinol binding protein 4. , Provide a test method for Kawasaki disease.

If at least one component selected from the group consisting of lipopolysaccharide binding protein, leucine-rich α2-glycoprotein, and angiotensinogen has a high level in the subject-derived specimen, it may be affected by Kawasaki disease When it is determined that the level is high and the level is low, it can be determined that the possibility of suffering from Kawasaki disease is low. The present inventors have confirmed that many of these proteins are expressed in the acute phase of Kawasaki disease (see Examples described later).
Retinol-binding protein 4 is judged to have a high possibility of suffering from Kawasaki disease when the level in the sample derived from the subject is low, and may be suffering from Kawasaki disease when the level is high Can be determined to be low. The present inventors have confirmed that the expression of this protein is decreased in the acute phase of Kawasaki disease (see Examples described later).
Therefore, the method of the present invention can be used for diagnosis of Kawasaki disease (determination of presence or absence of Kawasaki disease).
The cut-off value (acute vs control) in Table 1 below in Examples described later can be used to determine whether or not to have Kawasaki disease, in particular, to make a diagnosis by distinguishing from other diseases. For example, when the serum concentration of lipopolysaccharide binding protein (LBP) is 40.49 μg / mL (concentration when the specificity is 95%) or more, it is determined that the possibility of suffering from Kawasaki disease is high, When the serum concentration is less than 40.49 μg / mL, it can be determined that the possibility of suffering from Kawasaki disease is low. For leucine-rich α2-glycoprotein (LRG1), if the serum concentration is 391.3μg / mL or higher (concentration when the specificity is 95%), it is determined that there is a high possibility of suffering from Kawasaki disease. When the serum concentration is less than 391.3 μg / mL, it can be determined that the possibility of suffering from Kawasaki disease is low. As for angiotensinogen (AGT), when the serum concentration is 68.83 μg / mL (concentration when the specificity is 95%) or more, it is determined that there is a high possibility of suffering from Kawasaki disease. When the concentration is less than 68.83 μg / mL, it can be determined that the possibility of suffering from Kawasaki disease is low. Regarding retinol binding protein 4 (RBP4), when the serum concentration is 4.575 μg / mL (concentration when the specificity is 95%) or less, it is determined that the possibility of suffering from Kawasaki disease is high. When the serum concentration is higher than 4.574 μg / mL, it can be determined that the possibility of suffering from Kawasaki disease is low. However, referring to the bottom of Table 1, the above cut-off value is best when the specificity is 95% based on the ROC curve, better when the specificity is 90%, and when the specificity is 80%. You may change the time as good.

In addition, if the subject is a patient undergoing treatment for Kawasaki disease, at least one component selected from the group consisting of lipopolysaccharide binding protein, leucine-rich α2-glycoprotein, and angiotensinogen is contained in the subject-derived specimen. Is measured once or multiple times at different times, and when the level is low or decreased, it is determined that treatment has recovered from Kawasaki disease. It can be determined that no recovery has occurred or recovery is insufficient. The present inventors compared the acute phase and the recovery phase of Kawasaki disease, and confirmed that these proteins decreased with the recovery (see Examples described later).
For retinol binding protein 4, the level in the subject-derived sample was measured once or multiple times at different times, and when the level was high or increased, it was determined that the treatment recovered from Kawasaki disease, and the level was When it is low or does not rise, it can be determined that treatment has not recovered from Kawasaki disease or recovery is insufficient. The present inventors compared the acute phase and the recovery phase of Kawasaki disease, and confirmed that these proteins increased with the recovery (see Examples described later).
Therefore, the method of the present invention can also be used for changes in the pathology of patients with Kawasaki disease, current pathological conditions, prognosis tests, and confirmation of the therapeutic effects of Kawasaki disease.
In order to determine recovery from Kawasaki disease, the cut-off value (acute vs recovery) shown in Table 1 below in Examples described later can be used. For example, if the subject is a patient undergoing treatment for Kawasaki disease, for lipopolysaccharide binding protein (LBP), the serum concentration in the subject-derived specimen is measured once or multiple times at different times, and the serum concentration is 56.54μg / mL (concentration at 95% specificity) or less, it was determined that treatment recovered from Kawasaki disease, and when the serum concentration was higher than 56.54μg / mL, It can be determined that no recovery has occurred or recovery is insufficient. For leucine-rich α2-glycoprotein (LRG1), the serum concentration in the subject-derived specimen is measured once or multiple times at different times, and the serum concentration is 369.7 μg / mL (concentration when the specificity is 95%) ) In the following cases, it was determined that the treatment recovered from Kawasaki disease, and when the serum concentration was higher than 369.7 μg / mL, the treatment did not recover from Kawasaki disease, or the recovery was insufficient Can be determined. For angiotensinogen (AGT), the serum concentration in the sample derived from the subject is measured once or multiple times at different times, and the serum concentration is 101.9μg / mL (concentration when the specificity is 95%) or less. In some cases, it is determined that treatment has recovered from Kawasaki disease, and when the serum concentration is higher than 101.9 μg / mL, it is determined that treatment has not recovered from Kawasaki disease or recovery is insufficient. Can do. For retinol binding protein 4 (RBP4), the serum concentration in the subject-derived specimen is measured once or multiple times at different times, and the serum concentration is 6.759 μg / mL (concentration when the specificity is 95%) or more Is determined to have recovered from Kawasaki disease by treatment, and if the serum concentration is lower than 6.759 μg / mL, it is determined that treatment has not recovered from Kawasaki disease or recovery is insufficient be able to. However, referring to the bottom of Table 1, the above cut-off value is best when the specificity is 95% based on the ROC curve, better when the specificity is 90%, and when the specificity is 80%. You may change the time as good.

  Examples of the specimen derived from the subject include serum and whole blood.

  The present invention specifically detects a reagent that can detect lipopolysaccharide-binding protein, a reagent that can specifically detect leucine-rich α2-glycoprotein, a reagent that can specifically detect angiotensinogen, and a retinol-binding protein 4 specifically. There is also provided a test kit for Kawasaki disease comprising at least one reagent selected from the group consisting of possible reagents.

  As a reagent, an antibody is preferable, an antibody that specifically binds to lipopolysaccharide-binding protein, an antibody that specifically binds to leucine-rich α2-glycoprotein, an antibody that specifically binds to angiotensinogen, and retinol-binding protein 4 It may be an antibody that specifically binds. Such antibodies are commercially available and can be used. The antibody may be either a monoclonal antibody or a polyclonal antibody. The antibody may be labeled with a radioisotope, enzyme, luminescent material, fluorescent material, biotin, and the like. In the case of detecting a target molecule by reacting a primary antibody that specifically binds to a target molecule (in the present invention, LBP, LRG1, ATG, RBP4) and then reacting with a secondary antibody that binds to the primary antibody. In some cases, the secondary antibody is labeled (the primary antibody is not labeled).

  In addition, the kit of the present invention includes a standard protein (LBP, LRG1, ATG, RBP4), buffer, substrate (when antibody is enzyme-labeled), reaction stop solution, washing solution, reaction vessel, instruction manual, etc. May be included.

Hereinafter, the present invention will be described in more detail with reference to examples.
[Example 1]
(Method)
Two types of protein using serum from 55 acute cases of Kawasaki disease and 51 cases of convalescence (provided by Yokohama City University Hospital, Yokohama City University Hospital, Kanagawa Children's Medical Center, Public Showa Hospital) The following operations were performed.

Confirmation of protein expression level in serum by Western blot 0.1 μL of each serum was mixed with sample buffer, heated at 95 ° C. for 5 minutes, and then centrifuged at 15,000 rpm at room temperature for 5 minutes. Using Perfect NT Gel (DRC), electrophoresis was performed at a constant voltage of 300 V to separate proteins. After electrophoresis, the DNA was transferred from Perfect NT Gel to a PVDF membrane using a semi-drive blotting device (Trans-Blot Turbo System (BioRad)). The PVDF membrane after transfer was immersed in a blocking solution, and then shaken at room temperature for 1 hour to perform a blocking treatment. The PVDF membrane after blocking treatment was diluted with an antibody dilution buffer (anti-LBP antibody (GeneTex) diluted 3,000 times, anti-LRG1 [EPR 12362] antibody (abcam) diluted 5,000 times) and 16 times at room temperature. Reacted for ~ 18 hours. After the reaction, each PVDF membrane was washed with TBS (TBS-T) containing 0.05% [v / v] Tween 20 for 10 minutes three times, and diluted to 5,000 times with a buffer for antibody dilution. React with HRP at room temperature for 1 hour. After the reaction, it was washed again with TBS-T for 10 minutes three times, and detected with LAS-4000 EP UV mini PRH (Fuji Film) using ECL Select Western Blotting Detection Reagent (GE Healthcare) as a substrate.

  Then, from the obtained image, the intensity of the band was quantified by MultiGauge Analysis Software (ver. 3.11, Fujifilm), and a commercially available recombinant protein (Recombinant Human LBP (R & D Systems) [2.5 ng], Recombinant Human LRG1 ( Novoprotein) [10 ng]) was used as a standard protein for quantitative analysis, and the relative amount of each band when the intensity of the standard protein band was assumed to be 100 was calculated. The obtained numerical values were graphed and the Mann-Whitney test was performed in the acute phase and the recovery phase using GraphPad Prism (ver. 5, MDF).

(result)
We examined whether these proteins are specifically expressed in the acute phase of Kawasaki disease by Western blotting using antibodies against LBP and LRG1. Usually, even in electrophoresis, a high concentration protein becomes an obstacle to separation of other proteins. However, since LBP and LRG1 focused on this time have high expression levels and antibodies with high specificity, the amount of serum used to detect the two proteins was as low as 0.1 μL. Therefore, in this experiment, without removing high-concentration proteins, all sera from Kawasaki disease patients with acute 55 cases and convalescent 51 cases were separated by SDS-PAGE gel and the expression level was examined (Figures 1 and 2). As a result, the expression levels of LBP and LRG1 were significantly different from p <0.0001 in both acute and recovery periods. The results are shown in FIG. 3 for LBP and FIG. 4 for LRG1.

(Discussion)
LBP is a protein present in blood at high concentrations during bacterial infection [International Immunology, 22: 271-280, 2010.] and has a high affinity for lipopolysaccharide (LPS), a component of the gram-negative bacterial cell membrane. Hold and form a complex. This LBP-LPS complex is transported to CD14 present on cell membranes such as macrophages, binds to Toll-like receptor 4 [Journal of Periodontal Research, 49: 1-9, 2014.], activates the signal transduction pathway, It has been shown to promote the secretion of various inflammatory cytokines. In addition, LBP has been reported to be upregulated in febrile urinary tract infection and sepsis [Pediatric Nephrology, 28, 1091-1097, 2013.]. Therefore, it was considered that bacteria and the like were also infected in Kawasaki disease, and as a result, the expression of LBP increased.

  On the other hand, LRG1 is also present in blood and has been identified as a novel inflammatory marker protein, and it has been reported that its expression increases due to rheumatoid arthritis, cancer, inflammatory bowel disease, macrophage activation by LPS administration, etc. [Annals of the Rheumatic Diseases, 69: 770-774, 2010., Biochem Biophys Res Commun, 382: 776-779, 2009., Proc Natl Acad Sci US A. 110, E2332-E2341, 2013.]. In recent years, it has been reported that angiogenesis is promoted through the regulation of transforming growth factor-β (TGF-β) signaling [Nature, 499: 306-311, 2013.]. TGF-β is also involved in VEGF expression, and VEGF is said to be present in high concentrations in the serum of patients with acute Kawasaki disease [Pediatric Research, 44: 596-599, 1998.]. There are reports of inflammation and angiogenesis in coronary aneurysms and myocardium in patients with Kawasaki disease [Pediatric Cardiology, 26: 578-584, 2005.]. Therefore, it is suggested that LRG1 may be involved in coronary aneurysm formation in the myocardium or inflammation in the myocardium.

  Furthermore, LBP and LRG1 have high concentrations in blood and can be easily detected. Therefore, by using the expression level of these two proteins, both of which are the criteria for diagnosis, in addition to diagnostic methods based on major symptoms that are influenced by the subjectivity and experience of doctors and may be missed or missed, it is accurate and prompt. I think there is a possibility of diagnosis.

[Example 2]
(Method)
Serum of 20 patients with Kawasaki disease acute phase and 20 cases of recovery phase (provided by Yokohama City University Hospital, Yokohama City University Hospital, Kanagawa Children's Medical Center, Public Showa Hospital) The following operations were performed.

Confirmation of expression level of each protein in serum by Western blot 0.05 μL of each serum was mixed with a sample buffer, heated at 95 ° C. for 5 minutes, and then centrifuged at 15,000 rpm at room temperature for 5 minutes. Using Perfect NT Gel, electrophoresis was performed at a constant voltage of 300 V to separate proteins. After electrophoresis, it was transferred from Perfect NT Gel to a PVDF membrane using a semi-drive blotting device (Trans-Blot Turbo System). The PVDF membrane after transfer was immersed in a blocking solution, and then shaken at room temperature for 1 hour to perform a blocking treatment. The PVDF membrane subjected to the blocking treatment was reacted with Anti-AGT antibody (IBL) diluted 100 times with an antibody dilution buffer at room temperature for 16 to 18 hours. After the reaction, each PVDF membrane was washed with TBS-T for 10 minutes three times, and reacted with standard anti-mouse IgG-HRP diluted 5,000 times with an antibody dilution buffer at room temperature for 1 hour. After the reaction, it was washed again with TBS-T for 10 minutes three times, and detected with LAS-4000 EP UV mini PRH using ECL Select Western Blotting Detection Reagent as a substrate. After that, the intensity of the band is digitized from the obtained image by MultiGauge Analysis Software, and the obtained numerical value is graphed, and the Mann-Whitney test in the acute phase and the recovery phase using GraphPad Prism (ver. 5, MDF) Went.

(result)
We examined whether this protein was specifically expressed in the acute phase of Kawasaki disease by Western blot using an antibody against AGT. As with LBP and LRG1, AGT was expressed in a large amount and there was a highly specific antibody, so the amount of serum used for detection was as low as 0.05 μL. Therefore, in this experiment, without removing high-concentration proteins, the sera of 20 patients in Kawasaki disease acute phase and 20 cases in recovery phase were all separated on SDS-PAGE gel and the expression level was examined (Fig. 5). As a result, the expression level of AGT was significantly different from p <0.0006 between the acute phase and the recovery phase (FIG. 6).

(Discussion)
AGT is a precursor of angiotensin and decomposes into angiotensin I and II in the renin-angiotensin system. AGT is increased in hypertension, diabetes, and chronic nephritis, and is considered to play an important role in the development and development of hypertension and kidney damage. However, the relationship with Kawasaki disease is not known, and is the first knowledge obtained in the present invention.

Example 3
(Method)
Serum was provided by Yokohama City University Hospital, Kanagawa Children's Medical Center, Public Showa Hospital, National Institute of Infectious Diseases, Kobe University Hospital, Japanese Red Cross Wakayama Medical Center, Yokohama City University Citizen General Medical Center (On Table 1). All samples have received comprehensive consent from the provider.
・ Acute phase sera of Kawasaki disease patients (acute): Serum during fever of 55 patients ・ Recovery phase during recovery of Kawasaki disease patients: Serum after fever of 51 patients ・ Sera from patients with viral infections (G1): 106 people
(21 RS viruses, 23 influenza A viruses, 20 influenza B viruses, 20 rotaviruses, 7 noroviruses, 3 adenoviruses, 12 throat adenoviruses)
・ Bacterial infection patient serum (G2): 21
(1 pneumococci, 1 pneumoniae, 1 gram-negative bacilli, 1 gram-negative bacilli, 1 streptococci, 3 E. coli, 2 Staphylococcus aureus, 1 epidermidis, 1 micrococcus, 1 serratia Name, 1 difficile)
・ Autoimmune disease patient serum (G3): 24
(3 idiopathic thrombocytopenic purpura, 2 pediatric rheumatism, 1 GVHD (graft-versus-host disease), 1 VAHS (virus-related hemophagocytic syndrome), 17 juvenile idiopathic arthritis)
・ Healthy serum (by collecting blood during allergy test) (Healthy): 13

Confirmation of protein expression level in serum by Western blot Serum was obtained from 10 Kawasaki disease patients (sera in the acute and convalescent phases of the same patient). These were diluted with PBS-T and mixed with an equal volume of diluted serum and 2 × sample buffer so that the serum contained 0.1 μL per well to a total volume of 10 μL with milliQ water. The sample for LRG1 quantitative analysis was used after being heated at 95 ° C. for 5 minutes. Thereafter, the supernatant obtained by centrifugation at 21,600 × g and room temperature for 5 minutes was used as a sample for electrophoresis. The prepared gel was placed in an electrophoresis tank and filled with an electrode solution. Samples were placed in each well and electrophoresed at a constant voltage of 300 V to separate proteins in serum.
After electrophoresis, it was transferred to a PVDF membrane using a transfer device. The PVDF membrane after transfer was immersed in a blocking solution, and then shaken at room temperature for 1 hour to perform a blocking treatment. The PVDF membrane subjected to the blocking treatment was reacted with a primary antibody diluted with an antibody dilution buffer for 16 to 18 hours (each antibody dilution ratio is as follows. Anti-LRG1 antibody: 5,000 times, anti- AGT antibody: 100 times, anti-RBP4 antibody: 1,000 times). After the reaction, each PVDF membrane was washed with TBS-T for 3 minutes for 10 minutes, and reacted with anti-rabbit IgG-HRP or anti-mouse IgG-HRP diluted 5000 times with antibody dilution buffer for 1 hour at room temperature. I let you. After the reaction, it was washed again with TBS-T for 10 minutes three times, and photographed with LAS-4000 EP UV mini PRH using the secondary labeled antibody detection reagent as a substrate. Band intensities were quantified by MultiGauge Analysis Software.

Verification of Kawasaki Disease Specificity by ELISA Method Regarding Kawasaki disease related proteins LBP, LRG1, AGT, RBP4, the concentrations in serum of Kawasaki disease, pediatric diseases other than Kawasaki disease, and healthy subjects are measured by ELISA method. A significant difference test was performed. Serum of Kawasaki disease patients (55 samples in acute phase, 51 samples in recovery phase), 13 samples of healthy healthy subjects as controls, serum of patients with other pediatric diseases (106 samples of viral infections, 21 samples of bacterial infections, 24 autoimmune diseases) Specimen) was used. Serum used was LBP diluted 4000 times, LRG1 diluted 5000 times, AGT diluted 10,000 times, and RBP4 diluted 2500 times. Reagents such as dilute solution, washing solution, detection reagent, and reaction time, etc., followed the protocol of each protein ELISA kit.

Statistical analysis
Kawasaki disease acute phase regarding serum levels of each protein obtained from the results of Western blot method, significant difference test between Kawasaki disease acute phase and recovery period regarding the expression level in patient serum The statistical analysis software Graph Pad Prism was used to test the significant difference between each group (Kawasaki disease recovery period, healthy children, and other pediatric diseases). In addition, in order to verify its usefulness as a biomarker for Kawasaki disease, the Kawasaki disease acute phase (55 samples) and convalescent phase (51 samples), as well as other childhood diseases other than Kawasaki disease acute phase and Kawasaki disease (previous item: 144 samples) ) And ROC analysis was performed to calculate AUC.

(result)
Detection of novel Kawasaki disease-related protein candidates by Western blot method Among the Kawasaki disease-related protein candidates obtained from the results of serum proteome analysis, the expression level varies specifically in the acute phase of Kawasaki disease using Western blot method Investigated the protein to be. As a result, it was found that the expression level of one new protein RBP4 was significantly decreased in the acute phase (p <0.002) (Fig. 7).

Verification of specificity of Kawasaki disease-related protein by ELISA method About LBP, LRG1, AGT and RBP4 newly found as Kawasaki disease-related protein, Kawasaki disease patient serum (acute phase, convalescent phase) and pediatric healthy Serum protein concentrations were measured by ELISA using the serum of pediatric patients other than Kawasaki disease (virus infection, bacterial infection, autoimmune disease) (Table 1 top). As a result of comparing the protein concentrations in the serum of Kawasaki disease patients and healthy individuals, the expression levels of LBP, LRG1, AGT, and RBP4 in the acute phase and recovery period of Kawasaki disease are all significantly changed. (P <0.001) (Figure 8). In particular, it was confirmed that LRG1 decreased with recovery in all patients (FIG. 9). In addition, for LBP, LRG1, and RBP4, significant differences were found when comparing Kawasaki disease acute patients and healthy subjects (p <0.001) (Figure 9).
In addition, the concentrations of LBP, LRG1, AGT, and RBP4 in sera of pediatric patients other than Kawasaki disease were examined by ELISA (FIG. 10, upper table 1). As a result, there was a significant difference in all proteins between Kawasaki disease acute stage and viral infection or immune disease patients. On the other hand, there was a significant difference between LRG1 and RBP4 between Kawasaki disease acute stage and bacterial infection, but there was no significant difference between LBP and AGT.

Verification of biomarker specificity / sensitivity by ROC analysis Kawasaki disease acute phase and recovery period, and Kawasaki disease to clarify the disease specificity and usefulness of Kawasaki disease related proteins LBP, LRG1, AGT, RBP4 in the diagnosis of Kawasaki disease ROC curves were created between the acute phase of the disease and other pediatric diseases (Figure 11). The diagnostic performance was judged by the magnitude of the AUC value. As a result, the AUC value of LRG1 was 0.9615 between Kawasaki disease acute phase and recovery phase, 0.9636 between Kawasaki disease acute phase and other diseases, and among the four proteins, LRG1 was found to be Kawasaki disease It was shown to be the best in diagnosis and differentiation from other diseases. In addition, the AUC value of LBP was 0.8966 between Kawasaki disease acute phase and recovery phase, and 0.8497 between Kawasaki disease acute phase and other diseases, indicating that LBP is useful for diagnosis after LRG1. It was.
Furthermore, the cut-off value, sensitivity, and specificity were calculated using the statistical analysis software Graph Pad Prism based on the data in FIG. 11 (bottom of Table 1).
Cut-off value (best); concentration at which the specificity reaches 95%.
Cut-off value (better); concentration at which specificity is 90%.
Cut-off value (good): concentration at which the specificity reaches 80%.
Sensitivity: The rate at which patients with true Kawasaki disease are diagnosed as positive.
100%-specificity; the rate of misdiagnosis of patients with Kawasaki disease as Kawasaki disease.

[Table 1]
Patient information used in ELISA, serum levels (average values) of LBP, LRG1, AGT, and RBP4 in each group determined by ELISA, and cutoff values (best, better, good)

(Discussion)
Retinol-binding protein (RBP) is a protein with a molecular weight of 21 kDa synthesized in the liver, which binds and secretes vitamin A (retinol) accumulated in the liver and transports it to the target organ (cell). There is. RRBP4 is an RBP produced in the liver or adipocytes and secreted into the blood (plasma), so it is also referred to as Plasma RBP (PRBP). RBP4 has been pointed out to be involved in diabetes and insulin resistance, and is used as a marker that quickly reflects the protein synthesis ability of nutritional status and liver image. However, the relationship with Kawasaki disease is not known.
The etiology of Kawasaki disease is still unclear, and it is suggested that some abnormality in the immune system may have caused the pathology. In addition, inflammatory proteins such as CRP and SAA are present in excess in the patient acute phase serum, and the serum concentration can be examined as a reference item in general blood tests. However, many of such inflammatory proteins reflect non-specific systemic inflammation and vasculitis, and do not specifically differentiate Kawasaki disease.
. Therefore, it is important to develop diagnostic markers other than such proteins that can be distinguished from other diseases. This study suggests that Kawasaki disease can be specifically diagnosed by examining the serum levels of Kawasaki disease-related proteins LBP, LRG1, AGT, and RBP4 in patients. In particular, the diagnostic performance of LBP and LRG1 was found to be good. The pathology of Kawasaki disease is very widespread, and many mechanisms are expected to be involved in the onset. Since all four types of proteins found in this study are present at high concentrations in the blood, it is considered possible to develop a simple and accurate Kawasaki disease diagnosis method using these proteins as indicators.

  All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

  The present invention can be used for diagnosis of Kawasaki disease and confirmation of therapeutic effects.

Claims (26)

A test method for Kawasaki disease, comprising measuring a level in a subject-derived specimen for at least one component selected from the group consisting of lipopolysaccharide-binding protein, leucine-rich α2-glycoprotein, and angiotensinogen. If at least one component selected from the group consisting of lipopolysaccharide binding protein, leucine-rich α2-glycoprotein, and angiotensinogen has a high level in the subject-derived specimen, it may be affected by Kawasaki disease showed high that, if the level is low, method of claim 1, wherein indicating a decreased likelihood suffering from Kawasaki Disease. The subject is a patient undergoing treatment for Kawasaki disease, and at least one component selected from the group consisting of lipopolysaccharide binding protein, leucine-rich α2-glycoprotein, and angiotensinogen, When measured at one time or multiple times at different times, the level is low or decreased , indicating that the treatment has recovered from Kawasaki disease. If the level is not high or decreased, the treatment has recovered from Kawasaki disease. The method of claim 1, wherein the method indicates no or insufficient recovery. Further, for retinol binding protein 4, the level in the subject-derived specimen was measured once or multiple times at different times, and when the level was high or increased, it was shown that the treatment recovered from Kawasaki disease. if no low or elevated, not recovered from Kawasaki Disease treatment, or method of claim 3 wherein indicating that the recovery is insufficient. The method according to any one of claims 1 to 4 , wherein the specimen derived from the subject is serum or whole blood. Comprising at least one reagent selected from the group consisting of a reagent capable of specifically detecting lipopolysaccharide-binding protein, a reagent capable of specifically detecting leucine-rich α2-glycoprotein, and a reagent capable of specifically detecting angiotensinogen, Test kit for Kawasaki disease. The kit according to claim 6, wherein the reagent is an antibody. The method according to claim 1, further comprising measuring the level of the retinol-binding protein 4 in the subject-derived specimen. Retinol-binding protein 4 indicates a high possibility of suffering from Kawasaki disease when the level in the subject-derived specimen is low, and if the level is high, the possibility of suffering from Kawasaki disease The method of claim 8 , wherein the method is low. The method according to claim 8 or 9 , wherein the specimen derived from the subject is serum or whole blood. Furthermore, the kit of Claim 6 or 7 containing the reagent which can detect retinol binding protein 4 specifically. The kit according to claim 11, wherein the reagent is an antibody. A test method for Kawasaki disease, comprising measuring a level in a specimen derived from a subject for at least one component selected from the group consisting of a lipopolysaccharide binding protein and an angiotensinogen. When at least one component selected from the group consisting of lipopolysaccharide binding protein and angiotensinogen has a high level in a specimen derived from a subject, it indicates that there is a high possibility of suffering from Kawasaki disease, and the level The method according to claim 13, which indicates that the possibility of suffering from Kawasaki disease is low when the value is low. The method according to claim 13 or 14 , further comprising measuring a level in a subject-derived specimen for leucine-rich α2-glycoprotein. For leucine-rich α2-glycoprotein, a high level in the subject-derived specimen indicates a high possibility of suffering from Kawasaki disease, and a low level indicates a possibility of suffering from Kawasaki disease the method of claim 15, wherein indicating that low. The method according to any one of claims 13 to 16 , further comprising measuring the level of retinol binding protein 4 in a subject-derived specimen. Retinol-binding protein 4 indicates a high possibility of suffering from Kawasaki disease when the level in the subject-derived specimen is low, and if the level is high, the possibility of suffering from Kawasaki disease The method of claim 17 , wherein the method is low. The subject is a patient who is being treated for Kawasaki disease, and at least one component selected from the group consisting of lipopolysaccharide binding protein and angiotensinogen has multiple levels in the subject-derived specimen at one time or at different times When the level is low or decreased, it shows that the treatment has recovered from Kawasaki disease. When the level is high or does not decrease, the treatment has not recovered from Kawasaki disease, or recovery has not been achieved. 14. The method of claim 13 , indicating sufficient. In addition, for leucine-rich α2-glycoprotein, the level in the subject-derived specimen was measured once or multiple times at different times, and when the level was low or decreased, it was shown that the treatment recovered from Kawasaki disease, if the level is not high or lowered, not recovered from Kawasaki Disease treatment, or method of claim 19, wherein indicating that the recovery is insufficient. Further, for retinol binding protein 4, the level in the subject-derived specimen was measured once or multiple times at different times, and when the level was high or increased, it was shown that the treatment recovered from Kawasaki disease. if no low or elevated, not recovered from Kawasaki Disease treatment, or method of claim 19, wherein indicating that the recovery is insufficient. The method according to any one of claims 13 to 21 , wherein the subject-derived specimen is serum or whole blood. A test kit for Kawasaki disease comprising at least one reagent selected from the group consisting of a reagent capable of specifically detecting a lipopolysaccharide-binding protein and a reagent capable of specifically detecting angiotensinogen. The kit according to claim 23 , further comprising a reagent capable of specifically detecting leucine-rich α2-glycoprotein. The kit according to claim 23 or 24 , further comprising a reagent capable of specifically detecting retinol-binding protein 4. The kit according to any one of claims 23 to 25 , wherein the reagent is an antibody.