JP4795037B2 - Kawasaki disease determination method and kit therefor - Google Patents

Description

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

  Kawasaki disease (sometimes abbreviated as KD) is an acute febrile rash disease frequently reported in infants, reported by Dr. Tomasaku Kawasaki in 1967. Pathologically, it is systemic acute vasculitis, which may form an aneurysm in the coronary artery in some cases, and rarely cause ischemic heart disease.

  As a method for diagnosing Kawasaki disease by a blood test, a method for measuring TNF (tumor necrosis factor) inhibitor activity in urine (Patent Document 1) or a blood VEGF (vascular endothelial growth factor: vascular endothelial growth factor). factor) method for measuring concentration (patent document 2) and other genetic investigations are known, but since the etiology is unknown, it is not a diagnosis by measuring and detecting the etiological agent, but clinical Kawasaki disease has been diagnosed from the condition.

  1. In the Kawasaki Disease Diagnosis Guide (5th edition, revised by the Ministry of Health, Labor and Welfare, Kawasaki Disease Research Group), fever lasts more than 1.5 days (including cases of fever less than 5 days due to treatment). 2. Congestion of bilateral eyeball conjunctiva 3. Lips, oral findings: flushing of the lips, strawberry tongue, diffuse redness of the oropharyngeal mucosa. 4. Atypical rash, Changes in extremities: (Acute stage) Hard edema of limbs, Erythema on palms, soles or toes, (Recovery stage) Pattern desquamation from fingertips, 6. Major symptoms, such as non-suppurative cervical lymphadenopathy in the acute phase, with 5 or more symptoms or even if only 4 symptoms are present, tomographic echocardiography or cardiovascular angiography during the course If the coronary aneurysm is confirmed by the law and other diseases are denied, Kawasaki disease is diagnosed.

  Because clinical symptoms are similar to superantigen-related diseases such as Toxic Shock Syndrome (TSS), Kawasaki disease may be associated with superantigens produced by S. aureus and streptococci It has been suggested (Non-Patent Document 1). A superantigen binds to a major histocompatibility antigen class II (hereinafter abbreviated as MHC class II) on an antigen-presenting cell without undergoing a processing process in the antigen-presenting cell, and a complex with this MHC class II Is a protein that stimulates T cells having a Vβ region of a specific T cell receptor to abnormally activate the immune system. Superantigens include S. aureus exotoxins SEA, SEB, SEC, SED, SEE, TSST-1 and group A β-hemolytic streptococcal exotoxins SPEA, SPEC, as well as viral and plant proteins Ten or more types have been confirmed (Non-Patent Document 2).

  In order to prove the involvement of the superantigen, it is necessary to isolate the superantigen-producing bacteria from the patient or to detect the superantigen from the patient, but sometimes it is difficult to prove the presence of the bacteria, Superantigens may not be produced at the time of collection.

  Known examples suggesting the relationship between Kawasaki disease and superantigens include literature (non-patent document 3) that S. aureus producing TSST-1 was isolated from Kawasaki disease patients at a high rate, and T cell in the acute phase. Among them, literature reporting that Vβ2 increases (Non-patent literature 4), T cell receptor (T Cell Receptor: hereinafter abbreviated as TCR) in the blood of Kawasaki disease patients Vβ6.5 or Vβ2.1 positive T cell count There is a document (Patent Document 3) that shows that the increase in is significant compared with healthy infants. There is also a method that shows the possibility of sensitization to a superantigen by observing changes in the T cell Vβ repertoire consistent with the specificity of the superantigen in patient blood.

  In addition, there is a document that reports the possibility that TSST-1, (Non-patent document 5), or SPEA (Non-patent document 6) is involved in Kawasaki disease by a method for investigating the anti-superantigen IgG antibody titer. . Furthermore, although there are Non-Patent Document 9 and Non-Patent Document 11 as publicly known documents regarding anti-superantigen antibodies and Kawasaki disease, the relationship between Kawasaki disease and anti-superantigen IgM antibodies is not described. In addition, as a result of comparing Kawasaki disease patients from which Staphylococcus aureus was isolated and healthy control infants, IgM corresponding to the produced superantigen was obtained from patients from whom SEA and SEB-producing bacteria in acute serum were isolated. Although there is a report that it was recognized (Non-Patent Document 10), it is a result only for a patient from which bacteria were isolated, and there is no disclosure or suggestion about determining Kawasaki disease by measuring anti-superantigen IgM.

JP 3-139294 JP-A-11-6832 JP2000-157297 Chemotherapy 2000: Vol.16, No.8. P109 Food hygiene 2005; 51; 4: 81-90 Lancet 1993; 342: 1385-8. Proceedings of the National Academy of Sciences of the United States of America 1992; 89: 4066-70. The Journal of Infectious Diseases 2002; 185: 1677-80. The Pediatric Infectious Diseases Journal 2003; 22: 794-8. Journal of Infectious Diseases 2002; 76: 195-202. Knowledge of immunology Ohm, 1998; 1st edition, 3rd edition: 22 The Journal of Infectious Diseases 1995; 172: 558-61. Abstracts of 20th Annual Meeting of Kawasaki Diseases of Japan 2000; 82 Pediatric Research 2003; 53: 403-10. Japan Clinical Hygiene Laboratory Engineer Library XV Clinical Laboratory Accuracy Management Textbook Japan Clinical Hygiene Laboratory Engineers Association, 2000; 2nd edition: 48 Microbiology and Immunology 2000; 44: 419-23.

  As described above, some relations between superantigens and Kawasaki disease have been reported so far. Therefore, the inventors of the present application conducted an experiment to determine whether Kawasaki disease could be determined by measuring anti-superantigen IgG antibodies in the serum of patients. However, the relationship between the amount of anti-superantigen IgG antibody in patient serum and Kawasaki disease is not clear, and it is considered difficult to determine Kawasaki disease using the anti-superantigen IgG antibody in patient serum as an index. It was.

Therefore, an object of the present invention is to provide a method for detecting Kawasaki disease and a kit therefor, which can easily detect Kawasaki disease using a body fluid such as blood collected from a patient as a sample.
Has been confirmed experimentally.

  As the reason why it is difficult to determine Kawasaki disease by measuring anti-superantigen IgG antibodies in the serum of patients, the present inventors have been given the administration of intravenous human immunoglobulin preparations (referred to as “IVIG preparations”). I thought it was influencing. In other words, in Japan, most cases of Kawasaki disease are treated with large doses of IVIG preparations at an early stage. The IVIG preparation is a human IgG preparation obtained by fractionating the blood collected by blood donation, and therefore may contain a large amount of various antibodies. The blood of adults who have had many chances of being infected with S. aureus or streptococci until they reach the blood donation age is likely to contain large amounts of IgG against the antigens produced by those bacteria . Then, a large amount of anti-superantigen IgG is contained in the IVIG preparation, and administration of the IVIG preparation may affect the measured anti-superantigen IgG value. In fact, it has been reported that IgG against TSST-1 is contained (Non-patent Document 7). Therefore, as described in detail later, when the amount of anti-superantigen antibody in the patient serum was measured before and after administration of the IVIG preparation to the patient, the anti-superantigen IgG antibody in the patient serum was obtained by administration of the IVIG preparation. It was experimentally confirmed that the amount changed.

  As a result of intensive studies, the present inventors have conceived that it may be possible to determine Kawasaki disease by measuring anti-superantigen IgM antibodies in patient serum. Although IgM is produced earlier than IgG in response to antigenic stimulation, the half-life of IgM is short (IgM is about 10 days compared to the half-life of IgG is about 20 days). It disappears quickly because it is about 10% (Non-patent Document 8). For this reason, it was thought that IgM may be hardly contained in the IVIG preparation. Therefore, when the amount of anti-superantigen antibody in the patient's serum was measured before and after administration of the IVIG preparation to the patient, it was experimentally confirmed that the amount of anti-superantigen IgM antibody in the patient's serum was not changed by administration of the IVIG preparation. Furthermore, the inventors have experimentally confirmed that it is possible to determine Kawasaki disease by measuring the amount of anti-superantigen IgM antibody in the patient's serum, and have reached the present invention.

That is, the present invention provides a method for detecting Kawasaki disease, which comprises measuring IgM against one or more superantigens in a body fluid separated from a subject. The present invention also provides a solid phase on which one or more superantigens are immobilized, a solid phase on which IgM is immobilized on one or more superantigens, a labeled anti-human IgM antibody or an antigen-binding fragment thereof, or a labeled anti-antigen. Provided is a kit for determining Kawasaki disease, comprising at least a superantigen antibody or an antigen-binding fragment thereof.

The present invention provides for the first time a detection method that can easily detect Kawasaki disease using a body fluid sample such as serum collected from a subject. According to the method of the present invention, Kawasaki disease can be diagnosed by an immunoassay method that can be easily collected from a subject using an immunoassay method that can be measured at each hospital or the like. In Kawasaki disease, there is also an atypical Kawasaki disease in which coronary artery abnormalities appear later even though the diagnostic criteria are not met. This is a Kawasaki disease that was overlooked because there are few relevant items in the diagnosis, and it is difficult to find by diagnosis. In Kawasaki disease, coronary aneurysms appear and life-threatening situations occur if appropriate treatment is not performed at the beginning of the onset. Therefore, providing a new Kawasaki disease determination method plays a very important role.

  As described above, in the method of the present invention, IgM against one or more superantigens in a body fluid separated from a subject is measured.

The body fluid is not particularly limited, but blood is preferable. The blood includes blood components such as whole blood, serum, and plasma. In the immunoassay for carrying out the detection method of the present invention, it is easy and preferable to use serum or plasma. The body fluid sample should be diluted as much as possible in order to remove contaminants in the sample, but if diluted too much, it may be below the measurement sensitivity. In the present invention, in order to remove the influence of contaminants and measure with good sensitivity, it is preferable to perform dilution of 2 or more, particularly preferably 10 to 1000 times, and 20 to 500. More preferably, it is doubled.

The IgM antibody measured in the detection method of the present invention is an IgM antibody against one or more superantigens. Here, the “superantigen” is not limited as long as it is a substance produced by Staphylococcus aureus or group A β-hemolytic streptococci, but preferably, it is not subjected to processing by antigen-presenting cells and treated with MHC class II molecules. It is a substance generally classified as a superantigen that binds to the outside of TCR Vβ to overactivate T cells beyond immunological specificity. Preferred superantigens include S. aureus exotoxins SEA (staphylococcal enterotoxin A), SEB (staphylococcal enterotoxin B), SEC (staphylococcal enterotoxin C), SED (staphylococcal enterotoxin D), SEE (staphylococcal enterotoxin E), SEG (staphylococcal enterotoxin G), SEH (staphylococcal enterotoxin H), SEI (staphylococcal enterotoxin I), SEJ (staphylococcal enterotoxin J), SEK (staphylococcal enterotoxin K), SEL (staphylococcal enterotoxin L), SEM (staphylococcal enterotoxin staphylococcal enterotoxin N), SEO (staphylococcal enterotoxin O), SEP (staphylococcal enterotoxin P), SEQ (staphylococcal enterotoxin Q), SELR (staphylococcal enterotoxin lR), SEU (staphylococcal enterotoxin U), TSST-1 (toxic shock syndrome toxin-1 ), Group A β-hemolytic streptococcal exotoxins such as SPEA (streptococcal pyrogenic exotoxin A), SPEC (streptococcal pyrogenic exotoxin C), SEG (staphylococcal enterotoxin G), and the like. Of these, SEA, SEB, SEC, TSST-1 and SPEA are particularly preferred. These superantigens are known per se and are also commercially available.

  An anti-superantigen IgM antibody may measure only an IgM antibody against one type of superantigen, but measures multiple types of IgM against multiple types of superantigens, as specifically described in the Examples below. This is preferable because the detection rate of Kawasaki disease increases. In particular, it is preferable to measure all five types of superantigens corresponding to the five types of superantigens SEA, SEB, SEC, TSST-1 and SPEA, since the detection rate of Kawasaki disease is greatly increased.

  In the method of the present invention, the term “measurement” is used to include any of detection, semi-quantification, and quantification. In the method of the present invention, it is preferable to measure the anti-superantigen IgM concentration.

The detection method of the present invention can also be performed on a bodily fluid sample separated only once from a subject at a certain point in time, but as specifically described in the following examples, in the bodily fluid of a Kawasaki disease patient Since the amount of anti-superantigen IgM increases with the passage of time from the onset, it is preferable to measure the amount of anti-superantigen IgM from a sample collected over time from one subject. By using the measured increase in the amount of anti-superantigen IgM as an index, Kawasaki disease can be detected without comparing with the control value, which is preferable. The sample collection time is, for example, a period from onset or initial diagnosis to 2 weeks to complete cure. For example, a sample collected at an interval of about once a week can be examined. In the following examples, serum is collected and tested every week for 4 weeks.

  The amount of IgM for each superantigen can be easily measured by a conventional immunoassay. Various immunoassay methods are known, but since it is necessary to measure IgM separately from other classes of antibodies such as IgG, a labeled antibody specific for human IgM (or an antigen-binding fragment thereof) is used. Since it is used, the sandwich method can be preferably employed. In particular, a sandwich enzyme immunosorbent assay (hereinafter abbreviated as ELISA method) using an enzyme as a label is preferably used because it does not require an expensive device and is an inexpensive and safe method. it can.

  Hereinafter, the ELISA method will be further described. Examples of the measurement of anti-superantigen IgM antibody titer by ELISA include the following methods. A substance A and a substance B that specifically bind to the anti-superantigen IgM antibody to be measured by antigen-antibody reaction are prepared, and one of them is labeled with an enzyme. In this case, the substance B is a substance different from the substance A and needs to be labeled with an enzyme. In addition, a substrate that reacts with the enzyme to develop color and an acid solution that stops color development are prepared. By immobilizing the substance A on the surface of a solid material (solid phase), adding serum or plasma thereto and incubating the substance A, the anti-superantigen IgM antibody of the item to be measured is bound (primary reaction). After washing away components that did not bind after the primary reaction, substance B is added and incubated to bind the antigen-antibody complex produced by the primary reaction and substance B (secondary reaction). After the secondary reaction, the unbound components are washed away and then the substrate is added (color development reaction). After the color development reaction, an anti-superantigen IgM antibody is detected by adding a solution such as an acid and measuring the absorbance of the solution. At this time, by measuring an anti-superantigen IgM antibody solution having a known concentration at the same time as the serum or plasma sample, the anti-superantigen IgM antibody concentration in the serum or plasma sample can be obtained, or the anti-superantigen IgM having a known concentration can be obtained. Even when the antibody solution is not measured simultaneously, the absorbance at the time of color development or when color development is stopped may be used as the antibody titer. However, when the anti-superantigen IgM antibody solution having a known concentration is not simultaneously measured, it is necessary to always keep measurement conditions such as reagent concentration and reaction time constant.

The sample used for the measurement of the anti-superantigen IgM antibody is preferably a fluid solution for smooth antigen-antibody reaction, but it does not have to be particularly liquid, for example, the sample is solid. In this case, it may be dissolved or dispersed in a suitable solvent, but a body fluid, particularly serum or plasma, is selected as a meaningful solution for measuring IgM for detection of Kawasaki disease. The amount of sample necessary for the measurement is not particularly limited as long as the sample can contact the portion where the substance recognizing the anti-superantigen is immobilized.

  Since the purpose is to detect an anti-superantigen IgM antibody, the substance A can be selected from a superantigen or an anti-human IgM antibody, but considering that it is labeled with an enzyme or biotin in order to develop a color, Preferably, substance A is a superantigen and substance B is an anti-human IgM antibody. If there are multiple items you want to measure. There is also a method of immobilizing a plurality of superantigens on a plastic surface. Note that the superantigen itself is well known and is commercially available, and can be easily obtained.

  When substance A or substance B is immobilized on the surface of a solid material (solid phase), a well-known method using a covalent bond, metal bond, ionic bond, hydrophobic bond, hydrogen bond, hydrophilic bond, or the like is used.

  After immobilizing substance A or substance B on the surface of the solid material, the anti-superantigen IgM antibody is adsorbed on the part where substance A or substance B is not immobilized by applying a treatment that inhibits nonspecific adsorption. It is possible to prevent and efficiently detect the anti-superantigen IgM antibody. The treatment method for inhibiting non-specific adsorption is not particularly limited as long as the gap where substance A or substance B has not bound can be covered (blocked) with another substance, but the three-dimensional structure of the immobilized substance is maintained. It is recommended to use a solution containing casein or bovine albumin.

  The material of the solid material (solid phase) on which substance A or substance B is immobilized is not particularly limited, but considering that the substance to be immobilized is a protein, a highly adsorbent polymer such as polystyrene or polymethyl methacrylate It is preferable that the surface is modified in order to make the substance A or the substance B have an affinity for the solid material. Although it does not specifically limit as a shape, The shape with a hollow is preferable from the need to make the solution react and to measure the light absorbency. For example, a well of a microtiter plate can be used. Alternatively, as the solid phase, beads made of a material mainly composed of the above-described polymer substance can be used.

  The antibody against the anti-superantigen IgM antibody used in the present invention is not particularly limited as long as it can recognize human anti-superantigen IgM, and an antibody purified from antiserum obtained by immunizing human IgM to mammals, and anti-superantigen IgM Any monoclonal antibody produced from a hybridoma obtained by fusing antibody-producing cells taken from a mammal immunized with an antibody with myeloma cells can be used.

  When an antibody against human IgM is labeled with an enzyme, the enzyme is combined with an antibody or antibody fragment (the antibody is labeled with an enzyme) for purposes such as immunohistochemical staining and immunoassay. You can use what is being used. There is no particular limitation on the enzyme labeled with the enzyme-labeled antibody, but horseradish peroxidase (hereinafter abbreviated as HRP) because of its high ability to convert a substrate to a detection substance and low deactivation of the enzyme. ), Alkaline phosphatase (hereinafter abbreviated as ALP), or β-D-galactosidase (hereinafter abbreviated as β-Gal) is preferably selected. Enzyme labeling methods can be broadly classified into two types: labeling methods using covalent bonds and labeling methods using non-covalent bonds. As a labeling method by covalent bond, a method of joining using the amino group of an antibody or an antibody fragment and an amino group of the enzyme, a method of regulating the enzyme-labeled antibody by binding the enzyme and the antibody at the gene level, There is a method of selectively using a thiol group of a hinge part produced by reducing an antibody or antibody fragment and linking it with an enzyme. As a non-covalent bonding method, an enzyme and an anti-enzyme antibody are reacted, and this and the target antibody are cross-linked with an antibody against the antibody. Also, the antibody is labeled with biotin, and this is labeled with the enzyme label. Enzyme-labeled antibodies can also be prepared by reacting avidin.

  In the present invention, the substrate for coloring the enzyme is not particularly limited. For example, when HRP labeling is selected as the enzyme-labeled antibody, the substrate may be 3-amino-9-ethylcarbazole, 5-aminosalicylic acid, 4-chloro- 1-naphthol, o-phenylenediamine, 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid), 3,3-diaminobenzidine, 3,3 ′, 5,5′-tetramethylbenzidine , O-dianisidine and 3,3-dimethoxybenzidine are preferred. When ALP labeling is selected as the enzyme-labeled antibody, a solution containing any of 5-bromo-4-chloro-3-indolyl phosphate, nitroblue tetrazolium, and p-nitrophenyl phosphate is used as a substrate. be able to. Furthermore, when β-Gal labeling is selected as the enzyme-labeled antibody, either o-nitrophenyl-β-D-galactoside or 5-bromo-4-chloro-3-indole-β-D-galactopyranoside A solution containing can be used.

  When determining the absorbance, an acid or the like may be added after the color development reaction to stop the color development and the absorbance may be measured, or the absorbance may be measured without adding an acid or the like.

  Incubation in the present invention is to stand at a certain temperature during antigen-antibody reaction and color development reaction. In the incubation, the longer the incubation time, the higher the ratio of the reaction between the anti-superantigen IgM antibody and the substance that recognizes it, and as a result, the degree of color development increases, but the antigen-antibody reaction saturates after a certain period of time. In addition, if incubation is performed at a high temperature for a long time, there is a possibility that correct measurement cannot be performed due to decay or activation or deactivation of blood components. When these are concerned, it is desirable to add a preservative as long as the measurement is not affected, or to control at a temperature that does not rot during incubation.

  The washing in the present invention is to wash away substances that did not undergo antigen-antibody reaction after incubation, and the washing material is not particularly limited as long as it has a washing effect and does not affect substances bound by antigen-antibody reaction. There is no. The reaction temperature in each step of the primary reaction, secondary reaction, washing, color development, and color development stop in the present invention is not particularly limited, but 2 to 40 ° C. so that the reaction solution does not freeze and evaporate and the enzyme is not deactivated. It is preferable that it is the range of these.

  Although the ELISA method has been described, the method of the present invention is not limited to the ELISA method, and as a label used in the sandwich method, in addition to the enzyme, a fluorescent label, a chemiluminescent label, a colloidal gold, a radiolabel Other labels can also be used. In addition, an immunochromatography method which is one form of the sandwich method and is widely used due to the simple measurement operation can be used. All of these are conventional methods.

Based on the measured amount of IgM, Kawasaki disease can be detected . Detection can be performed based on whether or not the measured IgM amount is significantly higher than that of non-Kawasaki disease patients of the same generation as the subject. That is, it can be set by setting a person of the same generation as the Kawasaki disease patient as a control group (control group), measuring the anti-superantigen IgM antibody titer of the control group, and setting a cutoff value. The cut-off value is a value set for the purpose of judging and diagnosing a disease, and the cut-off value in the present invention is an anti-superantigen IgM antibody titer set to know whether there is a suspicion of Kawasaki disease. It is. The method for setting the cut-off value in the present invention is not particularly defined, but when the antibody titer is normally distributed, it is preferable to set the mean value ± 2SD value. In addition, 95% CI (confidence intervals) can be adopted. Furthermore, a cut-off value may be set according to the purpose by an ROC (receiver-operating characteristic) curve.

  There are various methods for setting the cut-off value, but in an examination in which a medically healthy group and a disease-bearing group are completely separated, the cut-off value may be set between the two. However, in most cases, there is an overlap between the values of the medically healthy group and the disease-bearing group, so it is easy to set a cut-off value that completely separates those who have the disease from those who do not Instead, it is often necessary to set the cutoff value somewhere in the overlapping range. In the case of a test in which a numerical value increases when a certain illness is obtained, setting a high cut-off value makes it possible to determine an abnormal subject as a illness, but also increases oversight. On the other hand, if the cut-off value is set low, there is less chance of overlooking, but there is a higher possibility of being determined to be ill while being healthy. Therefore, the value to be set as the cut-off value is set in consideration of the purpose of the examination and the seriousness of the disease.

  Conventionally, a method of determining an abnormality (positive) by deviation from the mean ± 2SD of the test value of a medically healthy group, or setting a cut-off value when 95% of patients in the disease group become positive In many cases, these methods can also be employed in the method of the present invention. However, since the degree of disadvantage due to oversight (false negatives) and oversight (false positives) varies depending on the disease, the nature of the disease should be increased to increase specificity for diseases with many disadvantages associated with false negatives. It is preferable to determine an optimum cutoff value according to the above.

  For example, in diabetes screening, a cut-off value is set at the highest efficiency. Although the standard range of hemoglobin A1c for normal subjects according to academic standards is up to 5.8%, the cutoff value of the Geriatric Health Law is set to 5.6%, and the cutoff value exists within the normal range . If the control of diabetes is successful, hemoglobin A1c often falls within the normal range, so it seems reasonable that the best cut-off value is about this level.

In some cases, a cut-off value is set when the probability of disease onset begins to increase. Such an approach can be adopted if the disease has a clear inflection point. For example, the higher the total cholesterol value, the higher the probability of developing ischemic heart disease, but a cut-off value is defined as a numerical value at the boundary where the risk of developing ischemic heart disease begins to increase.
In some cases, a value theoretically theoretically pathologically or physiologically is used as a cutoff value. For example, the maximum amount of uric acid dissolved in the blood is 7 mg / dl, and gout and uric acid calculus are generated when the maximum amount is exceeded.

  In the case where the above-described numerical value cannot be theoretically determined and the cut-off value is statistically determined, the reference range can be used as it is. As a standard, it is ideal if a fluctuation range that is acceptable for a person is obtained from data when the patient is healthy, and this can be used as a standard, but it is actually difficult. Therefore, the reference range usually collects the measured values of medically healthy individuals (reference value), and obtains a numerical value in the central portion including 95% of them. In the case of normal distribution, the average value ± 2 standard deviation (2SD) of the reference individual is set as a cutoff value (Non-patent Document 12). This range stochastically is a range in which 95.4% of the healthy group is normal, in other words, about 5% are determined to be abnormal. In addition, as a statistical method for calculating the normal value (range), various methods such as the probability paper method, the Hoffmann method, the Usui method, and the maximum likelihood conversion method can be used.

  The Usui method is an iterative cutting method in which values from ± 2SD to ± 3SD or more are repeatedly rounded down. In biological experiments, unexpected abnormal values may be mixed, but the data will be removed. The iterative cutting method is one of the methods for deleting abnormal data, and it repeatedly deletes abnormal data with a specified cutting SD value. When removing an outlier with a small SD, there is a high risk that data that should not be excluded will be erroneously set as an outlier, and with a large SD, there is a high risk of mixing an extreme value. In the case of the Usui method, 2.6SD is said to be appropriate. Since the iterative cutting method should be performed on a normal distribution, it is necessary to check the distribution type before cutting.

  ROC is one of methods for evaluating diagnostic techniques for clinical laboratory tests. ROC analysis is to compare the accuracy of each test method by simultaneously examining the sensitivity and specificity of multiple test methods. Some of the test values are positive / negative as cut-off values. Obtained by plotting sensitivity (or true positive rate: TPR) on the vertical axis and 1-specificity (or false positive rate: FPR) on the horizontal axis. Analysis based on ROC curve. The true positive rate in the present invention is a rate at which a Kawasaki disease patient can be determined as Kawasaki disease, and the false positive rate is a rate at which a person who is not Kawasaki disease is erroneously determined as Kawasaki disease. The ROC analysis can further provide a judgment material for determining an optimal cutoff value according to the nature of the disease.

In the present invention, the control group set for detecting Kawasaki disease is preferably composed of a medically healthy group, and it is preferable that there is no significant difference in age from the Kawasaki disease group as a control. Medically healthy means that it does not meet the diagnostic criteria for Kawasaki disease and is not sensitized to superantigens. In the present invention, “not sensitized with a superantigen” preferably means that there is no suspicion of infection with Staphylococcus aureus or group A β-hemolytic streptococci within 2 months, and more preferably an infection within 6 months is confirmed. It means not. In the present invention, the control group for comparison with the Kawasaki disease group is preferably composed of 10 years of age or younger with frequent occurrence of Kawasaki disease.

When Kawasaki disease is detected by comparison with the control group, there is a significant difference between the control group and the Kawasaki disease group, that is, when the antibody in the Kawasaki disease group is significantly higher than the control group, It is preferable to employ a detection method. The method for testing the significant difference is not particularly limited as long as it is a commonly used method. As a significant difference test method, for example, there is a method of testing whether or not the variances are equal and then performing a t test to obtain a p value. A significant difference in the present invention means that the p value is less than 0.05.

  In the present invention, as a method for determining the cut-off value of the anti-superantigen IgM antibody titer, when the measured value is normally distributed, the cut-off value is preferably within a range of 5% of the population. If the measured value does not have a normal distribution, the natural logarithm of the anti-superantigen IgM antibody titer is taken. If the natural logarithm has a normal distribution, the natural logarithmic value is 5% of the population. A method of cutting off may be used. When the natural logarithm of the measured value is not normally distributed, people who exceed the average value of natural logarithm plus 3SD are excluded. It is preferable to perform recalculation after the exclusion and use the average value of natural logarithms plus 2SD as a cutoff value. Or. You may employ | adopt 95% CI which is the range which can take the true value of a population with a significance level of 5%.

  When setting cut-off values using ROC curves created by measuring anti-superantigen IgM antibody titers in Kawasaki disease group and control group, we want to detect Kawasaki disease with what degree of sensitivity or specificity The cut-off value can be set for the purpose of whether or not. That is, the anti-superantigen IgM antibody titer at the point where desired sensitivity or specificity is obtained on the ROC curve may be used as the cut-off value. When creating an ROC curve, one item may be used, or a plurality of items may be combined.

As a method of detecting Kawasaki disease using the cut-off value of anti-superantigen IgM antibody titer, a method in which an item having a measured anti-superantigen IgM antibody titer exceeds the cut-off value, The method by which the item exceeds the cut-off value, and further, a method of detecting a new cut-off value by combining a plurality of measured anti-superantigen IgM antibody titers and using the value Conceivable.

As a method for setting a new cut-off value by combining a plurality of anti-superantigen IgM antibody titers, for example, there is the following method. When it is desired to evaluate the five types of anti-superantigen IgM antibody titers, the cutoff values of the anti-superantigen antibody titers are obtained by the above-described method (the obtained five types of cutoff values are a, b, c, d, e). And the value obtained by dividing all the measured items by itself (a ÷ a + b ÷ b + c ÷ c + d ÷ d + e ÷ e = 5) is set as a “new cut-off value”. When detecting Kawasaki disease, anti-superantigen IgM antibody titers of patients suspected of having Kawasaki disease (the five obtained anti-superantigen IgM antibody titers are a ′ and b ′, and the cutoff values are a ′ and b ′. , C ′, d ′, e ′) divided by the respective cut-off values and adding all (a ′ ÷ a + b ′ ÷ b + c ′ ÷ c + d ′ ÷ d + e ′ ÷ e) It is compared with “5” which is an “off value”. Here, the number of items of the anti-superantigen IgM antibody titer is exemplified by 5 items, but the number of items may be more or less than this.

In order to detect Kawasaki disease using the above method, it is necessary that the anti-superantigen IgM antibody titers of the Kawasaki disease group and the control group are not influenced by factors other than Kawasaki disease. For example, the control group needs to be composed of persons who are not sensitized to the superantigen. The control group is preferably a person who does not suffer from S. aureus or group A β-hemolytic streptococcal infection within 2 months, more preferably a person who has not been confirmed to have an infection within half a year. preferable.

In addition, as described above, the amount of anti-superantigen IgM in the body fluid of Kawasaki disease patients increases with the passage of time from the onset, so for samples collected over time from one subject, By measuring the amount of anti-superantigen IgM, it is possible to detect Kawasaki disease without using a comparison with a control or a cut-off value. For example, Kawasaki disease patients can observe Kawasaki disease group and control group by observing the behavior of anti-superantigen IgM antibody titer by dividing the disease day every week from the day when Kawasaki disease develops, and comparing Kawasaki disease group and control group. Diseases can also be detected . The anti-superantigen IgM antibody titer behavior includes anti-superantigen IgM antibody titer that increases or decreases daily, or temporarily increases or decreases temporarily. An increase or decrease in the antigen IgM antibody titer is observed.

The present invention further provides a kit for determining Kawasaki disease used for carrying out the method of the present invention. The kit includes a solid phase on which one or more superantigens are immobilized, a solid phase on which IgM for one or more superantigens is immobilized, a labeled anti-human IgM antibody or an antigen-binding fragment thereof, or a labeled anti-superantigen antibody. Or at least an antigen-binding fragment thereof and, as described above, at least a solid phase on which one or more superantigens are immobilized and a labeled anti-human IgM antibody or an antigen-binding fragment thereof. preferable. The “antigen-binding fragment” means an antibody fragment that has an ability to bind to an antigen, such as a Fab fragment or F (ab ′) ₂ fragment. The kit may contain other components such as a reaction buffer solution and a washing solution. These are the same as in the case of a normal immunoassay kit.

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

Reference Example 1: Measurement of anti-superantigen IgG antibody titer and anti-superantigen IgM antibody titer in IVIG preparation
In order to investigate the anti-superantigen IgG antibody titer contained in IVIG preparation, PBS (hereinafter referred to as Solution A) containing 0.25% IVIG preparation and 0.05% bovine serum albumin and 0.05% Tween20 (trade name) is referred to as SEA, SEB. , IgG antibody titers against SEC, TSST-1 and SPEA were measured. The antibody titer was measured by the ELISA method described in Non-Patent Document 13. However, the IVIG preparation was measured by diluting 1000 times with Solution A at the time of measurement. Absorbance was measured with a corona microplate reader MTP-300 (Corona Electric). Those having an absorbance exceeding 3.0 were further diluted with solution A and measured, and the obtained value was multiplied by the dilution factor to obtain the antibody titer.

  That is, the ELISA method was specifically performed as follows. SEA, SEB, SEC, TSST-1 and SPEA superantigens (commercially available from Toxin Technology, Florida, USA) were incubated overnight at 4 ° C. in wells of a 96-well microplate. After washing, blocking was performed overnight with PBS containing 0.5% fetal calf serum. After washing, serum diluted 1000-fold with PBS containing 0.25% fetal bovine serum and 0.05% Tween 20 (trade name) was added to each well and incubated at 25 ° C. for 1 hour. After washing, an HRP-labeled anti-human IgG antibody (from mouse, Zymed) was added and incubated at 25 ° C. for 30 minutes. After washing, 3,3 ′, 5,5′-tetramethylbenzidine was added and incubated at 25 ° C. for 30 minutes. The absorbance at 450 nm of the solution in each well was measured.

  Table 1 shows the average anti-superantigen IgG antibody titers for 4 lots of IVIG preparation (Mitsubishi) and 5 lots of Benilon (Teijin). As a result of comparing the measured values of IVIG preparation and solution A, there was a significant difference in all five items (SEA, SEB, SEC, TSST-1, and SPEA) (all P <0.01). That is, it was found that the anti-superantigen IgG was contained in the IVIG preparation. It was also found that there was almost no lot-to-lot difference or manufacturer difference in anti-superantigen IgG antibody titers.

  In addition, in order to investigate the anti-superantigen IgM antibody titer contained in the IVIG preparation, the IgM antibody titer against the SEA, SEB, SEC, TSST-1, and SPEA was measured for the IVIG preparation and Solution A. The IgM antibody titer was measured according to Non-Patent Document 12, but specifically, the IVIG preparation was diluted 100-fold with Solution A, and the above-mentioned IgG immunoassay (Non-Patent Document 13) was used for HRP-labeled anti-antibody. A human IgG antibody (derived from mouse, Zymed) was used as an HRP-labeled anti-human IgM antibody (derived from goat, Capel), which was diluted 2000 times and used. Furthermore, the reaction time with the chromogenic substrate was 15 minutes. Absorbance was measured with a corona microplate reader MTP-300 (Corona Electric). Table 1 shows the average values of anti-superantigen IgM antibody titers for 4 lots of IVIG preparation (Mitsubishi) and 5 lots of Benilon (Teijin). As a result of comparing the measured values of IVIG preparation and solution A, there was no significant difference in all five items (SEA, SEB, SEC, TSST-1, and SPEA) (all P> 0.1). That is, IgM for these superantigens was not significantly contained in the IVIG preparation.

Reference Example 2 Measurement of Total IgG Level in Serum of Kawasaki Disease Patients Treated with IVIG Kawai Disease Patients (44 Men, 21 Women, Total 65, Age 23.0 ± 19.5) Before and After IVIG Preparation (Days Before Administration) And the next day after administration) serum total IgG levels were measured. All patients with Kawasaki disease are treated with IVIG given by Venoglobulin or Benilone.

  Table 2 divides patients into groups (group A) where the IVIG formulation dose per kg is 1 g and groups (group B) where the IVIG formulation dose per kg body weight is 2 g. The increase in the total IgG value on the day before and after administration was shown. As a result, in both groups, it was found that the total IgG level in the serum increased immediately after receiving treatment with the IVIG preparation. In addition, Table 2 shows the ratio of the A group and the B group with respect to the increase in the total IgG value.

Comparative Example 1 Measurement of anti-superantigen IgG antibody titer in the serum of Kawasaki disease patients who received IVIG The anti-superantigen IgG antibody titer in serum before and after IVIG preparation administration of Kawasaki disease patients was measured. The subject was the same patient as in Reference Example 2. The anti-superantigen IgG antibody titer was measured by the same method as in Reference Example 1 with the same items as in Reference Example 1. Serum was diluted 1000 times with solution A and measured. As in Reference Example 2, the patients were divided into Group A and Group B, and the amount of increase in anti-superantigen IgG antibody titer on the day before and after IVIG preparation administration was calculated (Table 2).

  As a result, it was found that IgG antibody titers against serum SEA, SEB, SEC, TSST-1, and SPEA increased immediately after receiving treatment with IVIG preparation in both groups. In addition, Table 2 shows the ratio of Group A and Group B with respect to the increase in anti-superantigen IgG antibody titer.

  From Reference Example 2 and Comparative Example 1, the ratio between Group A and Group B regarding the increase in the total IgG value in the serum of patients treated with IVIG preparations is the ratio between Group A and Group B regarding the increase in anti-superantigen IgG antibody titer. It was found to be approximately equal to the group ratio. In other words, since the anti-superantigen IgG antibody titer in the serum of patients who received IVIG preparation is linked to the total IgG value, the anti-superantigen IgG antibody in the serum of Kawasaki disease patients who received IVIG preparation is almost Proved to be due to the IVIG formulation. In addition, Reference Examples 1 and 2 and Comparative Example 1 show that IgG against the superantigen in the blood of Kawasaki disease patients is increased by IVIG administration, so that the measurement of IgG cannot discuss the relationship between Kawasaki disease patients and superantigens. It was done.

Example 1 Measurement of total IgM value and anti-superantigen IgM antibody titer in serum of Kawasaki disease patients before and after IVIG administration By the ELISA method described in Reference Example 1, serum anti-superantigen IgM before and after IVIG preparation administration The antibody titer was measured. The subject was the same patient as in Reference Example 2.

  In patients with Kawasaki disease, there was no change in total IgM values before and after IVIG treatment (before administration: 171.4 ± 52.9 mg / dl, within 48 hours after administration of 2 g / kg body weight: 171.3 ± 48.4 mg / dl). In addition, the serum anti-superantigen IgM antibody titer on the day before and after administration of the IVIG preparation was unchanged (Table 3).

  From Reference Example 1 and Example 1, it is considered that the total IgM value is not affected by IVIG treatment, and the IVIG preparation contains almost no IgM against the superantigen, and the anti-superantigen IgM antibody titer It was found that the IVIG preparation had no effect on the measurement.

Reference Example 3 Measurement of anti-superantigen IgM antibody titer in non-Kawasaki disease patients and setting of cut-off value Serum was received from the control group (70 males, 50 females, total 120, age 24.1 ± 21.2). The anti-superantigen IgM antibody titer was measured. The control group consists of those who visited the hospital for screening and those who did not undergo surgery, were not treated with immunoglobulin preparations, and involved S. aureus and group A β-hemolytic streptococcal infections It has been confirmed that there is no infection with S. aureus and streptococci within 6 months. Furthermore, it is shown that there is no significant difference in age from the Kawasaki disease patient group. The anti-superantigen IgM antibody titer was measured in the same manner as in Reference Example 1.

  Since the measured value was converted into a natural logarithm, there was normality, and when 95% CI of the natural logarithm of the antibody titer in the control group was defined as a positive cut-off value, the positive cut-off values were SEA = 0.059, SEB = 0.108, SEC = 0.198, TSST-1 = 0.094, and SPEA = 0.098.

Example 2 Measurement and Positive Rate of Anti-Superantigen IgM Antibody Titers in Kawasaki Disease Patients Serum provided from Kawasaki disease patient group was the first week of onset of Kawasaki disease (onset 1-7 days, n = 128), 2 weeks. Serum resistance is divided into eyes (onset days 8-14, n = 113), week 3 (onset days 15-21, n = 38), and week 4 (onset days 22-28, n = 14). The superantigen IgM antibody titer was measured in the same manner as in Reference Example 1. The subjects were the same patients as in Reference Example 2 and Comparative Example 1.

  The ratio of the number of people exceeding the cut-off value set in Reference Example 3 is shown as “positive rate” in FIG. Moreover, the time-dependent change of each superantigen IgM antibody titer is shown in FIG. In FIG. 4, “*” and “**” indicate that there is a significant difference from that of an unmarked or different number of rice marks. As shown in FIG. 4, the anti-superantigen IgM antibody titer was statistically significantly different from the control group except for the first week of SEC (first week of SEA, second SEC). 3rd week; P <0.01, SEA second, third, fourth week, SEB first, second, third, fourth week, SEC fourth week, TSST-1, first, second, third, third Week 4, SPEA Weeks 1, 2, 3, and 4; p <0.001).

  Furthermore, the positive rate increased with each week, which was also demonstrated by the ROC curve (FIGS. 2a-e). Moreover, since the anti-superantigen IgM antibody titer was low at the early stage of infection, it was found that Kawasaki disease can also be determined by the daily increase of the individual anti-superantigen IgM antibody titer.

Example 3 Determination of Kawasaki Disease Using Multiple Items For the anti-superantigen IgM antibody titer measured in Reference Example 3 and Example 2, the positive rate was calculated according to the number of positive items, The ratio that was positive only for any one item, the ratio that was positive for any one or two items, the ratio that was positive for any one to three items, the ratio that was positive for any one to four items, any one It was plotted in FIG. 1 when ˜5 items were positive. In all cases, the percentage of Kawasaki disease group detected as positive was higher than that of the control group. When 1 to 5 items, that is, any one of them became positive, the rate of detection as positive was particularly high.

  From Examples 2 and 3, it was found that Kawasaki disease can be determined by measuring the anti-superantigen IgM antibody titer, and the reliability tends to increase every week. Furthermore, it was also found that the probability of detecting Kawasaki disease tends to increase when multiple items are used for the determination of Kawasaki disease. Moreover, since the anti-superantigen IgM antibody titer was low at the early stage of infection, it was found that Kawasaki disease can also be determined by the daily increase of the individual anti-superantigen IgM antibody titer.

Example 4 Method of using cut-off value of each item for determination of Kawasaki disease using a plurality of items The anti-superantigen IgM antibody titer measured in Reference Example 3 and Example 2 was newly evaluated by the following method.

  The anti-superantigen IgM antibody titer of each item of the Kawasaki disease group and the control group was divided by the cut-off value of each item determined in Example 2 (formulas (1) to (5)). The values obtained from the formulas (1) to (5) were added to obtain total SAg-IgM (formula (6)). Since the cut-off value of the values calculated by the formulas (1) to (5) is 1, the positive cut-off value of the total SAg-IgM calculated by the formula (6) is set to 5, and the positive rate is developed. Plotted in FIG. 3 by the number of weeks elapsed. All items were more positive in the Kawasaki disease group than in the control group. In the Kawasaki disease group, the positive rate increased with each week. Even in the method using the cut-off value of each item for the determination of Kawasaki disease using a plurality of items, since the total SAg-IgM is low at the early stage of infection, the individual's total SAg-IgM increases daily. It was found that Kawasaki disease can also be determined by doing.

  It was found that Kawasaki disease can be determined even when cut-off values are set for different items, and the reliability increases with each week. This was also proved by the ROC curve (Fig. 2f).

The anti-superantigen IgM antibody titer positive rate according to several items in a Kawasaki disease patient is shown. The anti-SEA-IgM antibody titer ROC curve in the serum sample of a Kawasaki disease patient is shown. The anti-SEB-IgM antibody titer ROC curve in the serum sample of a Kawasaki disease patient is shown. The anti-SEC-IgM antibody titer ROC curve in the serum sample of a Kawasaki disease patient is shown. The anti-TSST-1-IgM antibody titer ROC curve in the serum sample of a Kawasaki disease patient is shown. The anti-SPEA-IgM antibody titer ROC curve in the serum sample of a Kawasaki disease patient is shown. The total anti-superantigen IgM antibody titer ROC curve in the serum sample of a Kawasaki disease patient is shown. The anti-antigen IgM antibody titer positive rate in the serum sample of a Kawasaki disease patient is shown. The time course of various anti-superantigen IgM antibodies in serum samples of Kawasaki disease patients is shown.

Claims (12)

A method for detecting Kawasaki disease, comprising measuring IgM against one or more superantigens in a body fluid separated from a subject.   The method according to claim 1, wherein the superantigen is produced by Staphylococcus aureus or Group A β-hemolytic streptococci.   The method according to claim 2, wherein the superantigen is at least one selected from the group consisting of SEA, SEB, SEC, TSST-1 and SPEA.   The method according to any one of claims 1 to 3, wherein the body fluid is blood.   The method according to any one of claims 1 to 4, wherein IgM against a plurality of superantigens is measured.   The method according to claim 1, comprising measuring the concentration of IgM.   The method according to any one of claims 1 to 6, wherein the IgM is a significantly higher value as compared with a non-Kawasaki disease patient of the same generation as the subject. The method according to claim 6, wherein the detection is based on whether the IgM concentration is equal to or higher than a cutoff value.   The method according to claim 6, wherein an increase in the IgM concentration measured for each of a plurality of body fluid samples separated from one subject over time is used as an index.   The method according to any one of claims 1 to 9, wherein the subject from whom the body fluid has been separated is a patient to whom an intravenous human immunoglobulin preparation has been administered.   A solid phase on which one or a plurality of superantigens are immobilized, a solid phase on which IgM for one or a plurality of superantigens is immobilized, a labeled anti-human IgM antibody or an antigen-binding fragment thereof, or a labeled anti-superantigen antibody or an antigen thereof A kit for determining Kawasaki disease, comprising at least a binding fragment.   The kit according to claim 11, comprising at least a solid phase on which one or a plurality of superantigens are immobilized, and a labeled anti-human IgM antibody or an antigen-binding fragment thereof.

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