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WO2018030270A1 - Procédé et spécimen pour tester la maladie de kawasaki - Google Patents

Procédé et spécimen pour tester la maladie de kawasaki Download PDF

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Publication number
WO2018030270A1
WO2018030270A1 PCT/JP2017/028236 JP2017028236W WO2018030270A1 WO 2018030270 A1 WO2018030270 A1 WO 2018030270A1 JP 2017028236 W JP2017028236 W JP 2017028236W WO 2018030270 A1 WO2018030270 A1 WO 2018030270A1
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Prior art keywords
antibody
kawasaki disease
lrg1
lbp
test piece
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English (en)
Japanese (ja)
Inventor
弥生 堀内
雅亮 森
平野　久
洋子 齋藤
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Yokohama City University
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Yokohama City University
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Priority to JP2018532985A priority Critical patent/JPWO2018030270A1/ja
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • the present invention relates to a test method and a test piece 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 (1.5 days or more of fever, 2. hyperemia of the conjunctiva of the eyeballs, 3. redness of the lips, tongue, 4. irregular rash, 5. stiffness of the fingers in the acute phase, palmarity. 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).
  • 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 if not treated, 25-30% of patients suffer from cardiac complications typified by 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.
  • 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.
  • VEGF vascular endothelial growth factor
  • the object of the present invention is to provide a method and a test piece for quickly and simply examining Kawasaki disease.
  • LBP lipopolysaccharide binding protein
  • LRG1 leucine-rich alpha-2-glycoprotein 1
  • the gist of the present invention is as follows.
  • a method for examining Kawasaki disease comprising measuring the levels of lipopolysaccharide binding protein (LBP) and leucine-rich ⁇ 2-glycoprotein (LRG1) in a subject-derived specimen.
  • LBP lipopolysaccharide binding protein
  • LRG1 leucine-rich ⁇ 2-glycoprotein
  • a test piece for detecting Kawasaki disease by immunochromatography comprising a carrier on which an anti-LBP antibody is immobilized and a carrier on which an anti-LRG1 antibody is immobilized.
  • the test piece according to (5), wherein the carrier on which the anti-LBP antibody is immobilized and the carrier on which the anti-LRG1 antibody is immobilized are contained in the same test piece.
  • the test piece according to (5), wherein the carrier on which the anti-LBP antibody is immobilized and the carrier on which the anti-LRG1 antibody is immobilized are contained in separate test pieces.
  • Kawasaki disease About 10,000 patients are diagnosed with Kawasaki disease each year. 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 initial screening tests and even severity determination, the use of expensive gamma globulin preparations as therapeutic agents can be avoided, leading to savings in medical costs.
  • 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.
  • 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. 2016-156241 which is the basis of the priority of the present application.
  • the serum levels of LBP and LRG1 were determined using sera from 55 acute patients, 13 healthy children (during allergy testing), and 24 pediatric patients (autoimmune disease).
  • Results of LBP and LRG1 ROC (Receiver Operating Characteristic curve) analysis On the vertical axis is sensitivity% (percentage that is positive when people who are truly Kawasaki disease are tested), and on the horizontal axis is 100% -specificity% (proportion of misdiagnosing diseases other than Kawasaki disease as Kawasaki disease) I took it.
  • the present invention provides a method for examining Kawasaki disease, characterized by measuring the levels of lipopolysaccharide binding protein (LBP) and leucine-rich ⁇ 2-glycoprotein (LRG1) in a specimen derived from a subject.
  • LBP lipopolysaccharide binding protein
  • LRG1 leucine-rich ⁇ 2-glycoprotein
  • Predetermined values can be used for the presence or absence of Kawasaki disease, particularly for the determination when making a distinction from other diseases.
  • Kawasaki disease can be diagnosed with high accuracy when LBP is 25 ng / mL or more and LRG1 is 300 ng / mL or more in the acute phase of Kawasaki disease. Therefore, the method of the present invention can be used for diagnosis of Kawasaki disease (determination of presence or absence of Kawasaki disease).
  • the threshold values of LBP and LRG1 are set one by one, it can be made binary (more / less than) with LBP and binary (more / less) with LRG1. For example, by assigning 1 or 0 to each binary value, it can be used for digital judgment by software.
  • the measured value may be analyzed by ROC (Receiver Operating Characteristic curve), and the threshold value may be set according to the specificity (see Table 1 described later).
  • ROC Receiveiver Operating Characteristic curve
  • the specimen derived from the subject may be a liquid clinical specimen such as serum, blood (whole blood) or plasma.
  • enzyme-linked immunosorbent assay ELISA
  • immunoblotting fluorescent antibody
  • RIA radioimmunoassay
  • fluorescent enzyme immunoassay FLEIA
  • chemiluminescence Any method such as enzyme immunoassay (CLEIA), chemiluminescence immunoassay (CLIA), electrochemiluminescence immunoassay (ECLIA), immunochromatography (ICA), western blot (WB) may be used.
  • CLIA enzyme immunoassay
  • CLIA chemiluminescence immunoassay
  • ELIA electrochemiluminescence immunoassay
  • ICA immunochromatography
  • WB western blot
  • the present invention also provides a test strip for detecting Kawasaki disease by immunochromatography, which includes a carrier on which an anti-LBP antibody is immobilized and a carrier on which an anti-LRG1 antibody is immobilized.
  • an antibody that specifically binds to LBP and an antibody that specifically binds to LRG1 are commercially available and available.
  • the antibody may be either a monoclonal antibody or a polyclonal antibody.
  • an antibody that specifically binds to the target molecule in the present invention, LBP, LRG1
  • an antibody that specifically binds to the target molecule unlabeled antibody
  • colloidal particles for example, metal colloidal particles such as gold, silver, and platinum
  • colored insoluble particles such as polystyrene particles colored with pigments or dyes such as red and blue. Can do.
  • Any carrier can be used as long as it can develop the detection target substance (target molecule) in a chromatographic manner and can immobilize an antibody that specifically binds to the detection target substance, and has high water absorption so that it can move by capillary action.
  • it is made of a porous material.
  • Specific examples include nylon, polysulfone, polyethersulfone, polyvinyl alcohol, polyester, polyolefin, cellulose, nitrocellulose, cellulose acetate, acetylcellulose, glass fiber, and mixed materials thereof.
  • nitrocellulose can be suitably used.
  • the carrier on which the anti-LBP antibody is immobilized and the carrier on which the anti-LRG1 antibody is immobilized may be included in the same test piece, or the carrier on which the anti-LBP antibody is immobilized and the anti-LRG1 antibody
  • the immobilized carrier may be included in a separate test piece.
  • a test piece is good to be comprised from the following members, for example.
  • Sample pad for dropping specimen (4 in Fig. 3)
  • a conjugate pad on which a labeled antibody that specifically binds to the target molecule (in the present invention, LBP, LRG1) is immobilized (5 in FIG. 3)
  • An unlabeled antibody that specifically binds to the target molecule is immobilized on the test line (6 in FIG. 3), and an unlabeled antibody that specifically binds to the labeled antibody is immobilized on the control line (7 in FIG. 3).
  • Membrane / absorption pad (8 in Fig. 3) ⁇ Base sheet (2 in Fig. 3)
  • the sample pad (4 in Fig.
  • a material having high water absorption such as sponge, glass fiber, nylon, cellulose, polyurethane, polyacetate, cellulose acetate or the like, or a nonwoven fabric thereof can be used.
  • the mesh size of the nonwoven fabric may be selected so as to have a function of filtering the sample liquid.
  • the thickness of the sample pad 4 is not limited, but 0.1 to 3 mm is preferably used.
  • the conjugate pad (5 in FIG. 3) contains a labeled antibody that specifically binds to the target molecule (in the present invention, LBP, LRG1).
  • the conjugate pad (labeled portion) 5 is made of a water-absorbing material, for example, a nonwoven fabric such as sponge or glass fiber, for fixing the labeled antibody.
  • the thickness is not limited, but 0.1 to 3 mm is preferably used.
  • the conjugate pad (labeling site) 5 needs to be provided on the downstream side of the sample pad 4, the upstream side of the test line 6, and the upstream side of the control line 7.
  • the labeling substance is added to an antibody that specifically binds to the target molecule after adjusting the labeling substance to an optimal concentration for detection of the target molecule (in the present invention, LBP, LRG1). It is good to make it soaked in pads, such as a glass fiber nonwoven fabric, and to dry enough after that.
  • the membrane 3 is made of a porous material as described above, specifically, nylon, polysulfone, polyethersulfone, polyvinyl alcohol, polyester, polyolefin, cellulose, nitrocellulose, cellulose acetate, acetylcellulose, glass fiber, and the like.
  • a porous material as described above, specifically, nylon, polysulfone, polyethersulfone, polyvinyl alcohol, polyester, polyolefin, cellulose, nitrocellulose, cellulose acetate, acetylcellulose, glass fiber, and the like.
  • Examples of the mixed material may be exemplified.
  • nitrocellulose can be suitably used.
  • the thickness is not limited, but 0.1 to 3 mm is preferably used.
  • an antibody (unlabeled) that specifically binds to the target molecule is immobilized.
  • the shape is linear, but is not limited to a line, and a plurality of dots may be formed in a linear shape.
  • the line is not limited to a straight line but may be an arc or a curve.
  • An antibody that specifically binds to the labeled antibody (unlabeled) is immobilized on the control line of membrane 3 (7 in FIG. 3).
  • the antibody immobilized on the control line include anti-mouse IgG antibody, anti-goat Go IgG antibody, anti-rabbit IgG antibody, and anti-rat rat IgG antibody.
  • the shape is linear, but is not limited to a line, and a plurality of dots may be formed in a linear shape.
  • the line is not limited to a straight line but may be an arc or a curve.
  • the absorption pad 8 can absorb the sample liquid and the labeled antibody that have flowed due to the capillary phenomenon, and can control the direction of the liquid flow.
  • the absorption pad 8 is provided at one end different from the sample pad 4 of the test piece 1, and positively absorbs the sample liquid moving on the test piece 1, thereby causing a uniform flow in the specimen liquid, resulting in a result.
  • upstream and downstream are formed (sample pad 4 is upstream and absorption pad 8 is downstream).
  • the absorbent pad 8 is made of a water-absorbing material so that a large amount of liquid can be absorbed.
  • a nonwoven fabric made of cellulose, cellulose acetate, glass fiber, or the like is used.
  • the thickness is not limited, but 0.1 to 3 mm is preferably used.
  • the base sheet 2 backs the membrane 3, the sample pad (sample dropping part) 4 ⁇ ⁇ ⁇ , the conjugate pad (labeling part) 5, and the absorption pad 8, and is a member that does not transmit liquid, for example, a synthetic resin sheet. There is also a function of holding members such as the membrane 3 together so as to maintain a certain strength and preventing the sample liquid from flowing out of the test piece 1.
  • polyethylene terephthalate (PET) can be used as the synthetic resin.
  • PET polyethylene terephthalate
  • the thickness is not limited, but 0.1 to 3 mm is preferably used.
  • the base sheet 2 and the membrane 3, the sample pad (sample dropping part) 4), the conjugate pad (labeling part) 5 and the absorption pad 8 are in close contact to constitute the test piece 1, but in order to prevent peeling during transportation.
  • An adhesive layer made of an adhesive may be provided at the interface between the base sheet 2 and another member (for example, the interface between the base sheet 2 and the absorption pad 4, the interface between the base sheet 2 and the conjugate pad 5, the base sheet 2 and the membrane). 3, the interface between the base sheet 2 and the absorbent pad 8).
  • the size of the test piece 1 is not limited. However, for example, when it is configured with a width of 0.5 to 20 mm and a length of 10 to 100 mm, it is easy to handle in diagnosis, easy to judge visually, and the amount of sample liquid used is also large. Less. Each component is cut in the same size as or smaller than the test piece 1 and assembled.
  • the test piece 1 may be used as it is as a dipstick-type strip, or may be used as a test stick accommodated in a plastic case in which the lower part of the specimen sample droplet and the determination part are opened.
  • an example of a method for producing the test piece 1 for detecting Kawasaki disease by the immunochromatography method of the present invention will be described.
  • An antibody (labeled antibody) to which a labeling substance that specifically binds to the antigen to be detected is bound is applied to the conjugate pad (labeled site) 5.
  • an antibody capable of specifically binding to the antigen to be detected in the present invention, LBP, LRG1
  • a substance that specifically binds to the labeled antibody (labeled antibody-specific antibody) is immobilized on the control line (second detection region) 7.
  • the conjugate pad (labeled portion) 5 and the membrane 3 are bonded to other members (sample pad 4 and absorption pad 8), and cut to an appropriate width to obtain a test piece 1 for immunochromatography.
  • the test piece 1 (the principle of immunochromatography) will be described.
  • the specimen sample solution is dropped on the specimen dropping site 4 ⁇ of the test piece 1
  • the specimen sample solution containing the antigen bound to the labeled antibody flows downstream on the membrane 3 using the capillary phenomenon and is absorbed by the absorbent pad 8.
  • the absorbent pad 8 As a result, a solid-phased antibody-substance to be detected (antigen) -labeled antibody complex is formed in the first detection region 6 on the test piece 1 and visualized.
  • the labeled antibody that has not been captured in the first detection region 6 reaches the second detection region 7 and binds to the labeled antibody in the second detection region 7.
  • a substance-labeled antibody complex is formed and visualized. This shows that the antigen-antibody reaction has progressed normally. Therefore, two lines, the test line and the control line, are observed in the positive sample (FIG. 4 (a) positive reaction), and only one control line is observed in the negative sample (FIG. 4 (b) negative reaction). .
  • the signal emitted from the labeled antibody can be observed visually or measured using a detection device corresponding to the labeled substance.
  • a detection device corresponding to the labeled substance.
  • the labeling substance is colored insoluble carrier particles such as colloidal particles or polystyrene particles
  • a densitometer can be used.
  • the labeling substance is a fluorescent dye
  • a fluorescence detection apparatus can be used.
  • a so-called immunochromatographic reader may be used.
  • the user's guide should describe the inspection procedure, determination method, precautions for use and handling, storage conditions and expiration date of the test piece 1, and the like. If it is determined that there is a high possibility of suffering from Kawasaki disease, treatment should be started.
  • the present invention by measuring the level in a subject-derived specimen for at least one component selected from the group consisting of lipopolysaccharide binding protein, leucine rich ⁇ 2-glycoprotein, angiotensinogen and retinol binding protein 4, A method for treating Kawasaki disease is also included, including testing for Kawasaki disease and treating the subject.
  • the treatment strategy for Kawasaki disease is how to suppress vasculitis before the onset of coronary aneurysms (CALs) without dealing with the various clinical symptoms seen in the acute phase. That is, [1] How to suppress inflammation by the 7th to 10th disease day, [2] How to deal with severe cases in which CALs develop by the 7th disease day, [3] After the 10th disease day How to deal with the case where inflammation continues until now becomes a practical problem.
  • the principle of treatment is to suppress early inflammation.
  • IVIG Intravenous immunoglobulin
  • Anticoagulant therapy with aspirin (3) Thrombus for aneurysm formation
  • Thrombus for aneurysm formation
  • IVIG Ultra-high-dose 2g / kg single dose was approved by the Ministry of Health, Labor and Welfare, As a result of post-use investigation, effectiveness and safety were demonstrated. This therapy has also been reported to be effective in the United States (https://www.ncbi.nlm.nih.gov/pubmed/14584002#).
  • IVIG additional administration If there is no improvement despite initial treatment, additional administration is often performed in the clinical setting. As described above, excessively injecting a large molecule protein raises the problem that blood consistency increases and thrombus formation is promoted.
  • Methylprednisolone (mPSL) / pulse therapy The Boston Group in the United States reported that mPSL pulse therapy was effective for refractory cases, and reports showing the effectiveness of this method have been seen in Japan.
  • the maintenance dose starts at 0.05 to 0.12 mg / kg / day1 and reaches the optimal range in 4 to 5 days.
  • Prothrombin time is a screening test for factors II, V, VII and X clotting factors and is useful for monitoring the anticoagulant action of warfarin.
  • PT Prothrombin time
  • INR the international standard ratio
  • KD adjusts the dose so that PT-INR is 1.6 to 2.5 (thrombotest: 10 to 25%).
  • the AHA guidelines recommend adjusting 0.05 to 0.34 mg / kg and PT / INR to 2.0 to 2.5 (Circulation 2004; 110 (17): 2747-2771).
  • meningitis For complications other than the cardiovascular system (meningitis), treatment is performed in accordance with the guidelines for aseptic meningitis ((http://www.mhlw.go.jp/stf/shingi/2r98520000013qef-att /2r98520000013r5u.pdf).
  • meningitis For complications other than the cardiovascular system (encephalopathy), treatment is performed in accordance with the Guidelines for Clinical Practice of Pediatric Acute Encephalopathy (http://minds4.jcqhc.or.jp/minds/child-acute-encephalopathy/child-acute- encephalopathy.pdf).
  • Example 1 (Method) Serum was provided by Yokohama City University Hospital, Kanagawa Children's Medical Center, Public Showa Hospital, and Yokohama City University Citizen General Medical Center. All samples have received comprehensive consent from the provider.
  • Acute sera of Kawasaki disease patients Fever serum of 55 patients ⁇ Sera of autoimmune disease patients (G3): 24 (3 idiopathic thrombocytopenic purpura, 2 pediatric rheumatism, GVHD ( (Graft-versus-host disease) 1), VAHS (virus-related hemophagocytic syndrome) 1), juvenile idiopathic arthritis 17) ⁇ Healthy serum (by collecting blood during allergy test) (Healthy): 13
  • Kawasaki disease-related proteins LBP and LRG1 were measured by ELISA. Serum used was LBP diluted 4000 times and LRG1 diluted 5000 times. Reagents such as dilute solution, washing solution, detection reagent, and reaction time, etc., followed the protocol of each protein ELISA kit.
  • LRG1 concentration was 300 ⁇ g / ml or more and LBP concentration was 25 ⁇ g / ml or more (group not including autoimmune disease patients and healthy children) 83.6% (46 samples), the majority of patients with illness, were found to be effective as biomarkers for diagnosing Kawasaki disease.
  • Cut-off value (best); concentration at which the specificity is 95% or more. Cutoff value (better); concentration at which specificity is 90% or more. Cutoff value (good): Concentration when specificity is 80% or more. Sensitivity: The rate at which patients with true Kawasaki disease are diagnosed as positive. 100%-specificity; the rate at which patients other than Kawasaki disease are misdiagnosed as having Kawasaki disease. [Table 1]
  • inflammatory proteins such as CRP are excessively present, and the serum concentration of these proteins is examined as a reference item.
  • many such inflammatory proteins reflect non-specific systemic inflammation and do not specifically differentiate Kawasaki disease.
  • Kawasaki disease was diagnosed by examining the serum levels of both LBP and LRG1, both in patients with autoimmune diseases, which are also inflammatory diseases, and healthy children, who are normal children. It was clarified that more specific diagnosis was possible. Since both of these two types of proteins are present in blood at high concentrations, development of a simple and highly accurate method for diagnosing Kawasaki disease by measuring the abundance in serum using antibodies against these proteins Is considered possible.
  • Example 2 The method for carrying out the present invention by immunochromatography will be specifically described below as examples. Two types of test pieces are prepared in advance, and the specimen sample solution is dropped onto a predetermined position of each test piece. 1. Preparation of immunochromatographic test piece 1a Preparation of anti-LBP polyclonal antibody Mice were immunized with an antigen protein (LBP), and blood was collected from mice bred for a certain period to obtain polyclonal antibodies. 2.
  • LBP antigen protein
  • Immobilization of anti-LBP polyclonal antibody and labeled antibody-specific antibody on membrane 3 A solution obtained by diluting purified anti-LBP polyclonal antibody with purified water to 1.0 mg / mL (liquid 6), anti-mouse A solution obtained by diluting IgG polyclonal antibody with purified water to 1.0 mg / mL (liquid 7) is linearly arranged at predetermined positions on membrane 3 (nitrocellulose membrane) lined with base sheet 2 (PET sheet). Apply and dry at 45 ° C. for 30 minutes to obtain an anti-LBP polyclonal antibody / anti-mouse IgGs polyclonal antibody-immobilized membrane (hereinafter referred to as antibody-immobilized membrane).
  • antibody-immobilized membrane an anti-LBP polyclonal antibody / anti-mouse IgGs polyclonal antibody-immobilized membrane
  • this step corresponds to the application of liquid 6 to the test line (first detection region) 6 and liquid 7 to the control line (second detection region) 7.
  • Immobilization of anti-LBP polyclonal antibody on colored polystyrene particles Dilute anti-LBP polyclonal antibody with purified water to 1.0 mg / mL, add colored polystyrene particles to this to 0.1%, and stir carbodiimide after stirring. Add to 1% and stir further. The supernatant is removed by centrifugation and resuspended in 50 mM Tris (pH 9.0) and 3% BSA to obtain an anti-LBP antibody (colored polystyrene particle-bound labeled antibody) to which colored polystyrene particles are bound. 4. 2.
  • a predetermined amount of 1.0 ⁇ g of the colored polystyrene particle-bound labeled antibody obtained in the above is applied to a glass fiber nonwoven fabric and dried at 45 ° C. for 30 minutes to obtain a dry pad (corresponding to conjugate pad 5). 5. Bonding with immobilization membrane, drying pad, and other members And 4. The antibody-immobilized membrane (membrane 3 having the test line 6 and control line 7) and the conjugate pad 5 prepared in 1 above were bonded to other members, ie, the sample pad 4 and the absorption pad 8, and cut to a width of 5 mm and subjected to the LBP test. A piece (test piece 1a) is obtained.
  • Immobilization of anti-LRG1 polyclonal antibody and labeled antibody-specific antibody on membrane 3 Diluted purified anti-LRG1 polyclonal antibody with purified water to 1.0 mg / mL (liquid 6), anti-mouse A solution (liquid 7) obtained by diluting IgG polyclonal antibody with purified water so as to be 1.0 mg / mL (liquid 7) is linearly arranged at predetermined positions on the membrane 3 (nitrocellulose membrane) lined with the base sheet 2 (PET sheet). Apply and dry at 45 ° C.
  • this step corresponds to the application of liquid 6 to the test line (first detection region) 6 and liquid 7 to the control line (second detection region) 7.
  • Immobilization of anti-LRG1 polyclonal antibody to colored polystyrene particles Dilute anti-LRG1 polyclonal antibody with purified water to 1.0 mg / mL, add colored polystyrene particles to this to 0.1%, and stir carbodiimide after stirring. Add to 1% and stir further.
  • the antibody-immobilized membrane (membrane 3 having the test line 6 and the control line 7) and the conjugate pad 5 prepared in the above are bonded to other members and cut to a width of 5 mm to obtain an LRG1 test piece (test piece 1b).
  • Step 1 The specimen sample solution is dropped on the sample pad 4 of the test piece 1a. If the first detection region 6 (test line) is colored and the second detection region 7 (control line) is colored, positive is determined.
  • Step 2 The specimen sample solution is dropped on the sample pad 4 of the test piece 1b. If the first detection region 6 (test line) is colored and the second detection region 7 (control line) is colored, positive is determined.
  • Step 3 If it is positive for step 1 and positive for step 2, it is determined that there is a possibility of “Kawasaki disease”.
  • test pieces (1a, 1b)
  • a configuration as shown in FIG. 5 can be considered.
  • the test piece 10 is roughly divided into two parts.
  • the function of the test piece 1a described above is provided on the upstream side
  • the function of the test piece 1b described above is provided on the downstream side.
  • only one sample pad 4 and one absorption pad 8 are provided at one end and the other end of the test piece 10 so that the specimen liquid is dropped only on the sample pad 4 and finally absorbed by the absorption pad 8. It is. That is, the sample pad 4 is provided on the upstream side of the test piece 10, and the absorption pad 8 is provided on the downstream side of the test piece 10.
  • sites related to the antigen-antibody reaction in the test piece 1a are provided in 5A, 6A, and 7A
  • sites related to the antigen-antibody reaction in the test piece 1b are provided as 5B, 6B, and 7B.
  • the function of the test piece 1a is provided on the upstream side
  • the function of the test piece 1b is provided on the downstream side.
  • the upstream side may be the function of the test piece 1b
  • the downstream side may be the function of the test piece 1a.
  • the capture unit 9A has a function of capturing the labeled particles immobilized on 5A. Specifically, an antibody that binds only to the labeled particles immobilized on 5A is immobilized at a high concentration.
  • the marker function in 6B or 7B may be affected. It is necessary to capture the labeled particles. Accordingly, the antibody that binds only to the labeled particles is immobilized at a high concentration on the capture portion 9A, or the length in the longitudinal direction (upstream-downstream direction) of 9A is set to be longer. Specifically, in the former case, the same antibody as that used for 7A is immobilized at a concentration higher than that at 7A (for example, 9A is 2 to 10 times the concentration of 7A).
  • the same antibody as that used for 7A is used at the same concentration, but it is longer than 7A and immobilized (for example, 9A is 2 to 10 times longer than 7A).
  • the antibody solution is applied to the membrane 3, dropped or sprayed, dried and adsorbed.
  • the control line 7A is originally a line for confirming whether or not the sample liquid has passed through the test line 6A. However, since the function is also provided in the test line 7B, the control line 7A can be omitted.
  • the present invention can be used for diagnosis of Kawasaki disease.
  • Test strip 1 (1a, 1b). Test strip 2. Base sheet 3. Membrane 4. Sample pad (sample dropping part) 5. Conjugate pad (labeled part) 6. Test line (first detection area) 7. Control line (second detection area) 8. Absorbent pad 10. Test strip 5A. Conjugate pad A (first labeling site for A) 6A. Test line A (first detection area for A) 7A. Control line A (second detection area for A) 9A. Capture unit 5B. Conjugate pad B (first labeling site for B) 6B. Test line B (first detection area for B) 7B. Control line B (second detection area for B)

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  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La présente invention concerne un procédé et un spécimen pour tester rapidement et aisément la maladie de Kawasaki. Un procédé de test de la maladie de Kawasaki caractérisé par la mesure des taux de proprotéine de liaison de lipopolysaccharide (LBP) et de α2-glycoproprotéine riche en leucine (LRG1) dans un échantillon provenant d'un sujet à tester. Un spécimen pour la détection de la maladie de Kawasaki par l'intermédiaire d'un procédé d'immunochromatographie, le spécimen contenant des supports sur lesquels des anticorps anti-LBP sont fixés, et des supports sur lesquels des anticorps anti-LRG1 sont fixés.
PCT/JP2017/028236 2016-08-09 2017-08-03 Procédé et spécimen pour tester la maladie de kawasaki Ceased WO2018030270A1 (fr)

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JP2018532985A JPWO2018030270A1 (ja) 2016-08-09 2017-08-03 川崎病の検査方法および試験片

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024085238A1 (fr) * 2022-10-21 2024-04-25 Ｃｒａｎｅｂｉｏ株式会社 Article vestimentaire pourvu d'une bandelette réactive servant à détecter un marqueur du liquide amniotique

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007205842A (ja) * 2006-02-01 2007-08-16 Kobeshi Chiiki Iryo Shinko Zaidan 川崎病の判定方法及びそのためのキット
JP2008167714A (ja) * 2007-01-15 2008-07-24 Yokohama City Univ 川崎病発症のリスク因子
WO2010025393A2 (fr) * 2008-08-28 2010-03-04 The Regents Of The University Of California Marqueurs biologiques protéiques et méthodes pour diagnostiquer la maladie de kawasaki
JP2013512434A (ja) * 2009-11-25 2013-04-11 ホロジック，インコーポレイテッド 羊膜内感染の検出
WO2015006515A1 (fr) * 2013-07-09 2015-01-15 Sri International Batterie de biomarqueurs pour évaluation de la dose associée à une affection radio-induite et microfiltre à plasma sanguin
WO2015168602A2 (fr) * 2014-05-02 2015-11-05 Momenta Pharmaceuticals, Inc. Procédés et compositions pour le diagnostic et le traitement de la maladie de kawasaki
WO2016129631A1 (fr) * 2015-02-10 2016-08-18 公立大学法人横浜市立大学 Procédé et kit pour détecter la maladie de kawasaki

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007205842A (ja) * 2006-02-01 2007-08-16 Kobeshi Chiiki Iryo Shinko Zaidan 川崎病の判定方法及びそのためのキット
JP2008167714A (ja) * 2007-01-15 2008-07-24 Yokohama City Univ 川崎病発症のリスク因子
WO2010025393A2 (fr) * 2008-08-28 2010-03-04 The Regents Of The University Of California Marqueurs biologiques protéiques et méthodes pour diagnostiquer la maladie de kawasaki
JP2013512434A (ja) * 2009-11-25 2013-04-11 ホロジック，インコーポレイテッド 羊膜内感染の検出
WO2015006515A1 (fr) * 2013-07-09 2015-01-15 Sri International Batterie de biomarqueurs pour évaluation de la dose associée à une affection radio-induite et microfiltre à plasma sanguin
WO2015168602A2 (fr) * 2014-05-02 2015-11-05 Momenta Pharmaceuticals, Inc. Procédés et compositions pour le diagnostic et le traitement de la maladie de kawasaki
WO2016129631A1 (fr) * 2015-02-10 2016-08-18 公立大学法人横浜市立大学 Procédé et kit pour détecter la maladie de kawasaki

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024085238A1 (fr) * 2022-10-21 2024-04-25 Ｃｒａｎｅｂｉｏ株式会社 Article vestimentaire pourvu d'une bandelette réactive servant à détecter un marqueur du liquide amniotique

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