WO2005108985A1 - Method of judging systemic arteriosclerosis and judgement reagent - Google Patents

Method of judging systemic arteriosclerosis and judgement reagent Download PDF

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Publication number
WO2005108985A1
WO2005108985A1 PCT/JP2005/008070 JP2005008070W WO2005108985A1 WO 2005108985 A1 WO2005108985 A1 WO 2005108985A1 JP 2005008070 W JP2005008070 W JP 2005008070W WO 2005108985 A1 WO2005108985 A1 WO 2005108985A1
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Prior art keywords
diabetic nephropathy
hasg
antibody
elastin
antibodies
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PCT/JP2005/008070
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French (fr)
Japanese (ja)
Inventor
Osamu Hotta
Masahiko Katayama
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Eisai R & D Management Co., Ltd.
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Priority to JP2006512971A priority Critical patent/JP4762135B2/en
Publication of WO2005108985A1 publication Critical patent/WO2005108985A1/en

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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

Definitions

  • the present invention relates to a method for determining diabetic nephropathy and a kit used for the method. More specifically, the present invention measures the amount of elastin hydrolyzate in the circulating fluid of a subject and, based on the measured value, determines the diabetes!
  • the present invention relates to a method for determining systemic arteriosclerosis, preferably diabetic nephropathy, such as dyspnea, acute progressive glomerulonephritis, amyloidosis or cholesterol embolism, and a kit for use in the method.
  • Non-Patent Document 1 Diabetes is a group of diseases characterized by chronic hyperglycemia due to insufficient insulin action and accompanied by various characteristic metabolic abnormalities. Both genetic and environmental factors contribute to its onset. Long-term persistence of metabolic abnormalities causes unique complications and immediately promotes arteriosclerosis (Non-Patent Document 1).
  • diabetic nephropathy is one of the microvascular complications of diabetes and is an important disease that affects the prognosis of diabetic patients.
  • the number of patients with diabetic nephropathy has been steadily increasing, and the success or failure of diagnosis and treatment is not only important for the prognosis and quality of life of many diabetic patients, but also for medical economics.
  • Non-Patent Document 2 The stages of diabetic nephropathy are classified into stage 1 (early nephropathy) to stage 5 (dialysis treatment stage), and the more severe the stage, the more severe the treatment is. The need comes out.
  • Non-Patent Document 3 Diagnosis of atherosclerosis in diabetic patients has also been shown to indicate that the severity of complications correlates well with atherosclerosis.
  • diagnosis of arterial stiffness is broadly based on pulse wave velocity (PWV), etc. It is implemented in general medical institutions.
  • the pulse wave velocity measures the degree of stiffness of the blood vessel wall by measuring the velocity of the pulse wave through the blood vessel wall at a specific site (Non-Patent Document 4).
  • This diagnostic technique does not involve the collection of bodily fluids, requires the installation of special equipment that has high diagnostic reproducibility, and requires the patient to be restrained for a certain period of time.
  • Ccr indicates the amount of plasma required to excrete specific components in plasma into the kidneys and urine for a certain period of time, and it is a test item that accurately diagnoses the glomerular filtration capacity of the kidneys. Since it is necessary to restrain the patient for hours or 24 hours and it is necessary to collect urine and plasma at the same time, it lacks versatility and is difficult to perform frequently on a large number of patients.
  • urinary protein and urinary microalbumin measurement and Ccr are both indicators that mainly diagnose renal function, and may be diagnosed as positive even in renal insufficiency limited to the kidneys. It is not a specific diagnostic indicator of the disease.
  • Non-Patent Document 6 Requires a renal biopsy (Non-Patent Document 6). The problem is that renal biopsy is difficult to perform frequently because of the heavy physical burden on the patient.
  • Aortic dissection is based on arteriosclerosis and is a disease in which the blood vessel wall ruptures and detaches due to an increase in blood pressure.
  • elastin molecules which are mainly localized in the blood vessel wall, are cleaved by various proteases, Degradants are expected to appear in the circulating fluid, which will lead to increased blood levels of the degradants.
  • Non-Patent Documents 8, 11, and 12 the relationship between the concentration of elastin hydrolyzate in blood and arteriosclerosis and atherosclerosis-related diseases has been suggested.
  • Non-Patent Document 8 the relationship between the concentration of elastin hydrolyzate in blood and diabetes.
  • Non-Patent Document 13 the concentration of elastin hydrolyzate in blood increases in complications of circulatory disorders in diabetes.
  • this report showed only a tendency for a group of patients with extremely severe diabetic circulatory disorder called foot ulcers and scarring symptoms.
  • Patent Document 1 JP-A-2002-350439
  • Non-Patent Document 1 Diabetes 42, 385 (1999)
  • Non-patent document 2 Japanese clinical practice 60, 260-269 (2002)
  • Non-patent document 3 Hypertension treatment guideline 2000 edition, 55-58 (2000) Kyorinsha
  • Non-patent document 4 Japanese clinical practice 62, 80-86 (2004)
  • Non-Patent Document 5 Diabetes 44, 623 (2001)
  • Non-patent document 6 Japanese clinical practice 60, 316-322 (2002)
  • Non-Patent Document 7 Meth.EnzymoL, 163, 656-673 (1988)
  • Non-Patent Document 8 Clin.Physiol.Biochem., 8, 273-282 (1990)
  • Non-Patent Document 9 J. Immunol.Methods, 164, 175-187 (1993)
  • Non-Patent Document 10 Eur.J. Vase.Endovasc.Surg., 14, 12-16 (1997)
  • Non-Patent Document 11 Atherosclerosis, 66, 163-168 (1987)
  • Non-Patent Document 12 Eur.J. Vase.Endovasc.Surg., 24, 440-444 (2002)
  • Non-Patent Document 13 Diabetologia Croatica, 26-3, 151-155 (1997)
  • Non-Patent Document 14 Atherosclerosis, 131, 73-78 (1997)
  • Non-Patent Document 15 General Pharmacology, 35, 59-64 (2000)
  • the present invention provides a method and kit for simply and accurately determining systemic arteriosclerosis such as diabetic nephropathy, acute progressive glomerulonephritis, amyloidosis or cholesterol embolism, preferably diabetic nephropathy. To provide.
  • systemic arteriosclerosis such as diabetic nephropathy, acute progressive glomerulonephritis, amyloidosis or cholesterol embolism, preferably diabetic nephropathy.
  • the present inventors considered that among the group of diseases other than aortic dissection based on systemic atherosclerosis, there is a disease in which the measured value of elastin hydrolyzate in the blood increases. The study was conducted on diabetic patients and other related diseases.
  • the present inventors compared the elastin hydrolyzate concentration in the blood of the diabetic patient group and the diabetic nephropathy patient group, and found that the blood concentration was extremely high only in the diabetic nephropathy group. And found that measurement of the amount of elastin hydrolyzate is useful for diagnosis of diabetic nephropathy.
  • acute glomerulonephritis, membranous proliferative glomerulonephritis, and purpura nephritis! / Puta primary glomerulonephritis (renal dysfunction of the kidney-limited type) also shows that blood levels do not increase. I found it.
  • the present invention provides a method for measuring diabetic nephropathy, acute progressive glomerulonephritis, amyloidosis or cholesterol embolism, and measuring systemic arteriosclerosis, preferably diabetic nephropathy. It provides a new diagnostic index for diagnosing disease.
  • the present invention measures the amount of elastin hydrolyzate in the circulating fluid of a subject, and based on the measured value, evaluates diabetic nephropathy, acute progressive glomerulonephritis, amyloidosis or cholesterol embolism.
  • determining systemic atherosclerosis preferably diabetic nephropathy
  • the determination in the present invention includes not only determining whether or not the patient has diabetic nephropathy but also determining the severity of diabetic nephropathy.
  • the antibody is preferably a monoclonal antibody.
  • one of the antibodies is immobilized on a solid phase, and the other is labeled with a labeling substance, using the first or second monoclonal antibody.
  • Examples of the monoclonal antibody include HASG-2 (FERM BP-08488), HASG-30 (FERM BP-08488).
  • the first and second monoclonal antibodies are preferably a combination of monoclonal antibodies produced by HASG-30 and HASG-61-1 hybridomas.
  • the present invention also includes one or more antibodies having specificity and affinity for elastin hydrolyzate, measures the amount of elastin hydrolyzate in the circulating fluid of a subject, and measures the measured value. Based on the criteria for diabetic nephropathy, acute progressive glomerulonephritis, amyloidosis or cholesterol embolism, to determine systemic atherosclerosis, preferably diabetic nephropathy (Hereinafter, also referred to as “the kit of the present invention!”).
  • FIG. 1 is a standard curve showing the relationship between aortic elastin hydrolyzate concentration in a test sample and absorbance.
  • FIG. 3 Among the diabetic patient group (129 cases), 53 diabetic patients (without nephropathy), 53 diabetic nephropathy patients (51 cases), diabetic nephropathy + chronic renal failure patients (25 cases), Group 4 shows the distribution of serum elastin hydrolyzate concentration in diabetic nephropathy + dialysis therapy-introduced patients (53 patients).
  • the method of the present invention is characterized in that the amount of elastin hydrolyzate in the circulating fluid of a subject is measured, and diabetic nephropathy is determined based on the measured value.
  • the circulating fluid in the present invention refers to serum, plasma, cerebrospinal fluid, ascites, and / or ivy, or a fraction thereof, and is not particularly limited as long as it is normally collected in a medical institution or the like. However, blood samples such as serum from a subject are particularly suitable.
  • any method can be used as long as the amount of elastin hydrolyzate in the circulating fluid can be measured.
  • Examples include immunochemical methods using antibodies having specificity and affinity.
  • the immunological method is not particularly limited as long as it is a method using an antibody against an elastin degradation product, preferably a human aortic elastin degradation product, and may be a latex agglutination method, a western plot method, a sandwich method, or the like. There is a method such as a competition method, which is not particularly limited, but a sandwich ELISA method is preferable.
  • the detection method examples include a method using a radioactive substance label, a method using a fluorescent label, a method using an enzyme label, and a method using electrochemiluminescence, and are not particularly limited. Preferably, it is a safe and simple enzyme immunoassay (ELISA) or an electrochemical immunoassay.
  • ELISA enzyme immunoassay
  • the antibody used is a human aortic elastin hydrolyzate
  • the polyclonal antibody is a serum obtained by immunizing rabbits, goats, sheep, etc.
  • the antibody is purified.
  • the monoclonal antibody is mouse, rat, hamster, etc.
  • the ability to use a monoclonal antibody produced by immunizing a rodent of the present invention is preferably a monoclonal antibody!
  • the animal species used for producing the monoclonal antibody is not particularly limited, but generally, Balb / C mice are most often used.
  • Monoclonal antibodies are usually monoclonal Can be prepared in the same manner as in the preparation of the internal antibody.
  • Antibodies are not limited to fragments of Fab, Fab ', F (ab'), etc., as long as they have predetermined properties and affinity.
  • the antibody may be labeled with a labeling substance.
  • Labeling substances include those used in ordinary sandwich methods for immunologically binding two antibodies to an antigen, such as enzymes, electrochemiluminescent complexes, radioactive substances, latex, fluorescent substances, and chemical substances. Light-emitting substances, metal colloid particles and the like can be mentioned. In the present invention, enzymes or electrochemiluminescent complexes are preferred.
  • the binding between the label and the antibody can be performed according to a usual method.
  • the antibody may be immobilized on a solid phase!
  • the shape and material of the solid phase are not particularly limited, and specific examples include a microtiter plate and beads.
  • the immobilization of the antibody on the solid phase can be performed according to a usual method.
  • HASG-2 hybridoma HASG-2 (FERM BP-08488), HASG-30 (FERM BP-08489), and HASG-61-1 (FERM BP-08490).
  • HASG-2 and HASG-30 are disclosed in JP-A-2002-350439.
  • HASG-2 and HASG-30 were registered at the National Institute of Advanced Industrial Science and Technology (AIST) Patent Microorganisms Depositary Center ( ⁇ 305-8566, 1-1 Tsukuba East, Ibaraki Pref. Deposited on March 18 and assigned accession numbers FERM P-18335 and FERM P-18336.On September 18, 2003, they were transferred to the International Deposit under the Budapest Treaty, and they were transferred to FERM BP-08488 and FERM BP- It has a 08489 accession number.
  • AIST Advanced Industrial Science and Technology
  • HASG-61-1 is an independent bank Deposited at the National Institute of Advanced Industrial Science and Technology Patent and Microorganisms Depositary Center on October 8, 2002, granted the accession number FERM P-19058, and transferred to an international deposit based on the Budapest Treaty on September 18, 2003.
  • FERM BP-08490 accession number accession number.
  • Specificity for human large artery elastin hydrolyzate equivalent to any of the monoclonal antibodies produced by HASG-2, HASG-30, and HASG-61-1 And antibodies having affinity can be selected by the following methods.
  • Antibodies having the same specificity and affinity can be selected by, for example, a competition test (Antibodies A Laboratory Manual, old bpnng Harbor Laboratory (1988) p.567). Specifically, a human aortic elastin hydrolyzate, which is an antigenic substance, is dissolved at an appropriate concentration in a physiological buffer and adsorbed to a solid phase of a microplate. After performing the blocking treatment, add the antibody to be evaluated together with the enzyme-labeled HASG-2, HASG-30 or HASG-61-1 monoclonal antibody in the same amount. Antibodies with similar specificity and affinity can be selected by confirming their ability to inhibit the reaction of enzyme-labeled HASG-2, HASG-30 or HASG-61-1 monoclonal antibodies.
  • selection can be made by a peptide mapping method.
  • human aortic elastin hydrolyzate which is an antigenic substance, is separated by a high-performance liquid chromatography device or the like according to differences in molecular weight, hydrophobicity, etc., and binding to various separated elastin antigen fragments is determined by HASG-2, The specificity and affinity are evaluated by comparing with HASG-30 and HASG-61-1 monoclonal antibodies. Antibodies that react equally to the antigen fragments to which the HASG-2, HASG-30 or HASG-61-1 monoclonal antibodies react are judged to have the same specificity.
  • the human aortic elastin hydrolyzate can be obtained by the method described in Biochem. J., 61, 11-21 (1955) and can be obtained as a commercial product.
  • ELISA for elastin hydrolyzate in a biological sample
  • various methods are known as a measuring method. Particularly, a simple and highly quantitative method, such as immobilized first antibody and peroxidase, is used. A sandwich method using a second antibody labeled with an enzyme is often used. In this case, the first and second antibodies react specifically to elastin degradation products.
  • the first antibody and the second antibody desired to be used may be the same.
  • an antibody (first antibody) recognizing an aortic elastin degradation product is bound to a solid phase such as a 96-well microphone plate.
  • the solid phase may be bound by a covalent bond or a non-covalent bond.
  • adsorb a blocking protein such as milk force zein in order to reduce non-specific adsorption to the solid phase.
  • a standard aortic elastin hydrolyzate solution with a clear concentration and a test biological sample, and leave it for a certain period of time.
  • washing is performed, and then another anti-aortic elastin degradation product antibody (second antibody) labeled with an enzyme such as peroxidase is added at an appropriate concentration.
  • the complex is left to stand for a certain period of time to form a complex of the first antibody, the aortic elastin degradation product, and the second antibody on the solid phase. Thereafter, the solid phase is washed, and if the enzyme substrate or the enzyme is peroxidase, a mixed solution of hydrogen peroxide and a coloring substrate such as TMBZ is added to obtain a color by the labeling enzyme. After terminating the enzyme reaction by adding an inhibitor or the like, the absorbance of the color is measured with a device such as a plate reader. It is also possible to use a luminescent substrate instead of a chromogenic substrate. In this case, the luminescence intensity is measured instead of the absorbance.
  • the amount of aortic elastin hydrolyzate in the test solution can be known with high accuracy.
  • Various methods are known for the electrochemiluminescence immunoassay, and a particularly simple and highly quantitative method is used for the first antibody, such as an antibody immobilized on magnetic beads or a second antibody.
  • the first antibody and the second antibody which desirably react specifically with the elastin degradation product, may be the same.
  • an antibody that recognizes an aortic elastin degradation product is bound to a solid phase such as magnetic beads.
  • the solid phase may be bound by a covalent bond or a non-covalent bond.
  • adsorb blocking proteins such as milk casein to reduce non-specific binding to the solid phase.
  • the solid phase is washed and Another anti-elastin hydrolyzate antibody labeled with a chemiluminescent complex, preferably a ruthenium complex
  • the solid phase is magnetic beads, it is preferably stirred to form a complex of the first antibody, the elastin degradation product antigen and the second antibody on the magnetic beads. Thereafter, the solid phase is washed, and a current is preferably passed between the electrodes in a dedicated device to cause the chemiluminescent complex, preferably a ruthenium complex, to emit light, and the emission intensity is measured.
  • a dedicated device to cause the chemiluminescent complex, preferably a ruthenium complex, to emit light, and the emission intensity is measured.
  • HASG-30 and HASG-61-1 are desirable.
  • the determination in the present invention includes not only determination of the power of diabetic nephropathy but also determination of the severity of diabetic nephropathy.
  • the determination as to whether or not the patient has diabetic nephropathy is made based on whether or not the measured value of elastin degradation product is higher than the cutoff value.
  • the cut-off value is usually determined by measuring the concentration of elastin hydrolyzate in the blood of diabetic patients diagnosed as not having diabetic nephropathy by the conventional method, and calculating the average + 3SD (standard deviation) . In the case of measuring 55 diabetic patients described in the examples, the cutoff value was 184.9 ng / mL.
  • the highest measured value of elastin degradation in blood of a diabetic patient diagnosed as not having diabetic nephropathy may be set as the cut-off value, and diabetic nephropathy may develop. If it is possible to determine whether or not the force is applied, another value may be set as the cutoff value.
  • the cut-off value for acute progressive glomerulonephritis, amyloidosis, or cholesterol embolism can be performed in the same manner as in diabetic nephropathy, but the measurement value of a healthy individual can be used instead of a diabetic patient.
  • the measured values of elastin hydrolyzate in the blood were measured for patients with diabetes, diabetic nephropathy who progressed to diabetic nephropathy and chronic renal failure, diabetic nephropathy patients who progressed to the introduction of dialysis therapy, and diabetes mellitus. Blood elastin degradation products are used as an indicator to monitor the progression of diabetic nephropathy even after it has been determined to be diabetic nephropathy, since it increases with the severity of dyspnea. It is also possible.
  • the present inventors have found that the measured value hardly increases in diabetic patients without nephropathy, is extremely high in diabetic nephropathy patients, and is high at a positive rate, as shown in Examples described later.
  • the slip rate is also high with a high positive rate.
  • elastin degradation product concentration increased according to the severity of diabetic nephropathy, and the severity of diabetic nephropathy could be determined from the elastin hydrolyzate concentration. Therefore, by measuring the amount of elastin degradation products in the circulating fluid, systemic atherosclerosis such as diabetic nephropathy, acute progressive glomerulonephritis, amyloidosis or cholesterol embolism, preferably diabetes It is possible to determine sexual nephropathy.
  • a means capable of measuring the amount of elastin hydrolyzate in the circulating fluid for example, an antibody against elastin hydrolyzate can be used for the production of a diagnostic agent for diabetic nephropathy.
  • the kit of the present invention is characterized by containing one or more antibodies having specificity and affinity for elastin degradation products.
  • the kit measures the amount of elastin hydrolyzate in the subject's circulating fluid and uses that measurement to determine systemic arterial disease such as diabetic nephropathy, acute progressive glomerulonephritis, amyloidosis or cholesterol embolism. It is used to determine sclerosis, preferably diabetic nephropathy.
  • the antibody is as described for the method of the invention.
  • the antibodies are preferably the first and second antibodies which are preferably monoclonal antibodies. More preferably, a second monoclonal antibody is used. Further, it is preferable that one of the first and second antibodies is immobilized on a solid phase, and the other is labeled with an enzyme or an electrochemiluminescent complex. Labels and are as described for the method of the invention.
  • the antibody in the kit of the present invention may be a solution or a lyophilized antibody.
  • the kit of the present invention may contain, in addition to the first and second antibodies, reagents commonly used in immunoassays.
  • reagents commonly used in immunoassays include a standard antigen (human aortic elastin digest) solution, a substrate solution, a sample diluent, and a washing solution.
  • a standard antigen human aortic elastin digest
  • the reagents described in the method for measuring elastin degradation products described above can be used.
  • the kit of the present invention can be used according to the method of the present invention.
  • the kit of the present invention may be used as a diagnostic agent for systemic arteriosclerosis such as diabetic nephropathy, acute progressive glomerulonephritis, amyloidosis or cholesterol embolism, preferably diabetic nephropathy. Can be.
  • systemic arteriosclerosis such as diabetic nephropathy, acute progressive glomerulonephritis, amyloidosis or cholesterol embolism, preferably diabetic nephropathy.
  • Human aortic elastin hydrolyzate (Elastin Products, Cat. No .: HA587) was intraperitoneally injected into Balb / C female mice (6 weeks of age) in an amount of 0.1 mg / animal in Complete 'Freund's Adjuvant (Difco). Co., Ltd.). Three weeks later, the same amount of the human aortic elastin hydrolyzate was administered intraperitoneally together with Incomplete 'Freund's adjuvant (manufactured by Difco). Three weeks later, the mouse was administered the human aortic elastin hydrolyzate alone (0.1 mg / mouse). Three days after the end of the final immunization, the mouse spleen was also removed.
  • the excised spleen was dispersed with a mesh, cultured briefly, mixed with Sp2 / 0-Agl4 mouse myeloma cells, and the cells were added in the presence of 50% polyethylene glycol 1500 (Roche's Diagnostics). It was fused.
  • the fused hybridoma cells are placed in several 96-well microphone plate culture plates. The cells were dispersed and cultured in RPMI1640 liquid medium (Sigma-Aldrich) containing 10% fetal bovine serum and HAT reagent (Sigma-Aldrich) for 1 to 2 weeks.
  • a peroxidase-labeled secondary antibody that reacts with mouse IgG (manufactured by Dako Japan) was added at an appropriate concentration. After a certain period of time, the plate was washed, and an ABTS substrate solution (manufactured by Roche's Diagnostics) was dried. The desired monoclonal antibody-producing hybridoma was selected based on the presence or absence of color development. Each of the selected strains was crawled several times. The three strains of hybridomas thus obtained were named HASG-2, HASG-30, and HASG-61-1.
  • HASG-30 monoclonal antibody HASG-61-1 monoclonal antibody
  • the HASG-30 monoclonal antibody and HASG-61-1 monoclonal antibody prepared in section (1) were prepared as purified IgG. These monoclonal antibodies were confirmed to not compete with each other by a competition test.
  • the HASG-30 monoclonal antibody was dissolved in phosphate buffered saline (PBS) to a final concentration of 0.01 mg / mL, and 0.1 mL of the solution was added to each well of a 96-well microphone port plate (Nalge Nunc).
  • TBS Tris-buffered saline
  • Tween-TBS TBS containing 0.05% Tween-20 (manufactured by Sigma-Aldrich) (hereinafter abbreviated as Tween-TBS) Wash) three times. Then, the periodic acid method [Antibodies: a Laboratory Manual, by Ed. Harlow & D. Lane, Cold bpnng Haroor Laboratory Press]
  • the total number of diabetic patients in the first group was 53 diabetic patients (without nephropathy), 51 patients with diabetic nephropathy as the second group, and diabetic nephropathy patients who progressed to chronic renal failure as the third group ( (See below and Fig. 3 as diabetic nephropathy + chronic renal failure) 25 patients, diabetic nephropathy who progressed until the introduction of dialysis therapy as group 4 (below and diabetic nephropathy + in Fig. 3) Dialysis therapy was introduced).
  • the measured values in the sera of 108 healthy subjects were also compared with these four groups.
  • FIG. This figure is called a box-and-whisker plot.
  • Elastin degradation products were measured at 44.2 ⁇ 19.9 ng / mL for healthy subjects, 80.2 ⁇ 35.8 ng / mL for group 1 diabetes, 224.6 ⁇ 149.6 ng / mL for group 2 diabetic nephropathy, and group 3
  • the measured value was 337.8, 170.4 ng / mL
  • the measured value was 590.7 ⁇ 267.6 ng / mL, and the measured value clearly increased as the disease progressed. The result was obtained.
  • diabetic nephropathy by measuring the amount of elastin hydrolyzate in a circulating fluid such as serum, diabetic nephropathy can be obtained quickly and simply without using a special device, with a high positive rate, acute With advanced glomerulonephritis, amyloidosis or cholesterol embolism! / Patients with systemic arteriosclerosis, preferably diabetic nephropathy, and diabetic nephropathy! Can determine the severity.

Abstract

An elastin decomposition product in a circulating fluid of a subject is quantified by an immunochemical method with the use of, for example, an antibody having specificity and affinity for the elastin decomposition product and then systemic arteriosclerosis such as diabetic nephropathy, rapidly progressive glomerulonephritis, amyloidosis or cholesterin embolism, preferably diabetic nephropathy, is judged based on the measurement data.

Description

明 細 書  Specification
全身性動脈硬化症の判定方法、及び判定試薬  Method for judging systemic arteriosclerosis, and judgment reagent
技術分野  Technical field
[0001] 本発明は、糖尿病性腎症の判定方法及びそれに用いるキットに関し、詳しくは、被 験者の循環液中のエラスチン分解物の量を測定し、その測定値に基づ!/、て糖尿病 性腎症、急性進行性糸球体腎炎、アミロイド一シスまたはコレステリン塞栓症といった 全身性動脈硬化症、好ましくは糖尿病性腎症を判定する方法及び同方法に用いる キットに関する。  The present invention relates to a method for determining diabetic nephropathy and a kit used for the method. More specifically, the present invention measures the amount of elastin hydrolyzate in the circulating fluid of a subject and, based on the measured value, determines the diabetes! The present invention relates to a method for determining systemic arteriosclerosis, preferably diabetic nephropathy, such as dyspnea, acute progressive glomerulonephritis, amyloidosis or cholesterol embolism, and a kit for use in the method.
背景技術  Background art
[0002] 糖尿病は、インスリン作用の不足による慢性高血糖を主徴とし、種々の特徴的な代 謝異常を伴う疾患群である。その発症には遺伝因子と環境因子がともに関与する。 代謝異常の長期間にわたる持続は特有の合併症を来たしやすぐ動脈硬化症をも促 進する (非特許文献 1)。  [0002] Diabetes is a group of diseases characterized by chronic hyperglycemia due to insufficient insulin action and accompanied by various characteristic metabolic abnormalities. Both genetic and environmental factors contribute to its onset. Long-term persistence of metabolic abnormalities causes unique complications and immediately promotes arteriosclerosis (Non-Patent Document 1).
[0003] また、糖尿病性腎症は糖尿病の細小血管合併症のひとつであり、糖尿病患者の予 後を左右する重要な疾患である。近年、糖尿病性腎症の患者数は増加の一途をた どり、その診断と治療の成否は多くの糖尿病患者の予後や生活の質に対してのみな らず、医療経済的にもますます重要性を増している (非特許文献 2)。糖尿病性腎症 の病期は第 1期（腎症前期)〜第 5期 (透析療法期）に分類されており、後期へ移行 するほど重篤性が増していき厳格な治療が施される必要性が出てくる。第 5期の透析 療法期ではもはや腎臓移植しか治療法がなぐ生命予後も極端に悪ィ匕する。すなわ ち、できるだけ早期に正確な診断を施し、適切な治療や食事制限により疾患の進行 を阻止することが望ま 、と考えられて！/、る。  [0003] Furthermore, diabetic nephropathy is one of the microvascular complications of diabetes and is an important disease that affects the prognosis of diabetic patients. In recent years, the number of patients with diabetic nephropathy has been steadily increasing, and the success or failure of diagnosis and treatment is not only important for the prognosis and quality of life of many diabetic patients, but also for medical economics. (Non-Patent Document 2). The stages of diabetic nephropathy are classified into stage 1 (early nephropathy) to stage 5 (dialysis treatment stage), and the more severe the stage, the more severe the treatment is. The need comes out. In the fifth stage of dialysis treatment, the prognosis of life, when treatment is limited to kidney transplants, is extremely poor. In other words, it is thought that it is desirable to make an accurate diagnosis as early as possible and to prevent the progress of the disease by appropriate treatment and dietary restriction! /
[0004] 糖尿病性腎症は前述の如く細小血管合併症のひとつであり、腎臓に限局しない全 身性動脈硬化症を基礎疾患としており、全身血圧降下治療が極めて効果的に糖尿 病性腎症を改善することは既に証明されている (非特許文献 3)。糖尿病患者の動脈 硬化度を診断し、合併症の重症度が動脈硬化度と良好に相関することも示されてい る。現在、動脈硬化度の診断は、脈波速度 (Pulse Wave Velocity: PWV)等により広く 一般の医療機関で実施されている。脈波速度は特定部位の血管壁に脈波を通じて その速度を計ることにより血管壁硬化度を測定するものである（非特許文献 4)。この 診断技術は体液の採取を伴わないものであり、診断再現性も高いものである力 特 定の機器の設置が必要不可欠であり、患者を一定時間拘束する必要もある。 [0004] As described above, diabetic nephropathy is one of the microvascular complications, and the underlying disease is systemic atherosclerosis, which is not limited to the kidney. Has already been proven to improve (Non-Patent Document 3). Diagnosis of atherosclerosis in diabetic patients has also been shown to indicate that the severity of complications correlates well with atherosclerosis. Currently, diagnosis of arterial stiffness is broadly based on pulse wave velocity (PWV), etc. It is implemented in general medical institutions. The pulse wave velocity measures the degree of stiffness of the blood vessel wall by measuring the velocity of the pulse wave through the blood vessel wall at a specific site (Non-Patent Document 4). This diagnostic technique does not involve the collection of bodily fluids, requires the installation of special equipment that has high diagnostic reproducibility, and requires the patient to be restrained for a certain period of time.
[0005] 実際の臨床現場における糖尿病性腎症のスクリーニング検査、及び早期診断は、 患者からの随時尿による試験紙法による尿蛋白の検出、ないしは尿中微量アルブミ ンの検出 '定量により行われている。この検査は簡便であるが、同一患者でも水分摂 取量などが日々変動することにより、尿中への蛋白'アルブミンの***量が一定しな Vヽことから、同一患者へも一定期間内に数回に渡る検査を施行して陽'陰性を確認 することが求められている。この検査法以外にも、糖尿病性腎症の病期を診断するた めに、腎機能検査を目的としてクレアチニンクリアランス測定 (以降「Ccr」と略記）がー 般に行われて 、る (非特許文献 5)。 Ccrは血漿中の特定成分を一定時間中に腎臓 力 尿中へ***されるのに必要な血漿量を示しており、腎臓の糸球体濾過能を正確 に診断する検査項目である力 患者を数時間、ないしは 24時間拘束する必要があり 、尿と血漿を同時に採取する必要性もあることから、汎用性に欠けており、多数の患 者へ頻繁に行うことは困難である。また、尿中蛋白'尿中微量アルブミン測定や Ccrは いずれも腎機能を中心として診断する指標であり、腎臓に限局した腎機能不全症で も陽性と診断されることがあり、必ずしも糖尿病性腎症の特異的診断指標とはなって いない。糖尿病患者においても、急性糸球体腎炎、膜性増殖性糸球体腎炎、紫斑 性腎炎等の原発性糸球体腎炎が合併する場合があり、それぞれにおいて治療法が 異なっており、正確な診断のためには腎生検が必要とされている（非特許文献 6)。腎 生検は患者への身体的負担が大きく頻繁に施行することが困難であることが問題と なっている。 [0005] Screening tests and early diagnosis of diabetic nephropathy in actual clinical settings are performed by detecting urine protein by the test strip method using random urine from patients, or detecting and quantifying trace amounts of albumin in urine. I have. Although this test is simple, even in the same patient, the amount of excretion of protein 'albumin in urine is not constant due to fluctuations in water intake etc. every day. It is required to perform several tests to confirm positive and negative. In addition to this test method, creatinine clearance measurement (hereinafter abbreviated as “Ccr”) is commonly performed for the purpose of renal function testing in order to diagnose the stage of diabetic nephropathy (non-patented). Reference 5). Ccr indicates the amount of plasma required to excrete specific components in plasma into the kidneys and urine for a certain period of time, and it is a test item that accurately diagnoses the glomerular filtration capacity of the kidneys. Since it is necessary to restrain the patient for hours or 24 hours and it is necessary to collect urine and plasma at the same time, it lacks versatility and is difficult to perform frequently on a large number of patients. In addition, urinary protein and urinary microalbumin measurement and Ccr are both indicators that mainly diagnose renal function, and may be diagnosed as positive even in renal insufficiency limited to the kidneys. It is not a specific diagnostic indicator of the disease. Even in diabetic patients, primary glomerulonephritis such as acute glomerulonephritis, membranous proliferative glomerulonephritis, purpura nephritis, etc. may be complicated, and treatment methods are different in each case. Requires a renal biopsy (Non-Patent Document 6). The problem is that renal biopsy is difficult to perform frequently because of the heavy physical burden on the patient.
[0006] 最近、発明者らは、血中のエラスチン分解物の特異的測定試薬を開発することに 成功し、その試薬を用いて、大動脈解離症の体液診断が可能となることを見出した（ 特許文献 1)。大動脈解離症は、動脈硬化症を基礎疾患としており、血圧の上昇によ り血管壁が破裂 ·剥離する疾患である。動脈硬化が進行するに従って、血管壁に主と して局在しているエラスチン分子が様々な蛋白質分解酵素により切断を受けて、その 分解物が循環液中に出現すると予想されており、ひいてはその分解物の血中濃度が 上昇することとなる。 [0006] Recently, the present inventors have succeeded in developing a reagent for specifically measuring elastin degradation products in blood, and have found that it is possible to diagnose body fluid of aortic dissection using the reagent ( Patent Document 1). Aortic dissection is based on arteriosclerosis and is a disease in which the blood vessel wall ruptures and detaches due to an increase in blood pressure. As atherosclerosis progresses, elastin molecules, which are mainly localized in the blood vessel wall, are cleaved by various proteases, Degradants are expected to appear in the circulating fluid, which will lead to increased blood levels of the degradants.
[0007] ところで、免疫学的測定方法によりヒト血中を循環しているエラスチン分解物量の測 定が可能であることが示されている (非特許文献 7〜10参照)。また、本発明者らは、 エラスチン分解物を免疫学的に測定する方法に好適なモノクローナル抗体として、ハ イブリドーマ HASG-2及び HASG- 30が産生するモノクローナル抗体を開示して!/、る ( 特許文献 1)。  [0007] By the way, it has been shown that the amount of elastin hydrolyzate circulating in human blood can be measured by an immunological measurement method (see Non-Patent Documents 7 to 10). Further, the present inventors have disclosed monoclonal antibodies produced by hybridomas HASG-2 and HASG-30 as monoclonal antibodies suitable for a method for immunologically measuring elastin degradation products! Reference 1).
[0008] これまで、血中エラスチン分解物の濃度と動脈硬化症および動脈硬化関連疾患と の関連は示唆されて来た (非特許文献 8、 11、 12参照)。この中で血中エラスチン分 解物の濃度と糖尿病との関連についても示唆されている（非特許文献 8)。最近では 、糖尿病における循環障害合併症において血中エラスチン分解物の濃度が上昇す ることが示された (非特許文献 13)。しかし、この報告では足部潰瘍'壊痕症状という 極めて重症な糖尿病性循環障害患者群にぉ 、ての傾向を示したのみであった。さら に最近では、血中エラスチン分解物の濃度はむしろ糖尿病患者では低値となるとい う報告や糖尿病性循環障害合併症の診断に使用することに困難性を示す報告がな されている（非特許文献 14、 15)。しかし、本発明者らが明らかにした大動脈解離症 との関連を除いて、具体的に血中エラスチン分解物の濃度がどの様な疾患に関連す るかは知られていない。  [0008] Hitherto, it has been suggested that the relationship between the concentration of elastin hydrolyzate in blood and arteriosclerosis and atherosclerosis-related diseases has been suggested (see Non-Patent Documents 8, 11, and 12). Among them, it has been suggested that the relationship between the concentration of elastin hydrolyzate in blood and diabetes is suggested (Non-Patent Document 8). Recently, it has been shown that the concentration of elastin hydrolyzate in blood increases in complications of circulatory disorders in diabetes (Non-Patent Document 13). However, this report showed only a tendency for a group of patients with extremely severe diabetic circulatory disorder called foot ulcers and scarring symptoms. More recently, there have been reports that the concentration of elastin hydrolyzate in blood is rather low in diabetic patients and that it is difficult to use it for diagnosis of diabetic circulatory disorder complications (non- Patent documents 14, 15). However, except for the association with aortic dissection revealed by the present inventors, it is not known specifically what kind of disease is associated with the concentration of elastin hydrolyzate in blood.
特許文献 1：特開 2002— 350439号公報  Patent Document 1: JP-A-2002-350439
非特許文献 1 :糖尿病 42, 385 (1999)  Non-Patent Document 1: Diabetes 42, 385 (1999)
非特許文献 2 :日本臨床 60, 260-269 (2002)  Non-patent document 2: Japanese clinical practice 60, 260-269 (2002)
非特許文献 3 :高血圧治療ガイドライン 2000年版， 55-58 (2000)杏林舎  Non-patent document 3: Hypertension treatment guideline 2000 edition, 55-58 (2000) Kyorinsha
非特許文献 4 :日本臨床 62, 80-86 (2004)  Non-patent document 4: Japanese clinical practice 62, 80-86 (2004)
非特許文献 5 :糖尿病 44, 623 (2001)  Non-Patent Document 5: Diabetes 44, 623 (2001)
非特許文献 6 :日本臨床 60, 316-322 (2002)  Non-patent document 6: Japanese clinical practice 60, 316-322 (2002)
非特許文献 7 : Meth. EnzymoL, 163, 656-673 (1988)  Non-Patent Document 7: Meth.EnzymoL, 163, 656-673 (1988)
非特許文献 8 : Clin. Physiol. Biochem., 8, 273-282 (1990)  Non-Patent Document 8: Clin.Physiol.Biochem., 8, 273-282 (1990)
非特許文献 9 : J. Immunol. Methods, 164, 175-187 (1993) 非特許文献 10 : Eur. J. Vase. Endovasc. Surg., 14, 12-16 (1997) Non-Patent Document 9: J. Immunol.Methods, 164, 175-187 (1993) Non-Patent Document 10: Eur.J. Vase.Endovasc.Surg., 14, 12-16 (1997)
非特許文献 11 : Atherosclerosis, 66, 163-168 (1987)  Non-Patent Document 11: Atherosclerosis, 66, 163-168 (1987)
非特許文献 12 : Eur. J. Vase. Endovasc. Surg., 24, 440-444 (2002)  Non-Patent Document 12: Eur.J. Vase.Endovasc.Surg., 24, 440-444 (2002)
非特許文献 13 : Diabetologia Croatica, 26-3, 151-155 (1997)  Non-Patent Document 13: Diabetologia Croatica, 26-3, 151-155 (1997)
非特許文献 14 : Atherosclerosis, 131, 73-78 (1997)  Non-Patent Document 14: Atherosclerosis, 131, 73-78 (1997)
非特許文献 15 : General Pharmacology, 35, 59-64 (2000)  Non-Patent Document 15: General Pharmacology, 35, 59-64 (2000)
発明の開示  Disclosure of the invention
[0009] 本発明は、簡便で精度良く糖尿病性腎症、急性進行性糸球体腎炎、アミロイドーシ スまたはコレステリン塞栓症といった全身性動脈硬化症、好ましくは糖尿病性腎症を 判定する方法及びキットを提供するものである。  The present invention provides a method and kit for simply and accurately determining systemic arteriosclerosis such as diabetic nephropathy, acute progressive glomerulonephritis, amyloidosis or cholesterol embolism, preferably diabetic nephropathy. To provide.
[0010] 本発明者らは、全身性動脈硬化症を基礎疾患とする大動脈解離症以外の疾患群 の中で、エラスチン分解物の血中測定値が上昇する疾患があるのではないかと考え 、糖尿病患者、及びその他の関連疾患を対象として研究を実施した。  [0010] The present inventors considered that among the group of diseases other than aortic dissection based on systemic atherosclerosis, there is a disease in which the measured value of elastin hydrolyzate in the blood increases. The study was conducted on diabetic patients and other related diseases.
そして本発明者らは、糖尿病患者群と糖尿病性腎症合併患者群で両患者群の血 中のエラスチン分解物濃度を比較測定したところ、糖尿病性腎症合併群でのみ極め て高い血中濃度を検出し、エラスチン分解物の量の測定が糖尿病性腎症診断に有 用であることを見出すに至った。また、急性糸球体腎炎、膜性増殖性糸球体腎炎や 紫斑性腎炎と!/ヽつた原発性糸球体腎炎（腎臓限局型の腎機能不全)では ヽずれも 血中濃度が上昇しないことも併せて見出した。さらに詳細な検討を加えたところ、急 性進行性糸球体腎炎、アミロイド一シス、コレステリン塞栓症といった糖尿病性腎症と 同様に全身性動脈硬化症を基礎疾患とする関連疾患においても血中濃度が上昇す ることも見出した。すなわち、本発明は、血中エラスチン分解物を測定することにより 糖尿病性腎症、急性進行性糸球体腎炎、アミロイド一シスまたはコレステリン塞栓症 t ヽつた全身性動脈硬化症、好ましくは糖尿病性腎症を診断する新規の診断指標を 提供するものである。  The present inventors compared the elastin hydrolyzate concentration in the blood of the diabetic patient group and the diabetic nephropathy patient group, and found that the blood concentration was extremely high only in the diabetic nephropathy group. And found that measurement of the amount of elastin hydrolyzate is useful for diagnosis of diabetic nephropathy. In addition, acute glomerulonephritis, membranous proliferative glomerulonephritis, and purpura nephritis! / Puta primary glomerulonephritis (renal dysfunction of the kidney-limited type) also shows that blood levels do not increase. I found it. A more detailed study revealed that blood levels of blood levels in related diseases based on systemic arteriosclerosis as well as diabetic nephropathy such as acute progressive glomerulonephritis, amyloidosis, and cholesterol embolism. Was found to rise. That is, the present invention provides a method for measuring diabetic nephropathy, acute progressive glomerulonephritis, amyloidosis or cholesterol embolism, and measuring systemic arteriosclerosis, preferably diabetic nephropathy. It provides a new diagnostic index for diagnosing disease.
[0011] 本発明は、被験者の循環液中のエラスチン分解物の量を測定し、その測定値に基 づいて糖尿病性腎症、急性進行性糸球体腎炎、アミロイド一シスまたはコレステリン 塞栓症といった全身性動脈硬化症、好ましくは糖尿病性腎症を判定することを含む、 全身性動脈硬化症、好ましくは糖尿病性腎症の判定方法 (以下「本発明方法」とも 、 う）を提供する。 [0011] The present invention measures the amount of elastin hydrolyzate in the circulating fluid of a subject, and based on the measured value, evaluates diabetic nephropathy, acute progressive glomerulonephritis, amyloidosis or cholesterol embolism. Including determining systemic atherosclerosis, preferably diabetic nephropathy, Provided is a method for determining systemic arteriosclerosis, preferably diabetic nephropathy (hereinafter, also referred to as “the method of the present invention”).
糖尿病性腎症を対象とした場合においては、本発明における判定は、糖尿病性腎 症であるか否力判定するのみならず、糖尿病性腎症の重症度を判定することまで含 むものである。  In the case of diabetic nephropathy, the determination in the present invention includes not only determining whether or not the patient has diabetic nephropathy but also determining the severity of diabetic nephropathy.
[0012] 本発明方法においては、エラスチン分解物に対する持つ抗体を用いる免疫化学的 方法によりエラスチン分解物の量を測定測定することが好ましい。  [0012] In the method of the present invention, it is preferable to measure and measure the amount of elastin degradation product by an immunochemical method using an antibody against elastin degradation product.
[0013] 前記抗体は、モノクローナル抗体であることが好ま 、。また、本発明方法は、第 1 又は第 2のモノクローナル抗体を用い、抗体の一方が固相に固定され、及び、もう一 方が標識物質により標識されて 、ることが好ま U、。 [0013] The antibody is preferably a monoclonal antibody. In the method of the present invention, it is preferable that one of the antibodies is immobilized on a solid phase, and the other is labeled with a labeling substance, using the first or second monoclonal antibody.
[0014] 前記モノクローナル抗体としては、 HASG- 2 (FERM BP- 08488)、 HASG- 30 (FERM[0014] Examples of the monoclonal antibody include HASG-2 (FERM BP-08488), HASG-30 (FERM BP-08488).
BP-08489)、 HASG-61-1 (FERM BP-08490)力 選ばれるハイプリドーマにより産生 されるモノクローナル抗体であることが好まし!/、。 BP-08489), HASG-61-1 (FERM BP-08490) The monoclonal antibody produced by the selected hybridoma is preferred!
本発明方法においては、前記第 1及び第 2のモノクローナル抗体は、 HASG-30及 び HASG- 61-1のハイブリドーマにより産生されるモノクローナル抗体の組み合わせで あることが好ましい。  In the method of the present invention, the first and second monoclonal antibodies are preferably a combination of monoclonal antibodies produced by HASG-30 and HASG-61-1 hybridomas.
[0015] 本発明はまた、エラスチン分解物に対する特異性及び親和性を持つ抗体の 1種又 は 2種以上を含み、被験者の循環液中のエラスチン分解物の量を測定し、その測定 値に基づいて糖尿病性腎症、急性進行性糸球体腎炎、アミロイド一シスまたはコレス テリン塞栓症と!/ヽつた全身性動脈硬化症、好ましくは糖尿病性腎症を判定するため の、全身性動脈硬化症の判定キット (以下「本発明キット」とも!、う）を提供する。  [0015] The present invention also includes one or more antibodies having specificity and affinity for elastin hydrolyzate, measures the amount of elastin hydrolyzate in the circulating fluid of a subject, and measures the measured value. Based on the criteria for diabetic nephropathy, acute progressive glomerulonephritis, amyloidosis or cholesterol embolism, to determine systemic atherosclerosis, preferably diabetic nephropathy (Hereinafter, also referred to as “the kit of the present invention!”).
図面の簡単な説明  Brief Description of Drawings
[0016] [図 1]被験検体中の大動脈エラスチン分解物濃度と吸光度との関係を示す標準曲線 である。  FIG. 1 is a standard curve showing the relationship between aortic elastin hydrolyzate concentration in a test sample and absorbance.
[図 2]膜性増殖性糸球体腎炎（17例）、急性糸球体腎炎（ 20例）、紫斑性腎炎（ 23例 )、糖尿病（腎症非合併例） 55例と糖尿病性腎症（ 172例)、急性進行性糸球体腎炎 (85例）、アミロイド一シス (26例）、コレステリン塞栓症（17例）、健常人（108例）におけ る血清中エラスチン分解物濃度の分布を示す。 [図 3]糖尿病患者群 (129例）中の、糖尿病患者 (腎症非合併例) 53例、糖尿病性腎症 患者 (51例)、糖尿病性腎症 +慢性腎不全患者 (25例)、第 4群として糖尿病性腎症 +透析療法導入患者 (53例）における血清中エラスチン分解物濃度の分布を示す。 発明を実施するための最良の形態 [Figure 2] Membranous proliferative glomerulonephritis (17 cases), acute glomerulonephritis (20 cases), purpura nephritis (23 cases), diabetes (55 cases without nephropathy) and diabetic nephropathy (172 cases) Example), distribution of serum elastin degradation products in acute progressive glomerulonephritis (85 cases), amyloidosis (26 cases), cholesterol embolism (17 cases), and healthy subjects (108 cases) . [Figure 3] Among the diabetic patient group (129 cases), 53 diabetic patients (without nephropathy), 53 diabetic nephropathy patients (51 cases), diabetic nephropathy + chronic renal failure patients (25 cases), Group 4 shows the distribution of serum elastin hydrolyzate concentration in diabetic nephropathy + dialysis therapy-introduced patients (53 patients). BEST MODE FOR CARRYING OUT THE INVENTION
[0017] < 1 >本発明測定法  [0017] <1> Measurement method of the present invention
本発明方法は、被験者の循環液中のエラスチン分解物の量を測定し、その測定値 に基づ 、て糖尿病性腎症を判定することを特徴とする。  The method of the present invention is characterized in that the amount of elastin hydrolyzate in the circulating fluid of a subject is measured, and diabetic nephropathy is determined based on the measured value.
[0018] 本発明における循環液とは、血清、血漿、髄液、腹水と!/、つた体液又はその画分を 示しており、医療機関等において通常採取されるものであれば特に限定されるもので はな 、が、被験者からの血清などの血液検体が特に好適である。  [0018] The circulating fluid in the present invention refers to serum, plasma, cerebrospinal fluid, ascites, and / or ivy, or a fraction thereof, and is not particularly limited as long as it is normally collected in a medical institution or the like. However, blood samples such as serum from a subject are particularly suitable.
[0019] 循環液中のエラスチン分解物の量の測定方法としては、循環液中のエラスチン分 解物の量を測定することができればどのような方法でも用いることができる力 例えば 、エラスチン分解物に対する特異性及び親和性を持つ抗体を用いる免疫化学的方 法が挙げられる。  [0019] As a method of measuring the amount of elastin hydrolyzate in the circulating fluid, any method can be used as long as the amount of elastin hydrolyzate in the circulating fluid can be measured. Examples include immunochemical methods using antibodies having specificity and affinity.
[0020] 免疫学的方法は、エラスチン分解物、好適にはヒト大動脈エラスチン分解物に対す る抗体を用いた方法であれば特に制限されず、ラテックス凝集方法、ウェスタンプロ ット法、サンドイッチ法、競合法などの方法があって、特に限定されるものではないが 、サンドイッチ ELISA法が好ましい。  [0020] The immunological method is not particularly limited as long as it is a method using an antibody against an elastin degradation product, preferably a human aortic elastin degradation product, and may be a latex agglutination method, a western plot method, a sandwich method, or the like. There is a method such as a competition method, which is not particularly limited, but a sandwich ELISA method is preferable.
その検出方法としては、放射性物質標識物を用いる方法、蛍光標識物を用いる方 法、酵素標識物を用いる方法や電気化学発光を用いる方法などがあり、特に限定さ れるものではな!/、が、安全で簡便な酵素免疫測定方法 (ELISA)または電気化学発 光免疫測定法であることが好まし 、。  Examples of the detection method include a method using a radioactive substance label, a method using a fluorescent label, a method using an enzyme label, and a method using electrochemiluminescence, and are not particularly limited. Preferably, it is a safe and simple enzyme immunoassay (ELISA) or an electrochemical immunoassay.
[0021] また、使用される抗体としては、ヒト大動脈エラスチン分解物を、ポリクローナル抗体 としてはゥサギ、ャギ、ヒッジ等に免疫した血清力 精製される抗体、モノクローナル 抗体としてはマウスゃラット、ハムスター等の齧歯類動物に免疫して作製されるモノク ローナル抗体を用いることが可能である力 好ましくはモノクローナル抗体が適して!/ヽ る。モノクローナル抗体の作製に用いる動物種は特に限定されるものではないが、一 般的には Balb/Cマウスが最もよく用いられる。モノクローナル抗体は、通常のモノクロ ーナル抗体の作製と同様の方法により作製することができる。 [0021] The antibody used is a human aortic elastin hydrolyzate, the polyclonal antibody is a serum obtained by immunizing rabbits, goats, sheep, etc. The antibody is purified. The monoclonal antibody is mouse, rat, hamster, etc. The ability to use a monoclonal antibody produced by immunizing a rodent of the present invention is preferably a monoclonal antibody! The animal species used for producing the monoclonal antibody is not particularly limited, but generally, Balb / C mice are most often used. Monoclonal antibodies are usually monoclonal Can be prepared in the same manner as in the preparation of the internal antibody.
[0022] 抗体は、所定の特性及び親和性を有する限り、 Fab, Fab'、 F(ab')などのフラグメン [0022] Antibodies are not limited to fragments of Fab, Fab ', F (ab'), etc., as long as they have predetermined properties and affinity.
2  2
トであってもよい。また、抗体は、標識物質により標識されていてもよい。標識物質とし ては、二つの抗体を抗原に免疫学的に結合させる通常のサンドイッチ法に使用され るものが挙げられ、例えば、酵素、電気化学発光錯体、放射性物質、ラテックス、蛍 光物質、化学発光物質、金属コロイド粒子などが挙げられる。本発明においては、酵 素又は電気化学発光錯体が好ましい。標識と抗体との結合は、通常の方法に従って 行うことができる。  May be used. Further, the antibody may be labeled with a labeling substance. Labeling substances include those used in ordinary sandwich methods for immunologically binding two antibodies to an antigen, such as enzymes, electrochemiluminescent complexes, radioactive substances, latex, fluorescent substances, and chemical substances. Light-emitting substances, metal colloid particles and the like can be mentioned. In the present invention, enzymes or electrochemiluminescent complexes are preferred. The binding between the label and the antibody can be performed according to a usual method.
[0023] また、抗体は、固相化されて!/、てもよ 、。固相の形状や材質は特に限定されず、具 体的には、マイクロタイタープレート、ビーズなどが挙げられる。抗体の固相への固定 は、通常の方法に従って行うことができる。  The antibody may be immobilized on a solid phase! The shape and material of the solid phase are not particularly limited, and specific examples include a microtiter plate and beads. The immobilization of the antibody on the solid phase can be performed according to a usual method.
ポリクローナル抗体及びモノクローナル抗体及びその Fab、 Fab'、 p(ab')などのフラ Polyclonal and monoclonal antibodies and their Fab, Fa b hula such ', p (ab')
2 グメントの作製、マイクロプレート、磁気ビーズ、ラテックスビーズなどの固相への抗体 の固定化、酵素、電気化学発光錯体、放射性物質、蛍光物質、化学発光物質、金属 コロイド粒子など標識物質の抗体への結合、ラテックス凝集方法、ウェスタンプロット 法、サンドイッチ法、競合法などの免疫学的測定は、 Antibodies A Laboratory Manual (Cold Spring Harbor Laboratory (1988))、酵素免疫測定法（石川栄治ら、医 学書院)などに記載の周知の方法に基づいて行うことができる。  2 preparation of antibodies, immobilization of antibodies on solid phases such as microplates, magnetic beads, latex beads, etc., to antibodies of labeled substances such as enzymes, electrochemiluminescent complexes, radioactive substances, fluorescent substances, chemiluminescent substances, and metal colloid particles. Immunoassays such as binding, latex agglutination, western blotting, sandwich and competition methods are described in Antibodies A Laboratory Manual (Cold Spring Harbor Laboratory (1988)), enzyme immunoassays (Eiji Ishikawa et al., Medical Science Institute). ) Can be performed based on a known method described in, for example,
[0024] 本発明に用いるモノクローナル抗体として具体的には、ハイプリドーマ HASG-2 ( FERM BP- 08488)、 HASG-30 (FERM BP- 08489)、 HASG- 61- 1 (FERM BP- 08490) が産生するモノクローナル抗体、又はこれらの抗体のいずれかと同等の、ヒト大動脈 エラスチン分解物に対する特異性及び親和性を持つ抗体が挙げられる。 HASG-2及 び HASG-30は、特開 2002-350439号公報に開示されて!ヽる。  [0024] Specific examples of the monoclonal antibody used in the present invention include hybridoma HASG-2 (FERM BP-08488), HASG-30 (FERM BP-08489), and HASG-61-1 (FERM BP-08490). Monoclonal antibodies, or antibodies having the same specificity and affinity for human aortic elastin degradation products as any of these antibodies. HASG-2 and HASG-30 are disclosed in JP-A-2002-350439.
[0025] HASG-2及び HASG-30は、独立行政法人産業技術総合研究所特許微生物寄託セ ンター ( τ 305-8566 日本国茨城県つくば巿東 1丁目 1番地 1中央第 6)に 2001年 5月 18日に寄託され、受託番号 FERM P-18335及び FERM P-18336が付与され、 2003年 9月 18日に、ブタペスト条約に基く国際寄託に移管され、それぞれ、 FERM BP-08488 及び FERM BP-08489の受託番号が付与されている。また、 HASG-61-1は、独立行 政法人産業技術総合研究所特許微生物寄託センターに 2002年 10月 8日に寄託され 、受託番号 FERM P-19058が付与され、 2003年 9月 18日に、ブタペスト条約に基く国 際寄託に移管され、 FERM BP-08490の受託番号が付与されている。 HASG-2により 産生されるモノクローナル抗体、 HASG- 30により産生されるモノクローナル抗体、及 び、 HASG- 61-1により産生されるモノクローナル抗体のいずれかと同等の、ヒト大動 脈エラスチン分解物に対する特異性及び親和性を持つ抗体は、以下に挙げるような 方法により選択することができる。 [0025] HASG-2 and HASG-30 were registered at the National Institute of Advanced Industrial Science and Technology (AIST) Patent Microorganisms Depositary Center (τ 305-8566, 1-1 Tsukuba East, Ibaraki Pref. Deposited on March 18 and assigned accession numbers FERM P-18335 and FERM P-18336.On September 18, 2003, they were transferred to the International Deposit under the Budapest Treaty, and they were transferred to FERM BP-08488 and FERM BP- It has a 08489 accession number. HASG-61-1 is an independent bank Deposited at the National Institute of Advanced Industrial Science and Technology Patent and Microorganisms Depositary Center on October 8, 2002, granted the accession number FERM P-19058, and transferred to an international deposit based on the Budapest Treaty on September 18, 2003. FERM BP-08490 accession number. Specificity for human large artery elastin hydrolyzate equivalent to any of the monoclonal antibodies produced by HASG-2, HASG-30, and HASG-61-1 And antibodies having affinity can be selected by the following methods.
[0026] 同等の特異性及び親和性を持つ抗体は、例えば競合試験により選択することがで さる (Antibodies A Laboratory Manual,し old bpnng Harbor Laboratory (1988) p.567) 。具体的には、抗原物質であるヒト大動脈エラスチン分解物を適当な濃度で生理緩 衝液に溶解し、マイクロプレート固相部に吸着させる。ブロッキング処理を行った後に 、酵素標識した HASG- 2、 HASG- 30又は HASG- 61-1モノクローナル抗体とともに評 価すべき抗体を同量で添加する。同様な特異性及び親和性を有する抗体は、酵素 標識した HASG- 2、 HASG- 30又は HASG- 61-1モノクローナル抗体の反応を阻害する 性能を確認することにより選択することができる。  Antibodies having the same specificity and affinity can be selected by, for example, a competition test (Antibodies A Laboratory Manual, old bpnng Harbor Laboratory (1988) p.567). Specifically, a human aortic elastin hydrolyzate, which is an antigenic substance, is dissolved at an appropriate concentration in a physiological buffer and adsorbed to a solid phase of a microplate. After performing the blocking treatment, add the antibody to be evaluated together with the enzyme-labeled HASG-2, HASG-30 or HASG-61-1 monoclonal antibody in the same amount. Antibodies with similar specificity and affinity can be selected by confirming their ability to inhibit the reaction of enzyme-labeled HASG-2, HASG-30 or HASG-61-1 monoclonal antibodies.
[0027] 別法としては、例えばペプチドマッピング法により選択することができる。具体的に は、抗原物質であるヒト大動脈エラスチン分解物を高速液体クロマトグラフィー装置等 により、分子量や疎水性等の違いにより分離し、分離された様々なエラスチン抗原断 片に対する結合を HASG- 2、 HASG- 30及び HASG- 61-1モノクローナル抗体と比較す ることにより、特異性及び親和性を評価するものである。 HASG- 2、 HASG-30又は HASG-61-1モノクローナル抗体が反応する抗原断片に同等に反応する抗体はそれ ぞれ同等の特異性を有すると判断される。  [0027] Alternatively, for example, selection can be made by a peptide mapping method. Specifically, human aortic elastin hydrolyzate, which is an antigenic substance, is separated by a high-performance liquid chromatography device or the like according to differences in molecular weight, hydrophobicity, etc., and binding to various separated elastin antigen fragments is determined by HASG-2, The specificity and affinity are evaluated by comparing with HASG-30 and HASG-61-1 monoclonal antibodies. Antibodies that react equally to the antigen fragments to which the HASG-2, HASG-30 or HASG-61-1 monoclonal antibodies react are judged to have the same specificity.
[0028] ヒト大動脈エラスチン分解物は、 Biochem. J., 61, 11-21(1955)に記載の方法によつ て得ることができるものであり、市販品として入手することができる。  [0028] The human aortic elastin hydrolyzate can be obtained by the method described in Biochem. J., 61, 11-21 (1955) and can be obtained as a commercial product.
[0029] 生体試料中のエラスチン分解物の ELISAにおいては、その測定方式としても様々な 方法が知られているが、特に簡便で定量性が高い方法として固相化した第 1抗体と ペルォキシダーゼなどの酵素を標識した第 2抗体を用いたサンドイッチ方式がよく用 いられる。この場合の第 1抗体と第 2抗体はエラスチン分解物に対して特異的に反応 することが望ましぐ第 1抗体と第 2抗体は同一のものであっても構わない。具体的に は、大動脈エラスチン分解物を認識する抗体 (第 1抗体)を 96ウェルマイク口プレート などの固相に結合させる。固相化は共有結合により結合させても非共有結合により 結合させても構わない。次いで、固相への非特異的吸着を低減するためにミルク力 ゼインなどのブロッキングタンパク質を吸着させることが好まし 、。そこへあらカゝじめ濃 度の明らかな標準大動脈エラスチン分解物溶液と被験生体試料を加えて一定時間 放置する。試料中の大動脈エラスチン分解物抗原を固相に吸着させた後に洗浄し、 今度はペルォキシダーゼなどの酵素標識した別の抗大動脈エラスチン分解物抗体（ 第 2抗体)を適当な濃度で加える。一定時間まで放置して第 1抗体と大動脈エラスチ ン分解物と第 2抗体の三者の複合体を固相上に形成させる。その後に固相を洗浄し て、酵素の基質、酵素がペルォキシダーゼであれば過酸ィ匕水素と TMBZ等の発色基 質の混合溶液を添加し、標識酵素による発色を得る。酵素反応を阻害剤等を添加し て停止させた後に、その発色の吸光度をプレートリーダー等の機器により測定する。 発色基質に代えて発光基質を使用することも許され、この場合は吸光度に代えて発 光強度を測定する。被験液の吸光度と標準品の吸光度を検量線等を用いて比較す ることにより精度良く被験液中の大動脈エラスチン分解物量を知ることができる。 電気化学発光免疫測定においては、その測定方式としても様々なものが知られて いるが、特に簡便で定量性が高いものとして第 1抗体に、例えば磁気ビーズに固相 化した抗体、第 2抗体に、化学発光性錯体好ましくはルテニウム錯体によって標識し た抗体を用いたサンドイッチ方式が好ましい。この場合の第 1抗体と第 2抗体はエラス チン分解物に対して特異的に反応することが望ましぐ第 1抗体と第 2抗体は同一の ものであっても構わない。具体的には、大動脈エラスチン分解物を認識する抗体 (第 1抗体)を磁気ビーズなどの固相に結合させる。固相化は共有結合により結合させて も非共有結合により結合させても構わない。次いで、固相への非特異結合を低減す るためにミルクカゼインなどのブロッキングタンパク質を吸着させることが好ま U、。そ こへあらかじめ濃度の明らかなエラスチン分解物溶液、または被験生体試料を加え て一定時間放置する。固相が磁気ビーズであれば撹拌することが好ましい。試料中 のエラスチン分解物抗原を抗体結合粒子表面に吸着させた後に固相を洗浄し、化 学発光性錯体好ましくはルテニウム錯体にて標識した別の抗エラスチン分解物抗体[0029] In ELISA for elastin hydrolyzate in a biological sample, various methods are known as a measuring method. Particularly, a simple and highly quantitative method, such as immobilized first antibody and peroxidase, is used. A sandwich method using a second antibody labeled with an enzyme is often used. In this case, the first and second antibodies react specifically to elastin degradation products. The first antibody and the second antibody desired to be used may be the same. Specifically, an antibody (first antibody) recognizing an aortic elastin degradation product is bound to a solid phase such as a 96-well microphone plate. The solid phase may be bound by a covalent bond or a non-covalent bond. Then, it is preferable to adsorb a blocking protein such as milk force zein in order to reduce non-specific adsorption to the solid phase. Add a standard aortic elastin hydrolyzate solution with a clear concentration and a test biological sample, and leave it for a certain period of time. After adsorbing the aortic elastin degradation product antigen in the sample to the solid phase, washing is performed, and then another anti-aortic elastin degradation product antibody (second antibody) labeled with an enzyme such as peroxidase is added at an appropriate concentration. The complex is left to stand for a certain period of time to form a complex of the first antibody, the aortic elastin degradation product, and the second antibody on the solid phase. Thereafter, the solid phase is washed, and if the enzyme substrate or the enzyme is peroxidase, a mixed solution of hydrogen peroxide and a coloring substrate such as TMBZ is added to obtain a color by the labeling enzyme. After terminating the enzyme reaction by adding an inhibitor or the like, the absorbance of the color is measured with a device such as a plate reader. It is also possible to use a luminescent substrate instead of a chromogenic substrate. In this case, the luminescence intensity is measured instead of the absorbance. By comparing the absorbance of the test solution with the absorbance of the standard using a calibration curve or the like, the amount of aortic elastin hydrolyzate in the test solution can be known with high accuracy. Various methods are known for the electrochemiluminescence immunoassay, and a particularly simple and highly quantitative method is used for the first antibody, such as an antibody immobilized on magnetic beads or a second antibody. Furthermore, a sandwich system using an antibody labeled with a chemiluminescent complex, preferably a ruthenium complex, is preferable. In this case, the first antibody and the second antibody, which desirably react specifically with the elastin degradation product, may be the same. Specifically, an antibody (first antibody) that recognizes an aortic elastin degradation product is bound to a solid phase such as magnetic beads. The solid phase may be bound by a covalent bond or a non-covalent bond. Next, it is preferable to adsorb blocking proteins such as milk casein to reduce non-specific binding to the solid phase. Then, add a solution of elastin hydrolyzate with a known concentration or a test biological sample in advance, and leave it for a certain period of time. If the solid phase is magnetic beads, it is preferable to stir. After adsorbing the elastin-decomposed antigen in the sample to the antibody-bound particle surface, the solid phase is washed and Another anti-elastin hydrolyzate antibody labeled with a chemiluminescent complex, preferably a ruthenium complex
(第 2抗体)を適当な濃度で加える。一定時間放置し、固相が磁気ビーズの場合は好 ましくは撹拌して、第 1抗体とエラスチン分解物抗原と第 2抗体の三者の複合体を磁 気ビーズ上に形成させる。その後に固相を洗浄して、好ましくは専用の装置中の電 極間にて電流を通し化学発光性錯体、好ましくはルテニウム錯体を発光させ、発光 強度を計測する。被験生体試料の発光量と標準品の発光量を検量線等を用いて比 較することにより精度良く被験生体試料中のエラスチン分解物量を知ることができる。 (Second antibody) at an appropriate concentration. The mixture is allowed to stand for a certain period of time, and if the solid phase is magnetic beads, it is preferably stirred to form a complex of the first antibody, the elastin degradation product antigen and the second antibody on the magnetic beads. Thereafter, the solid phase is washed, and a current is preferably passed between the electrodes in a dedicated device to cause the chemiluminescent complex, preferably a ruthenium complex, to emit light, and the emission intensity is measured. By comparing the luminescence of the test biological sample with the luminescence of the standard product using a calibration curve or the like, the amount of elastin degradation product in the test biological sample can be known with high accuracy.
[0031] 上記サンドイッチ法のように、第 1抗体及び第 2抗体を用いる場合、 V、かなる組合せ も許されるが、好ましい各々の抗体の組み合わせとしては、具体的には以下ノ、イブリ ドーマにより産生されるモノクローナルの組合せが例示される。 [0031] When the first antibody and the second antibody are used as in the above sandwich method, a combination of V and such a combination is also allowable. Specific combinations of the respective antibodies are specifically described below. The combinations of monoclonals produced are exemplified.
(a) HASG- 30及び HASG- 61- 1、  (a) HASG-30 and HASG-61-1,
(b) HASG- 61- 1及び HASG- 2、  (b) HASG-61-1 and HASG-2,
(c)いずれも HASG- 2、又は  (c) HASG-2, or
(d) HASG- 30及び HASG- 2。  (d) HASG-30 and HASG-2.
更に好ましくは、 HASG-30及び HASG-61-1の組合せが望ましい。  More preferably, a combination of HASG-30 and HASG-61-1 is desirable.
[0032] 本発明における判定とは、糖尿病性腎症である力否か判定するのみならず、糖尿 病性腎症の重症度を判定することまで含むものである。 [0032] The determination in the present invention includes not only determination of the power of diabetic nephropathy but also determination of the severity of diabetic nephropathy.
糖尿病性腎症であるか否かの判定は、エラスチン分解物の測定値がカットオフ値よ り高いか否かにより行われる。カットオフ値は通常、従来の方法により糖尿病性腎症 ではないと診断された糖尿病患者の血中エラスチン分解物の濃度を測定し、その平 均値 + 3SD (標準偏差)の値として設定される。実施例に記載した 55例の糖尿病患 者を測定した例においては、カットオフ値は 184.9 ng/mLであった。  The determination as to whether or not the patient has diabetic nephropathy is made based on whether or not the measured value of elastin degradation product is higher than the cutoff value. The cut-off value is usually determined by measuring the concentration of elastin hydrolyzate in the blood of diabetic patients diagnosed as not having diabetic nephropathy by the conventional method, and calculating the average + 3SD (standard deviation) . In the case of measuring 55 diabetic patients described in the examples, the cutoff value was 184.9 ng / mL.
場合によっては、糖尿病性腎症ではないと診断された糖尿病患者の血中エラスチ ン分解物の測定値の最高値をカットオフ値と定めても良ぐまた糖尿病性腎症を発症 して 、る力否かを判定できれば、他の値をカットオフ値と定めることも許される。  In some cases, the highest measured value of elastin degradation in blood of a diabetic patient diagnosed as not having diabetic nephropathy may be set as the cut-off value, and diabetic nephropathy may develop. If it is possible to determine whether or not the force is applied, another value may be set as the cutoff value.
急性進行性糸球体腎炎、アミロイド一シスまたはコレステリン塞栓症のカットオフ値 は、糖尿病性腎症と同様に行うことができるが、糖尿病患者に代えて健常人の測定 値を用いることちできる。 [0033] 血中エラスチン分解物の測定値は、糖尿病、糖尿病性腎症、慢性腎不全まで進行 した糖尿病性腎症患者、透析療法を導入するまで進行した糖尿病性腎症患者と、糖 尿病性腎症の重症度に従って増大するため、血中エラスチン分解物の測定値は、糖 尿病性腎症であると判定された後も、糖尿病性腎症の進行をモニターする指標とし て使用することも可能である。 The cut-off value for acute progressive glomerulonephritis, amyloidosis, or cholesterol embolism can be performed in the same manner as in diabetic nephropathy, but the measurement value of a healthy individual can be used instead of a diabetic patient. [0033] The measured values of elastin hydrolyzate in the blood were measured for patients with diabetes, diabetic nephropathy who progressed to diabetic nephropathy and chronic renal failure, diabetic nephropathy patients who progressed to the introduction of dialysis therapy, and diabetes mellitus. Blood elastin degradation products are used as an indicator to monitor the progression of diabetic nephropathy even after it has been determined to be diabetic nephropathy, since it increases with the severity of dyspnea. It is also possible.
[0034] 本発明者らは、後述する実施例に示すように、腎症を合併していない糖尿病患者 ではほとんど測定値が上昇せず、糖尿病性腎症の患者で極めて高 、陽性率で高 、 血中エラスチン分解物濃度を示すことを初めて発見した。また、健常人ではほとんど 陽性となるケースはなく、腎症を合併しない糖尿病や原発性糸球体腎炎の患者にお いても陽性となった症例はわずかであった。また、糖尿病性腎症と同じく全身性動脈 硬化症を基礎疾患とする各種疾患 (急性進行性糸球体腎炎、アミロイド一シス、コレ ステリン塞栓症）にお 、ても 、ずれも高い陽性率で高 、エラスチン分解物濃度を示 すことを初めて発見した。また、エラスチン分解物濃度は、糖尿病性腎症の重症度に 従って上昇し、エラスチン分解物の濃度により糖尿病性腎症の重症度を判定すること ができた。したがって、循環液中のエラスチン分解物の量を測定することにより、糖尿 病性腎症、急性進行性糸球体腎炎、アミロイド一シスまたはコレステリン塞栓症といつ た全身性動脈硬化症、好ましくは糖尿病性腎症を判定することが可能である。  [0034] The present inventors have found that the measured value hardly increases in diabetic patients without nephropathy, is extremely high in diabetic nephropathy patients, and is high at a positive rate, as shown in Examples described later. The first time to show that the concentration of elastin hydrolyzate in the blood. In addition, there were almost no positive cases in healthy subjects, and few cases were positive in patients with diabetes or primary glomerulonephritis without nephropathy. In addition, as in diabetic nephropathy, in various diseases based on systemic atherosclerosis (acute progressive glomerulonephritis, amyloidosis, cholesterol embolism), the slip rate is also high with a high positive rate. For the first time, it was found that it showed elastin degradation product concentration. The elastin hydrolyzate concentration increased according to the severity of diabetic nephropathy, and the severity of diabetic nephropathy could be determined from the elastin hydrolyzate concentration. Therefore, by measuring the amount of elastin degradation products in the circulating fluid, systemic atherosclerosis such as diabetic nephropathy, acute progressive glomerulonephritis, amyloidosis or cholesterol embolism, preferably diabetes It is possible to determine sexual nephropathy.
また、循環液中のエラスチン分解物の量を測定することができる手段、例えば、エラ スチン分解物に対する抗体は、糖尿病性腎症の診断薬の製造に用いることができる  In addition, a means capable of measuring the amount of elastin hydrolyzate in the circulating fluid, for example, an antibody against elastin hydrolyzate can be used for the production of a diagnostic agent for diabetic nephropathy.
[0035] < 2 >本発明キット <2> Kit of the Present Invention
本発明キットは、エラスチン分解物に対する特異性及び親和性を持つ抗体の 1種 又は 2種以上を含むことを特徴とする。同キットは、被験者の循環液中のエラスチン 分解物の量を測定し、その測定値に基づいて糖尿病性腎症、急性進行性糸球体腎 炎、アミロイド一シスまたはコレステリン塞栓症といった全身性動脈硬化症、好ましく は糖尿病性腎症を判定するために用いられる。前記抗体は、本発明方法に関して記 載した通りである。  The kit of the present invention is characterized by containing one or more antibodies having specificity and affinity for elastin degradation products. The kit measures the amount of elastin hydrolyzate in the subject's circulating fluid and uses that measurement to determine systemic arterial disease such as diabetic nephropathy, acute progressive glomerulonephritis, amyloidosis or cholesterol embolism. It is used to determine sclerosis, preferably diabetic nephropathy. The antibody is as described for the method of the invention.
[0036] 本発明キットにおいては、抗体はモノクローナル抗体であることが好ましぐ第 1及び 第 2のモノクローナル抗体を用いることがより好ましい。また、第 1及び第 2の抗体の一 方が固相に固定され、及び、もう一方が酵素又は電気化学発光錯体により標識され ていることが好ましい。標識及びは、本発明方法に関して記載したとおりである。 [0036] In the kit of the present invention, the antibodies are preferably the first and second antibodies which are preferably monoclonal antibodies. More preferably, a second monoclonal antibody is used. Further, it is preferable that one of the first and second antibodies is immobilized on a solid phase, and the other is labeled with an enzyme or an electrochemiluminescent complex. Labels and are as described for the method of the invention.
[0037] 本発明キットにおける抗体は溶液とされたものでも、凍結乾燥されたものであっても よい。 [0037] The antibody in the kit of the present invention may be a solution or a lyophilized antibody.
本発明キットは、第 1及び第 2の抗体の他に、免疫学的測定法で通常に使用される 試薬を含んでいてもよい。この様な試薬としては、標準抗原 (ヒト大動脈エラスチン分 解物)液、基質溶液、検体希釈液、洗浄液などが挙げられる。これらの試薬としては、 上述のエラスチン分解物を測定する方法で記載した試薬を用いることができる。 本発明キットは、本発明方法に従って使用することができる。具体的には、本発明 キットは、糖尿病性腎症、急性進行性糸球体腎炎、アミロイド一シスまたはコレステリ ン塞栓症といった全身性動脈硬化症、好ましくは糖尿病性腎症の診断薬として使用 することができる。  The kit of the present invention may contain, in addition to the first and second antibodies, reagents commonly used in immunoassays. Examples of such a reagent include a standard antigen (human aortic elastin digest) solution, a substrate solution, a sample diluent, and a washing solution. As these reagents, the reagents described in the method for measuring elastin degradation products described above can be used. The kit of the present invention can be used according to the method of the present invention. Specifically, the kit of the present invention may be used as a diagnostic agent for systemic arteriosclerosis such as diabetic nephropathy, acute progressive glomerulonephritis, amyloidosis or cholesterol embolism, preferably diabetic nephropathy. Can be.
実施例  Example
[0038] 以下、実施例を示し、本発明を具体的に説明するが、本発明がこれに限定されるも のではない。  Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited thereto.
[0039] <実施例 1 > <Example 1>
1)大動脈エラスチン分解物に対するモノクローナル抗体の作製  1) Preparation of monoclonal antibody against aortic elastin degradation product
ヒト大動脈エラスチン分解物（エラスチン ·プロダクツ社製、カタログ番号： HA587)を 、 Balb/C雌マウス（6週齢）の腹腔内へ、一匹当たり 0.1 mgの量でコンプリート'フロイ ント ·アジュバント (ディフコ社製)とともに投与した。 3週間後に同量の当該ヒト大動脈 エラスチン分解物をインコンプリート'フロイント ·アジュバント（ディフコ社製）とともに腹 腔内へ投与した。さらに 3週間後に同マウスへ当該ヒト大動脈エラスチン分解物のみ を一匹当たり 0.1 mg投与した。最終の免疫感作が終了した 3日後にマウス力も脾臓を 摘出した。以降の作業は無菌クリーンベンチ内で実施した。摘出した脾臓をメッシュ で分散させ、あら力じめ培養してぉ 、た Sp2/0- Agl4マウスミエローマ細胞と混合し、 50%ポリエチレングリコール 1500 (ロッシュ 'ダイァグノステイク社製）存在下で細胞融 合した。融合したハイプリドーマ細胞は、 96ウェルマイク口カルチャープレート数枚に 分散させ、 10%牛胎児血清と HAT試薬 (シグマ ·アルドリッチ社製)を含む RPMI1640 液体培地 (シグマ'アルドリッチ社製）中で 1〜2週間培養した。この間にモノクローナ ル抗体を安定的に産生するハイプリドーマ細胞のみが生存し、融合しな力つたミエ口 一マ細胞やマウス脾臓細胞は死滅した。当該ヒト大動脈エラスチン分解物に対する モノクローナル抗体を産生する細胞の選択は抗原固相プレートによる ELISAにより実 施した。すなわち、ハイプリドーマ細胞のコロニーが十分に育った時点でその培養上 清を採取し、免疫抗原を固相吸着した 96ウェルマイク口プレート（ナルジェ'ヌンク社 製)へ添加して、その上清中のモノクローナル抗体を免疫抗原に反応させた。その後 に、マウス IgGへ反応する 2次抗体のペルォキシダーゼ標識物 (ダコ 'ジャパン社製） を適当な濃度で添加した。一定時間後にプレートを洗浄し、 ABTS基質溶液 (ロシュ' ダイァグノスティック社製)をカ卩えた。その発色の有無により目的のモノクローナル抗 体産生ハイプリドーマを選択した。選択された株は、それぞれ数回のクローユングを 施した。こうして得られた 3株のハイプリドーマは、 HASG- 2、 HASG-30、 HASG-61-1 と命名された。それぞれのモノクローナル抗体の大量製造は、常法に従いマウス腹 水からプロテイン A固定化セファロースゲル（フアルマシア社製）を用いたァフィニィテ ィークロマトグラフィーにより行った。以下、 HASG- 30及び HASG- 61-1が産生するモノ クローナル抗体を、それぞれ「HASG- 30モノクローナル抗体」及び「HASG- 61- 1モノ クローナル抗体」と呼ぶことがある。 Human aortic elastin hydrolyzate (Elastin Products, Cat. No .: HA587) was intraperitoneally injected into Balb / C female mice (6 weeks of age) in an amount of 0.1 mg / animal in Complete 'Freund's Adjuvant (Difco). Co., Ltd.). Three weeks later, the same amount of the human aortic elastin hydrolyzate was administered intraperitoneally together with Incomplete 'Freund's adjuvant (manufactured by Difco). Three weeks later, the mouse was administered the human aortic elastin hydrolyzate alone (0.1 mg / mouse). Three days after the end of the final immunization, the mouse spleen was also removed. Subsequent work was performed in a sterile clean bench. The excised spleen was dispersed with a mesh, cultured briefly, mixed with Sp2 / 0-Agl4 mouse myeloma cells, and the cells were added in the presence of 50% polyethylene glycol 1500 (Roche's Diagnostics). It was fused. The fused hybridoma cells are placed in several 96-well microphone plate culture plates. The cells were dispersed and cultured in RPMI1640 liquid medium (Sigma-Aldrich) containing 10% fetal bovine serum and HAT reagent (Sigma-Aldrich) for 1 to 2 weeks. During this time, only the hybridoma cells that stably produced the monoclonal antibody survived, and the non-fused myeloma cells and mouse spleen cells died. Selection of cells producing a monoclonal antibody against the human aortic elastin hydrolyzate was performed by ELISA using an antigen solid phase plate. That is, when the colony of the hybridoma cells has grown sufficiently, the culture supernatant is collected, added to a 96-well mic opening plate (manufactured by Narge Nunc) on which the immunizing antigen has been solid-phase-adsorbed, and the supernatant is added to the supernatant. Were reacted with the immunizing antigen. Thereafter, a peroxidase-labeled secondary antibody that reacts with mouse IgG (manufactured by Dako Japan) was added at an appropriate concentration. After a certain period of time, the plate was washed, and an ABTS substrate solution (manufactured by Roche's Diagnostics) was dried. The desired monoclonal antibody-producing hybridoma was selected based on the presence or absence of color development. Each of the selected strains was crawled several times. The three strains of hybridomas thus obtained were named HASG-2, HASG-30, and HASG-61-1. Mass production of each monoclonal antibody was carried out from mouse ascites by affinity chromatography using protein A-immobilized Sepharose gel (Pharmacia) according to a conventional method. Hereinafter, the monoclonal antibodies produced by HASG-30 and HASG-61-1 may be referred to as “HASG-30 monoclonal antibody” and “HASG-61-1 monoclonal antibody”, respectively.
2)血清中大動脈エラスチン分解物の酵素免疫測定法による測定 2) Measurement of serum aortic elastin degradation product by enzyme immunoassay
(1)項で作製された HASG- 30モノクローナル抗体と HASG- 61-1モノクローナル抗体 を精製 IgGとして用意した。これらのモノクローナル抗体は、競合試験により互いに競 合しな 、ことを確認した。 HASG-30モノクローナル抗体を最終濃度 0.01mg/mLとして リン酸緩衝生理食塩水（PBS)に溶解して 96ウェルマイク口プレート（ナルジェ'ヌンク 社製)の各ゥエルに O.lmLずつ添加した。含有するモノクローナル抗体を吸着させた 後に溶液を捨てて、ブロッキングを目的として 1%スキムミルクを含有するトリス緩衝生 理食塩水（以降「TBS」と略記)溶液を各ゥエルに 0.2mLずつ添加した。 1時間後にス キムミルク溶液を廃棄し、 TBSにて洗浄した。洗浄したゥエルに、あらカゝじめ濃度を設 定した標準ヒト大動脈エラスチン分解物、または被験血清を添加した。この際に被検 血清は 1%スキムミルクを含有する PBS溶液にて 10倍に希釈したのちに測定に供した 。そのプレートをフィルムでカバーして 1時間室温にて静置した後に溶液をすベて廃 棄して、 0.05%Tween- 20 (シグマ'アルドリッチ社製）を含む TBS (以降、 Tween- TBSと 略記する）にて 3回洗浄した。その後に、過ヨウ素酸法 [Antibodies: a Laboratory Manual, by Ed. Harlow & D. Lane, Cold bpnng Haroor Laboratory Press The HASG-30 monoclonal antibody and HASG-61-1 monoclonal antibody prepared in section (1) were prepared as purified IgG. These monoclonal antibodies were confirmed to not compete with each other by a competition test. The HASG-30 monoclonal antibody was dissolved in phosphate buffered saline (PBS) to a final concentration of 0.01 mg / mL, and 0.1 mL of the solution was added to each well of a 96-well microphone port plate (Nalge Nunc). After adsorbing the contained monoclonal antibody, the solution was discarded, and 0.2 mL of a Tris-buffered saline (hereinafter abbreviated as “TBS”) solution containing 1% skim milk was added to each well for the purpose of blocking. One hour later, the skim milk solution was discarded and washed with TBS. To the washed well, a standard human aortic elastin hydrolyzate or a test serum at a predetermined concentration was added. At this time Serum was diluted 10-fold with a PBS solution containing 1% skim milk and used for measurement. After the plate was covered with a film and allowed to stand at room temperature for 1 hour, all the solution was discarded, and TBS containing 0.05% Tween-20 (manufactured by Sigma-Aldrich) (hereinafter abbreviated as Tween-TBS) Wash) three times. Then, the periodic acid method [Antibodies: a Laboratory Manual, by Ed. Harlow & D. Lane, Cold bpnng Haroor Laboratory Press]
(1988)]によりペルォキシダーゼ（ロシュ'ダイァグノスティックス社）にて標識された HASG- 61-1モノクローナル抗体を含む 1%スキムミルク含有 TBS溶液をそれぞれ別々 のゥエルに O.lmLずつ添カ卩した。その後、フィルムでカバーして室温にて 1時間静置 した。反応が終了した後に、プレート中の溶液をすベて廃棄し、 Tween-TBSにて 3回 洗浄した。洗浄が終了した後に、過酸ィ匕水素含有 TMBZ (テトラメチルベンチジン)塩 酸基質溶液 (シグマ社製)を各ゥエルに O.lmLずつ添加した。そのままプレートを静置 して 10分間発色させた。その後にプレートの各ゥエルへ 2N塩酸溶液を O.lmLずつ添 加し、よく混和して反応を停止させた。反応停止後にマイクロプレートリーダー（モレキ ユラ一デバイス社製）にてプレート各ゥエルの 450應波長における吸光度を測定し、 発色の強さを数値化した。巿販解析プログラムソフト（SOFTmax-J Ver.2.1 和光純薬 工業社製)を用いて、標準ヒト大動脈エラスチン分解物溶液の抗原濃度と吸光度値 から標準曲線を作製し (図 1)、被験血清中の抗原濃度を算定した。上記測定方法に よって、以下の被験患者の血清中の抗原濃度を測定した。結果を図 2に示す。 (1988)], a 1% skim milk-containing TBS solution containing HASG-61-1 monoclonal antibody labeled with peroxidase (Roche's Diagnostics) was added to separate wells in O.lmL volumes. . Then, it covered with the film and left still at room temperature for 1 hour. After the reaction was completed, all the solutions in the plate were discarded and washed three times with Tween-TBS. After the washing was completed, 0.1 mL of TMBZ (tetramethylbenzidine) hydrochloride substrate solution (manufactured by Sigma) containing hydrogen peroxide was added to each well. The plate was allowed to stand for 10 minutes to develop color. Thereafter, 0.1 mL of a 2N hydrochloric acid solution was added to each well of the plate and mixed well to stop the reaction. After the reaction was stopped, the absorbance of each well of the plate at 450 wavelength was measured with a microplate reader (manufactured by Molecular Devices) to quantify the intensity of color development.標準 Using a sales analysis program software (SOFTmax-J Ver.2.1, manufactured by Wako Pure Chemical Industries, Ltd.), a standard curve was prepared from the antigen concentration and absorbance value of the standard human aortic elastin hydrolyzate solution (Figure 1), Was calculated. The antigen concentration in the serum of the following test patients was measured by the above measurement method. The result is shown in figure 2.
3)糖尿病患者における血中エラスチン分解物の測定値 3) Measured value of elastin degradation product in blood in diabetic patients
上記図 2のヒト大動脈エラスチンの測定値につ 、て解析を行った。腎症合併のな ヽ 糖尿病患者 55例の測定値の平均値と標準偏差は 79.2ng/mL± 35.2であり、糖尿病 患者群における測定値分布上限 (カットオフ値）を平均値 + 3SDの値 184.9ng/mLと 設定した。これを目安とすると、 172例の糖尿病性腎症患者群 (平均値と標準偏差は 363.8士 252.1ng/mL)では 172例中 129例が陽性（陽性率 75.0 %、以下同様）となつ た。健常人 108例における血中エラスチン分解物濃度を調査したところ 44.2士 19.9ng/mLとなり全例陰性となった。これに対して、糖尿病（腎症非合併例） 55例で は 1例のみ陽性（ 1.8%)となった。膜性増殖性糸球体腎炎 17例では 2例のみ陽性（ 11.8%)、急性糸球体腎炎 20例では全例陰性 (0%)、紫斑性腎炎 23例においても 1 例のみ陽性（ 4.3%)となり、腎臓限局型の腎炎である原発性糸球体腎炎ではいずれ もほとんど陰性という結果が得られた。また、糖尿病性腎症と同様に全身性動脈硬化 症を基礎疾患とする急性進行性糸球体腎炎、アミロイド一シス、及びコレステリン塞 栓症ではそれぞれ 85例中 33例（38.8 %)、 26例中 6例（23.1 %)、 17例中 11例（64.7 %) が陽性となって判定された。 Analysis was performed on the measured values of human aortic elastin in FIG. 2 above. The mean value and standard deviation of the measured values of 55 diabetic patients without nephropathy were 79.2 ng / mL ± 35.2, and the upper limit of the measured value distribution (cutoff value) in the diabetic patient group was the mean + 3SD value of 184.9. ng / mL was set. Using this as a guide, 129 of 172 diabetic nephropathy patients (mean and standard deviation were 363.8 persons and 252.1 ng / mL) were positive in 129 of the 172 patients (positive rate 75.0%, and so on). The elastin hydrolyzate concentration in the blood of 108 healthy subjects was investigated. In contrast, only 55 cases of diabetes (without nephropathy) were positive (1.8%). Membranous proliferative glomerulonephritis in 17 cases was positive in only 2 cases (11.8%), acute glomerulonephritis in 20 cases was negative in all cases (0%), and purpura nephritis was 1 in 23 cases Only positive cases (4.3%) were positive, and almost all were negative in primary glomerulonephritis, a type of nephritis limited to the kidney. Similarly to diabetic nephropathy, acute progressive glomerulonephritis, amyloidosis, and cholesterol embolism, which are based on systemic arteriosclerosis, are 33 out of 85 cases (38.8%) and 26 cases, respectively. Six (23.1%) of the 17 cases and 11 (64.7%) of the 17 cases were determined to be positive.
以上の結果より、ヒト大動脈エラスチンの測定値により腎臓限局型の腎炎から全身 性動脈硬化の起きている糖尿病性腎症を分別することが可能であり、また他の全身 性動脈硬化症を基礎疾患とする急性進行性糸球体腎炎、アミロイド一シス、及びコレ ステリン塞栓症を診断することも可能であることが示された。  Based on the above results, it is possible to distinguish diabetic nephropathy with systemic atherosclerosis from renal-limited nephritis by measuring human aortic elastin, and to diagnose other systemic atherosclerosis It has been shown that it is also possible to diagnose acute progressive glomerulonephritis, amyloidosis, and cholesterol embolism.
[0042] 4)糖尿病性腎症患者の進行度と血中エラスチン分解物の測定値 [0042] 4) Progress of diabetic nephropathy patients and measured values of elastin degradation products in blood
これらの糖尿病患者群 129例を疾患の進行度別に 3段階に分類した。糖尿病患者 群全体は、第 1群として糖尿病患者 (腎症非合併例) 53例、第 2群として糖尿病性腎 症患者 51例、第 3群として慢性腎不全まで進行した糖尿病性腎症患者 (以下、及び 図 3で糖尿病性腎症 +慢性腎不全と記載) 25例、第 4群として透析療法を導入するま で進行した糖尿病性腎症患者 (以下、及び図 3で糖尿病性腎症 +透析療法導入と 記載) 53例に分類した。また、健常人 108例の血清における測定値もこれらの 4群と比 較した。  These 129 diabetic patients were classified into three stages according to disease progression. The total number of diabetic patients in the first group was 53 diabetic patients (without nephropathy), 51 patients with diabetic nephropathy as the second group, and diabetic nephropathy patients who progressed to chronic renal failure as the third group ( (See below and Fig. 3 as diabetic nephropathy + chronic renal failure) 25 patients, diabetic nephropathy who progressed until the introduction of dialysis therapy as group 4 (below and diabetic nephropathy + in Fig. 3) Dialysis therapy was introduced). The measured values in the sera of 108 healthy subjects were also compared with these four groups.
結果を図 3にまとめた。この図は箱ヒゲ図と言われる図であり、各群を測定値順に並 ベた時の中央値（ボックス中央の横線）、 25%〜75%が含まれる範囲（ボックスの上端と 下端の横線）、 10%〜90%が含まれる範囲 (バーの上端と下端の横線)を表して 、る。 エラスチン分解物の測定値は健常人では、 44.2 ± 19.9ng/mL、第 1群の糖尿病では 80.2 ±35.8ng/mL、第 2群の糖尿病性腎症では 224.6± 149.6ng/mL、第 3群の糖尿病 性腎症 +腎不全では 337.8士 170.4ng/mL、第 4群の糖尿病性腎症 +透析導入では 590.7±267.6ng/mLとなり、疾患の進行に伴って明らかに測定値が上昇するという結 果が得られた。  The results are summarized in FIG. This figure is called a box-and-whisker plot. The median (horizontal line at the center of the box) when each group is arranged in the order of measured values, the range including 25% to 75% (horizontal line at the top and bottom of the box) ), 10% to 90% is included in the range (the horizontal line at the top and bottom of the bar). Elastin degradation products were measured at 44.2 ± 19.9 ng / mL for healthy subjects, 80.2 ± 35.8 ng / mL for group 1 diabetes, 224.6 ± 149.6 ng / mL for group 2 diabetic nephropathy, and group 3 In the case of diabetic nephropathy + renal insufficiency, the measured value was 337.8, 170.4 ng / mL, and in group 4 diabetic nephropathy + dialysis, it was 590.7 ± 267.6 ng / mL, and the measured value clearly increased as the disease progressed. The result was obtained.
産業上の利用の可能性  Industrial potential
[0043] 本発明によれば、血清などの循環液中のエラスチン分解物の量を測定することによ り、特別な装置を用いることなぐ迅速で簡便に、高い陽性率で糖尿病性腎症、急性 進行性糸球体腎炎、アミロイド一シスまたはコレステリン塞栓症と!/ヽつた全身性動脈 硬化症、好ましくは糖尿病性腎症と判定することが可能になり、また糖尿病性腎症に つ!、ては重症度を判定することが可能となる。 According to the present invention, by measuring the amount of elastin hydrolyzate in a circulating fluid such as serum, diabetic nephropathy can be obtained quickly and simply without using a special device, with a high positive rate, acute With advanced glomerulonephritis, amyloidosis or cholesterol embolism! / Patients with systemic arteriosclerosis, preferably diabetic nephropathy, and diabetic nephropathy! Can determine the severity.

Claims

請求の範囲 The scope of the claims
[I] 被験者力 採取した循環液中のエラスチン分解物の量を測定し、その測定値に基づ Vヽて全身性動脈硬化症を判定することを含む、全身性動脈硬化症の判定方法。  [I] Subject power A method for determining systemic arteriosclerosis, comprising measuring the amount of elastin hydrolyzate in a collected circulating fluid, and determining systemic arteriosclerosis based on the measured value.
[2] 全身性動脈硬化症が糖尿病性腎症、急性進行性糸球体腎炎、アミロイド一シスまた はコレステリン塞栓症である請求項 1に記載の判定方法。  [2] The method according to claim 1, wherein the systemic arteriosclerosis is diabetic nephropathy, acute progressive glomerulonephritis, amyloidosis or cholesterol embolism.
[3] 全身性動脈硬化症が糖尿病性腎症である請求項 1に記載の判定方法。  [3] The method according to claim 1, wherein the systemic arteriosclerosis is diabetic nephropathy.
[4] 糖尿病性腎症、急性進行性糸球体腎炎、アミロイド一シスまたはコレステリン塞栓症 の判定が、糖尿病性腎症を発症している力否かの判定である、請求項 2に記載の方 法。  [4] The method according to claim 2, wherein the determination of diabetic nephropathy, acute progressive glomerulonephritis, amyloidosis or cholesterol embolism is a determination of whether or not diabetic nephropathy is developing. Method.
[5] 糖尿病性腎症の判定が、糖尿病性腎症の重症度の判定である、請求項 3に記載の 方法。  5. The method according to claim 3, wherein the determination of diabetic nephropathy is a determination of the severity of diabetic nephropathy.
[6] エラスチン分解物に対する抗体を用いる免疫化学的方法によりエラスチン分解物の 量を測定する、請求項 1〜5のいずれか一項に記載の方法。  [6] The method according to any one of claims 1 to 5, wherein the amount of the elastin degradation product is measured by an immunochemical method using an antibody against the elastin degradation product.
[7] 前記抗体がモノクローナル抗体である、請求項 6に記載の方法。 [7] The method according to claim 6, wherein the antibody is a monoclonal antibody.
[8] 第 1及び第 2のモノクローナル抗体を用い、抗体の一方が固相に固定され、及び、も う一方が標識物質により標識されている請求項 7に記載の方法。 [8] The method according to claim 7, wherein the first and second monoclonal antibodies are used, one of the antibodies is immobilized on a solid phase, and the other is labeled with a labeling substance.
[9] モノクローナル抗体力 HASG-2 (FERM BP- 08488)、 HASG- 30 (FERM BP- 08489)、[9] Monoclonal antibody potency HASG-2 (FERM BP-08488), HASG-30 (FERM BP-08489),
HASG-61-1 (FERM BP-08490)力 選ばれるハイプリドーマにより産生される請求項HASG-61-1 (FERM BP-08490) Claims Produced by selected hybridomas
8に記載の方法。 8. The method according to 8.
[10] 前記第 1及び第 2のモノクローナル抗体力 HASG- 30及び HASG- 61-1のハイブリド 一マにより産生されるモノクローナル抗体の組み合わせである請求項 9に記載の方 法。  [10] The method according to claim 9, wherein the first and second monoclonal antibodies are a combination of monoclonal antibodies produced by a hybrid of HASG-30 and HASG-61-1.
[II] エラスチン分解物に対する抗体の 1種又は 2種以上を含み、被験者の循環液中のェ ラスチン分解物の量を測定し、その測定値に基づ!、て糖尿病性腎症を判定するため の、全身性動脈硬化症の判定キット。  [II] Determine the diabetic nephropathy based on the amount of elastin degradation product in the circulating fluid of the subject, which contains one or more antibodies against elastin degradation product, and based on the measured value! For determining systemic arteriosclerosis.
[12] 全身性動脈硬化症が糖尿病性腎症、急性進行性糸球体腎炎、アミロイド一シスまた はコレステリン塞栓症である請求項 11に記載の判定キット。  12. The determination kit according to claim 11, wherein the systemic arteriosclerosis is diabetic nephropathy, acute progressive glomerulonephritis, amyloidosis or cholesterol embolism.
[13] 全身性動脈硬化症が糖尿病性腎症である請求項 11に記載の判定キット。  13. The determination kit according to claim 11, wherein the systemic arteriosclerosis is diabetic nephropathy.
PCT/JP2005/008070 2004-05-06 2005-04-27 Method of judging systemic arteriosclerosis and judgement reagent WO2005108985A1 (en)

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Citations (2)

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Publication number Priority date Publication date Assignee Title
JP2002537775A (en) * 1999-02-26 2002-11-12 ハイバージェン・リミテッド Identification of a gene having a role in presenting diabetic nephropathy
JP2002350439A (en) * 2001-05-30 2002-12-04 Eisai Co Ltd Measuring method and measuring kit of elastin decomposition product and detection method and detection kit of aortic dissection

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002537775A (en) * 1999-02-26 2002-11-12 ハイバージェン・リミテッド Identification of a gene having a role in presenting diabetic nephropathy
JP2002350439A (en) * 2001-05-30 2002-12-04 Eisai Co Ltd Measuring method and measuring kit of elastin decomposition product and detection method and detection kit of aortic dissection

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