WO1997008549A1 - Method for detecting kidney diseases, diagnostic drug therefor, and diagnostic kit therefor - Google Patents

Method for detecting kidney diseases, diagnostic drug therefor, and diagnostic kit therefor Download PDF

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Publication number
WO1997008549A1
WO1997008549A1 PCT/JP1996/002461 JP9602461W WO9708549A1 WO 1997008549 A1 WO1997008549 A1 WO 1997008549A1 JP 9602461 W JP9602461 W JP 9602461W WO 9708549 A1 WO9708549 A1 WO 9708549A1
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Prior art keywords
integrin
antibody
renal disease
urine
complex
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PCT/JP1996/002461
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French (fr)
Japanese (ja)
Inventor
Naomasa Yamamoto
Kenjiro Tanoue
Koichi Utsumi
Hiroshi Saito
Yasuhiro Katagiri
Masaharu Kotani
Shuichi Miyaura
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Seikagaku Corporation
The Tokyo Metropolitan Institute Of Medical Science
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Priority to JP51013097A priority Critical patent/JP3870242B2/en
Publication of WO1997008549A1 publication Critical patent/WO1997008549A1/en

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893Chemical 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

Definitions

  • the present invention relates to a method for detecting a renal disease, a diagnostic agent and a diagnostic kit, and more particularly, to a method for measuring a renal disease by measuring integrin ⁇ 3 in a body fluid. It relates to the method of detecting BACKGROUND ART
  • diagnosis has been made based on examination of leakage of blood components (cells such as red blood cells and white blood cells and plasma proteins) into urine by urine sediment, urine protein and the like.
  • urine / 3 2 - concentration microglobulin (microglobulin) and N- ⁇ cetyl glucosaminoglycans NIDA one peptidase (NAG) is to show the extent of injury of tubular.
  • the activated complement system is thought to deposit on glomerular epithelial cells and damage the glomerulus, resulting in urinary complement. Detection of a component or measurement of its activity has been referred to.
  • the activated complement system binds to vitronectin (VN), a plasma protein that inactivates it, and forms a VNZCb5-9 complex to be excreted in urine. Measuring vitronectin has also been one method of diagnosing renal disease.
  • Enzymim Assay A method is known in which a sample urine is added to a plate for EIA), and then the antibody is added to measure normal ⁇ tissue antigen in the urine to diagnose renal injury (JP-A-63-112994).
  • integrin is an adhesion molecule on the surface of a cell membrane in which a complex of one subunit and three subunits forms a one-to-one complex
  • various types of molecules are known from combinations of different subunits and three subunits.
  • ⁇ subunit against the / 3 3 Sabuyunitto £ ⁇ and 0: 1 of 2 types are known Till.
  • I i b 3 3 Complex known to be a major integrin present on platelets, alpha [nu 3 3 double coalescence is known to be a receptor for vitronectin, also a the tissue by connexion yarn ball body such as immunostaining It has been reported that the vy S 3 complex is frequently found (N mark hron, 68 (1994) p. 87-96). It is known that the urine of patients with renal disease excretes platelets and cells from the kidney, such as glomerular cells, but integrin 3 is present in the body fluids of patients with renal disease, specifically, urine. Its presence and the association between bodily fluids, specifically urinary integrins, and renal disease have not been studied.
  • the present invention has been made in view of the above, and is a method for easily detecting the presence or absence of renal disease, particularly glomerular injury, or the degree of renal tissue damage. It is an object to provide a diagnostic agent and a diagnostic kit.
  • the present inventors have found extensive research result, the body fluid of a patient having renal disease, the presence of integrin / 8 3 More specifically urine, and more specifically in the urine sediment in order to solve the above problems and, finding a way to measure this integrin 3 3 easily and accurately, dull to the present invention.
  • detection method preferably urine, more preferably renal disease and measuring the integrin yS 3 in urine sediment,
  • a solid phase body to which an antibody against 3 integrin) 3 3 is fixed a kit for diagnosing renal disease comprising the antibody labeled with or capable of being labeled with a labeling substance
  • antibodies against I integrin yS 3 (integrin ⁇ 3 and antibodies specifically reactive, hereinafter referred to as "anti-integrin / 3 3 antibody” ) By an antigen-antibody reaction.
  • a specific method for measuring the integrin / 3 3 by antigen-antibody reaction for example, subjected to electrophoresis Solubilized urine sediment as a specimen, followed by transferring the analyte after electrophoresis to a membrane, anti-integrin on the film; S 3 antibody was added, integrase phosphorus by antigen-antibody reaction; and measuring the S 3, the integrin urinary sediment was solubilized, anti integrin Glynn; is reacted with S 3 antibody, antibody has one integrin / 3 3 - antibody and a method of measuring the integrin y3 3 by detect the formation of the complex, but is not limited thereto.
  • a kidney disease refers to a disease associated with damage to kidney glomeruli and the like, such as nephritis and nephropathy (nephrosis).
  • Such renal diseases are known to be caused by various causes, such as those caused by binding or deposition of immune complexes. Specifically, for example, systemic lupus erythematosus (SLE), IgA ⁇ disease , Diabetic nephropathy, membranous nephropathy, hypertensive renal sclerosis, membranous proliferative nephritis, acute glomerulonephritis, focal glomerulosclerosis, purpura nephritis and the like.
  • SLE systemic lupus erythematosus
  • IgA ⁇ disease Diabetic nephropathy, membranous nephropathy, hypertensive renal sclerosis, membranous proliferative nephritis, acute glomerul
  • Integrin S 3 of the present invention of the integrin; molecule having a 3 3 Sabuyunitto (alpha [nu S 3 complex, nb / 8 3 complex, etc.),; as 3 sub Buyunitto 3 3 Sabuyunitto itself, And fragments thereof.
  • Integrin / 93 is excreted in the urine of patients with renal disease, but is not present in the urine in a soluble form such as urine protein, but is excreted in the urine. It is thought that it exists in a form bound to fragments. That is, the integrin / 3 3 to be discharged into diuresis by the injury of glomeruli like kidney, measured as injury marker ⁇ tissue Thereby, renal disease can be detected. This detection of renal disease includes the determination of the presence or absence and degree of renal disease, as well as prognostic determination such as evaluation of therapeutic effects.
  • Anti-integrin used in the present invention the 9 3 antibody, / 3 3 subunits, three sub Buyuni' integrin molecule comprising Bok ( ⁇ complex Ya ⁇ composite) or antibodies such as recognizing and these fragments Can be mentioned.
  • alpha [nu / 3 3 complex (glomerular origin) of ⁇ 3 Sabuyunitto and a I Ib; S 3 complex (derived platelets) of; 6 3 follows the line if you wish to measure and differentiate between Sabuyunitto Just do it. That a, lb; 8 3 a l lb Sabuyuni' Bok specifically reactive with monochromatic one monoclonal antibody use the t, was a by antigen-antibody reaction complex (platelet-derived),! »Sabuyunitto amount measured to determine the 3 3 Sabuyunitto amount of platelet-derived. Then by subtracting the / 3 3 Sabuyunitto amount from platelet from all / 3 3 Sabuyunitto amount can be determined integrin> 3 3 content derived from glomeruli.
  • the method for detecting a renal disease of the present invention uses a body fluid, preferably urine, more preferably a urine sediment or a solubilized urine sediment (hereinafter, unless otherwise specified, collectively referred to as “urine sediment”) as a specimen, It is performed by measuring 9 3; integrin in the sample.
  • a body fluid preferably urine, more preferably a urine sediment or a solubilized urine sediment (hereinafter, unless otherwise specified, collectively referred to as “urine sediment”) as a specimen, It is performed by measuring 9 3; integrin in the sample.
  • a method for measuring integrin ⁇ 3 in urine sediment there is a method in which an anti-integrin;
  • Anti-integrin used in the present invention the S 3 antibody, modified / 3 3 Sabuyunitto, a v yS 3 antibody has high reactivity with complex or a iib S 3 complexes are preferred Te cowpea in unmodified or solubilized .
  • anti-integrin 83 antibodies for example, Ant i -Human Integrin ⁇ (clone number VNR3, VNR5, Takara Shuzo Co., Ltd.) can be used such as , prepared monoclonal antibody of the present inventors (2 J 40 antibody, see example), even in the native / 3 3 sub Yunitto, also modified with sodium dodecyl sulfate (SDS) / 3 3 Sabuyuni Tsu DOO Is particularly preferred because of its high reactivity.
  • SDS sodium dodecyl sulfate
  • anti-integrin 5 3 antibodies can be also child prepared as below, for example according to a conventional method.
  • Purified 3 3 Sabuyunitto used as an antigen it is possible to obtain in a known manner.
  • normal human platelets are solubilized and converted to a peptide having the amino acid sequence of Arg-Gly-Asp or a peptide having the amino acid sequence of Gly-Arg-Gly-Asp-Ser_Pro-Lys (SEQ ID NO: 1).
  • Poly Skr Norre anti-integrin S 3 antibodies such as mice, rats, guinea pigs, Usagi, catcher formic
  • the animals to be immunized such as Hijji immunized with the above antigens may be obtained by removing a serum from these animals . It is preferable to use an auxiliary agent (adjuvant) in immunizing the animal to be immunized, since it activates antibody-producing cells. From the obtained antiserum, the immunoglobulin fraction may be purified by a conventional method.
  • a monoclonal anti-integrin antibody can be obtained, for example, as follows. That is, the antigen is administered intraperitoneally, subcutaneously, or to the footpad of an animal to be immunized, such as a mouse, rat, guinea pig, rabbit, goat, or sheep, and then the spleen or popliteal lymph node is removed. The cells collected from these cells are fused with myeloma cells, which are tumor cell lines, to establish hybridomas. The resulting hybridomas are continuously grown, and the specific antibodies against the above antigens are continuously obtained from the obtained hybridomas. Select the cell line that will produce. By culturing the selected strain in a suitable medium, a monoclonal anti-integrin antibody can be obtained in the medium. Alternatively, by culturing the High Priestess dormer in vivo, such as mouse peritoneal cavity it can be mass production of monoclonal anti Integurin [delta] 3 antibody.
  • Cells used for cell fusion include lymph node cells and peripheral blood Lymphocytes and the like can be used. Further, the myeloma cell line is preferably derived from the same cell line as compared to that derived from a heterologous cell type, and a stable antibody-producing hybridoma can be obtained.
  • Methods for purifying the obtained polyclonal and monoclonal antibodies include salting out with sodium sulfate, ammonium sulfate, etc., low-temperature alcohol precipitation, selective precipitation fractionation using polyethylene glycol or isoelectric point, electrophoresis, DEAE Ion-exchange chromatography using an ion exchanger such as tylaminoethyl) mono-derivative and CM (carboxymethyl) mono-derivative; affinity chromatography using protein A or protein G; Examples include tight chromatography, immunosorbent chromatography in which an antigen is immobilized, gel filtration, ultracentrifugation, and the like.
  • anti-integrin; S 3 antibodies can be used as it is, it is also possible to use those obtained by Furagume cement of. Preservation of the antibody's antigen binding site (F ab) during antibody fragmentation is essential for binding the antigen to the antibody, so proteases that do not degrade the antigen binding site (eg, plasmin, pepsin, papain) Etc.), a fragment containing Fab obtained by treating the antibody can also be used.
  • fragment preparative Ya chimeric antibodies e.g., anti Integurin including genetic engineering F ab; 8 3 antibody F chimeric antibodies containing an ab portion
  • fragments and chimeric antibodies can also be used in the present invention.
  • anti-integrin 3 3 antibody forms of the antigen-antibody reaction using the anti Integurin / 9 3 antibody as above is not particularly limited, the measurement system used for ordinary Imunoassi (immunoassays) can be applied to the present invention regardless of the type.
  • the measurement system include an immunoblotting method (for example, a Western plotting method), a labeled immunoassay method (for example, an EIA method, an enzyme-linked immunosorbent assay (ELISA method), and a radioimmunoassay). Assay method, fluorescence immunoassay method, etc.), flow cytometry method and the like. It is.
  • These measurement methods are known methods [S. Irie, edited by "Radio Simnoassy",
  • electrophoresis is performed using a body fluid, preferably urine, more preferably a solubilized urine sediment as a sample, and then the sample after electrophoresis is transferred to a membrane.
  • the anti-integrin 3 antibody is added, can be done by measuring the integrin) S 3 by antigen-antibody reaction.
  • the electrophoresis is preferably performed by SDS-polyacrylamide gel electrophoresis.
  • the conditions for SDS-polyacrylamide electrophoresis and the conditions for transfer to a membrane can be appropriately determined by those skilled in the art according to the known conditions used for the Western blotting method.
  • the membrane may be a polyvinylidene difluoride (PVDF) membrane, a nitrocellulose membrane, a nylon membrane, or the like.
  • PVDF polyvinylidene difluoride
  • Measurement of the antigen-antibody reaction in the Western blotting technique leave labeled in advance with a labeling substance anti integrase phosphorus 3 3 antibodies, and Inkyubeto the labeled antibody and the membrane, the labeled antibody bound to the integrin / [delta] 3 of the H ⁇
  • the detection can be performed by detecting the labeling substance by a method suitable for the labeling substance.
  • anti-integrin unlabeled; and S 3 Inkyubeto antibody the membrane and, Integurin; 5 3 anti Integurin; after forming the conjugates of S 3 antibody, anti-integrin; be used in the preparation of 3 3 antibody
  • a labeled secondary antibody obtained by labeling an antibody against immunoglobulin of an immunized animal (secondary antibody) with a labeling substance is bound to the conjugate, and the labeling substance in the secondary antibody bound to the conjugate is detected.
  • the antigen-antibody reaction can be measured. Is the anti-integrin 5 3 antibody was modified with the modifying agent (eg SDS, etc.); 3 3 Sabuyuni' antibody is highly reactive to Bok is preferred.
  • Labeling immunoassay for example Sanditsuchi EI Alpha method, a body fluid, preferably urine, is Ri preferably good integrin urine sediment was solubilized; and 9 3 is reacted with anti-integrin 3 3 antibodies, sandwich complex , i.e. anti-integrin; 8 3 antibody has one Integrated Darrin; 9 3 - can and this carried out by detecting the formation of a complex of anti-integrin / 3 3 antibody.
  • the complex is formed by using a labeled antibody as one of the antibodies forming the complex and using an antibody capable of binding or binding to the solid phase on the other side, and using the labeled substance in the complex as the labeled substance. Can be detected by the method described above.
  • the labeled secondary antibody is bound to the complex, and the labeling substance of the labeled secondary antibody bound to the complex is measured to form the complex. Can be detected. More specifically, examples of the sandwich EIA method include the following methods, but are not limited thereto.
  • the solid phase body examples include a microtiter plate (imnoplate), a ball of polystyrene, latex particles, a test tube wall, and a metal colloid.
  • a method for immobilizing an antibody on these solid phase bodies include a physical adsorption method and a covalent binding method. 75, published by Kodansha, pp. 9-75).
  • the physical adsorption method is preferred because it is simple and widely used as a method for fixing an antibody to a solid phase.
  • the portion to which the antibody is not bound may be blocked by serum albumin, gelatin, milk protein, or the like.
  • the solid phase and the liquid phase are separated, and the integrin in the specimen sample can also be measured by measuring the labeled substance in the liquid phase. it can.
  • the integrin / S 3 labeled with a labeling substance, anti-integrin was immobilized on a solid phase with a test sample; mixed with 9 3 antibody to measure the amount of labeled substance bound to the immobilized antibody, so-called competitive method as well, integrin specimen sample; it is possible to measure the 3 3.
  • the antigen-antibody reaction is not limited to the solid phase method as described above, and may be performed by a so-called liquid phase method without using an immobilized antibody. That is, anti-integrin; mixing 3 3 antibody and the test sample, integrin Glynn yS 3 was further added a labeled secondary antibody - anti-integrin; to precipitate the complex of 9 3 antibody has one labeled secondary antibody, the precipitation This is a method for detecting the contained label.
  • the labeling substance used for labeling the antibody enzyme (Peruokishidaze, alkaline phosphatase, ⁇ , such as single-galactosidase), a radioactive isotope (1 25 1, 1 3 1 I, 3H , etc.), fluorescent substances (Full O receptacle in Isothiocarbazate Xia And chemiluminescent substances (such as noreminol), and other substances (such as biotin and avidin).
  • enzyme Peruokishidaze, alkaline phosphatase, ⁇ , such as single-galactosidase
  • a radioactive isotope (1 25 1, 1 3 1 I, 3H , etc.
  • fluorescent substances Flul O receptacle in Isothiocarbazate Xia And chemiluminescent substances (such as noreminol)
  • other substances such as biotin and avidin.
  • the method of labeling the antibody may be a known method suitable for the labeling substance, for example, when labeling an enzyme.
  • the glutaraldehyde method the periodic acid crosslinking method, the maleimide crosslinking method, the carpoimide method, and the like.
  • the chloramine T method the lactoperoxidase method, etc. (Seismic Chemistry Laboratory Course 5 Immunobiochemistry Research method, Tokyo Kagaku Dojin, published in 1986).
  • the urinary sediment is not particularly limited as long as it is insoluble in urine.
  • insoluble proteins, cells, cell fragments, etc. in urine are separated by centrifugation or ultrafiltration. What was concentrated by a membrane etc. is mentioned.
  • centrifugal separation is simple and therefore preferable.
  • the centrifugation conditions include, for example, the conditions of centrifugation at 400 X g or more for 5 minutes or more.
  • a urinary sediment is solubilized by adding a surfactant, a denaturant (solubiliser) such as guanidine hydrochloride or urea that denatures proteins.
  • the denaturant (solubiliser) is preferably a surfactant, and more preferably an ionic surfactant.
  • an alkyl sulfate such as sodium dodecyl sulfate (SDS) is used among ionic surfactants, its concentration is, for example, about 0.1 to 5% based on urine sediment.
  • a urine sediment solubilized by adding a surfactant or the like is usually used as a specimen sample.
  • the type and concentration of the surfactant can be appropriately selected within a range that does not affect the antigen-antibody reaction.
  • surfactants include polyoxetylene alkylphenyl ethers (Triton-based surfactants) (Triton X-100, etc.), polyoxyethylene sorbitan alkyl esters (Tween-based surfactants) c body fluid non-ionic surfactant such as agent) and the like, preferably urine, more preferably in the urinary sediment integrin / 3 3 of quantification, using a known amount integrin) 3 3 as a standard substance
  • the calibration can be performed by preparing a calibration curve based on the relationship between the concentration of the standard substance and the amount of the detected label, and comparing with the calibration curve. In the competition method, a certain amount of standard substance is added to the antigen-antibody reaction system.
  • integrin s 3 With unlabeled integrin s 3 of a known amount is added, the relationship between the amount of label is detected to a standard concentration Nitsu L, Te it is sufficient to create a calibration curve.
  • anti-integrin by measuring the integrin in body fluids by using a 3 3 antibody, it is possible to perform detection of renal disease. That is, anti-integrin Glynn; 9 3 antibodies can be used as a diagnostic agent for renal diseases.
  • a diagnostic kit may be added by adding substances, solubilizing agents (denaturing agents) that do not affect the antigen-antibody reaction. Solid-phase body may be allowed to secure the advance anti-integrin / 9 3 antibody, and favored arbitrary. By using such a kit, integrin 3 can be easily measured.
  • the affinity column is a CNBr-activated Sepharose (manufactured by Pharmacia, Inc.) to which the Gly-Arg-Gly-Asp_Ser-Pro-Lys (SEQ ID NO: 1) peptide is used as a ligand, and this peptide is bound in a conventional manner. A column packed with)) was used.
  • This complex was identified as two nodes by silver staining after SDS-polyacrylamide gel electrophoresis. Obtained a l lb / S 3 complex in SDS- polyacrylamide gel electrophoresis, alpha ,, b Sabuyunitto and; fractionated separated fraction into a 8 3 Sabuyunitto was isolated yS 3 Sabuyunitto.
  • a solution containing promophenol blue hereinafter, abbreviated as “solubilizing solution”) 400 ⁇ 1 was added and dissolved (solubilized), and heated at 100 for 5 minutes.
  • the obtained urine sediment sample was stored at 20 ⁇ until measurement, and dissolved and used at the time of measurement.
  • SDS-polyacrylamide gel electrophoresis was performed with 20 urine sediment samples per lane.
  • the electrophoresis was performed using Resep Ge 1 (5-20% gradient) (manufactured by Washimori Mori Co., Ltd.) at 30 mA for about 1 hour until the bromophenol buffer reached the tip of the gel.
  • proteins were transferred from the gel after electrophoresis using Immobilon TM (PVDF membrane, Millipore ⁇ ⁇ ) according to a conventional method.
  • Monoclonal antibody 2J40 (1: 400 dilution) was added to the membrane after the transfer, and incubated at room temperature for 1 hour. Washing was performed twice with PBS—0.05% Tween 20 for 5 minutes and then for 10 minutes.
  • HRP horseradish peroxidase
  • the above-mentioned healthy subjects and patients with renal disease were 23 healthy subjects and 48 patients with various renal diseases.
  • the breakdown of the disease was 19 cases of systemic lupus erythematosus (SLE) (6 cases of active (acute), Active (chronic) 13 cases), Ig A nephropathy 10 cases, diabetic nephropathy (DM) 6 cases, minimal change nephropathy 6 cases, membranous nephropathy (MN) 4 cases, membranous proliferative
  • SLE systemic lupus erythematosus
  • DM diabetic nephropathy
  • MN membranous nephropathy
  • MPGN membranous nephropathy
  • HTN hypertensive renal sclerosis
  • the healthy subjects were 13 males and 10 females, aged between 22 and 59 years.
  • Patients with various renal diseases included 20 males and 28 females, ages 23-68.
  • Table 1 shows the results.
  • Table 1 Urinary integrin; 3 3 of excretion
  • integrin S3 was hardly excreted in urine in healthy subjects.
  • quantitative results of integrin ⁇ 3 showed that its S output varied depending on the type of kidney disease, and also varied depending on the activity in the same kidney disease group. That is, the amount of urinary integran ⁇ 3 was much higher in active SLE than in inactive 3 £ compared to 3 inactive. Also, slight changes in which no histopathological glomerular injury was observed in nephropathy, integrin from urinary sediment; 3 3 was detected.
  • P 2 antibody as a comparative example ( "M b / S 3 A monoclonal antibody against the complex, Cosmo Bio) was added to each, and a FITC-labeled secondary antibody was further added.
  • Flow cytometry was performed using a FACS can (BECTON DICKINSON) according to a conventional method. It was. As a result, 2 J 40 antibodies and cells, etc. of the urine sediment for LM609 antibody stained positively, although integrin / 3 3 was detected, there was negative for P2 antibodies. platelet derived alpha, , b 3 3 complexes were not detected.
  • eXAMPLE 3 anti-integrin was prepared by the method described in preparation> 3 3 antibody (2 J 40 antibodies) phosphate buffered saline (PH7.2 ⁇ 7.5 , Without divalent ion) (hereinafter referred to as PBS (—) ), Add 50 1 (lg / ⁇ ) of this solution to each well of Imnoplate (trade name: Maxisorp, manufactured by Nunc), and store at 4 for 16 hours. Coated evenly.
  • the plate is then washed twice with PBS (-), and 3% Pseudoserum albumin (BSA) (BSA) is used as a blocking substance to cover the parts of the wells that are not coated with anti-integrin / 333 antibody. (Industry Co., Ltd.) containing PBS (-) solution was added and left at room temperature for 2 hours.
  • PBS Pseudoserum albumin
  • a body fluid preferably urine, more preferably urine sediment, very preferably
  • solubilizers e.g., T Riton based or T ween surfactant
  • integrin e.g., T Riton based or T ween surfactant

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Abstract

Kidney diseases are detected by assaying integrin β3 in solubilized urinary sediment by the Western blotting technique, labeling immunoassay method, etc. with the use of an antibody against integrin β3.

Description

明細書 腎疾患の検知法、 診断薬及び診断用キット 技術分野 本発明は、 腎疾患の検知法、 診断薬及び診断用キットに関し、 詳しくは、 体液 中のインテグリン ^ 3を測定することにより腎疾患を検知する方法に関するもので あ 。 背景技術 腎疾患の検査法として、 尿沈渣、 尿蛋白等により血液成分 (赤血球、 白血球な どの細胞および血漿蛋白) の尿への漏出の検査を基に診断がなされてきた。 また、 尿中の /3 2-ミクログロブリン (microglobulin) と N—ァセチルグルコサミニダ一 ゼ (N A G ) の濃度は、 尿細管の傷害の程度を示すとされている。 TECHNICAL FIELD The present invention relates to a method for detecting a renal disease, a diagnostic agent and a diagnostic kit, and more particularly, to a method for measuring a renal disease by measuring integrin ^ 3 in a body fluid. It relates to the method of detecting BACKGROUND ART As a method for examining renal disease, diagnosis has been made based on examination of leakage of blood components (cells such as red blood cells and white blood cells and plasma proteins) into urine by urine sediment, urine protein and the like. Also, urine / 3 2 - concentration microglobulin (microglobulin) and N- § cetyl glucosaminoglycans NIDA one peptidase (NAG) is to show the extent of injury of tubular.
さらに、 全身性エリテマトーデス (S L E ) や膜性腎症においては、 活性化さ れた補体系が糸球体の上皮細胞に沈着し、 糸球体を損傷すると考えられているの で、 尿中の補体成分の検出、 あるいはその活性測定が参考とされてきた。 活性化 された補体系は、 これを不活性化する血漿蛋白であるビトロネクチン (V N) と 結合し、 V NZC b 5- 9複合体を形成して尿中に排出されることから、 尿中のビト ロネクチンを測定することも腎疾患の診断の 1つの方法とされてきた。  In addition, in systemic lupus erythematosus (SLE) and membranous nephropathy, the activated complement system is thought to deposit on glomerular epithelial cells and damage the glomerulus, resulting in urinary complement. Detection of a component or measurement of its activity has been referred to. The activated complement system binds to vitronectin (VN), a plasma protein that inactivates it, and forms a VNZCb5-9 complex to be excreted in urine. Measuring vitronectin has also been one method of diagnosing renal disease.
また、 ホモジナイズしたヒト正常腎組織を免疫原として作製した、 尿細管内腔 壁または糸球体および尿細管基底膜に特異的に反応するモノクローナル抗体を使 用して、 ェンザィムィムノアッセィ (E I A) 用プレートに検体尿を加え、 つい で該抗体を加えることで尿中の正常肾組織抗原を測定し、 腎傷害を診断する方法 が知られている (特開昭 63- 112994) 。  Also, using a monoclonal antibody that specifically reacts with the tubule lumen wall or glomerulus and tubule basement membrane prepared from homogenized normal human kidney tissue as an immunogen, Enzymim Assay ( A method is known in which a sample urine is added to a plate for EIA), and then the antibody is added to measure normal 肾 tissue antigen in the urine to diagnose renal injury (JP-A-63-112994).
ところで、 インテグリンはなサブュニッ卜と 3サブュニットとが 1対 1で複合 体を形成する細胞膜表面の接着分子であり、 異なる サブュニッ 卜と 3サブュニ ットとの組み合わせから多種の分子が知られている。 例えば、 /33サブュニットに 対する αサブユニッ トとしては、 £^と0:1 の2種類が知られてぃる。 な i I b 33 複合体は、 血小板に存在する主要なインテグリンであることが知られ、 α ν 33複 合体はビトロネクチンの受容体であることが知られ、 また免疫染色によつて糸球 体等の組織に a v yS 3複合体が多く見られることが報告されている (N印 hron, 68 (1994) p. 87-96) 。 なお、 腎疾患患者の尿に血小板の排出や糸球体細胞など腎由 来の細胞の排出がみられることは知られているが、 腎疾患患者の体液、 具体的に は、 尿にインテグリン 3が存在すること、 および体液、 具体的には、 尿中のイン テグリン と腎疾患との関連については検討されていない。 By the way, integrin is an adhesion molecule on the surface of a cell membrane in which a complex of one subunit and three subunits forms a one-to-one complex, and various types of molecules are known from combinations of different subunits and three subunits. For example, as the α subunit against the / 3 3 Sabuyunitto, £ ^ and 0: 1 of 2 types are known Till. I i b 3 3 Complex, known to be a major integrin present on platelets, alpha [nu 3 3 double coalescence is known to be a receptor for vitronectin, also a the tissue by connexion yarn ball body such as immunostaining It has been reported that the vy S 3 complex is frequently found (N mark hron, 68 (1994) p. 87-96). It is known that the urine of patients with renal disease excretes platelets and cells from the kidney, such as glomerular cells, but integrin 3 is present in the body fluids of patients with renal disease, specifically, urine. Its presence and the association between bodily fluids, specifically urinary integrins, and renal disease have not been studied.
従来の、 ビトロネクチンや補体成分のような血液成分の尿中での測定は、 腎全 体の傷害もしくは免疫反応の関与を示唆するものであるが、 腎組織内の傷害部位 を同定することは困難であった。 また上記の正常腎組織抗原を測定する方法では、 抗体を作製する際にヒト正常腎組織を使用するため、 抗原の入手が極めて困難で あること、 および E I A用プレー卜に固着される尿中の抗原量が一定でなく、 測 定結果が不正確である等の欠点を有していた。 さらに従来知られる方法や臨床検 査では、 腎疾患を詳細に確定するには不十分で、 糸球体損傷あるいは腎組織損傷 の確定診断を行うには腎の生検を行う必要があり、 患者に苦痛を与えるものであ つた o 発明の開示 本発明は、 上記観点からなされたものであり、 腎疾患、 特に糸球体の傷害の有 無あるいは腎組織損傷の程度を簡便に検知することができる方法、 診断薬および 診断キットを提供することを課題とする。  Conventional measurement of blood components, such as vitronectin and complement components, in urine suggests damage to the whole kidney or involvement of the immune response, but it is not possible to identify the site of damage in kidney tissue. It was difficult. In addition, in the above method for measuring normal kidney tissue antigen, it is extremely difficult to obtain the antigen because human normal kidney tissue is used when producing the antibody, and it is difficult to obtain the antigen in urine fixed to the EIA plate. There were disadvantages such as the amount of antigen was not constant and the measurement results were inaccurate. In addition, conventional methods and clinical tests are not sufficient to define kidney disease in detail, and a kidney biopsy must be performed to make a definitive diagnosis of glomerular damage or renal tissue damage. DISCLOSURE OF THE INVENTION The present invention has been made in view of the above, and is a method for easily detecting the presence or absence of renal disease, particularly glomerular injury, or the degree of renal tissue damage. It is an object to provide a diagnostic agent and a diagnostic kit.
本発明者らは、 上記課題を解決するために鋭意研究した結果、 腎疾患患者の体 液、 具体的には尿、 より具体的には尿沈渣中にインテグリン /8 3が存在することを 発見し、 このインテグリン 3 3を簡便かつ正確に測定する方法を見いだし、 本発明 に生った。 The present inventors have found extensive research result, the body fluid of a patient having renal disease, the presence of integrin / 8 3 More specifically urine, and more specifically in the urine sediment in order to solve the above problems and, finding a way to measure this integrin 3 3 easily and accurately, dull to the present invention.
すなわち本発明は、  That is, the present invention
1 )体液中、 好ましくは尿中、 より好ましくは尿沈渣中のインテグリン yS 3を測定 することを特徴とする肾疾患の検知法、 1) in body fluids, detection method preferably urine, more preferably renal disease and measuring the integrin yS 3 in urine sediment,
2 ) インテグリン ^ 3に対する抗体を含み、 体液中、 好ましくは尿中、 より好まし くは尿沈渣中のィンテグリン 5 3を測定して腎疾患を検知するための診断薬である ことを特徴とする腎疾患診断薬、 及び 2) Contains antibodies to integrin ^ 3 , and is more preferable in body fluids, preferably urine Ku renal disease diagnostic agent which is a diagnostic agent for detecting a renal disease by measuring Integurin 5 3 in the urine sediment, and
3 ) インテグリン) 3 3に対する抗体を固着させた固相体と、 標識物質で標識された、 又は標識されうる該抗体とを含む腎疾患診断用キット、 3) a solid phase body to which an antibody against 3 integrin) 3 3 is fixed, and a kit for diagnosing renal disease comprising the antibody labeled with or capable of being labeled with a labeling substance,
である。 It is.
前記腎疾患の検知法において、 体液中のインテグリン /9 3の測定法としては、 ィ ンテグリン yS 3に対する抗体(インテグリン ^ 3と特異的に反応する抗体、 以下、 「抗インテグリン /3 3抗体」 という) を用いた抗原抗体反応によって行う方法が挙 げられる。 In the detection method of the renal disease, as the method for measuring integrin / 9 3 in body fluids, antibodies against I integrin yS 3 (integrin ^ 3 and antibodies specifically reactive, hereinafter referred to as "anti-integrin / 3 3 antibody" ) By an antigen-antibody reaction.
抗原抗体反応によってインテグリン /3 3を測定する具体的方法としては、 例えば 可溶化した尿沈渣を検体として電気泳動を行い、 次いで電気泳動後の検体を膜に 転写し、 この膜に抗インテグリン; S 3抗体を加え、 抗原抗体反応によってインテグ リン; S 3を測定する方法や、 可溶化した尿沈渣中のインテグリン と、抗インテ グリン; S 3抗体とを反応させ、 抗体一インテグリン /3 3—抗体の複合体の形成を検 出することによりインテグリン y3 3を測定する方法等が挙げられるが、 これに限定 されるものではない。 As a specific method for measuring the integrin / 3 3 by antigen-antibody reaction, for example, subjected to electrophoresis Solubilized urine sediment as a specimen, followed by transferring the analyte after electrophoresis to a membrane, anti-integrin on the film; S 3 antibody was added, integrase phosphorus by antigen-antibody reaction; and measuring the S 3, the integrin urinary sediment was solubilized, anti integrin Glynn; is reacted with S 3 antibody, antibody has one integrin / 3 3 - antibody and a method of measuring the integrin y3 3 by detect the formation of the complex, but is not limited thereto.
本発明において腎疾患とは、 腎炎ゃ腎症 (ネフローゼ) など、 腎臓の糸球体等 の傷害を伴う疾患をいう。 このような腎疾患としては、 免疫複合体などの結合や 沈着によるものなど種々の原因による疾患が知られているが、 具体的には、 例え ば全身性エリテマトーデス (S L E )、 I g A肾症、 糖尿病性腎症、 膜性腎症、 高血圧性腎硬化症、 膜性増殖性腎炎、 急性糸球体腎炎、 巣状糸球体硬化症、 紫斑 病性腎炎などが挙げられる。  In the present invention, a kidney disease refers to a disease associated with damage to kidney glomeruli and the like, such as nephritis and nephropathy (nephrosis). Such renal diseases are known to be caused by various causes, such as those caused by binding or deposition of immune complexes. Specifically, for example, systemic lupus erythematosus (SLE), IgA 肾 disease , Diabetic nephropathy, membranous nephropathy, hypertensive renal sclerosis, membranous proliferative nephritis, acute glomerulonephritis, focal glomerulosclerosis, purpura nephritis and the like.
なお本発明におけるインテグリン S 3という用語には、 インテグリンのうち ;3サ ブュニットとして ;3 3サブュニットを有する分子 ( α ν S 3複合体、 n b /8 3複合体 等) 、 ;3 3サブュニット自体、 およびこれらの断片等を包含する。 インテグリン /9 3は、 腎疾患患者の尿中に排出されるが、 尿蛋白の如く可溶性の形態で尿中に存在 するのではなく、 尿中に排出される糸球体ゃ肾由来の細胞または細胞断片等に結 合した形態で存在するものと考えられる。 すなわち、 腎臓の糸球体等の傷害によ り尿中に排出されるインテグリン /3 3を、 肾臓組織の傷害マーカーとして測定する ことにより、 腎疾患を検知することができる。 この腎疾患の検知には、 腎疾患の 有無や程度の判定の他、 治療効果の評価等、 予後の判定も含まれる。 Note that the term integrin S 3 of the present invention, of the integrin; molecule having a 3 3 Sabuyunitto (alpha [nu S 3 complex, nb / 8 3 complex, etc.),; as 3 sub Buyunitto 3 3 Sabuyunitto itself, And fragments thereof. Integrin / 93 is excreted in the urine of patients with renal disease, but is not present in the urine in a soluble form such as urine protein, but is excreted in the urine. It is thought that it exists in a form bound to fragments. That is, the integrin / 3 3 to be discharged into diuresis by the injury of glomeruli like kidney, measured as injury marker肾臓tissue Thereby, renal disease can be detected. This detection of renal disease includes the determination of the presence or absence and degree of renal disease, as well as prognostic determination such as evaluation of therapeutic effects.
本発明に使用される抗インテグリン; 93抗体としては、 /33サブユニット、 3サ ブュニッ卜を含むインテグリン分子( ^複合体ゃ ^複合体等) または これらの断片などを認識する抗体等を挙げることができる。 Anti-integrin used in the present invention; the 9 3 antibody, / 3 3 subunits, three sub Buyuni' integrin molecule comprising Bok (^ complex Ya ^ composite) or antibodies such as recognizing and these fragments Can be mentioned.
なお、 αν/33複合体 (糸球体由来) の^ 3サブュニットと aI Ib;S3複合体 (血小 板由来) の; 63サブュニットとを区別して測定したい場合は次の通り行えばよい。 すなわち a ,lb;83複合体 (血小板由来) の al lbサブュニッ卜に特異的に反応する モノクロ一ナル抗体を用 t、た抗原抗体反応によって a ,! »サブュニット量を測定し、 血小板由来の 33サブュニット量を求める。 次いで全 /33サブュニット量から血小 板由来の /33サブュニット量を差し引くことで、 糸球体由来のインテグリン >33量 を求めることができる。 Incidentally, alpha [nu / 3 3 complex (glomerular origin) of ^ 3 Sabuyunitto and a I Ib; S 3 complex (derived platelets) of; 6 3 follows the line if you wish to measure and differentiate between Sabuyunitto Just do it. That a, lb; 8 3 a l lb Sabuyuni' Bok specifically reactive with monochromatic one monoclonal antibody use the t, was a by antigen-antibody reaction complex (platelet-derived),! »Sabuyunitto amount measured to determine the 3 3 Sabuyunitto amount of platelet-derived. Then by subtracting the / 3 3 Sabuyunitto amount from platelet from all / 3 3 Sabuyunitto amount can be determined integrin> 3 3 content derived from glomeruli.
また、 同様に /S 3複合体を認識する抗体を用いて糸球体由来のインテグリン /33量を測定し、 次いで全 /33サブュニット量から糸球体由来のインテグリン /33量 を差し引くことで、 血小板由来のインテグリン /83量を求めることができる。 Moreover, using an antibody that recognizes the same manner as / S 3 complex measures integrin / 3 3 content derived from glomeruli and then by subtracting the integrin / 3 3 content derived from glomeruli from all / 3 3 Sabuyunitto amount , it can be determined integrin / 8 3 amount of platelet-derived.
以下に、 本発明を詳細に説明する。  Hereinafter, the present invention will be described in detail.
本発明の腎疾患の検知法は、 体液、 好ましくは尿、 より好ましくは尿沈渣また は可溶化した尿沈渣(以下、 特に指定しないかぎり両者を合わせて 「尿沈渣」 と いう) を検体とし、検体中のインテグリン;93を測定することにより行われる。 尿 沈渣中のィンテグリン β 3を測定する方法としては、 抗インテグリン; 33抗体を用 I、た抗原抗体反応によって行う方法が挙げられる。 The method for detecting a renal disease of the present invention uses a body fluid, preferably urine, more preferably a urine sediment or a solubilized urine sediment (hereinafter, unless otherwise specified, collectively referred to as “urine sediment”) as a specimen, It is performed by measuring 9 3; integrin in the sample. As a method for measuring integrin β 3 in urine sediment, there is a method in which an anti-integrin;
本発明に用いる抗インテグリン; S 3抗体としては、 未変性もしくは可溶化によつ て変性した /33サブュニット、 avyS3複合体または aiib S3複合体と反応性が高い 抗体が好ましい。 抗インテグリン; S 3抗体として、 例えば市販のウェスタンブロッ ティング用の抗インテグリン ;83抗体、 例えば Ant i -Human Integrin βζ (クロー ン番号 VNR3、 VNR5、 宝酒造 (株) 製) 等を用いることができるが、 本発明者らの 作製したモノクローナル抗体 (2 J 40抗体、 実施例参照) は、 未変性の /33サブ ュニットにも、 またドデシル硫酸ナトリウム (SDS) で変性された /33サブュニ ットにも反応性が高いので特に好ましい。 2 J 40抗体を産生するハイプリ ドー マ株 RINSHOKEN-2J40は、 平成 7年 8月 2 9日に通商産業省工業技術院生命工学ェ 業技術研究所 (郵便番号 3 0 5 日本国茨城県つくば市東一丁目 1番 3号) に F E RM P - 1 5 1 3 9の受託番号で寄託され、 平成 8年 8月 7日にブダぺスト条 約に基づく国際寄託に移管されて、 F E RM B P— 5 6 1 9の受託番号で寄託 されている。 Anti-integrin used in the present invention; the S 3 antibody, modified / 3 3 Sabuyunitto, a v yS 3 antibody has high reactivity with complex or a iib S 3 complexes are preferred Te cowpea in unmodified or solubilized . As S 3 antibodies, such as the commercially available anti-integrin for Western blotting; anti-integrin 83 antibodies, for example, Ant i -Human Integrin βζ (clone number VNR3, VNR5, Takara Shuzo Co., Ltd.) can be used such as , prepared monoclonal antibody of the present inventors (2 J 40 antibody, see example), even in the native / 3 3 sub Yunitto, also modified with sodium dodecyl sulfate (SDS) / 3 3 Sabuyuni Tsu DOO Is particularly preferred because of its high reactivity. Hybrids that produce 2 J40 antibodies The RINSHOKEN-2J40 was established on August 29, 1995 by the Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology, Institute of Biotechnology and Industrial Technology (zip code: 30-5, 1-3 1-3 Higashi, Tsukuba, Ibaraki, Japan). Deposited with accession number RM P-1515, transferred to international deposit under the Budapest Agreement on August 7, 1996 and deposited under accession number FE RM BP—56 19 Have been.
また、 抗インテグリン )5 3抗体は、 常法に従って例えば以下のように作製するこ とも可能である。 Moreover, anti-integrin) 5 3 antibodies can be also child prepared as below, for example according to a conventional method.
抗原として用いる精製された) 3 3サブュニットは、 公知の方法で得ることができ る。 例えば、 正常ヒト血小板を可溶化して、 これを Arg- Gly-Aspのアミノ酸配列を 有するペプチドまたは Gly-Arg-Gly-Asp-Ser_Pro-Lys (配列番号 1 ) のアミノ酸配 列を有するぺプチド等をリガンドとするァフィ二ティー クロマトグラフィ一によ りな, ' b /3 3複合体を精製し、 ついで S D S—ポリアクリルアミ ドゲル電気泳動を 行って、 ;3 3サブュニットを分離精製する方法等が挙げられる。 Purified) 3 3 Sabuyunitto used as an antigen, it is possible to obtain in a known manner. For example, normal human platelets are solubilized and converted to a peptide having the amino acid sequence of Arg-Gly-Asp or a peptide having the amino acid sequence of Gly-Arg-Gly-Asp-Ser_Pro-Lys (SEQ ID NO: 1). the Rina by the Afi two tea chromatography one the ligand, purified the 'b / 3 3 complex, carried out followed SDS- polyacrylamide gel electrophoresis; and a method of separating and purifying the 3 3 Sabuyunitto .
ポリクローナノレ抗インテグリン S 3抗体は、 例えばマウス、 ラット、 モルモット、 ゥサギ、 ャギ、 ヒッジ等の被免疫動物を上記の抗原で免疫し、 これらの動物から 血清を採取することによって得ることができる。 被免疫動物を免疫する際に、 補 助剤 (アジュバント) を併用することは抗体産生細胞を賦活するので望ましい。 得られた抗血清から、 常法によってィムノグロプリン分画を精製してもよい。 Poly Skr Norre anti-integrin S 3 antibodies, such as mice, rats, guinea pigs, Usagi, catcher formic, the animals to be immunized such as Hijji immunized with the above antigens may be obtained by removing a serum from these animals . It is preferable to use an auxiliary agent (adjuvant) in immunizing the animal to be immunized, since it activates antibody-producing cells. From the obtained antiserum, the immunoglobulin fraction may be purified by a conventional method.
モノクローナル抗インテグリン^抗体は、 例えば次のようにして得られる。 す なわち、 上記抗原をマウス、 ラット、 モルモット、 ゥサギ、 ャギ、 ヒッジ等の被 免疫動物の腹腔内、 皮下あるいは足踱 (footpad) に投与した後に脾臓又は膝窩リ ンパ節を摘出し、 これらから採取した細胞と腫瘍細胞株であるミエローマ細胞と を細胞融合させてハイプリ ドーマを樹立し、 得られたハイプリ ドーマを連続増殖 させ、 さらに得られたハイプリ ドーマから上記抗原に対する特異抗体を継続的に 産生する細胞株を選別する。 こうして選別された株を好適な培地で培養すること によって、 培地中にモノクロ一ナノレ抗インテグリン 抗体が得られる。 あるいは、 マウスの腹腔などの生体内にて前記ハイプリ ドーマを培養することによって、 モ ノクローナル抗ィンテグリン δ 3抗体を大量に製造することができる。 A monoclonal anti-integrin antibody can be obtained, for example, as follows. That is, the antigen is administered intraperitoneally, subcutaneously, or to the footpad of an animal to be immunized, such as a mouse, rat, guinea pig, rabbit, goat, or sheep, and then the spleen or popliteal lymph node is removed. The cells collected from these cells are fused with myeloma cells, which are tumor cell lines, to establish hybridomas.The resulting hybridomas are continuously grown, and the specific antibodies against the above antigens are continuously obtained from the obtained hybridomas. Select the cell line that will produce. By culturing the selected strain in a suitable medium, a monoclonal anti-integrin antibody can be obtained in the medium. Alternatively, by culturing the High Priestess dormer in vivo, such as mouse peritoneal cavity it can be mass production of monoclonal anti Integurin [delta] 3 antibody.
細胞融合に用いる細胞としては、 脾細胞以外にリンパ節細胞および末梢血中の リンパ細胞等を用いることができる。 また、 ミエローマ細胞株は、 異種細胞種由 来のものに比べ同種細胞株由来のものが望ましく、 安定な抗体産生ハイプリ ドー マを得ることができる。 Cells used for cell fusion include lymph node cells and peripheral blood Lymphocytes and the like can be used. Further, the myeloma cell line is preferably derived from the same cell line as compared to that derived from a heterologous cell type, and a stable antibody-producing hybridoma can be obtained.
得られたポリクローナル抗体およびモノクローナル抗体の精製法としては、 硫 酸ナトリウム、 硫酸アンモニゥム等による塩析、 低温アルコール沈澱およびポリ エチレングリコールまたは等電点による選択的沈澱分別法、 電気泳動法、 D E A E (ジェチルアミノエチル) 一誘導体、 C M (カルボキシメチル) 一誘導体等の イオン交換体を用いたイオン交換クロマトグラフィー、 プロテイン Aまたはプロ ティン Gを用いたァフィ二ティークロマトグラフィー、 ハイドロキシァノヽ。タイト クロマトグラフィー、 抗原を固定化した免疫吸着クロマトグラフィー、 ゲルろ過 法および超遠心法等を挙げることができる。  Methods for purifying the obtained polyclonal and monoclonal antibodies include salting out with sodium sulfate, ammonium sulfate, etc., low-temperature alcohol precipitation, selective precipitation fractionation using polyethylene glycol or isoelectric point, electrophoresis, DEAE Ion-exchange chromatography using an ion exchanger such as tylaminoethyl) mono-derivative and CM (carboxymethyl) mono-derivative; affinity chromatography using protein A or protein G; Examples include tight chromatography, immunosorbent chromatography in which an antigen is immobilized, gel filtration, ultracentrifugation, and the like.
なお、 抗インテグリン; S 3抗体は、 そのまま使用することもできるが、 フラグメ ント化したものを使用することもできる。 抗体のフラグメント化の際、 抗体の抗 原結合部位 (F a b ) が保存されていることが抗原と抗体との結合に必須である ので、 抗原結合部位を分解しないプロテアーゼ (例えばプラスミン、 ペプシン、 パパィン等) で抗体を処理して得られる F a bを含むフラグメントを使用するこ ともできる。 また、 抗インテグリン 3抗体をコードする遺伝子の塩基配列もしく は抗体のァミノ酸配列が決定されれば、 遺伝子工学的に F a bを含むフラグメン トゃキメラ抗体 (例えば抗ィンテグリン; 83抗体の F a b部分を含むキメラ抗体等) を作製することができ、 このようなフラグメントやキメラ抗体も本発明において 用いることができる。 Incidentally, anti-integrin; S 3 antibodies can be used as it is, it is also possible to use those obtained by Furagume cement of. Preservation of the antibody's antigen binding site (F ab) during antibody fragmentation is essential for binding the antigen to the antibody, so proteases that do not degrade the antigen binding site (eg, plasmin, pepsin, papain) Etc.), a fragment containing Fab obtained by treating the antibody can also be used. Further, if the anti-integrin 3 nucleotide sequence also properly of the gene encoding the antibody determined Amino acid sequence of the antibody, fragment preparative Ya chimeric antibodies (e.g., anti Integurin including genetic engineering F ab; 8 3 antibody F chimeric antibodies containing an ab portion), and such fragments and chimeric antibodies can also be used in the present invention.
なお、 これら抗ィンテグリン; 33抗体の F a b部分を含むフラグメントやキメラ 抗体も、 本発明における 「抗インテグリン; 33抗体」 という用語に包含される。 上記のような抗ィンテグリン /93抗体を用いた抗原抗体反応の形態は特に制限さ れず、通常のィムノアッセィ (免疫測定法) に用いられる測定系は種類に関わら ず本発明に適用できる。 測定系として具体的には、 ィムノブロッティング法(例 えば、 ウェスタンプロッティング法等) 、 標識化免疫測定法 (例えば、 E I A法、 酵素結合ィムノソルベントアツセィ法 (E L I S A法) 、 ラジオィムノアッセィ 法、 蛍光ィムノアツセィ法等) 、 フローサイトメ トリーによる方法などが挙げら れる。 これらの測定法は、 公知の方法 [入江 實編、 「続ラジオィムノアッセィ」、Note that these anti Integurin; fragments and chimeric antibodies that contain F ab portion of the 3 3 antibodies, in the present invention; are encompassed by the term "anti-integrin 3 3 antibody". Form of the antigen-antibody reaction using the anti Integurin / 9 3 antibody as above is not particularly limited, the measurement system used for ordinary Imunoassi (immunoassays) can be applied to the present invention regardless of the type. Specific examples of the measurement system include an immunoblotting method (for example, a Western plotting method), a labeled immunoassay method (for example, an EIA method, an enzyme-linked immunosorbent assay (ELISA method), and a radioimmunoassay). Assay method, fluorescence immunoassay method, etc.), flow cytometry method and the like. It is. These measurement methods are known methods [S. Irie, edited by "Radio Simnoassy",
(株)講談社、 1 9 7 9年 5月 1日発行、 石川栄治ら編、 「酵素免疫測定法」 、 第 2版、 (株) 医学書院、 1 9 8 2年 1 2月 1 5日発行、 Method in Enzymology, Vol. 92 (1983), Immunochemical Techniques, Part E: Monoclonal Antibodies and General Immunoassay Methods, ァカデミックプレス社発行 参照] に準じ て実施することができる。 Published by Kodansha on May 1, 1997, Eiji Ishikawa et al., "Enzyme Immunoassay", 2nd edition, Medical Shoin Co., Ltd., published on Feb. 15, 1982 Method in Enzymology, Vol. 92 (1983), Immunochemical Techniques, Part E: Monoclonal Antibodies and General Immunoassay Methods, published by Academic Press Co.].
ィムノブロッティング法、 例えばウェスタンブロッティング法は、 体液、 好ま しくは尿、 より好ましくは可溶化した尿沈渣を検体として電気泳動を行い、 次い で電気泳動後の検体を膜に転写し、 この膜に抗インテグリン 3抗体を加え、 抗原 抗体反応によってインテグリン) S 3を測定することによって行うことができる。 電 気泳動は、 S D S—ポリアクリルアミ ドゲル電気泳動により行うことが好ましい。 In the immunoblotting method, for example, the Western blotting method, electrophoresis is performed using a body fluid, preferably urine, more preferably a solubilized urine sediment as a sample, and then the sample after electrophoresis is transferred to a membrane. the anti-integrin 3 antibody is added, can be done by measuring the integrin) S 3 by antigen-antibody reaction. The electrophoresis is preferably performed by SDS-polyacrylamide gel electrophoresis.
S D S—ポリアクリルアミ ド電気泳動の条件および膜への転写の条件は、 公知の ウェス夕ンブロッティング法に用いられる条件に準じて当業者が適宜決定するこ とができる。 例えば、 膜としてはポリビニリデンジフルオリ ド (polyvinylidene dif luoride: P V D F ) 膜、 ニトロセルロース膜あるいはナイロン膜等が挙げら れる。 ウェスタンブロッテイング法における抗原抗体反応の測定は、 抗インテグ リン 3 3抗体を予め標識物質で標識しておき、 膜とこの標識抗体をィンキュベート し、 H±のインテグリン/ δ 3に結合した標識抗体の標識物質を、 該標識物質に適し た方法で検出することによって行うことができる。 また、 未標識の抗インテグリ ン; S 3抗体を膜とィンキュベートし、 ィンテグリン; 5 3と抗ィンテグリン; S 3抗体の 結合体を形成させた後に、 抗インテグリン; 3 3抗体の調製に用いた被免疫動物のィ ムノグロプリンに対する抗体 (二次抗体) を標識物質で標識した標識化二次抗体 を前記結合体に結合させ、 結合体に結合した二次抗体中の標識物質を検出するこ とによっても、 抗原抗体反応を測定することができる。 抗インテグリン 5 3抗体と しては、 変性剤 (例えば S D S等) で変性された; 3 3サブュニッ卜に対して反応性 が高い抗体が好ましい。 The conditions for SDS-polyacrylamide electrophoresis and the conditions for transfer to a membrane can be appropriately determined by those skilled in the art according to the known conditions used for the Western blotting method. For example, the membrane may be a polyvinylidene difluoride (PVDF) membrane, a nitrocellulose membrane, a nylon membrane, or the like. Measurement of the antigen-antibody reaction in the Western blotting technique, leave labeled in advance with a labeling substance anti integrase phosphorus 3 3 antibodies, and Inkyubeto the labeled antibody and the membrane, the labeled antibody bound to the integrin / [delta] 3 of the H ± The detection can be performed by detecting the labeling substance by a method suitable for the labeling substance. Moreover, anti-integrin unlabeled; and S 3 Inkyubeto antibody the membrane and, Integurin; 5 3 anti Integurin; after forming the conjugates of S 3 antibody, anti-integrin; be used in the preparation of 3 3 antibody A labeled secondary antibody obtained by labeling an antibody against immunoglobulin of an immunized animal (secondary antibody) with a labeling substance is bound to the conjugate, and the labeling substance in the secondary antibody bound to the conjugate is detected. The antigen-antibody reaction can be measured. Is the anti-integrin 5 3 antibody was modified with the modifying agent (eg SDS, etc.); 3 3 Sabuyuni' antibody is highly reactive to Bok is preferred.
標識化免疫測定法、 例えばサンドィツチ E I Α法は、 体液、 好ましくは尿、 よ り好ましくは可溶化した尿沈渣中のインテグリン; 9 3と、 抗インテグリン 33抗体 とを反応させ、 サンドイッチ状複合体、 すなわち抗インテグリン; 83抗体一インテ ダリン; 9 3—抗インテグリン /3 3抗体の複合体の形成を検出することにより行うこ とができる。 この複合体の形成は、 複合体を形成する抗体の一方に標識抗体を用 い、 他方に固相体に結合もしくは結合しうる抗体を用いて、 複合体中の標識物質 を該標識物質に適した方法で検出することにより、 検出することができる。 また、 複合体を形成させた後に、 標識化二次抗体を複合体に結合させ、 複合体に結合し た標識化二次抗体の標識物質を測定することによつても、 複合体の形成を検出す ることができる。 さらに具体的にサンドイッチ E I A法としては、 例えば次のよ うな方法が挙げられるが、 これに限定されない。 Labeling immunoassay, for example Sanditsuchi EI Alpha method, a body fluid, preferably urine, is Ri preferably good integrin urine sediment was solubilized; and 9 3 is reacted with anti-integrin 3 3 antibodies, sandwich complex , i.e. anti-integrin; 8 3 antibody has one Integrated Darrin; 9 3 - can and this carried out by detecting the formation of a complex of anti-integrin / 3 3 antibody. The complex is formed by using a labeled antibody as one of the antibodies forming the complex and using an antibody capable of binding or binding to the solid phase on the other side, and using the labeled substance in the complex as the labeled substance. Can be detected by the method described above. Also, after forming the complex, the labeled secondary antibody is bound to the complex, and the labeling substance of the labeled secondary antibody bound to the complex is measured to form the complex. Can be detected. More specifically, examples of the sandwich EIA method include the following methods, but are not limited thereto.
( 1 ) インテグリン /33を含む可溶化した尿沈渣、 標識された抗インテグリン; 8 3 抗体、 および固相体に結合しているかあるいは結合しうる抗インテグリン 3 3抗体 を同時に混合して反応させて、 インテグリン; δ 3を該抗体と標識された該抗体とで 挟んだサンドィツチ状複合体を形成させるか、 Is 8 3 antibodies, and binding to and or an anti-integrin 3 3 antibody capable of binding simultaneously mixed to a solid body reaction; that (1) integrin / 3 3 solubilized urine sediment containing, anti-integrin labeled Forming a sandwich-like complex in which integrin; δ 3 is sandwiched between the antibody and the labeled antibody,
( 2 ) インテグリン /3 3を含む可溶化した尿沈渣と標識された抗インテグリン 抗体とを混合し、 インテグリン yS 3と標識された該抗体との結合体を形成させた後、 この結合体と、 抗インテグリン; 9 3抗体を固着した固相体とを混合して、 該結合体 のインテグリン 3 3をさらに該固相体に固着した該抗体に結合させて、 インテグリ ン Q 3を、 固相体に固着した該抗体と標識された該抗体とで挟んだサンドイッチ状 複合体を形成させるか、 または (2) After mixing the integrin / 3 3 urine sediment and labeled anti-integrin antibody solubilized comprising, to form a conjugate of integrin yS 3 and labeled antibody, and the conjugate, anti-integrin; 9 3 antibody by mixing solid phase and body which is fixed to, be bound to the antibody fixed to the further solid state body integrin 3 3 conjugate, the integrin Q 3, Kataaikarada Forming a sandwich-like complex sandwiched between the antibody fixed to and the labeled antibody, or
( 3 ) 固相体に固着された抗インテグリン S 3抗体と、 インテグリン yS 3を含む可 溶化した尿沈渣とを混合して、 該抗体とインテグリン 3 3とを結合させ、 必要に応 じて固相を洗浄し、 さらに予め標識物質で標識された抗インテグリン ;3 3抗体と混 合して、 インテグリン を、 該抗体と標識された該抗体とで挟み、 サンドイッチ 状複合体を形成させるかした後、 (3) solid phase anti-integrin S 3 antibodies secured to the body, was mixed with urinary sediment solubilized containing integrin yS 3, by coupling the antibody and the integrin 3 3, solid depending needs phases are washed, anti-integrin was further labeled beforehand with a labeling substance; 3 3 engages antibody confused, integrins, sandwiched between the antibody and the labeled antibody, after or to form a sandwich complex ,
固相に結合したサンドィツチ状複合体と結合しなかった該標識された抗ィンテ グリン 3 3抗体とを分離し、 固相と液相のいずれかの相の標識物質を該標識物質に 応じた方法(例えば酵素の場合、 酵素反応による基質の変化を測定する等) で測 定する方法を挙げることができる。  Separating the sandwich-like complex bound to the solid phase and the unlabeled anti-integrin 33 antibody which did not bind, a method according to the labeling substance in either the solid phase or the liquid phase (For example, in the case of an enzyme, a change in a substrate due to an enzyme reaction is measured, etc.).
サンドィツチ法において抗ィンテグリン /3 3抗体としてモノクローナル抗体を用 いる場合には、 ェピトーブの異なる二種類のモノクローナル抗体を用いるか、 あ るいはサンドィッチ状複合体を形成する抗体の一方にポリクローナノレ抗体を用い ることが好ましい。 If it is use of monoclonal antibodies as an anti Integurin / 3 3 antibody in Sanditsuchi method, or using two kinds of monoclonal antibodies of Epitobu, Oh Alternatively, it is preferable to use a polyclonal antibody as one of the antibodies forming the sandwich complex.
固相体としては、 マイクロタイタープレート (ィムノプレート) のゥヱル、 ポ リスチレンなどのボール、 ラテックス粒子、 試験管壁、 金属コロイドなどが挙げ られる。 これらの固相体に抗体を固着する方法としては、 物理的吸着法、 共有結 合法等が挙げられ、 さらには包括法など固定化酵素の調製法として一般的な方法 (固定化酵素、 1 9 7 5年、 講談社発行、 第 9〜 7 5頁参照) を応用することも できる。 特に物理的吸着法は簡便であり、 また抗体を固相体に固着する方法とし て広く用いられていることから好ましい。 なお、 抗体が結合していない部分は、 血清アルブミン、 ゼラチン、 乳タンパクなどによってブロッキングしてもよい。 また、 上記のサンドイッチ状複合体を固相に固着させた後、 固相と液相とを分 離し、 液相の標識物質を測定することによつても検体試料中のインテグリン を 測定することができる。  Examples of the solid phase body include a microtiter plate (imnoplate), a ball of polystyrene, latex particles, a test tube wall, and a metal colloid. Examples of a method for immobilizing an antibody on these solid phase bodies include a physical adsorption method and a covalent binding method. 75, published by Kodansha, pp. 9-75). In particular, the physical adsorption method is preferred because it is simple and widely used as a method for fixing an antibody to a solid phase. The portion to which the antibody is not bound may be blocked by serum albumin, gelatin, milk protein, or the like. In addition, after the above sandwich-like complex is immobilized on a solid phase, the solid phase and the liquid phase are separated, and the integrin in the specimen sample can also be measured by measuring the labeled substance in the liquid phase. it can.
また、 標識物質で標識化したインテグリン /S 3を、 検体試料とともに固相に固着 した抗インテグリン ;9 3抗体と混合し、 固相化抗体に結合した標識物質の量を測定 する、 いわゆる競合法によっても、検体試料中のインテグリン; 3 3を測定すること ができる。 Further, the integrin / S 3 labeled with a labeling substance, anti-integrin was immobilized on a solid phase with a test sample; mixed with 9 3 antibody to measure the amount of labeled substance bound to the immobilized antibody, so-called competitive method as well, integrin specimen sample; it is possible to measure the 3 3.
本発明の腎疾患の検知法においては、 抗原抗体反応は上記のような固相法に限 らず、 固相化抗体を使用しない、 いわゆる液相法で行ってもよい。 すなわち、 抗 インテグリン ;3 3抗体と検体試料を混合し、 さらに標識化二次抗体を加えてインテ グリン yS 3—抗インテグリン ;9 3抗体一標識化二次抗体の複合体を沈殿させ、 沈殿 に含まれる標識を検出する方法である。 In the method for detecting a renal disease of the present invention, the antigen-antibody reaction is not limited to the solid phase method as described above, and may be performed by a so-called liquid phase method without using an immobilized antibody. That is, anti-integrin; mixing 3 3 antibody and the test sample, integrin Glynn yS 3 was further added a labeled secondary antibody - anti-integrin; to precipitate the complex of 9 3 antibody has one labeled secondary antibody, the precipitation This is a method for detecting the contained label.
抗体の標識に用いる標識物質としては、 酵素 (ペルォキシダーゼ、 アルカリホ スファターゼ、 ^一ガラクトシダーゼなど) 、 放射性同位元素 (1 25 1、 1 3 1 I、 3Hなど) 、蛍光物質 (フルォレセインイソチオシァネート、 ゥンベリフエロン、 7—アミノー 4一メチルクマリン一 3—酢酸など) 、 化学発光物質 (ノレミノール など) 、 または他の物質 (ピオチン、 アビジンなど) 等が挙げられる。 尚、 固相 体として金属コロイド等を用いた場合は、 標識は必ずしも必要ではない。 The labeling substance used for labeling the antibody, enzyme (Peruokishidaze, alkaline phosphatase, ^, such as single-galactosidase), a radioactive isotope (1 25 1, 1 3 1 I, 3H , etc.), fluorescent substances (Full O receptacle in Isothiocarbazate Xia And chemiluminescent substances (such as noreminol), and other substances (such as biotin and avidin). When a metal colloid or the like is used as the solid phase, labeling is not necessarily required.
抗体の標識方法は、 標識物質に適した公知の方法、 例えば、 酵素を標識する際 にはグルタルアルデヒド法、 過ヨウ素酸架橋法、 マレイミ ド架橋法、 カルポジィ ミ ド法など、 放射性同位元素で標識する際にはクロラミン T法、 ラクトペルォキ シダーゼ法など(続生化学実験講座 5 免疫生化学研究法、 東京化学同人、 1 9 8 6年発行参照) から適宜選択できる。 The method of labeling the antibody may be a known method suitable for the labeling substance, for example, when labeling an enzyme. For example, the glutaraldehyde method, the periodic acid crosslinking method, the maleimide crosslinking method, the carpoimide method, and the like. When labeling with a radioisotope, the chloramine T method, the lactoperoxidase method, etc. (Seismic Chemistry Laboratory Course 5 Immunobiochemistry Research method, Tokyo Kagaku Dojin, published in 1986).
さらに、 本発明の別の測定法として、 可溶化していない尿沈渣に緩衝液 (例え ばリン酸緩衝生理食塩液(P B S )) 等を加え浮遊液を調製し、 抗インテグリン β 3抗体あるいは標識された該抗体を用いてフローサイトメ トリーにより尿中のイン テグリン ;33を測定する方法を挙げることができる。 Furthermore, another as a measuring method, to prepare a solubilized in buffer to the urine sediment is not (for example phosphate-buffered saline (PBS)) or the like was added suspension, anti-integrin beta 3 antibody or labeling of the present invention and a method of measuring a 3 3; has been the antibody in integrin in urine by flow cytometry using.
一方、 検体試料として尿沈渣を用いる場合、 尿沈渣は、 尿中に含まれる不溶物 であれば特に限定されず、 例えば尿中の不溶性蛋白、 細胞、 細胞断片等を遠心分 離または限外濾過膜等によって濃縮したものが挙げられる。 濃縮法としては、 遠 心分離が簡便であるので好ましい。遠心分離の条件としては、 例えば 4 0 0 X g 以上で 5分間以上遠心する条件が例示される。  On the other hand, when urine sediment is used as a sample, the urinary sediment is not particularly limited as long as it is insoluble in urine.For example, insoluble proteins, cells, cell fragments, etc. in urine are separated by centrifugation or ultrafiltration. What was concentrated by a membrane etc. is mentioned. As an enrichment method, centrifugal separation is simple and therefore preferable. Examples of the centrifugation conditions include, for example, the conditions of centrifugation at 400 X g or more for 5 minutes or more.
ウェスタンブロッテイング法においては、 通常、 電気泳動に先だって、 尿沈渣 に界面活性剤、 塩酸グァニジン、 尿素等の蛋白の変性を起こす変性剤 (可溶化剤) を加えて可溶化する。 変性剤 (可溶化剤) としては、 好ましくは界面活性剤であ り、 さらに好ましくはイオン性界面活性剤である。 イオン性界面活性剤のうちド デシル硫酸ナトリウム (S D S ) 等のアルキル硫酸塩を使用する場合、 その濃度 としては尿沈渣に対して 0. 1〜5 %程度が例示される。  In the Western blotting method, usually, prior to electrophoresis, a urinary sediment is solubilized by adding a surfactant, a denaturant (solubiliser) such as guanidine hydrochloride or urea that denatures proteins. The denaturant (solubiliser) is preferably a surfactant, and more preferably an ionic surfactant. When an alkyl sulfate such as sodium dodecyl sulfate (SDS) is used among ionic surfactants, its concentration is, for example, about 0.1 to 5% based on urine sediment.
また、 標識化免疫測定法においても、 尿沈渣に界面活性剤等を加えて可溶化し たものが検体試料として通常用いられる。 界面活性剤の種類や濃度は、 抗原抗体 反応に影響を与えない範囲で適宜選択することができる。 界面活性剤としては、 例えばポリォキシェチレンアルキルフヱニルエーテル類(Triton系界面活性剤) (トライトン (Triton) X— 1 0 0等) 、 ポリオキシエチレンソルビタンアルキ ルエステル類 (Tween系界面活性剤) などの非イオン性界面活性剤等が例示される c 体液、 好ましくは尿、 より好ましくは尿沈渣中のインテグリン /33量の定量は、 既知量のインテグリン )33を標準物質として用い、 標準物質濃度と検出される標識 量との関係につ t、て検量線を作成しておき、 この検量線と比較することにより行 うことができる。 また、 競合法においては、 抗原抗体反応系に一定量の標準物質 とともに既知量の未標識インテグリン s 3を加え、 標準物質濃度と検出される標識 量との関係につ L、て検量線を作成しておけばよい。 上記のようにして、 抗インテグリン ;3 3抗体を用いて体液中のインテグリン を測定することにより、 腎疾患の検知を行うことができる。 すなわち、 抗インテ グリン ;9 3抗体は、 腎疾患の診断薬として用いることができる。 また、 抗インテグ リン; S 3抗体の他に、 固相体、 標識抗体、 必要に応じて標識化二次抗体、 標識物質 を検知するための試薬、 緩衝液、 標識物質、 インテグリン; S 3標準物質、 抗原抗体 反応に影響を与えない可溶化剤 (変性剤) などを加えて診断用キットとしてもよ い。 固相体は、 予め抗インテグリン /93抗体を固着させておいてもよく、 かつ好ま しい。 このようなキットを用いると、 インテグリン 3の測定を簡便に行うことが できる。 図面の簡単な説明 図 1は、 調製例に記載の方法で製造した抗インテグリン /3 3抗体をコーティング したィムノプレートを用いて、 各種濃度のィンテグリン ;9 3標準液を測定した時の 標準曲線を示す図である。 発明を実施するための最良の形態 以下に実施例を挙げるが、 本発明は何等これに限定されるものではない。 調製例 抗インテグリン; S 3抗体の作製 モノクローナル抗インテグリン /8 3抗体の作製を、 Koehler. G.らの方法 (Natur e, 256 (1975) p. 495) に準じて行った。 Also in the labeled immunoassay, a urine sediment solubilized by adding a surfactant or the like is usually used as a specimen sample. The type and concentration of the surfactant can be appropriately selected within a range that does not affect the antigen-antibody reaction. Examples of surfactants include polyoxetylene alkylphenyl ethers (Triton-based surfactants) (Triton X-100, etc.), polyoxyethylene sorbitan alkyl esters (Tween-based surfactants) c body fluid non-ionic surfactant such as agent) and the like, preferably urine, more preferably in the urinary sediment integrin / 3 3 of quantification, using a known amount integrin) 3 3 as a standard substance The calibration can be performed by preparing a calibration curve based on the relationship between the concentration of the standard substance and the amount of the detected label, and comparing with the calibration curve. In the competition method, a certain amount of standard substance is added to the antigen-antibody reaction system. With unlabeled integrin s 3 of a known amount is added, the relationship between the amount of label is detected to a standard concentration Nitsu L, Te it is sufficient to create a calibration curve. As described above, anti-integrin; by measuring the integrin in body fluids by using a 3 3 antibody, it is possible to perform detection of renal disease. That is, anti-integrin Glynn; 9 3 antibodies can be used as a diagnostic agent for renal diseases. Moreover, anti-integrase phosphorus; the other S 3 antibody, Kataaikarada, labeled antibody, reagents for detecting labeled secondary antibody, the labeled substance if necessary, buffer, labeled substance, integrin; S 3 standard A diagnostic kit may be added by adding substances, solubilizing agents (denaturing agents) that do not affect the antigen-antibody reaction. Solid-phase body may be allowed to secure the advance anti-integrin / 9 3 antibody, and favored arbitrary. By using such a kit, integrin 3 can be easily measured. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1, using the Imunopureto coated with anti-integrin / 3 3 antibody prepared by the method described in Preparation, Integurin various concentrations; shows the standard curve when measuring 9 3 standard solution FIG. BEST MODE FOR CARRYING OUT THE INVENTION Examples will be given below, but the present invention is not limited thereto. Preparation anti-integrin; Preparation of S 3 antibody of making monoclonal anti-integrin / 8 3 antibody, was carried out in accordance with Koehler G. et al. Method (Natur e, 256 (1975) p 495.)..
使用可能期限をすぎた濃縮ヒト血小板浮遊液から白血球成分を除き、 血小板を H E P E S—タイロード緩衝液 (HEPES- Tyrode buffer) で洗浄後、 2 mM C a z M g 2 +を含む 5 O mM ;8—才クチルグルコシドで可溶化した。 超遠心後、 2 mM M n 2 +を添加し、 はじめにセファロース (Sepharose) 4 Bカラム (ファ ルマシア社製) でゲルろ過後、 続いてァフィ二ティークロマトグラフィーを行な つた。 ァフィ二ティーカラムには、 Gly- Arg-Gly- Asp_Ser- Pro-Lys (配列番号 1 ) ペプチドをリガンドとし、 このペプチドを常法によって結合させた CNB r活性 化セファロース (CNBr activated Sepharose (フアルマシア社製) ) を充填した カラムを用いた。 After removing the leukocyte component from the concentrated human platelet suspension that has passed the expiration date, wash the platelets with HEPES-Tyrode buffer, then add 5 mM mM containing 8 mM CazMg2 + ; —Solubilized with octyl glucoside. After ultracentrifugation, 2 mM Mn2 + was added, gel filtration was performed first on a Sepharose 4B column (Pharmacia), and then affinity chromatography was performed. I got it. The affinity column is a CNBr-activated Sepharose (manufactured by Pharmacia, Inc.) to which the Gly-Arg-Gly-Asp_Ser-Pro-Lys (SEQ ID NO: 1) peptide is used as a ligand, and this peptide is bound in a conventional manner. A column packed with)) was used.
3 OmM ;9—ォクチルダルコシドと 0. 5 M N a C 1を含む溶液で十分洗浄 後、 1 mM Gly-Arg-Gly-Asp-Ser-Pro (配列番号 2 ) ぺプチドで α , , b /33複合体 画分を溶出した。 さらにこの al lb/S3複合体画分を、 セフアデックス (Sephadex) G— 50カラム (フアルマシア社製) でゲルろ過し、 Gly- Arg_Gly-Asp-Ser-Pro (配列番号 2) ペプチドと Mn2+を除き、 精製された al lbJ83複合体を得た。 な お、 この複合体は S D S—ポリアクリルアミ ドゲル電気泳動後の銀染色で 2本の ノ ンドとして確認された。 得られた a l lb/S3複合体を SDS—ポリアクリルアミ ドゲル電気泳動で、 α,, bサブュニットと; 83サブュニットとに分離分画し、 yS 3サブュニットを単離した。 3 Omm; 9-O click tilde Turkey Sid and washed thoroughly with a solution containing 0. 5 MN a C 1, at 1 mM Gly-Arg-Gly- Asp-Ser-Pro ( SEQ ID NO: 2) peptide alpha,, b / 3 3 to elute the complex fraction. Furthermore the a l lb / S 3 complex fraction, gel filtration with Sephadex (Sephadex) G-50 column (Pharmacia, Inc.), Gly- Arg_Gly-Asp-Ser -Pro ( SEQ ID NO: 2) peptide and Mn 2+ was removed and to obtain a purified a l lbJ 8 3 complex. This complex was identified as two nodes by silver staining after SDS-polyacrylamide gel electrophoresis. Obtained a l lb / S 3 complex in SDS- polyacrylamide gel electrophoresis, alpha ,, b Sabuyunitto and; fractionated separated fraction into a 8 3 Sabuyunitto was isolated yS 3 Sabuyunitto.
この /93サブユニットを精製抗原として、 アジュバントと処理した後、 BALB Zcマウスの両後肢の足踱に、 1足当たり 10 1を 4〜5日ごとに免疫した。 免疫開始 3週間後、 精製抗原のみを同じ部位に注射し、 その 3日後に膝窩リンパ 節より、 リンパ球を調製し、 以下常法に従って、 PA I細胞 (ジャパニーズ 'キ ャンサー · リサーチ · リソース 'バンクの登録番号: J CRB— 01 1 3) とポ リエチレングリコールを用いて融合し、 HAT培地 (ヒポキサンチン、 アミノブ テリン、 チミジンを含む培地) で選択後、増殖したハイプリ ドーマの上清を用い てスクリーニングを行った。 As the purified antigen this / 9 3 subunit, after processing with an adjuvant, in both hind legs踱of BALB Zc mice were immunized 10 1 per pair every 4-5 days. Three weeks after the start of immunization, the purified antigen alone was injected into the same site, and three days later, lymphocytes were prepared from the popliteal lymph nodes. PAI cells (Japanese 'Cancer Research Resources') Bank registration number: J CRB-01 13) Fused with polyethylene glycol, selected in HAT medium (medium containing hypoxanthine, aminobuterin, and thymidine), and grown using the supernatant of the hybridoma. Screening was performed.
ヒト血小板、 および C 32メラノーマ細胞 (大日本製薬 (株) 販売 ; α,β ζ を発現して α , , b ;33は発現していない細胞株) の浮遊液にそれぞれ最終濃度 2 % SDSを加えて可溶化したものを抗原するィムノブロット法によって、 得られた ハイプリ ドーマの中から、 該抗原に反応性の高い抗体を産生するハイブリ ドーマ 株を選択した。 このハイプリ ドーマ株 RINSH0KEN- 2J40は、 平成 7年 8月 29日に 通商産業省工業技術院生命工学工業技術研究所 (郵便番号 305 日本国茨城県つ くば市東一丁目 1番 3号) に FERM P— 1 5 1 39の受託番号で寄託され、 平成 8年 8月 7日にブダぺス卜条約に基づく国際寄託に移管されて、 FERM BP— 5619の受託番号で寄託されている。 この株が産生するモノクローナル 抗インテグリン) 33抗体を 2 J 40抗体と命名した。 このハイプリ ドーマ株の培養 液から常法に従ってモノクロ一ナル抗体を精製した。 該抗体は、 変性していない ;83サブュニッ卜よりも SDSで変性された /33サブュニットに対して反応が高か つ 実施例 1 健常者および腎疾患患者の随時尿 10mlをそれぞれ 400 X g (1, 500 回転/分、 KUB OTA製遠心機 KN— 70)で、 5分間遠心し、 上澄を除いた。 Human platelets, and C 32 melanoma cells (Dainippon Pharmaceutical Co., Ltd. sold; alpha, expressing β ζ α,, b; 3 3 do not express cell line) final concentrations 2% SDS in suspension of A hybridoma strain producing an antibody highly reactive with the antigen was selected from the obtained hybridomas by an immunoblot method using the antigen solubilized by the addition of the antigen. On August 29, 1995, this high-priority dorma strain, RINSH0KEN-2J40, was transferred to the Institute of Biotechnology, Institute of Industrial Technology, Ministry of International Trade and Industry (zip code: 305, 1-3 1-3 Higashi 1-3-chome, Tsukuba, Ibaraki, Japan). — Deposited with accession number 1 5 1 39, It was transferred to an international deposit under the Budapest Treaty on August 7, 1996 and deposited under the accession number FERM BP-5569. This strain was designated as a monoclonal anti-integrin) 3 3 antibody 2 J 40 antibody produced. A monoclonal antibody was purified from the culture of the hybridoma strain according to a conventional method. The antibody modified non; 8 3 Sabuyuni' than Bok modified with SDS / 3 3 each 400 reaction high or One Example 1 healthy subjects and renal disease patients at any time urine 10ml respect Sabuyunitto X g (1,500 revolutions / minute, KUB OTA centrifuge KN-70) for 5 minutes, and the supernatant was removed.
500 ^ 1のリン酸緩衝生理食塩液 (PB S)を加えて懸濁し、 15, 00 Ox g (15, 000回転 Z分、 トミー精ェ (株)製遠心機 MRX- 150)で、 1 5分間遠心し、 上澄を除去した。 沈渣に 4 % S D Sを含む可溶化溶液 (トリス 1. 393 g、 20% SDS 20ml、 グリセロール 20ml、 EDTA 0. 07 4gを水に溶解し、 100m 1とした後、 塩酸で pH6. 8に調整し、 プロモフ ノールブルーを加えた溶液:以下、 「可溶化溶液」 と略す) 400 ^ 1を加え て溶解(可溶化) し 100 で 5分間加熱した。 得られた尿沈渣試料は測定まで 一 20^で保存し、 測定時に溶解して使用した。  Add 500 ^ 1 phosphate buffered saline (PBS) and suspend. Add 15,000 Ox g (15,000 rpm, min., Centrifuge MRX-150, manufactured by Tommy Seie Co., Ltd.) After centrifugation for minutes, the supernatant was removed. Solubilization solution containing 4% SDS in the sediment (Tris 1.393 g, 20% SDS 20 ml, glycerol 20 ml, EDTA 0.04 g was dissolved in water, adjusted to 100 ml, adjusted to pH 6.8 with hydrochloric acid). And a solution containing promophenol blue: hereinafter, abbreviated as “solubilizing solution”) 400 ^ 1 was added and dissolved (solubilized), and heated at 100 for 5 minutes. The obtained urine sediment sample was stored at 20 ^ until measurement, and dissolved and used at the time of measurement.
1レーン当たり尿沈渣試料 20 1づっをのせ、 SDS—ポリアクリルアミ ド ゲル電気泳動を行った。 電気泳動は、 R e s e p G e 1 ( 5— 20 %グラジェ ント) (和科盛 (株)製) を用い、 30mAで約 1時間、 ブロモフエノールブル 一がゲルの先端に達するまで行った。 ついで、 ィモビロン TM (PVDF膜、 ミリ ポアネ ±¾) を用いて、 常法に従って泳動後のゲルからタンパク質を転写した。 転 写後の該膜にモノクローナノレ抗体 2 J 40 (1 : 400希釈) を加え、 室温にて 1時間インキュベートした。 PBS— 0.05% Twe en20で 5分間、 2回 ついで 10分間、 2回洗浄をおこなった。 SDS-polyacrylamide gel electrophoresis was performed with 20 urine sediment samples per lane. The electrophoresis was performed using Resep Ge 1 (5-20% gradient) (manufactured by Washimori Mori Co., Ltd.) at 30 mA for about 1 hour until the bromophenol buffer reached the tip of the gel. Then, proteins were transferred from the gel after electrophoresis using Immobilon (PVDF membrane, Millipore ± ネ) according to a conventional method. Monoclonal antibody 2J40 (1: 400 dilution) was added to the membrane after the transfer, and incubated at room temperature for 1 hour. Washing was performed twice with PBS—0.05% Tween 20 for 5 minutes and then for 10 minutes.
この膜に、 HRP (ホースラディッシュ 'ペルォキシダーゼ) 標識抗マウス抗 体 (1 : 2000希釈) を加え、 室温で 45分間インキュベートし、 次いで上記 と同じ洗浄を行った。 ECLTM試薬(アマシャム社製) を加え、 2分間インキュ ペートした後、 X線フィルム (アマシャム社製) に 2分間暴露した。 デンシトメ 一夕で染色された部分の黒化度を測定した。 To this membrane, HRP (horseradish peroxidase) -labeled anti-mouse antibody (1: 2000 dilution) was added, incubated at room temperature for 45 minutes, and then washed as above. Add ECL TM reagent (Amersham) and incubate for 2 minutes. After coating, they were exposed to X-ray film (Amersham) for 2 minutes. Densitome The degree of blackening of the portion stained overnight was measured.
可溶化溶液で可溶化した洗浄された血小板 1 0 3個を試料としたときのィンテグ リン; S 3量を、 尿沈渣のインテグリン^ 3の 1ユニットとした。 既知量の、 可溶化 した洗浄された血小板を用いて、 デンシトメ一夕で測定される黒化度とインテグ リン) S 3量との関係について調べ、 0. 4〜 6 3. 0ュニッ卜の範囲で直線に回帰さ れた検量線を作成した。 なお、 インテグリン /33量は、 尿中のクレアチニン量で補 正して算出した。 Integu phosphorus when platelets 1 0 3 substituents washed and solubilized in solubilization solution was used as a sample; the S 3 content, was defined as 1 unit of the urine sediment integrin ^ 3. A known quantity, by using the washed platelets were solubilized, examined the relationship between blackness and integrase phosphorus) S 3 amount measured by Denshitome Isseki, from 0.4 to 6 3.0 Yuni' range Bok A calibration curve regressed to a straight line was created. It should be noted, integrin / 3 3 amount was calculated correctly complement in the amount of creatinine in the urine.
上記の健常者および腎疾患患者は、 健常者 2 3例と各種腎疾患患者 4 8例であ り、 疾患の内訳は全身性エリテマトーデス (S L E ) 1 9例 (活動性 (急性) 6 例、 非活動性 (慢性) 1 3例) 、 I g A腎症 1 0例、 糖尿病性腎症 (DM) 6例、 微小変化腎症 6例、膜性腎症 (MN) 4例、 膜性増殖性腎炎 (M P G N) 1例、 高血圧性腎硬化症(H T N) 2例であった。 健常者は男性 1 3例、 女性 1 0例で 年齢は 2 2歳〜 5 9歳であった。 また、 各種腎疾患患者は、 男性 2 0例、 女性 2 8例で、 年齢は 2 3〜 6 8歳であった。  The above-mentioned healthy subjects and patients with renal disease were 23 healthy subjects and 48 patients with various renal diseases. The breakdown of the disease was 19 cases of systemic lupus erythematosus (SLE) (6 cases of active (acute), Active (chronic) 13 cases), Ig A nephropathy 10 cases, diabetic nephropathy (DM) 6 cases, minimal change nephropathy 6 cases, membranous nephropathy (MN) 4 cases, membranous proliferative One case was nephritis (MPGN) and two cases were hypertensive renal sclerosis (HTN). The healthy subjects were 13 males and 10 females, aged between 22 and 59 years. Patients with various renal diseases included 20 males and 28 females, ages 23-68.
結果を表 1に示す。 表 1 尿中インテグリン; 33の排泄量 Table 1 shows the results. Table 1 Urinary integrin; 3 3 of excretion
Figure imgf000016_0001
測定の結果、 インテグリン; S 3と、 尿蛋白、血尿の程度、尿中 2—ミクログロ ブリン、 N—ァセチルグルコサミニダーゼ (NAG) との相関は認められなかつ
Figure imgf000016_0001
As a result of the measurement, there was no correlation between integrin; S3 and urinary protein, degree of hematuria, urinary 2-microglobulin, N-acetylglucosaminidase (NAG) and
/ /
また本測定により、 インテグリン S 3は健常者では尿中にほとんど排出されない ことがわかった。 さらにインテグリン ^ 3の定量的な測定の結果では、 その S出量 は腎臓疾患の種類によって変動し、 かつまた同一腎臓疾患群においてもその活動 性によって変動が見られた。 すなわち、 5し£にぉぃて非活動性3 £に比べ活 動性 S L Eのほうが尿中ィンテグ " ン β 3量が極めて多かった。 また組織病理的に 糸球体の傷害を認めなかった微少変化腎症では、 尿沈渣由来のインテグリン; 33は 検出されなかった。 In addition, the measurement showed that integrin S3 was hardly excreted in urine in healthy subjects. Furthermore, the quantitative results of integrin ^ 3 showed that its S output varied depending on the type of kidney disease, and also varied depending on the activity in the same kidney disease group. That is, the amount of urinary integran β 3 was much higher in active SLE than in inactive 3 £ compared to 3 inactive. Also, slight changes in which no histopathological glomerular injury was observed in nephropathy, integrin from urinary sediment; 3 3 was detected.
以上のように、 尿中インテグリン /33を測定することによって、 糸球体の傷害の 有無およびその程度を含めて腎疾患の検出ならびにその予後を簡便に判定するこ とが可能である。 実施例 2 活動性 S L Ε患者の 24時間畜尿 1, 000m lを 400 x gで 5分間遠心し 上澄を除いた。 沈澱を PB S 20m lで 2回洗浄し、 PBS 3m lを加えて浮 遊液を調製した。 これに 2 J 40抗体、 LM609抗体 (a V;S3複合体に対する モノクローナノレ抗体、 ケミコン (CHEMICON) ¾tM、 商品番号 [MAB 1976] ) 、 比較例として P 2抗体(" M b/S 3複合体に対するモノクローナル抗体、 コスモバ ィォ社製) を各々加え、 さらに F I TC標識二次抗体を加え、 以下常法にしたが つて FACS c a n (BECTON DICKINSON社製) を用いてフローサイトメ トリーを 行った。 この結果、 2 J 40抗体および LM609抗体に対しては尿沈渣の細胞 等は陽性に染色され、 インテグリン /33が検出されたが、 P2抗体には陰性であり. 血小板由来の α , , b 33複合体は検出されなかった。 実施例 3 調製例に記載された方法で作製した抗インテグリン >33 抗体 (2 J 40抗体) をリン酸緩衝生理食塩水 (PH7.2〜7.5、 二価ィォン不含) (以下、 P B S (—)と 略す) で 20mg/mlに希釈し、 この溶液の 50 1 (l g/ゥエル) ずつをィムノブレ ート (商品名マキシソープ、 ヌンク社製) の各ゥエルに加え、 4 で 16時間保 存することにより、 均一にコーティングした。 As described above, by measuring the urinary integrin / 3 3, including the presence and extent of injury glomeruli are possible and detection and simply determine this the prognosis of kidney diseases. Example 2 Active SL IV patients' 1,000-ml 24-hour urine was centrifuged at 400 xg for 5 minutes, and the supernatant was removed. The precipitate was washed twice with 20 ml of PBS, and 3 ml of PBS was added to prepare a suspension. This 2 J 40 antibody, LM609 antibody (a V; S 3 monochrome over nano-les antibody to complex, Chemicon (CHEMICON) ¾tM, Item No. [MAB 1976]), P 2 antibody as a comparative example ( "M b / S 3 A monoclonal antibody against the complex, Cosmo Bio) was added to each, and a FITC-labeled secondary antibody was further added. Flow cytometry was performed using a FACS can (BECTON DICKINSON) according to a conventional method. It was. As a result, 2 J 40 antibodies and cells, etc. of the urine sediment for LM609 antibody stained positively, although integrin / 3 3 was detected, there was negative for P2 antibodies. platelet derived alpha, , b 3 3 complexes were not detected. eXAMPLE 3 anti-integrin was prepared by the method described in preparation> 3 3 antibody (2 J 40 antibodies) phosphate buffered saline (PH7.2~7.5 , Without divalent ion) (hereinafter referred to as PBS (—) ), Add 50 1 (lg / で) of this solution to each well of Imnoplate (trade name: Maxisorp, manufactured by Nunc), and store at 4 for 16 hours. Coated evenly.
次いで、 このプレートを PBS (—)で 2回洗浄し、 ゥエルの抗インテグリン /3 3 抗体が被覆されていない部分を被覆するために、 ブロッキング物質として 3% ゥシ血清アルブミン (B S A) (生化学工業株式会社販売) を含む P B S (―)溶 液を加え、 室温で 2時間静置した。  The plate is then washed twice with PBS (-), and 3% Pseudoserum albumin (BSA) (BSA) is used as a blocking substance to cover the parts of the wells that are not coated with anti-integrin / 333 antibody. (Industry Co., Ltd.) containing PBS (-) solution was added and left at room temperature for 2 hours.
次いで、 このプレートを洗浄液 (0.0596トウィーン 20を含む PBS (—)) で 3 回洗浄し、 抗インテグリン 33 抗体が固着されたィムノプレートを作製した。 Then the plate (PBS containing 0.0596 Tween 20 (-)) washings were washed three times, to prepare a Imunopureto that anti-integrin 3 3 antibody is fixed.
抗インテグリン; 33 抗体が固着されたィムノプレートに、 各濃度のインテグリ ン ^3標準液 (0.4、 0.8、 1.6、 3.2、 6.25、 12.5、 25、 50および 100ng/ml) をそれ ぞれ 50^1ずつ添加し、 37 で 1時間静置して反応させた。 なお、 インテグリン 3標準液の溶媒としては、 1%85八を含む?83(—) (以下、 「反応希釈液」 という) を用いた。 Anti-integrin; 3 3 Imunopureto the antibody is fixed, integrin ^ 3 standard solution of each concentration (0.4, 0.8, 1.6, 3.2, 6.25, 12.5, 25, 50 and 100 ng / ml) it respectively 50 ^ 1 The reaction was allowed to stand at 37 ° C. for 1 hour. Does the solvent for Integrin 3 standard solution contain 1% 858? 83 (—) (hereinafter referred to as “reaction diluent”) was used.
この後、 このプレートを前記洗浄液にて 3回洗浄し、 1ゥエルあたり、 0.7 zg /mlのピオチン標識抗インテグリン; 33 抗体 25// 1及び 5000倍希釈したペルォキシ ダーゼ標識ストレブトアビジン (生化学工業株式会社販売) 25 1を添加し、 37 で 1時間静置して反応させた。 更に、 このプレートを前記洗浄液で 3回洗浄し、 ペルォキシダーゼの基質としてテトラメチノレべンジジン溶液 (モス社製、 以下 Τ MBと略す) を 50 1加え、 37 で 15分間反応させ、 発色させた。 Thereafter, washing 3 times the plate in the washing liquid, per Ueru, 0.7 zg / ml of Piochin labeled anti-integrin; 3 3 antibody 25 // 1 and 5000-fold diluted Peruokishi Daze labeled stress but-avidin (Biochemistry (Industry Co., Ltd.) 25 1 was added, and allowed to stand at 37 for 1 hour to react. Further, the plate was washed three times with the above-mentioned washing solution, and 501 of a tetramethinolebenzidine solution (manufactured by Moss, hereinafter abbreviated as MB) was added as a substrate for peroxidase, and the plate was reacted at 37 for 15 minutes to develop color.
発色後、 プレートに 1Mの HC 1を 50^1加えて反応を停止させ、 TMBの分解 による着色液の波長 450 nmの吸光度(対照波長 630 nm) をゥヱルリーダ 一 (生化学工業株式会社販売) にて測定した。 得られた結果を図 1に示す。  After color development, the reaction was stopped by adding 50 ^ 1 of 1M HC1 to the plate, and the absorbance at 450 nm wavelength (comparative wavelength: 630 nm) of the coloring solution due to the decomposition of TMB was transferred to Perleida I (produced by Seikagaku Corporation). Measured. The results obtained are shown in FIG.
この結果から、 抗インテグリン /53 抗体を固着させた固相体 (ここではマイク ロタイタ一プレートを用いているが、 これに限定されるものではない) を用いた ELISA法によっても尿中のインテグリン β3 の定量が可能であることが示唆された c またこの結果から、 ELISA法による尿中のィンテグリン 33定量時の検量線の作成 が可能であることが示された。 This result anti-integrin / 5 3 (although here uses a microphone Rotaita first plate, which is not limited to) antibodies solid-phase body which is fixed to the integrin in the urine by ELISA using from been c the result suggested that beta 3 of quantification is possible, it was shown that can create Integurin 3 3 quantified during the calibration curve in the urine by ELISA.
同様の方法で、 体液、 好ましくは尿、 より好ましくは尿沈渣、 極めて好ましく は可溶化剤 (例えば、 T r i t o n系や T w e e n系界面活性剤) により可溶化 した尿沈渣中のインテグリン) δ 3を測定することにより、 腎疾患の検知をすること ができる。 産業上の利用性 本発明の診断薬、 診断キットおよび測定法によれば、 腎疾患、 特に糸球体の傷 害の有無および程度を簡便に検出することができる。 In a similar manner, a body fluid, preferably urine, more preferably urine sediment, very preferably By measuring solubilizers (e.g., T Riton based or T ween surfactant) integrin) [delta] 3 of the urine sediment was solubilized by, it is possible to detect the renal disease. INDUSTRIAL APPLICABILITY According to the diagnostic agent, diagnostic kit and assay method of the present invention, the presence / absence and degree of renal disease, particularly glomerular damage, can be easily detected.
配列直 Array
(1)一般情報 (1) General information
(i) 出願人:  (i) Applicant:
(ii) 発明の名称  (ii) Title of invention
(iii) 配列数:  (iii) Number of sequences:
(iv) 連絡先:  (iv) Contact:
(A)宛名  (A) Address
(B)番地  (B) Address
(C)市  (C) City
(D)州  (D) State
(E)国  (E) Country
(F) ZIP:  (F) ZIP:
(V) コンピュータ読取り可能形式  (V) Computer readable format
(A)媒体:フロッピーディスク (A) Medium: floppy disk
(B)コンピュータ IBM PC互換(B) Computer IBM PC compatible
(C)操作システム PC-DOS/MS-DOS(C) Operation system PC-DOS / MS-DOS
(D)ソフトウエア FastSEQ Version 1. 5 (vi) 現行出願データ (D) Software FastSEQ Version 1.5 (vi) Current application data
(A)出願番号  (A) Application number
(B)出願日  (B) Filing date
(。分類  (.Classification
(viii)代理人ノ事務所情報  (viii) Agent information
(A)名前:  (A) Name:
(B)登録番号:  (B) Registration number:
(C)整理番号:  (C) Reference number:
(ix)通信情報  (ix) Communication information
(A)電話番号:  (A) Phone number:
(B)ファクシミリ番号  (B) Facsimile number
(2) 配列番号 1の配列の情報: (2) Sequence information of SEQ ID NO: 1
(i) 配列の性質:  (i) Properties of the array:
(A) 配列の長さ: 7 amino acids (A) Sequence length: 7 amino acids
(B) 配列の型: アミノ酸 (B) Sequence type: amino acid
(D) トポロジー: 直鎖状 (ii) 配列の種類: ペプチド (D) Topology: linear (ii) Sequence type: peptide
(xi) 配列: SEQ ID N0: 1 :  (xi) Sequence: SEQ ID N0: 1:
Gly Arg Gly Asp Ser Pro Lys Gly Arg Gly Asp Ser Pro Lys
1 5  1 5
(2) 配列番号 2の配列の情報: (2) Sequence information of SEQ ID NO: 2
(i) 配列の性質:  (i) Properties of the array:
(A) 配列の長さ: 6 amino acids (A) Sequence length: 6 amino acids
(B) 配列の型: アミノ酸 (D) トポロジー: 直鎖状(B) Sequence type: amino acid (D) Topology: linear
(ii) 配列の種類: ペプチド (ii) Sequence type: peptide
(xi) 配列: SEQ ID N0:2:  (xi) Sequence: SEQ ID N0: 2:
Gly Arg Gly Asp Ser Pro Gly Arg Gly Asp Ser Pro
1 5  1 5

Claims

請求の範囲 The scope of the claims
1 . 尿沈渣中のインテグリン) S 3を測定することを特徴とする腎疾患の検知法 c1. A method for detecting renal disease characterized by measuring integrin) S3 in urine sediment c
2 . 尿沈渣中のインテグリン yS 3を、 インテグリン; 3 3に対する抗体を用いた 抗原抗体反応によって測定することを特徴とする請求項 1記載の腎疾患の検知法。 . 2 integrin yS 3 of urinary sediment, integrin; detection method for renal disease of claim 1, wherein the measuring the antigen-antibody reaction using an antibody against 3 3.
3 . 可溶化した尿沈渣を検体として電気泳動を行 ゝ、 次 ゝで電気泳動後の検 体を膜に転写し、 この膜にインテグリン に対する抗体を加え、 抗原抗体反応に よってインテグリン^ 3を測定することを特徴とする請求項 2記載の腎疾患の検知 3. Perform electrophoresis using the solubilized urine sediment as a sample, transfer the electrophoresed sample to a membrane in the next step, add an antibody against integrin to this membrane, and measure integrin ^ 3 by antigen-antibody reaction. 3. Detection of renal disease according to claim 2, wherein
4 . 可溶化した尿沈渣中のインテグリン /9 3と、 インテグリン ^ 3に対する抗 体とを反応させ、 抗体一インテグリン ;8 3—抗体の複合体の形成を検出することに よりインテグリン) S 3を測定することを特徴とする、 請求項 2記載の腎疾患の検知 . 4 and solubilization integrin / 9 3 urinary sediment that is reacted with antibody to integrin ^ 3, Antibodies In one integrin; 8 3 - more integrin) S 3 to detect the formation of a complex between the antibody 3. Detection of renal disease according to claim 2, wherein the measurement is performed.
5 . インテグリン /3 3に対する抗体を含み、 尿沈渣中のインテグリン) S 3を測 定して腎疾患を検知するための診断薬であることを特徴とする腎疾患診断薬。 5. Comprising an antibody to integrin / 3 3, renal disease diagnostic agent which is a diagnostic agent for detecting a renal disease and measure the integrin) S 3 in urine sediment.
6 . ィンテグリン /3 3に対する抗体を固着させた固相体と、 標識物質で標識さ れた、 又は標識されうる該抗体とを含む腎疾患診断用キット。 6. Integurin / 3 3 solid phase body was fixed antibodies to, labeled with a labeling substance, or labeled Kit renal disease diagnosis including the antibody can.
7 . 構成試薬として尿沈渣の可溶化剤をさらに含む請求項 6記載のキット。 7. The kit according to claim 6, further comprising a solubilizing agent for urine sediment as a constituent reagent.
PCT/JP1996/002461 1995-08-31 1996-08-30 Method for detecting kidney diseases, diagnostic drug therefor, and diagnostic kit therefor WO1997008549A1 (en)

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WO2002037099A1 (en) * 2000-10-27 2002-05-10 International Reagents Corporation Method of diagnosing nephropathy
JP2008122410A (en) * 2000-10-27 2008-05-29 Masanori Hara Method of diagnosing nephropathy
WO2009041577A1 (en) * 2007-09-27 2009-04-02 Niigata University Urine pretreatment agent for urinary protein determination, urine pretreatment method, and urinary protein determination method
WO2014023819A1 (en) * 2012-08-10 2014-02-13 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for predicting the survival time of a patient suffering from a glioblastoma
JP2014517276A (en) * 2011-05-13 2014-07-17 キングス・カレッジ・ロンドン Methods and compositions related to platelet sensitivity
CN115667921A (en) * 2020-05-26 2023-01-31 东洋纺株式会社 Staining solution for urinary sediment

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002037099A1 (en) * 2000-10-27 2002-05-10 International Reagents Corporation Method of diagnosing nephropathy
US6969591B2 (en) 2000-10-27 2005-11-29 Masanori Hara Method for diagnosing nephropathy
JP2008122410A (en) * 2000-10-27 2008-05-29 Masanori Hara Method of diagnosing nephropathy
WO2009041577A1 (en) * 2007-09-27 2009-04-02 Niigata University Urine pretreatment agent for urinary protein determination, urine pretreatment method, and urinary protein determination method
EP2196803A1 (en) * 2007-09-27 2010-06-16 Niigata University Urine pretreatment agent for urinary protein determination, urine pretreatment method, and urinary protein determination method
EP2196803A4 (en) * 2007-09-27 2010-12-22 Univ Niigata Urine pretreatment agent for urinary protein determination, urine pretreatment method, and urinary protein determination method
JPWO2009041577A1 (en) * 2007-09-27 2011-01-27 国立大学法人 新潟大学 A urine pretreatment agent for urine protein quantification, a urine pretreatment method, and a urine protein quantification method.
US8105840B2 (en) 2007-09-27 2012-01-31 Niigata University Urine pretreatment agent for urinary protein quantitation, urine pretreatment method, and urinary protein quantitation method
JP5515740B2 (en) * 2007-09-27 2014-06-11 国立大学法人 新潟大学 A urine pretreatment agent for urine protein quantification, a urine pretreatment method, and a urine protein quantification method.
JP2014517276A (en) * 2011-05-13 2014-07-17 キングス・カレッジ・ロンドン Methods and compositions related to platelet sensitivity
WO2014023819A1 (en) * 2012-08-10 2014-02-13 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for predicting the survival time of a patient suffering from a glioblastoma
CN115667921A (en) * 2020-05-26 2023-01-31 东洋纺株式会社 Staining solution for urinary sediment

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