WO2001048019A1 - Methode permettant de diagnostiquer la nephrite proliferative a cellules mesangiales chez le rat - Google Patents

Methode permettant de diagnostiquer la nephrite proliferative a cellules mesangiales chez le rat Download PDF

Info

Publication number
WO2001048019A1
WO2001048019A1 PCT/JP2000/009251 JP0009251W WO0148019A1 WO 2001048019 A1 WO2001048019 A1 WO 2001048019A1 JP 0009251 W JP0009251 W JP 0009251W WO 0148019 A1 WO0148019 A1 WO 0148019A1
Authority
WO
WIPO (PCT)
Prior art keywords
megsin
antibody
rat
protein
cells
Prior art date
Application number
PCT/JP2000/009251
Other languages
English (en)
Japanese (ja)
Inventor
Toshio Miyata
Original Assignee
Kurokawa, Kiyoshi
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kurokawa, Kiyoshi filed Critical Kurokawa, Kiyoshi
Priority to AU22260/01A priority Critical patent/AU2226001A/en
Publication of WO2001048019A1 publication Critical patent/WO2001048019A1/fr

Links

Classifications

    • 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 diagnosing mesangial proliferative nephritis in rats, an antibody recognizing rat megsin useful for this method, and use thereof.
  • Renal failure is the condition that ultimately leads to kidney disease patients. causes and history are not uniform, and renal failure occurs due to non-renal lesions such as drug addiction, infectious disease, malignancy, diabetes mellitus, and systemic lupus erythematosus (SLE). Be looked at.
  • urinary protein is transiently increased in healthy people even during extreme exercise, mental stress, heavy carnivores, and premenstrual periods.
  • urinary proteins that are not derived from renal diseases, such as orthostatic proteinuria, which is common in young people (about 0.5% of healthy people).
  • Urinary protein is also observed in urinary tract diseases, bladder diseases, and female genital diseases. Therefore, it is difficult to make a definitive diagnosis of renal damage only by urine protein testing.
  • Urine sediment is obtained by centrifuging urine and observing the sediment with a microscope.Since erythrocyte sediment is also found in healthy people and may be derived from organs related to the urinary system other than renal impairment, This is also not sufficient for a definitive diagnosis of renal impairment.
  • renal biopsy is an invasive test and is always associated with complications such as bleeding and infection.
  • the patient in order to perform the test, the patient must be hospitalized in a well-equipped facility with a specialist, and the physical and social burden on the patient cannot be ignored.
  • urinalysis is a simple and excellent test method that can process a large amount of samples, but is not satisfactory from the viewpoint of definitive diagnosis of renal impairment.
  • the use of renal biopsy although reliable in diagnosing renal damage and assessing its severity, has to be extremely limited. Against this background, there has been a demand for a method for diagnosing renal impairment that combines the simplicity of urine analysis and the accuracy of renal biopsy.
  • proteins that are specifically expressed not only in the kidney but also in specific tissues are often used as indicators of dysfunction of the organ.
  • an enzyme protein such as LDHA GTP is widely used as a liver function marker.
  • kidney-specific protein that can be used as an indicator of its function in the kidney.
  • megsin a gene that is particularly strongly expressed in mesangial cells by large-scale DNA sequencing and database analysis. Then, they succeeded in obtaining megsin protein, a novel protein consisting of 380 amino acids encoded by the full-length cDNA clone of megsin. Furthermore, when an amino acid homology search was performed by the FASTA program using the Swiss Prot data base, human megsin protein was found to be SERPIN (serine protease inhibitor) superfamily (R. Carrell et al., Trends Biochem. Sci., Vol. 10, p. 20, 1985; R. Carre 11 et al., Cold Spring Harbor Symp. Quant. Biol., Vol.
  • SERPIN serine protease inhibitor
  • Reproductive and developmental toxicity tests are animal tests performed to obtain information on whether or not the application of a drug to a living body causes any adverse effects during the course of its reproductive development.
  • the test results obtained are extrapolated to humans and used to evaluate the safety (danger) of the drug against human reproductive development.
  • Rats have a long history of laboratory animals, and their knowledge of reproductive physiology and general metabolic patterns are fairly well known. In addition, the time to maturity, gestation and lactation are relatively short, and spontaneous malformations are relatively low. These advantages make rats suitable as test animals for reproductive and developmental toxicity studies. It is the species with the most experience in reproductive and developmental studies and has a lot of background data. In reproductive and developmental toxicity tests, various markers are measured according to the organ in which the disorder is to be observed. As the primary functions of renal function, blood creatinine and urinary albumin and globulin are known. However, these markers are for comprehensive evaluation of renal function. Therefore, it does not reflect the pathology of mesangial proliferative nephritis.
  • Mesangial cells play a central role in maintaining the structure and function of renal glomeruli.
  • the proliferation of mesangial cells and the accumulation of extracellular mesangial matrices are associated with the two major causes of end-stage renal failure, such as chronic glomerulonephritis and diabetic nephropathy.
  • end-stage renal failure has been described as the first step in causing glomerulosclerosis in patients with various glomerular disorders (D. Schlondorff, Kidney Int., 49, 1583-1585, 1996; RB Sterzel et al. , Glomerular mesangial cells. Immunologic Renal Diseases, 595-626, 1997). Therefore, it is desirable to provide an index that reflects the pathology of mesangial proliferative nephritis in rats. Disclosure of the invention
  • An object of the present invention is to solve the above problems and provide a method for diagnosing mesangial proliferative nephritis and determining the severity of the disease in rats, and to provide a reagent therefor.
  • mesangial cells (mesangial eel 1) present in glomeruli.
  • Mesangium is located in the center of the lobule of the capillary loop of the renal glomerulus, and is the core tissue that connects the lobules.
  • the mesangium is covered by the glomerular basement membrane, and the capillary lumen is separated from the cells separated by endothelial cells (mesangial cells) and the inner transparent layer in the glomerular basement membrane consisting of three layers. It is composed of form substances (mesangial matrix).
  • mesangial cells In rats as well, to identify genes specifically expressed in mesangial cells, and to clarify the mechanism of their expression and their relevance to the pathology of renal disease, the biological properties of mesangial cells It is considered to be effective for elucidation of mesangial cells, elucidation of the cause of diseases related to mesangial cells, and treatment and diagnosis of diseases associated with mesangial cells.
  • the present inventors have proposed that, even in rats, if the expression of the megsin protein gene is increased in association with the onset and enhancement of renal disease, and consequently the production of megsin protein is increased, it may be expressed in the urine or blood. We suspected that the megsin protein leaked and that the amount of the leak increased with the progress of the disease state. In order to confirm this mechanism, we tried to measure and compare the concentration of megsin protein in various biological samples from rats and the amount of megsin protein, and evaluated the state of renal disease involving megsin protein based on the measured values. The inventors have found that the present invention can be performed, and arrived at the present invention.
  • the present invention relates to the following methods for diagnosing mesangial proliferative nephritis, reagents therefor, antibodies against rat 'megsin, and uses thereof (hereinafter, in the present specification, the megsin protein may be simply referred to as megsin. is there) .
  • a composition comprising a peptide comprising the amino acid sequence of SEQ ID NO: 3 and an adjuvant.
  • a method for measuring rat megsin comprising the step of reacting a megsin protein in a rat-derived biological sample with the antibody according to [1] to detect a product of an antigen-antibody reaction.
  • [5] A composition comprising the antibody of [1].
  • a method for diagnosing mesangial proliferative nephritis in rats comprising the following steps:
  • the present invention relates to the use of a peptide comprising the amino acid sequence of SEQ ID NO: 3 in the production of an antibody that recognizes rat megsin.
  • Rat megsin protein was isolated as a protein (SEQ ID NO: 2) encoded by a homolog (SEQ ID NO: 1) in the rat of a gene highly expressed in human renal mesangial cells.
  • human megsin has been shown to be an indicator of renal function, it is not known to be useful as an indicator of mesangial proliferative nephritis in rats.
  • Megsin protein in the present invention This includes not only the protein having the amino acid sequence shown in SEQ ID NO: 2 (rat 'megsin protein) but also its functionally equivalent protein.
  • Functionally equivalent proteins include proteins encoded by the following DNAs:
  • amino acid sequence of SEQ ID NO: 2 one or several amino acids encode a protein consisting of an amino acid sequence having substitutions, deletions, no insertions, and / or insertions, and are encoded in a rat mesangial cell. Highly expressed DNA
  • Such DNA can be obtained, for example, by performing RT-PCR using mRNA extracted from rat cultured mesangial cells as type III. Primers required for RT-PCR can be set based on the nucleotide sequence of SEQ ID NO: 1.
  • the method for measuring megsin protein used in the present invention is not limited.
  • an immunological assay using an immunological reaction between an antibody against the megsin protein and the megsin protein is superior in specificity and sensitivity. Examples include immunoprecipitation, radioimmunoassay, immunofluorescence analysis, enzymymnoassay, chemiluminescence analysis, and immunohistochemist analysis.
  • the megsin protein can also be measured by the Western blot method using an antibody against the megsin protein.
  • these immunoassays can be used in combination, for example, by immunoprecipitation and subsequent Western blotting. These means of analysis are known in the art.
  • Immunohistochemistry refers to isolated rat cells or their lysates, tissues or their lysates, serum, pleural effusion, ascites, eye fluid, etc. After reacting the antibody, and reacting with a fluorescent substance such as fluorescin isothiocyanate (FITC), an anti-mouse IgG antibody or a binding fragment labeled with an enzyme such as peroxidase, observation using a microscope How to Each labeling substance can also be indirectly labeled by binding a streptavidin-binding labeling substance to a biotinylated antibody.
  • FITC fluorescin isothiocyanate
  • an anti-mouse IgG antibody or a binding fragment labeled with an enzyme such as peroxidase an enzyme such as peroxidase
  • the origin and preparation method of the antibody necessary for the immunoassay of megsin protein is not limited as long as it can recognize the megsin protein to be detected. Therefore, polyclonal antibodies, monoclonal antibodies, or mixtures thereof can be used. In addition, a fragment containing the variable region of an antibody molecule can also be used.
  • An antibody against the megsin protein can be obtained, for example, as follows.
  • the antibodies used in the present invention include, for example, antibodies against a protein having the amino acid sequence of SEQ ID NO: 2.
  • Antibodies against the megsin protein or its partial amino acid sequence are megsin protein, an oligopeptide containing the partial amino acid sequence, or c-myc- (His) 6-
  • a fusion protein such as Tag-megsin protein or MBP-megsin protein as an antigen
  • a monoclonal antibody can be produced according to the method described below.
  • the present invention provides an antibody that is particularly advantageous in the immunological measurement of rat and megsin. That is, the present invention provides an amino acid sequence represented by SEQ ID NO: 3 (ESNIVEKLLPESTV)
  • SEQ ID NO: 3 an antibody recognizing a peptide consisting of This region in rat megsin contains an amino acid sequence that is specifically found in rat megsin in the SERPIN superfamily to which megsin belongs. Therefore, an antibody that recognizes this region can be said to have excellent specificity for rat 'megsin.
  • SERP IN superfamily also has many proteins commonly found in biological fluids, such as plasminogen 1 or 2 or 1. Therefore, low cross-reactivity with these proteins is a useful feature for immunologically measuring megsin as a diagnostic indicator.
  • the active site of the megsin protein is not always clear at present, if the peptide antibody of the present invention binds to a region other than the active site of the megsin protein, the megsin protein and the peptide-2 antibody
  • the compound can be subjected to a test such as a measurement of physiological activity while the compound is bound. Therefore, the isolated and purified megsin protein Beptide 2 antibody conjugate can be used immediately in the test, and therefore, speeding up of the test can be expected.
  • the present invention relates to an immunogenic composition that can be used for preparing the antibody.
  • the composition of the present invention comprises a peptide comprising the amino acid sequence of SEQ ID NO: 3 together with an adjuvant.
  • the peptide includes at least the amino acid sequence of SEQ ID NO: 3.
  • the peptide may include, in addition to the sequence of SEQ ID NO: 3, an amino acid residue adjacent to the amino acid sequence of SEQ ID NO: 3 among the amino acid sequences of SEQ ID NO: 2.
  • the peptide may further have an inactive amino acid sequence added to the amino acid sequence selected from SEQ ID NO: 2.
  • the number of amino acids constituting a peptide further containing an additional amino acid sequence with respect to the amino acid sequence described in SEQ ID NO: 3 is usually 100 or less, preferably 50 or less, more preferably 3 or less. It is 0 or less, particularly preferably 20 or less.
  • the adjuvant any substance having an immunopotentiating effect on an immunized animal can be used. Specifically, a carrier protein, a bacterial toxin, or a bacterial cell component described later is used as an adjuvant. A When a carrier protein is used as the adjuvant, it may be bound to the above-mentioned peptide.
  • the peptide having a partial amino acid sequence of the rat megsin protein according to the present invention is administered to a warm-blooded animal by itself or together with a carrier and a diluent at a site capable of producing an antibody by administration.
  • any peptide can be used as long as it contains the amino acid sequence shown in SEQ ID NO: 3. Therefore, a partial peptide containing the amino acid sequence shown in SEQ ID NO: 3 or a peptide obtained by adding an inactive amino acid sequence to this amino acid sequence is included in the present invention.
  • the immunogen of the present invention can be obtained by cutting the rat megsin protein.
  • a peptide having the required amino acid sequence can be chemically synthesized.
  • the peptide is used as an immunogen by binding it to a carrier protein such as thyroglobulin or keyhole limpet mosquisin (hereinafter abbreviated as KLH).
  • KLH keyhole limpet mosquisin
  • it can be administered together with complete Freund's adjuvant / incomplete Freund's adjuvant. Administration is usually performed once every 1 to 6 weeks, for a total of about 2 to 10 times.
  • Examples of the warm-blooded animal to be used include monkeys, egrets, dogs, guinea pigs, mice, sheep, goats, and chickens, and mice are preferably used.
  • monoclonal antibody-producing cells When preparing monoclonal antibody-producing cells, select an individual with an antibody titer from a warm-blooded animal immunized with the antigen, such as a mouse, and collect the spleen or lymph node 2 to 5 days after the final immunization and include the spleen or lymph node. By fusing the antibody-producing cells obtained with myeloma cells, a monoclonal antibody-producing hybridoma can be prepared. The measurement of the antibody titer in the antiserum is performed, for example, by reacting a labeled megsin protein described below with the antiserum, and then measuring the activity of the labeling agent bound to the antibody.
  • the monoclonal antibody according to the present invention can be made not to cross with other proteins by selecting an antibody that recognizes an epitope specific to the megsin protein.
  • an ebitope represented by an amino acid sequence of at least seven or more consecutive amino acid residues, preferably 10 to 20 amino acids shows an epitope unique to the protein.
  • a monoclonal antibody that recognizes an ebitope composed of a peptide selected from the amino acid sequence shown in SEQ ID NO: 2 and having an amino acid sequence consisting of at least 7 consecutive amino acid residues is a rat antibody. It can be said that it is a monoclonal antibody specific to megsin protein.
  • Separation and purification of the anti-megsin protein monoclonal antibody is carried out in accordance with the method for separation and purification of immunoglobulin in the same manner as ordinary polyclonal antibodies.
  • Known purification methods include, for example, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, adsorption / desorption method using ion exchanger (for example, DEAE), ultracentrifugation method, gel filtration method, antigen binding Techniques such as a specific purification method in which only the antibody is collected by a solid phase or an active adsorbent such as protein A or protein G and the bond is dissociated to obtain the antibody can be shown.
  • the monoclonal antibody or polyclonal antibody recognizing the rat megsin protein thus obtained can be used for diagnosis of mesangial proliferative nephritis according to the present invention. That is, the present invention relates to a composition comprising an antibody that recognizes a peptide containing the amino acid sequence of SEQ ID NO: 3.
  • the antibody is preferably labeled or immobilized on a carrier.
  • the composition of the present invention is useful for immunoassay of rat 'megsin.
  • a sandwich complex formed by reacting a megsin protein with an antibody bound to an insoluble carrier and a labeled antibody bound to a labeled molecule is used.
  • Rat 'megsin protein in a sample can be measured using a competitive method for measurement.
  • Immobilized antibody In the measurement of rat megsin protein by the sandwich method, first, the immobilized antibody is allowed to react with rat megsin protein, and then unreacted substances are sufficiently removed by washing, and the labeled antibody is added.
  • Immobilized antibody A two-step method of forming a rat megsin protein-labeled antibody, or a one-step method of simultaneously mixing the immobilized antibody, the labeled antibody and the rat megsin protein can be used.
  • Insoluble carriers used for measurement include, for example, synthetic resins such as polystyrene, polyethylene, polypropylene, polyvinyl chloride, polyester, polyacrylate, nylon, polyacetal, and fluororesins, polysaccharides such as cellulose and agarose, glass, and metals. And the like.
  • synthetic resins such as polystyrene, polyethylene, polypropylene, polyvinyl chloride, polyester, polyacrylate, nylon, polyacetal, and fluororesins, polysaccharides such as cellulose and agarose, glass, and metals.
  • shape of the insoluble carrier various shapes such as a particle shape, a tray shape, a spherical shape, a fiber shape, a rod shape, a disk shape, a container shape, a cell, a test tube and the like can be used.
  • the antibody-adsorbed carrier is stored in a cool place in the presence of a preservative such as sodium azide as appropriate.
  • a reagent for evaluating the fertility of human sperm granules for detecting sperm fertility, which are obtained by binding a monoclonal antibody that specifically reacts with human sperm after the acrosome reaction to the surface of solid granules, are used. It is known (Patent No. 2 615 1 249). In the detection granules, a monoclonal antibody that specifically reacts with human sperm after the acrosome reaction is bound to the solid granules. By binding sperm to this and counting the bound spermatozoa, the fertilizing ability of human sperm can be evaluated.
  • a known chemical bonding method or physical adsorption method can be used.
  • a chemical bonding method for example, a method using glutaraldehyde, N-succinimide Maleimide method using Jil-4- (N-maleidomethyl) cyclohexane-to-carboxylate and N-succiniimidyl-2-maleidoacetate, tolethyl-3- (3-dimethylaminopropyl) carposimid
  • a calposimid method using hydrochloric acid or the like can be given.
  • the labeling substance is not particularly limited as long as it can be used for an immunological assay. Specifically, enzymes, fluorescent substances, luminescent substances, radioactive substances, metal chelates and the like can be used. Preferred labeling enzymes include, for example, peroxidase, alkaline phosphatase,? -D-galactosidase, malic acid dehydrogenase, staphylococcal nuclease, Dermal-5-steroid isomerase, and glycerol phosphate dehydrogenase.
  • Triose phosphatase isomerase horseradish peroxidase, asparaginase, glucose oxidase, ribonuclease, urease, force codulase, glucose 16-phosphate dehydrogenase, glucoamylase, acetylcholinesterase, etc.
  • Preferred fluorescent substances include, for example, fluorescein isocyanate, phycobiliprotein, oral damine, phycoerythrin, phycocynin, arophycocynin, and orthophthalaldehyde.
  • Preferred luminescent substances include isoluminol, lucigenin, luminol, aromatic acridinium ester, imidazole, acridinium salt and its modified ester, luciferin, luciferase, and aequorin.
  • Preferred radioactive material include 125 1, 127 1, 131 1 , 14, 32 P S or 35 S, or the like. Techniques for binding the label to an antibody are known. Specifically, direct and indirect signs can be used. As a direct labeling method, a method of chemically covalently bonding an antibody or an antibody fragment and a label with a crosslinking agent is generally used.
  • Crosslinking agents include ⁇ , ⁇ '-orthophenylenedimaleide, 4- ( ⁇ -maleimide methyl) cyclohexanoate ⁇ ⁇ -succinimide ester, 6-maleimide hexanoate ⁇ ⁇ -succinimide ester, 4, 4'-dithiopyridine and other known crosslinking agents can be used.
  • the reaction between these cross-linking agents and enzymes and antibodies may be performed according to known methods depending on the properties of the respective cross-linking agents.
  • a method in which a low-molecular-weight hapten such as piotin, dinitrophenyl, pyridoxal, or fluorescamine is bound to the antibody, and a method of indirectly labeling with a binding component that recognizes this can be used.
  • Streptavidin is used as a recognition ligand.
  • dinitrophenyl, pyridoxal or fluorescamine an antibody that recognizes these haptens is labeled.
  • horseradish peroxidase can be used as a labeling enzyme.
  • This enzyme is advantageous because it can react with many substrates and can be easily bound to an antibody by the periodate method.
  • a fragment thereof for example, Fab ′, Fab F (ab,) 2 is used as the antibody.
  • an enzyme receptor can be obtained by the same treatment. If the enzyme-labeled product obtained using the above-mentioned cross-linking agent is purified by a known method such as affinity chromatography, a more sensitive immunoassay system can be obtained.
  • the purified enzyme-labeled antibody is preserved by adding Thimerosal as a preservative and glycerin as a stabilizer.
  • the labeled antibody can be stored for a longer period by freeze-drying and storing in a cool dark place.
  • the labeling agent is an enzyme
  • a substrate and, if necessary, a color former are used to measure its activity.
  • peroxidase is used as the enzyme
  • the substrate solution 2,2, the 3 ⁇ 40 2 using, as a color-developing agent as - azino - di - [3- E chill benz thiazoline sulfonic acid]
  • Anmoniumu salt (ABTS) 5-Aminosarichiru acid, Orutofue two Ren Jiamin, 4- Aminoanchi Villin, 3,3,5,5-tetramethylbenzidine and the like can be used.
  • alkaline phosphatase When alkaline phosphatase is used as the enzyme, orthoditrophenyl phosphate, paraditrophenyl phosphate, or the like can be used as a substrate.
  • /?-D-galactosidase As the enzyme, use fluorescein-di-(?-D-galactobilanoside), 4-methylpumbellifenyl-5-D-galactobilanoside, etc. as the substrate be able to.
  • the present invention also provides a reagent for immunoassay of megsin protein, which is obtained by labeling or immobilizing the above-mentioned monoclonal antibody or polyclonal antibody. It also includes a kit of an outgoing indicator, a control sample, and the like.
  • the measurement target of rat megsin protein in the present invention is not limited as long as it is a biological sample containing rat megsin protein, or a precursor or fragment thereof.
  • Specific measurement targets include, for example, plasma, serum, blood, urine, tissue fluid, and body fluids such as cerebrospinal fluid.
  • megsin protein is detected at a high frequency with the proliferation and activation of mesangial cells. Therefore, measurement of urinary megsin protein is useful as a marker for mesangial proliferative nephritis such as IgA nephropathy.
  • Diagnosis of mesangial proliferative nephritis in the present invention means to grasp the state of mesangial cells, which are important cells constituting the kidney tissue, and to know whether or not there is a renal disease that causes abnormalities in mesangial cells. I do.
  • the mesangial proliferative nephritis in the present invention includes all nephritis accompanied by proliferation of mesangial cells. Specific examples include IgA nephropathy, acute glomerulonephritis, focal glomerulosclerosis, membranous proliferative glomerulonephritis, diabetic nephritis, and lupus nephritis, etc.
  • the status of renal function can be assessed by the method of the present invention.
  • the method for evaluating renal function according to the present invention includes mesangial diseases such as IgA nephropathy among these diseases. It is particularly useful as a marker for proliferative nephritis.
  • the method for evaluating renal function of the present invention can be applied to the evaluation of the therapeutic effect and the determination of prognosis, in addition to the determination of the presence or absence and degree of renal disease.
  • the method for evaluating renal function by measuring the amount of megsin protein in a sample that is positive in a urine protein test, acute pyelonephritis or chronic pyelonephritis not caused by the proliferation of mesangial cells Diseases such as minimal change nephrotic syndrome, chronic glomerulonephritis, and renal amyloidosis can be excluded.
  • the mesangial proliferative nephritis of the present invention includes artificially induced nephritis in addition to the above diseases.
  • Artificially induced nephritis may include mesangial proliferative nephritis induced in the kidneys of rats by various toxic substances.
  • Such artificially induced nephritis rats can be used for various toxicity tests and the like, but more accurate judgment can be made by using megsin as an evaluation index.
  • mesangial proliferative nephritis can be diagnosed accurately and easily by using megsin as an index. The results of toxicity studies in rats can be easily extrapolated to humans.
  • the physiological state of rats is similar to that of humans, and the megsin to be measured as an indicator is a common factor in humans and rats. ⁇ is easy.
  • the present invention is of great significance in that it has been found that megsin in rats is not only a structural homolog but also useful as an indicator of mesangial proliferative nephritis.
  • nephritis induced by administration of an anti-Thyl antibody may be referred to as ATS.
  • the present invention is used to track changes in the pathogenesis of mesangial proliferative nephritis in rats in which mesangial cells have been abnormally altered by genetic manipulation, such as antisense DNA and transgenic rats. be able to.
  • a biological sample of an individual to be diagnosed is collected and the concentration of the megsin protein contained therein is measured based on the method described above. I do.
  • the amount of megsin protein is determined from the concentration and the volume of the body fluid, and compared with the value of a normal rat.
  • To determine the amount of megsin protein when using urine as a sample, for example, pooling one day's urine and measuring the amount of urine reveals the amount of megsin protein per day in urine can do. Alternatively, even if urine is used as a sample at any time, a value similar to the amount can be estimated by creatinine correction.
  • Creatinine correction is a technique for correcting the effect of dilution (or concentration) of the analyte due to fluctuations in urine volume based on the concentration of creatinine. Based on the constant amount of creatinine excreted in urine per day, the ratio of urine to total urinary excretion in one day is calculated based on the concentration of creatinine, and the measurement target obtained from the same urine Component concentrations can be converted to total excretions per day. For blood, the amount can be estimated by applying numerical corrections commonly used in renal function diagnosis such as weight correction. Weight correction is a technique for calculating the amount of blood components based on the volume of blood estimated from the weight of the individual from whom the blood was collected.
  • a normal value of the megsin protein concentration of a body fluid sample in a population of a specific species or the like is set in advance, and the megsin protein concentration of the specific individual (and The amount can also be used to determine the presence or absence of mesangial proliferative nephritis.
  • urine or blood can be used as a biological sample.
  • urine is a preferred sample that can be collected non-invasively and directly reflects the status of renal function.
  • blood sampling is somewhat invasive, abnormalities in the measurements are closely related to abnormalities in renal function, as the megsin protein is a protein specific to the kidney. Therefore, high specificity can be expected as an index of renal function.
  • a specific operation for measuring megsin protein in urine and evaluating renal function based on an immunoassay for megsin protein using a monoclonal antibody will be described in detail.
  • the animal is immunized, for example, as follows.
  • a partial peptide of rat megsin protein (for example, a peptide consisting of the amino acid sequence of SEQ ID NO: 3) is synthesized and combined with KLH to produce an immunogen.
  • This immunogen is immunized to a mammal such as a mouse.
  • a mammal such as a mouse.
  • an animal of the same strain as the partner of the cell in which the cells are fused.
  • the age of the animal is preferably, for example, 8 to 10 weeks in mice. Sex may be either male or female.
  • the method of immunization is to mix the above immunogen with an appropriate adjuvant (eg, Freund's complete adjuvant or aluminum hydroxide gel-pertussis vaccine) to form an emulsion, and then subcutaneously, intraperitoneally, or intravenously into the animal. Give. Thereafter, this immunization is performed 2 to 5 times at intervals of 1 to 2 weeks. Final immunization is performed by intraperitoneally administering 0.5 to 2 zg of the immunogen to the animal. A polyclonal antibody is obtained from the body fluid of the immunized animal.
  • an appropriate adjuvant eg, Freund's complete adjuvant or aluminum hydroxide gel-pertussis vaccine
  • the protein A rosette atsay method is, for example, a method in which a human erythroid cell line K562 (Japanese 'Cancer' Research Resources Bank '(JCRS)) is placed on a 72-well Terasaki plate (Falcon).
  • PBS phosphate Ninato potassium 2.90 g, Ichiriki phosphate potassium 0.20 g, chloride Natoriumu 8 g, chloride force Riu beam 0.2 g
  • the sample diluted with distilled water 1 had added, 3 7 ° in the C0 2 incubator Leave it for 30 minutes at C. Then, wash with PBS, add sheep erythrocytes coated with Protein A (manufactured by Amersham Pharmacia Biotech), and observe the formation of rosette with a microscope. .
  • Antibody-producing cells are collected from the animal immunized with the synthetic peptide as described above.
  • Antibody-producing cells can be obtained from spleen, lymph nodes, peripheral blood, etc., with spleen being particularly preferred. For example, 3-4 days after the final immunization, the spleen is aseptically removed, shredded in Minimal Essential Medium (MEM) medium (manufactured by Nissui Pharmaceutical Co., Ltd.), dissected with tweezers, and subjected to conditions of 120 Orpm x 5 minutes.
  • MEM Minimal Essential Medium
  • any cells having permanent proliferation can be used, but myeloma cells are generally used.
  • Permanently proliferating cells are preferably derived from animals of the same species as the antibody-producing cells.
  • the following cell lines are known as bone tumor cell lines derived from 8-azaguanine-resistant mice (BALB / c).
  • P3-X63Ag8-Ul (P 3 -U 1) (Current. Topics in Microbiol. Immu nol., Vol. 81, pp. 1-7, 1978),
  • SP-2 / 0-A 14 SP-2 (Nature, Vol. 276, pp. 269-270, 1978), P 3-X63-Ag 8653 (653) (J. Immunol., Vol. 123, pp. 1548-1550, 1979), and
  • P3-X63-Ag8 (X63) (Nature, Vol. 256, pp. 495-497, 1975) These permanently proliferating cell lines were cultured in 8-azaguanine medium (RPMI-1640 medium with glutamate). 1.5m), 2-mercaptoethanol (5 x 10 " 5 ), gentamicin (10 ⁇ g / mL) and fetal calf serum (FCS, CLS) (10%), and an additional 8%.
  • - subcultured Azaguanin (15 / g / mL) and the mixture was culture land), and they are subcultured in the normal medium 3 or 4 days before cell fusion to ensure the 2 XI 0 7 or more cell number day of fusion .
  • Cell fusion is performed, for example, as follows.
  • the antibody-producing cells obtained in (1) and the permanently proliferating cells prepared in (2) are thoroughly washed with MEM medium or PBS to reduce the cell number to 5 to 10.
  • the supernatant was removed by centrifugation (150 Orpm x 5 minutes), the cells were gently dissociated, and 10 OmL of normal medium (RPMI-1640 medium, 10% FCS) was added. Gently suspend cells by pitting. The suspension was dispensed by 100 ⁇ L / well to the culture plate for 96 Ueru, in 5% C0 2 incubator base Isseki primary, cultured for 3-5 days at 37 ° C. HAT medium 10 0 ⁇ L / well in a culture plate (normal medium hypoxanthine (10- 4 M), thymidine
  • the anti-rat megsin protein monoclonal bound to the solid phase is prepared by adding the hybridoma culture supernatant to the solid phase to which immunoglobulin antibody or protein A is adsorbed, adding rat megsin protein labeled with radioactive substances, enzymes, etc. It can also be confirmed by a method for detecting a nal antibody.
  • Cloning was repeated four times by the limiting dilution method for the rat in which production of an antibody reactive with the rat megsin protein was observed. Select as a pre-doma strain.
  • the hybridoma obtained as described above is cultured in vitro and in vivo to produce a monoclonal antibody.
  • the hybridoma is transplanted to any animal, but it is preferable to use an animal of the same species as the animal from which the spleen cells used for cell fusion were collected.
  • pristane treatment (2,6,10,14-tetramethylpentanedecane-0.5 ml of pristane is intraperitoneally administered, Raise for 2 weeks.
  • ascites containing a high concentration of monoclonal antibody accumulates in the abdominal cavity of the mouse and the abdomen grows.
  • the ascites is collected from the mouse, centrifuged (300 Orpm x 5 minutes) to remove solids, and IgG is purified.
  • culturing in vitro of the hybridoma is preferably performed in a serum-free medium, and an optimal amount of the antibody is given to the supernatant. Salt out the ascites fluid and culture supernatant using 50% ammonium sulfate and dialyze against PBS for 1-2 weeks.
  • the dialyzed fraction is passed through a Protein II Sepharose column, and the IgG fraction is collected to obtain a purified monoclonal antibody.
  • the antibody isotype was determined by the Oku Yuguchi Nii (double immunodiffusion) method (Introduction to Immunology Experiments, Biological Chemistry Experiment Method 15, published by Gakkai Shuppan Center, p. 74, 1981).
  • the characteristics of the monoclonal antibody obtained as described above include, for example, (1) Immunoprecipitation reaction using a human lymphocyte-derived cell line such as HSB-2 or K562 with iodine-labeled cell surface (J. Immunol. , Vol. 138, pp. 2850-3855, 1987) and
  • the purified monoclonal antibody obtained is obtained by the glutaraldehyde method (I-Cho unochem., Vol. 6, p. 43, 1969), the periodic acid method (J. Histochem. Cytochem., Vol. 22, 1084, 1974). Enzymatic methods such as the maleimide method (J. Biochem., Vol. 79, p. 233, 1976) and the pyridyl disulfide method (Biochem. J., vol. 173, p. 723, 1978). Can be labeled.
  • periodate method 50 ⁇ L of periodate (38.5 mg / mL) is added to the peroxidase solution (4 mg / mL) with stirring, and the mixture is reacted at room temperature for 20 minutes.
  • the buffer with PD-10 (Amersham-Pharmacia-Biotech) replaced with ImM acetate buffer (pH 4.5).
  • 40 L of 0.2 M sodium hydroxide add 1 Om of the monoclonal antibody dialyzed against 1 OmM carbonate buffer (pH 9.5) and react for 2 hours at room temperature. After the reaction is completed, cool on ice, add 100 ⁇ L sodium borohydride solution (4mg / mL), and react for 2 hours.
  • the granules for detection used in the present invention can be produced by physically or chemically binding an anti-megsin protein antibody to appropriate granules, for example, a gel for chromatography.
  • the method of binding the antibody used in the present invention to chemically activated granules is a desirable binding method because binding stability can be expected. Specifically, a method of binding the antibody used in the present invention to granules tosylated by p-toluenesulfonyl chloride is shown.
  • the granules As the granules, granules made of glass, agarose, sepharose, agarose-filled porous diatomaceous earth, hydrophilic copolymerized acrylic gel, polystyrene and the like are used. If the granules having magnetism are used, the granules can be focused by using a magnet or the like, so that a small amount of sample can be measured. For example it is possible to granulate which gave superparamagnetic by including magnetizable material (e.g., Fe 2 0 3) in the core. Such granules are commercially available as solid phases for immunological analysis.
  • magnetizable material e.g., Fe 2 0 3
  • the shape of the granules Any shape such as a spherical shape and an irregularly crushed shape is preferable, but a spherical shape is preferable.
  • the particle size is not particularly limited, and for example, can show an average particle size of 5 to 100 m. Also, when granules having a specific gravity higher than the specific gravity of the reaction solution (about 1) are used, the convergence of the granules becomes easy, and the same effect as when using magnetic granules is obtained. Furthermore, in this case, the conditions for centrifugation for condensing the granules can be moderated, which is advantageous when using an antibody that is easily dissociated.
  • the binding of the anti-rat megsin protein antibody to the granules can utilize not only direct binding but also indirect binding.
  • an antibody recognizing mouse IgG can be bound to the granules, and the mouse antibody can be bound to the granules indirectly.
  • Such antibodies are called secondary antibodies.
  • secondary antibodies protein A or protein G that binds the immunoglobulin constant region, or a method of capturing a biotinylated antibody with avidin-immobilized granules Can also.
  • protein A or protein G to the granules as described above, it is preferable to activate the granules and then bind them.
  • any activation method for binding proteins to this kind of granules can be selected.
  • Examples of such an activation method include the tosyl mouth method, the Bromcian method, the bromacetyl method, and the glutaraldehyde method.
  • Some activated granules are commercially available.
  • Such activation and binding of the activated granules to a protein such as a secondary antibody, protein A or protein G can be performed by a conventional method.
  • granules to which a secondary antibody, protein A, protein G, or the like has been bound are already on the market.
  • the following are known as commercially available granules, for example. Imported by Nippon Dinal Co., Ltd., Dynabeads sold by Veritas Co., Ltd.
  • the suspended granules are treated with a protein solution in a suitable medium to prevent non-specific adsorption, and then the antibody-containing ascites or purified antibody Mix the solution.
  • blood or urine is collected from a test rat, and the supernatant after centrifugation is used as a specimen.
  • the urine is centrifuged to separate the precipitate, and the supernatant after standing or the urine sample from which the precipitate has been removed by filtration can be used.
  • the diluted sample and the labeled conjugated antibody obtained in (7) are added to the granules for detection produced as described above, and incubated at room temperature for 2 hours. After the completion of the reaction, wash, add a base solution, color, centrifuge to remove granules, transfer the supernatant to a microplate, and measure the absorbance. Similarly, the samples of normal subjects are measured and the values are compared.
  • the absolute amount of rat megsin protein in the body fluid which can be obtained by multiplying the volume of the body fluid of the individual, Alternatively, a comparison based on a similar correction value can be performed.
  • kits Materials needed to perform the above tests can be supplied as kits.
  • a kit can include a detection granule and a magnet on which the above-described antibody is immobilized. Further, it may contain an antibody to which a label molecule is bound.
  • the kit according to the present invention can include test tubes, centrifuge tubes, other similar containers, pipettes or similar suction devices, or microscopes. Alternatively, an enzyme substrate necessary for detecting the label, a positive or negative standard sample, and the like can be combined.
  • a combination of solid granules which are raw materials for the production thereof, and an antibody can be used.
  • FIG. 1 is a graph showing the titer of an anti-rat megsin peptide-2 antibody.
  • the horizontal axis represents the dilution ratio, and the vertical axis represents the absorbance (0D490 nm).
  • 1 to 3 each represent a peony individual.
  • Figure 2 shows the results obtained by examining the reactivity of a polyclonal antibody obtained by using the partial amino acid sequence of rat megsin peptide-2 (SEQ ID NO: 1) as an immunogen by the stamp lot method. Gel photo shown. Each lane corresponds to the following proteins. 1: MBP human megsin protein fusion protein
  • FIG. 3 shows a micrograph of a normal rat kidney tissue obtained by tissue immunostaining.
  • FIG. 4 shows a comparison of megsin amino acid sequences in different species. A shows comparison between human and rat, B shows comparison between human and mouse, and C shows comparison between rat and mouse. Matched regions are shown with diagonal lines. Sequences were aligned using Protein Scoring Matrix Pam 250 with a window size of 8, minimum% score of 60, and hash value of 2.
  • FIG. 5 is a photograph showing the identification of megsin-expressing cells in the glomerulus and the change in expression of megsin confirmed by semi-quantitative PCR using a rat anti-Thyl nephritis model.
  • FIG. 6 is a photograph showing changes in the expression of megsin mRNA in a rat anti-Thyl nephritis model by Northern blot analysis. Total A 10 was separated by electrophoresis and transferred to a nitrocellulose filter. Megsin mRNA was clearly detected on day 8. The labels on the left correspond to the sizes of 28S and 18S liposome RNA, respectively (5.1 and 1.9 kb, respectively).
  • FIG. 7 is a photograph showing the specificity of the anti-rat megsin antibody. Recombinant megsin or protein (2 g each) was analyzed by 10% SDS-PAGE, and immunoblot was performed using an anti-rat megsin antibody (lanes 1-3).
  • Lanes 1, 4, and 7 c-myc-histidine-labeled megsin derived from CH0 cells
  • Lane 4-6 Anti Rattomegushin antibodies plain-incubated Ichito synthetic rat Meg Shin peptide P 2 of excess
  • each recombinant protein is indicated by an arrow.
  • FIG. 8 is a photograph showing the results of immunohistochemical analysis of a kidney derived from an anti-Thyl nephritis model rat.
  • (B) shows that megsin protein is accumulated on day 8. Up to 2 days after induction of nephritis, there was no significant change in the expression of megsin protein. Megushi On the fourth day, the accumulation of nappa increased slightly. The mesangium region showed a marked positive reaction to megsin protein staining on day 8. Megsin protein accumulation increased until day 14 and returned to basal levels on day 28. X200 magnification.
  • FIG. 9 is a photograph showing the expression of megsin and sperm-smooth muscle actin using a series of sections. FITC-labeled anti-rat megsin antibody and anti-smooth muscle actin antibody were immunofluorescently stained. Megsin was mainly localized in the splenic-smooth muscle actin-positive region, but was also observed in other regions. BEST MODE FOR CARRYING OUT THE INVENTION
  • Rat Megsin protein A peptide containing cysteine at the C-terminus of the C-terminal of the 341-354 peptide from the N-terminus of the protein “H 2 NESNI-VEK-LLPES-TV-C-C00H / SEQ ID NO: 3” as a solid phase Synthesized by the peptide method (Perkin-Elmer, model 432A), purified by high-performance liquid chromatography, and purified by MBS (m-maleimidobenzoyl-N-hydroxylsuccinimide ester) to keyhole limpet to mosyanin (KLH: Calbio chem-Novabiochem).
  • a primary reaction was performed by adding 100 ⁇ of antiserum serially diluted to a 96-well plate on which 150 ng / well of antigen was immobilized to each well, followed by washing. ⁇ A heron IgG (manufactured by Institute for Immunochemistry) was reacted. After washing, color was developed using orthophenylenediamine (manufactured by Wako Pure Chemical Industries, Ltd.) as a substrate, and the absorbance was measured at 490 nm (SPECTRAmax250 manufactured by Nippon Molecular Devices). Table 1 shows the absorbance at each dilution ratio. Fig. 1 shows the measured values as a graph.
  • the antibody titer was measured 32 days, 46 days, 60 days, 79 days, and 95 days later. As a result, it was confirmed that the antibody titer was sufficiently increased. It was confirmed that the obtained antibody reacted with rat megsin protein by Western plot.
  • Example 2 Reactivity of anti-rat 'megsin antibody to human megsin protein
  • a polyclonal antibody against rat megsin synthetic peptide can be prepared by a known method (Cell Engineering Separate Volume Experimental Protocol Series, Anti-peptide Experimental Protocol, And purified by immunoaffinity chromatography according to the method described in Jun. The operation is as follows. The synthetic peptide was immobilized on FMP (2-fluoro-l-methylpyridinium toluene-4-sulfonate) activated cell mouth fine (manufactured by Seikagaku Corporation) to prepare an affinity column. The antibody was isolated from rat megsin as described in Example 1.
  • Egret serum with an increased antibody titer after immunization with the main peptide was diluted with PBS (-), and then affinity-purified using a peptide column.
  • the purified antibody obtained was reacted with the following sample using the estamp lot to confirm the antigen specificity (FIG. 2).
  • This antibody reacted slightly with human megsin, confirming strong reactivity with lysates of rat mesangial cells.
  • Lane 1 Human 'megsin protein fusion protein (W099 / 15652), Lane 2: Human megsin protein expressed in CH0 cells (CH0-megsin protein: T. Miyata et al., J. Clin. Invest. 120, 828-836, 1998, W0
  • Lane 3 Light 'mesangeum cell lysate'
  • Kidney tissue was collected from normal rats. Kidney tissue was embedded using a frozen tissue embedding medium (0. C. T. compound, Miles Laboratories) according to a conventional method. 4 / m frozen sections were prepared from the frozen-embedded tissues using a Frios kit. The frozen section was mounted on a slide coated with 3-aminopropyltriethoxysilane (manufactured by Sigma) (fixed with 4% paraformaldehyde, 15 minutes).
  • the frozen sections were washed with PBS containing 0.5% Tween20, blocked with 4% skim milk, and then incubated with an anti-rat megsin antibody in a humidified chamber at 4 ° C for 1 d.
  • the tissue section was washed and incubated at room temperature for 2 hours using a peroxidase-labeled goat anti-Peacock IgG antibody (manufactured by DAK0) diluted 1: 100.
  • a peroxidase-labeled goat anti-Peacock IgG antibody manufactured by DAK0
  • a 3,3-, diaminobenzidine solution containing 0.003% aqueous hydrogen peroxide was used.
  • Cell nuclei were stained with hematoxylin. Hematoxylin / eosin staining was performed by a known method.
  • FIG. 3 shows a micrograph (Nikon ECLIPSE E400: 80 ⁇ magnification) of tissue immunostaining of rat kidney tissue.
  • rat kidney glomerular tissue As can be seen from the figure, rat kidney glomerular tissue
  • remarkable positive staining was observed in the cells within the mesangial region and in the mesangial matrix, but not in the renal tubules.
  • MRNA was extracted from cultured rat mesangial cells at passage 14 using ISOGEN (Nippon Gene) and oligotex. This mRNA was subjected to a reverse transcription reaction with reverse transcriptase Superscript II (manufactured by GIBCO), and the obtained cDNA was designated as type III.
  • PCR was performed using DNA Thermal Cycler (Perkin Elmer Cetus) under the conditions of 94 ° C for 45 seconds (denaturation), 50 ° C for 45 seconds (annealing), 72 ° C for 2 minutes (amplification), and 35 cycles.
  • a PCR product close to the expected size (576 bp) was inserted into a pCRI I vector (Clonetech), and the nucleotide sequence was determined by the dideoxy method using a DNA autosequencer.
  • gene-specific primers were prepared from the cloned fragment of rat megsin, and degenerate PCR was performed again.
  • PCR was performed using RM-CtermCl and RM-MR-A2 under the conditions of 45 cycles of 94 ° C for 45 seconds, 55 ° C for 45 seconds, 72 ° C for 1 minute.
  • nested PCR was performed using RM-CtermCl and RM-MR-A2 under conditions of 94 ° C 45 seconds, 55 ° C 45 seconds, 72 ° C 1 minute, and 25 cycles. went.
  • PCR was repeated under the conditions of 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 40 seconds and 25 cycles using the same primers.
  • the obtained PCR product was incorporated into pGEM-T-easy vector (Promega).
  • the nucleotide sequence was determined by the dideoxy method using a DNA automatic sequencer.
  • the gene-specific sense primer RM-MR-S3 GAGGTCTCAGMGMGGCACTGAGGCAACTGCTGCC / sequence number: 15 was used for 3, -RACE. Based on the sequence thus obtained, the nucleotide sequence of the full-length cDNA of rat megsin consisting of 1229 bp shown in SEQ ID NO: 1 was finally determined.
  • RNA sequence CTCTATAGGAGACACTTGG / SEQ ID NO: 16 ( Sense primer 1) and 3, -UTR-A1: GAAACAAATCAAAGCAAAC / SEQ ID NO: 17 (antisense primer 1) were used.
  • PCR was performed under the following conditions: 45 seconds at 94 ° C (denaturation), 45 seconds at 50 ° C (annealing), 1 minute 30 seconds at 72 ° C (amplification), and 35 cycles. Of the expected size (about 1300bp) The PCR product was integrated into the pCRI I vector to isolate a clone containing the open reading frame of rat megsin.
  • Rat and mouse megsin contain 380 amino acids, and at the amino acid level were 75.3% and 73.9% identical to human megsin, respectively ( Figures 4A and 4A).
  • the present inventors evaluated changes in urinary protein excretion following mesangial injury using anti-Thyl nephritis, a representative model of mesangial proliferative glomerulonephritis in rats.
  • the 24-hour urine protein concentration was measured using a commercially available pyrogallol red kit (Wako Pure Chemical Industries, Tokyo, Japan), and the total protein amount in the 24-hour urine sample was calculated. Serum was separated from the blood sample by centrifugation and blood urea nitrogen (BUN) was measured using a commercially available Perylase-Indophenol kit (Wako).
  • the anti-Thyl-treated rats significantly increased urinary protein excretion compared to control animals (272 ⁇ 13.1 vs. 86.3 ⁇ 13.6 mg / kg body weight, p ⁇ 0.05 ).
  • the extent and histology of proteinuria in the kidneys of the anti-Thyl-treated animals returned to normal on day 28 (66.8 ⁇ 6.45 mg / kg body weight).
  • Example 8 Semi-quantitative RT-PCR of megsin mRNA in rat glomerular cells and anti-Thyl nephritis model rat
  • the present inventors extracted MA from glomeruli and performed semi-quantitative RT-PCR analysis.
  • CDNA was synthesized using SuperScript11 (Gibco BRL) according to the manufacturer's protocol, using l / g of total glomerular cells of cultured cells or total glomerular RNA of each rat.
  • the reverse transcription reaction mixture was used for subsequent semi-quantitative RT-PCR.
  • Rat megsin primers include rat megsin S27 forward primer corresponding to 34-65 bp (AGA ATT TGG CTT CGA CTT ATT CAG AGA GAT GG / SEQ ID NO: 4), rat megsin AS506 corresponding to 481-513 bp A reverse primer (ATG ACA GCT GAT GAG CTG AGG CTG CTG TCC CCC / SEQ ID NO: 5) was used. PCR was performed in which each cycle consisted of a denaturation at 94 ° C for 1 minute, an incubation at 60 ° C for 1 minute and an extension incubation at 72 ° C for 1 minute.
  • rat 5-actin protein primer corresponding to 1455 to 1484 bp (GTG TGA TGG TGG GTA TGG GTC AGA AGG ACT / SEQ ID NO: 6) and rat 5-actin reverse corresponding to 2318 to 2288 Primers (ATG GCA TGA GGG AGC GCG TAA CCC TCA TAG / SEQ ID NO: 7) were constructed.
  • the amplification product was visualized by agarose gel electrophoresis. Amplification of ⁇ -actin mRNA was used as a control for cDNA integrity, allowing approximately equal amounts of cDNA to be distributed for each megsin amplification reaction.
  • the actin and megsin mRNA amplification reactions were further tested to ensure that the number of cycles used was outside the reaction plateau, ie, within the range of increased amplification. This test was performed by visually observing the progress of each amplification reaction in 25 to 40 cycles. Therefore, the amount of cDNA used for each amplification was normalized to obtain a signal comparable to /?-Actin mRNA, and the reaction parameters were selected for detection outside the reaction plateau. Atsey was semiquantitative. Negative controls for RT-PCR include parallel amplification reactions lacking reverse transcriptase or lacking cDNA type II.
  • Multiple Tissue Northern Blots (trade name: manufactured by Clontech) was used. Multiple Northern Tissue Blots contain 2 ⁇ poly + RM from heart, brain, spleen, lung, liver, skeletal muscle, kidney and testis.
  • megsin could not be detected in all cases.
  • the up-regulation of megsin mRNA expression in rat anti-Thyl nephritis was confirmed again by Northern blot analysis in two separate experimental animal groups.
  • Kidneys were removed before (day 0) and 2, 4, 8, 14, and 28 days after treatment to perform Northern plots. At each time point, six rats were euthanized and the experiments in this set were performed twice. In order to separate glomeruli, one kidney was cut into pieces by a sieving method (Salant DJ, et al., J. Clin. Invest., 66: 71-81, 1980), and IS0GEN (manufactured by Nippon Gene) was used. MA was purified from the glomeruli isolated using.
  • RNA isolated from rats at each time point was combined, 10 zg of total RNA was separated by electrophoresis in a 1% agarose containing 2.2 mol / L formaldehyde denaturing gel, and then The trocellulose fill was transferred to the capillaries in the evening.
  • a partial cDNA of rat 'megsin of clone 27.2c was used.
  • the membrane was hybridized in Rapid Hyb (Amersham) solution at 65 ° C for 3 hours. The plot was washed at 60 ° C. in a final stringency of 0.1 ⁇ SSC / 0.1% SDS.
  • FIG. 6 shows a photograph of a representative Northern blot. Megsin mRNA expression was not detected at basal levels, but prolonged exposure detected only a small band of megsin on day 8. This supports the data from semi-quantitative RT-PCR. The band megsin c which was not observed at any point in the other Example 1 0] Immunohistochemical analysis
  • Proteins are not necessarily co-localized with mRNA.
  • megsin is a member of the SERPIN superfamily and may therefore act as a secretory factor, but its target is still unknown.
  • the present inventors formed a polyclonal antibody against a synthetic peptide of rat megsin and examined the localization of the megsin protein by an immunohistochemical method.
  • Antigenic synthetic peptides represent amino acids 341-353 of rat megsin and are conserved between humans and rodents but do not share sequence homology with other members of the SERPIN superfamily.
  • the treated tissues were stained with a 4- ⁇ m section using a periodic acid-Schiff reagent and double-stained with hematoxylin.
  • the number of glomerular cells and the number of proliferating cells per glomerular section were blindly counted. 30 glomeruli per rat randomly selected were examined, and the average number of cells per glomerulus was calculated. Glomerular sections containing only a small amount of glomerular tufts ( ⁇ 20 distinct capillary segments per section) were not used.
  • each glomerulus was semi-quantitatively graded (Yoshimura A., et al., J. Am. Soc. Nephrol., 9: 2027-2039, 1998). 0) no staining, 1; mesangial staining of 1-25% glomerular tufts, 2; positive staining 25-50% glomerular tufts, 3; positive staining 50- 75% glomerular tufts, 4;> 75% glomerular tufts are strongly stained.
  • mesangiolysis on day 2 showed a decrease in glomerular cells (59.3 ⁇ 1.9 cells per glomerulus versus 78.3 ⁇ 2.0 on day 0). Thereafter, on day 4 (69.5 ⁇ 3.2) and day 8 (92.6 ⁇ 2.9), total cells increased. On day 28, glomerular cell numbers returned to basal levels (76.5 ⁇ 3.4). Expression of splenic-smooth muscle actin in glomeruli was first seen on day 4 after injection of 0X-7 (1.6 ⁇ 0.2) and increased on day 8 (3.4 ⁇ 0.2). Low level expression of splenic-smooth muscle actin was also observed in glomeruli on day 28 (1.3 ⁇ 0.2).
  • Example 8 To perform immunohistochemical analysis, the kidney is removed as in Example 8, embedded in OCT (Lab-Tek Products, Miles Laboratories, Naperville, II), and quickly frozen in acetone using liquid nitrogen. To obtain frozen sections.
  • OCT Lab-Tek Products, Miles Laboratories, Naperville, II
  • Rat kidney tissue samples were sectioned at 4 ⁇ m and fixed with 4% paraformaldehyde in PBS. After washing with PBS, the sections were dehydrated with graded ethanol and incubated in methanol supplemented with 0.3% H202 at room temperature for 20 minutes to block endogenous oxidase. After washing three times with PBS containing 0 and 25% Tween 20, the sections were pre-incubated with blocking buffer (Block AceTM, Snow Bland Tokyo, Japan) at room temperature, and then the immunized rabbits, egrets, anti-ratmegsin IgG Alternatively, the cells were subjected to 1-incubation at 4 ° C together with pre-immune serum IgG.
  • blocking buffer Block AceTM, Snow Bland Tokyo, Japan
  • megsin deposition was mainly observed in the mesangium area.
  • megsin-positive cells were as low as control animals at all time points (data not shown).
  • Megsin staining was slight even on day 2, probably due to the progression of mesangiolysis.
  • Megsin accumulation increased slightly on day 4 and was most pronounced on day 8.
  • megsin deposition increased, some glomerular epithelial cells also showed positive staining. The increase in megsin deposition was evident in the 14th but returned to the 28th. Immunity Staining was negative when preserum was used or when the antibody was preincubated with an excess of megsin peptide ( Figure 8A).
  • megsin The relationship between megsin and mesangial cell activation was further investigated.
  • the present inventors used an anti-megsin antibody and an anti-smooth muscle actin antibody to identify the expression localization of sperm-smooth muscle actin, which is a marker for activation of megsin and mesangial cells.
  • an anti-megsin antibody and an anti-smooth muscle actin antibody to identify the expression localization of sperm-smooth muscle actin, which is a marker for activation of megsin and mesangial cells.
  • FITC-labeled 1A4 monoclonal antibody Sigma
  • FITC-labeled bushu anti-peregin IgG Dako was used at a 50-fold dilution.
  • Megsin was mainly localized in the splenic-smooth muscle actin-positive region, but was also observed in other regions (FIG. 9). Industrial applicability
  • Rat mesangial proliferative nephritis is representative of an artificially caused renal dysfunction, such as a reproductive and developmental toxicity test. Rats are said to be physiologically similar to humans as experimental animals.
  • a homolog of megsin rat which is an indicator of renal function in humans, is useful as a marker for mesangial proliferative nephritis. Therefore, it is considered that the behavior of megsin in rats can be extrapolated to humans as it is, and it can be said that it greatly enhances the value of toxicity tests using megsin as an index.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne une méthode permettant de diagnostiquer la néphrite proliférative à cellules mésangiales chez le rat, à l'aide de la protéine megsine comme indicateur dans un échantillon biologique. L'invention concerne également un anticorps et un réactif de dosage immunologique utilisés pour doser la protéine megsine du rat dans un échantillon biologique.
PCT/JP2000/009251 1999-12-28 2000-12-26 Methode permettant de diagnostiquer la nephrite proliferative a cellules mesangiales chez le rat WO2001048019A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU22260/01A AU2226001A (en) 1999-12-28 2000-12-26 Method of diagnosing mesangium proliferative nephritis in rat

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP11/373677 1999-12-28
JP37367799 1999-12-28

Publications (1)

Publication Number Publication Date
WO2001048019A1 true WO2001048019A1 (fr) 2001-07-05

Family

ID=18502577

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2000/009251 WO2001048019A1 (fr) 1999-12-28 2000-12-26 Methode permettant de diagnostiquer la nephrite proliferative a cellules mesangiales chez le rat

Country Status (2)

Country Link
AU (1) AU2226001A (fr)
WO (1) WO2001048019A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006121047A1 (fr) * 2005-05-09 2006-11-16 Tokai University Educational System Procede de determination d’une megsine dans un echantillon biologique
JP2010237001A (ja) * 2009-03-31 2010-10-21 Sysmex Corp 腎疾患診断支援装置およびコンピュータプログラム

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0583884A1 (fr) * 1992-07-17 1994-02-23 Suntory Limited Facteur de différentiation de mégakaryocytes
WO1999015652A1 (fr) * 1997-09-22 1999-04-01 Kurokawa, Kiyoshi Proteine megsine
WO2000057189A1 (fr) * 1999-03-19 2000-09-28 Kurokawa, Kiyoshi Procede de detection de proteine megsine et son utilisation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0583884A1 (fr) * 1992-07-17 1994-02-23 Suntory Limited Facteur de différentiation de mégakaryocytes
WO1999015652A1 (fr) * 1997-09-22 1999-04-01 Kurokawa, Kiyoshi Proteine megsine
WO2000057189A1 (fr) * 1999-03-19 2000-09-28 Kurokawa, Kiyoshi Procede de detection de proteine megsine et son utilisation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TOSHIO MIYATA ET AL.: "A mesangium-predominant gene, megsin, is a new serpin upregulated in IgA nephropathy", J. CLIN. INVEST., vol. 102, no. 4, 1998, pages 828 - 836, XP002937823 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006121047A1 (fr) * 2005-05-09 2006-11-16 Tokai University Educational System Procede de determination d’une megsine dans un echantillon biologique
JP2010237001A (ja) * 2009-03-31 2010-10-21 Sysmex Corp 腎疾患診断支援装置およびコンピュータプログラム

Also Published As

Publication number Publication date
AU2226001A (en) 2001-07-09

Similar Documents

Publication Publication Date Title
US8399207B2 (en) Monoclonal antibodies against osteopontin
KR100606388B1 (ko) 멕신단백질의 검출방법 및 그 용도
JP3259768B2 (ja) 腎疾患の検査方法
US5534431A (en) Hybridomas and monoclonal antibodies specific for unique determinants of nephropathy-related immunoglobulin G and complexes thereof
CA2088354C (fr) Anticorps specifique de l'elastase pancreatique 1; methode d'obtention et trousse d'essai renfermant des anticorps de ce type
JPH05501204A (ja) 糸球体腎炎の診断
JP2000515854A (ja) 脳タンパク質ｓ―１００の存在の測定方法
US11827696B2 (en) Antibody composition specifically recognizing an immunogenic fragment peptide of EN2 protein
US9291629B2 (en) Adiponectin receptor C-terminal fragments (CTF)-immunoglobulin
WO2001048019A1 (fr) Methode permettant de diagnostiquer la nephrite proliferative a cellules mesangiales chez le rat
US20070184485A1 (en) Method of diagnosing diseases relating to endometriosis
JP3870242B2 (ja) 腎疾患の検知法、診断薬及び診断用キット
US5196307A (en) Cloned human centromere autoantigen
JP2915530B2 (ja) ラミニン フラグメント
JP4327436B2 (ja) セミノジェリンの精子運動抑制因子（ｓｐｍｉ）部分を認識するモノクローナル抗体、及び、これを用いる検出方法
JP2022110557A (ja) Ａｈアミロイドーシス用の診断用抗重鎖抗体
WO2006121047A1 (fr) Procede de determination d’une megsine dans un echantillon biologique
JPH058678B2 (fr)
JPH06189786A (ja) 抗オキシトシン受容体抗体およびその製法
JPH03156369A (ja) ヒトアルブミンの測定方法
JP2005049343A (ja) 子宮内膜症関連疾患の診断方法
JP2000002703A (ja) ヒト補体制御因子の検出方法及びその用途
JP2005261244A (ja) 腎糸球体上皮細胞局在タンパク質特異性抗体

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
ENP Entry into the national phase

Ref country code: JP

Ref document number: 2001 548558

Kind code of ref document: A

Format of ref document f/p: F

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
DPE2 Request for preliminary examination filed before expiration of 19th month from priority date (pct application filed from 20040101)