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WO2006121047A1 - Procede de determination d’une megsine dans un echantillon biologique - Google Patents

Procede de determination d’une megsine dans un echantillon biologique Download PDF

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Publication number
WO2006121047A1
WO2006121047A1 PCT/JP2006/309326 JP2006309326W WO2006121047A1 WO 2006121047 A1 WO2006121047 A1 WO 2006121047A1 JP 2006309326 W JP2006309326 W JP 2006309326W WO 2006121047 A1 WO2006121047 A1 WO 2006121047A1
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Prior art keywords
megsin
antibody
monoclonal antibody
protein
biological sample
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Japanese (ja)
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Toshio Miyata
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Tokai University Educational System
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Tokai University Educational System
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/38—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against protease inhibitors of peptide structure
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy

Definitions

  • the present invention relates to a hyperpridoma Msl2a and a monoclonal antibody produced by the hyperidoma Msl2a.
  • the present invention also relates to a method for measuring megsin protein contained in a biological sample using the monoclonal antibody.
  • Renal failure is a pathological condition that ultimately leads to kidney disease patients. causes and history are not uniform.Non-natural kidney lesions such as drug addiction, infectious diseases, malignant tumors, diabetes, and systemic lupus erythematosus (SLE) can cause kidney damage and often lead to renal failure. Be looked at.
  • SLE systemic lupus erythematosus
  • Kidney transplantation is the only treatment for end-stage renal failure where the blood filtration and detoxification of the kidneys do not function at all.
  • Japan the system for supplying transplanted kidneys is well established. It's hard to say. Also, social awareness of transplantation therapy itself has not progressed.
  • the number of kidney transplant cases in Japan is only 700 cases per year, and this figure has not increased in recent years. Therefore, dialysis is the only treatment for renal replacement therapy.
  • the present inventors have paid attention to renal mesangial cells as a tissue deeply related to functions such as the onset and progress of renal diseases. It is well known that mesangial cells are organ-specific cells that are not found outside the kidney and play an important role in maintaining the structure and function of the glomeruli. Yes.
  • the present inventors considered that it is possible to detect a highly expressed gene group specific to mesangial cells by clarifying the profile of the gene group expressed in mesangial cells. From there, it is also possible to determine the gene group involved in the state of renal disease, and to find clues to elucidate the molecular mechanism of renal disease, making it possible to develop diagnostic methods and treatments for new renal diseases based on it. I thought. Therefore, the present inventors have clarified the gene expression pattern of mesangial cells and attempted to analyze the cell characteristics at the gene level.
  • Non-patent Document 1 First, for the purpose of quantitatively analyzing genes expressed in mesangial cells, the present inventors extracted cultured human mesangial cell force mRNA to prepare a 3'-directed cDNA library. Large-scale DNA sequencing and database analysis of gene fragments inserted into clones were performed (Non-patent Document 1).
  • megsin a gene consisting of 2,249 bp in total length, named megsin, was isolated as a gene that is particularly strongly expressed in mesangial cells. Then, we succeeded in isolating and obtaining megsin protein, a novel protein with the power of 380 amino acids that encodes the full-length cDNA clone of megsin.
  • amino acid homology search by FASTA program was performed using SwissProt amino acid sequence database.
  • amino acid sequence of the megsin protein includes the physiology of the serine protease inhibitor (SERPIN) superfamily (Non-Patent Documents 2 to 6). It was found that there is a sequence (EEGTEATAATZ SEQ ID NO: 3) similar to the consensus sequence (EEGTEAAAATZ SEQ ID NO: 2) in the reactive loop region that is important as the active center site.
  • SERPIN serine protease inhibitor
  • megsin has a structural feature of serpin and, like other serpins, there is a reactive loop region (P17-P5 ': EEGTEATAATGSNIVEKQLPQSZ SEQ ID NO: 4) that is an active site (non-patented). Reference 7). From these facts, it was clarified that the protein belongs to human megsin protein serine (Non-patent Document 7). A patent application was filed for these findings (Patent Document 1).
  • megsin has a biologically important effect on the function of mesangyum.
  • single genetic manipulation of megsin can generate early mesangial lesions present in experimental and human glomerulonephritis.
  • megsin has also been reported to be involved in the development of mesangial proliferative glomerulonephritis in animal individuals.
  • the present inventors considered that it is necessary to measure a specific protein closely related to a disease state in order to determine a definite diagnosis and severity of a renal disease. Therefore, the present inventors focused on the megsin gene and megsin protein, and established a renal function evaluation method comprising measuring megsin protein in a biological sample using a megsin peptide antibody (Patent Literature). 3).
  • Patent Document 1 International Publication No. 99/15652
  • Patent Document 2 International Publication No. 01/24628
  • Patent Document 3 International Publication No. 00/57189
  • Non-patent literature l Yasuda, Y. et al., Kidney Int., 53: 154-158 (1998)
  • Non-Patent Document 2 Carrell, R.W. et al., Trends Biochem. Sci "10: 20 (1985)
  • Non-Patent Document 3 Carrell, R.W. et al, Cold Spring Harbor Symp.Quant. Biol, 52: 527 (1987)
  • Non-Patent Document 4 Kruithof, E.K.O. et al., Blood, 86: 4007 (1995)
  • Non-Patent Document 5 Potempa, J. et al., J. Biol. Chem., 269: 15957 (1994)
  • Non-Patent Document 6 Remold-0'Donnell. E., FEBS Lett., 315: 105 (1993)
  • Non-Patent Document 7 Miyata, T. et al., J. Clin. Invest., 102: 828-836 (1998)
  • Non-Patent Document 8 Suzuki, D. et al "J. Am. Soc. Nephrol, 10: 2606 (1999)
  • Non-Patent Document 9 Nangaku, M. et al., Kidney Int., 60: 641 (2001)
  • Non-Patent Document 10 Miyata, T. et al., J. Clin. Invest., 109: 585 (2002)
  • Non-Patent Document l l Inagi, R. et. Al "Biochem Biophys Res Commun., 286: 1098-106 (20
  • An object of the present invention is to provide a megsin protein measurement method useful for diagnosis of renal dysfunction.
  • the present inventors have previously measured megsin protein in biological samples by ELISA. A method has been established (WO 00/57189). However, in the conventional measurement method, in order to measure megsin protein with sufficient detection sensitivity, it is necessary to perform pretreatment such as concentration of a biological sample. It was. It would be useful to provide a method that can directly measure a biological sample without requiring such pretreatment.
  • the present inventors obtained a monoclonal antibody that recognizes the megsin protein.
  • the inventors have found that megsin protein in a biological sample can be directly measured by using the monoclonal antibody in an ELISA method, and the present invention has been completed.
  • the present invention relates to the following monoclonal antibody produced by Ibridoma and a method for measuring megsin protein contained in a biological sample using the monoclonal antibody.
  • a method for producing a fragment containing a monoclonal antibody or an antigen-binding region thereof comprising culturing the hybridoma Msl2a deposited as FERM BP-10598 and recovering the immunoglobulin contained in the culture.
  • a method for measuring megsin protein contained in a biological sample comprising the following steps.
  • the present invention provides a method for directly measuring the megsin protein content in a biological sample.
  • the method of the present invention makes it possible to measure the megsin protein content even when a non-concentrated biological sample is used.
  • FIG. 1 is a diagram showing the reactivity of monoclonal antibody Msl2a with various serpins.
  • FIG. 2 shows the reactivity of monoclonal antibody Msl2a with various megsins.
  • FIG. 3 shows the reactivity of monoclonal antibody Msl2a with plasmin.
  • FIG. 4 shows the reactivity of monoclonal antibody Msl2a with human urine.
  • the upper figure shows the case of detection using the monoclonal antibody Msl2a, and the lower figure shows the case of detection using only the secondary antibody.
  • FIG. 5 shows the reactivity of monoclonal antibody Msl2a with human plasma.
  • the left figure shows the case of detection using the monoclonal antibody Msl2a, and the right figure shows the case of detection using only the secondary antibody.
  • FIG. 6 is a diagram showing a calibration curve when urinary megsin is detected by ELISA.
  • the vertical axis represents absorbance, and the horizontal axis represents megsin concentration (ng / ml).
  • FIG. 7 shows the results of examining the change in reactivity due to pH when a monoclonal antibody Msl2a was reacted with a urine sample.
  • FIG. 8 ELISA measurement of urinary megsin levels in normal subjects and patients with various renal diseases.
  • FIG. The vertical axis shows the megsin concentration per creatine lg (g / g Cr), and the horizontal axis shows the type of kidney disease.
  • the present inventors have found that it reacts with the monoclonal antibody-potential megsin protein produced by Hypridoma Msl2a. That is, the present invention relates to a monoclonal antibody produced by Hypridoma Msl2a and a method for measuring megsin protein contained in a biological sample using the monoclonal antibody.
  • a monoclonal antibody against megsin can be obtained by a known method using human megsin or its domain peptide as an immunogen. The method for obtaining the monoclonal antibody will be specifically described later.
  • Hybridoma Msl2a is 1st chome, Tsukuba Sagahito, Ibaraki, Japan, February 9, 2005
  • the H chain isotype of the monoclonal antibody produced by this hyperidoma Msl2a cell line was IgGl.
  • the present invention also encompasses class switch variants of the above antibodies, such as variants belonging to isotype IgG3, IgGl, IgG2b, IgG2a and other immunoglobulin subclasses, such variants are described by Martin et al. It can be made by a method (Martin. C. et al .: J. Immunol. Methods., 145: 1118, 1991).
  • fragment containing an antigen-binding region means a fragment having a partial force containing the antigen-binding region of a monoclonal antibody.
  • F (ab '), Fab', Fab, Fv (v) means a fragment having a partial force containing the antigen-binding region of a monoclonal antibody.
  • Antibody fragments such as /, ⁇ or dA b (single domain antibody) are included in the “fragment containing the antigen-binding region”.
  • F (ab ') and Fab' refer to immunoglobulin (monoclonal antibody), protein component
  • Pepsin which is a degrading enzyme, means an antibody fragment produced by treatment with nopain or the like and digested before and after disulfide bonds existing between two H chains in the hinge region.
  • V L chain variable region
  • C L chain constant region
  • a fragment containing an antigen-binding region can also be obtained by expressing DNA encoding the fragment.
  • cDNA encoding the antigen-binding region of an antibody can be amplified by PCR using the mRNA of the hyperidoma secreting the monoclonal antibody of the present invention as a saddle type.
  • the antigen-binding region of an antibody is composed of a highly variable complementarity determining region (CDR) and a relatively conserved frame region (FR)! Four FRs are arranged across three CDRs. Therefore, a cDNA encoding the entire variable region can be amplified using a primer encoding the N-terminal FR and a primer having a base sequence close to the variable region of the constant region and complementary to the portion.
  • the amino acid sequence encoded by the cDNA thus recovered may have a different length from the antibody fragment obtained by the enzymatic digestion described above. Furthermore, each molecule of VL and VH can be expressed as a single peptide via a linker to obtain scFv.
  • the structure of scFv is different from natural antibodies. However, if it contains an amino acid sequence including CDR and FR, the ability to bind to an antigen is maintained. Therefore, even fragments that have different amino acid sequence capabilities from antibody fragments obtained by enzymatic digestion, or fragments that have different structures, can be used as antibodies as long as they maintain the ability to bind antigen. can do. That is, such an antibody fragment is also included in the fragment containing the antigen-binding region of the present invention.
  • the antibody fragment containing the antigen-binding region in the present invention can be bound to a labeling substance, an affinity substance, or can be used as a fusion protein, as long as the antigen-binding ability is maintained.
  • labeled antibody fragments and fusion proteins are included in the monoclonal antibody of the present invention.
  • a monoclonal antibody against megsin protein can be prepared using megsin protein as an immunogenic antigen.
  • Megsin protein as an antigen can be obtained using cultured cells, for example, megsin protein-producing cells. Examples of megsin protein-producing cells include human kidney-derived cells. This megsin protein-producing cell is cultured in a culture medium or culture medium known in the art or substantially similar to them, and megsin protein produced in the culture supernatant is ionized, for example. It can be purified by exchange chromatography and affinity chromatography using Z or polyclonal antibodies.
  • a recombinant megsin protein can also be used. Specifically, after transforming a host cell with a recombinant vector containing a gene fragment containing a base sequence encoding the amino acid sequence of megsin protein, this transformed host is cultured to obtain the amino acid sequence of megsin protein. Is produced, and the polypeptide is used as an immunogen.
  • Recombinant vectors containing megsin cDNA are prepared by ordinary gene recombination techniques, for example by insertion into plasmid vectors. As vectors, viruses such as vaccinia virus and baculovirus can be used in addition to plasmids and phages.
  • Examples of hosts include prokaryotes such as Escherichia coli, Bacillus subtilis, and actinomycetes, and commercially available cell lines such as various cells such as animal cells and CHO cells, and eukaryotes such as yeast, plant cells, and insect cells. Can be used.
  • Examples of promoters that can be used in prokaryotes include tributophan synthase operon and ratatosoperon.
  • promoters that can be used in eukaryotes include viral promoters, promoters for alcohol dehydrogenase, and promoters for glycolytic enzymes. Etc.
  • vectors or plasmids having a multicloning site, promoter, resistance gene, replication origin, terminator, ribosome binding site, etc. can also be used. Resistance genes include those for tetracycline, ampicillin, and neomycin.
  • the megsin protein thus prepared may be further used as an immunogenic conjugate, but can be used as it is to immunize animals by mixing it with an appropriate adjuvant.
  • antigens can be obtained from various raw materials such as cultured cells, cultured tissues, transformed raw materials such as transformed cells, and conventionally known methods, for example, salting out such as ammonium sulfate precipitation method, gels by sephadetus etc. Purification by filtration chromatography, ion exchange chromatography, hydrophobic chromatography, dye gel chromatography, electrophoresis, dialysis, ultrafiltration, affinity chromatography, high performance liquid chromatography, etc. You can get it.
  • the megsin protein is a fragmented product thereof, or a characteristic sequence region is selected based on the amino acid sequence of the megsin protein, the polypeptide is designed and chemically synthesized, and the obtained polypeptide fragment. It may be.
  • Polypeptide fragments can be combined with various carrier proteins via an appropriate condensing agent to form hapten-protein immunoconjugates, which can be used to obtain monoclonal antibodies that recognize specific sequences. Cysteine residues and the like can be added in advance to the polypeptide to be designed to facilitate preparation of an immunogenic conjugate.
  • the present invention provides at least one monoclonal antibody that specifically binds to megsin protein.
  • the monoclonal antibody used in the present invention is obtained by immunizing an animal using recombinant megsin as an immunogen, cell fusion between myeloma cells and antibody-producing cells, selection and monocloning of a hyperidoma, production of a monoclonal antibody, If necessary, it can be produced by a process such as ascites.
  • immunization of animals is performed as follows. Mammals such as rats and mice are immunized with human megsin protein purified according to a known method (Miyata, T. et al., J. Clin. Invest., 120: 828 (1998)). It is preferable to use an animal of the same strain as the partner's permanent proliferating cell for cell fusion. The age of animals is, for example, 8-10 weeks for mice. Is preferred. Sex may be either male or female.
  • a human immunoglobulin can be produced by using a transgenic animal in which an immunoglobulin gene is recombined with a human gene.
  • a method for obtaining a target reactive antibody using a transgenic animal in which the immunoglobulin gene is recombined with a human gene is known.
  • Immunoglobulins that can be obtained in this way are fully human immunoglobulin molecules, although they have gained animal power.
  • the immunization is carried out by mixing purified human megsin protein with an appropriate adjuvant (for example, Freund's complete adjuvant or hydroxyaluminum gel-pertussis vaccine, etc.) to form an emulsion, and then subcutaneously or abdominally Intravenous or intravenous administration. Thereafter, this immunization operation is performed 2 to 5 times at intervals of 1 to 2 weeks.
  • Final immunization is carried out by administering 0.5 to 2 g of human megsin protein into the peritoneal cavity of animals.
  • Polyclonal antibodies are obtained from the body fluids of animals immunized in this manner. Three to seven days after each immunization, blood is collected from the fundus venous plexus, and the antibody titer of the serum is measured. When the antibody titer rises sufficiently, antibody or antibody-producing cells are collected. The antibody titer against megsin can be measured by a technique such as ELISA. ELISA for measuring the antibody titer can be performed by adding serum to a plate coated with megsin and further collecting the labeled antibody against IgG of the immunized animal.
  • Examples of the adjuvant used together with the antigen include Freund's complete adjuvant, Ribi adjuvant, pertussis vaccine, BCG, ribosome, aluminum hydroxide, silica gel and the like.
  • animals such as mice such as Balb / c mice and FI mice can be used.
  • antibody-producing cells are also collected from animal forces immunized with human megsin protein.
  • Antibody-producing cells can be obtained from the spleen, lymph nodes, peripheral blood, etc., and the spleen is particularly preferable.
  • the spleen is aseptically removed, shredded in Minimal Essentia 1 Medium (MEM) medium (manufactured by Nissui Pharmaceutical), disassembled with tweezers, and l, 200rpm x 5 minutes
  • MEM Minimal Essentia 1 Medium
  • the tumor cell line used prior to cell fusion can be selected, for example, from cell lines that do not produce immunoglobulins.
  • the tumor cell line used prior to cell fusion can be selected, for example, from cell lines that do not produce immunoglobulins.
  • the ability to use any cell having permanent proliferation is generally used.
  • Myeloma cells myeloma cells
  • Permanently proliferating cells are preferably derived from the same animal species as the antibody-producing cells.
  • mice the following cell lines are known as bone tumor cell lines derived from 8-azaguanine resistant mice (Balb / c).
  • P3-X63Ag8-Ul P3-U1 (Current. Topics in Microbiol. Immunol, 81: 1, (1978))
  • SP2 / 0-Agl4 SP-2 / 0-Agl4 (SP-2) (Nature, 276: 269 (1978))
  • Cell fusion between antibody-producing cells and myeloma cells is performed, for example, as follows.
  • the antibody-producing cells and the permanently proliferating cells obtained above are thoroughly washed with MEM medium or PBS, and mixed so that the number of cells is 5 to 10: 1.
  • HAT medium 100 ⁇ L / well to the culture plate (normal medium hypoxanthine (10- 4 M), thymidine (1.5 X 10- 5 M) and aminopterin (4 X 1 0 "7 M ) medium supplemented with) Incubate for another 3 days, remove half the volume of the culture supernatant every 3 days, add the same amount of HAT medium, and in a 5% CO incubator at 37 ° C.
  • a hyperidoma culture supernatant is added to a solid phase on which megsin protein antigen is adsorbed directly or together with a carrier, and then an anti-immunoglobulin antibody labeled with a radioactive substance or an enzyme is added.
  • the antibody titer can be measured by measuring the label.
  • a microplate or the like is used for the solid phase.
  • the anti-immunoglobulin antibody when the cell used for cell fusion is a mouse, an anti-mouse immunoglobulin antibody is used.
  • protein A can be added in place of the labeled antibody, and the anti-megsin protein monoclonal antibody bound to the solid phase can be detected.
  • the antibody titer is measured by adding hypridoma culture supernatant to a solid phase adsorbed with anti-immunoglobulin antibody or protein A, and adding megsin protein labeled with a radioactive substance or enzyme. Monkey.
  • the monoclonal antibody is produced by culturing the hyperidoma obtained as described above in vitro and in vivo.
  • the desired monoclonal antibody can be cultured in a suitable medium such as an FCS-containing MEM medium or RPMI-1640 medium, and the culture supernatant strength can be obtained.
  • a suitable medium such as an FCS-containing MEM medium or RPMI-1640 medium
  • In vitro culture of the hyperidoma is preferably performed in a serum-free medium, and an optimal amount of antibody is given to the supernatant.
  • any animal is transplanted with a hybridoma.
  • the host animal for transplantation is preferably an animal of the same kind as the animal from which the spleen cells used for cell fusion were collected.
  • 2 to 4 ⁇ 10 6 anti-megsin protein monoclonal antibody-producing hybridoma cells obtained above are intraperitoneally administered to 8-10 week old Balb / c female mice treated with pristane.
  • the pristane treatment is performed, for example, by intraperitoneally administering 2,6,10,14-tetramethylpentadecane-pristane-0.5 mL and rearing for 2 weeks.
  • Ascites or culture supernatant is salted with 50% ammonium sulfate and dialyzed against PBS for 1-2 weeks.
  • the dialysis fraction is passed through a protein A sepharose column, and the IgG fraction is collected to obtain a purified monoclonal antibody. This monoclonal antibody reacts specifically with the megsin protein.
  • the antibody isotype a commercially available kit (Gibco BRL, Mouse Antibody Isotyping Kit, etc.) is used, or the Octa Mouth (double immunodiffusion) method (introduction to immunological experiments, biochemical experimental methods 15, academic societies) Published by Publishing Center, p. 74, 1981).
  • the amount of protein is calculated by the phosphorin method and absorbance at 280 nm (1.4 (OD280) immunoglobulin 1 mg / mL).
  • hybridoma In order to obtain a large amount of monoclonal antibody, ascitic fluidization of hybridoma can be used.
  • each hybridoma is transplanted into the abdominal cavity of an animal having the same tissue compatibility as that of an animal derived from myeloma cells and allowed to proliferate, or each mouse and ibridoma are transplanted into nude mice, etc.
  • Monoclonal antibodies can be obtained.
  • the animal can be administered intraperitoneally with a mineral oil such as pristane before transplanting the hyperpridoma.
  • Ascites fluid can be purified as it is or by a conventional method. For example, salting out such as ammonium sulfate precipitation, gel filtration using cefadex, ion exchange chromatography, electrophoresis, dialysis, ultrafiltration, and affinity chromatography. 1. It can be purified by high performance liquid chromatography. The characteristics of the monoclonal antibody obtained as described above can be clarified by, for example, enzyme immunoassay (ELISA method) or the like.
  • ELISA method enzyme immunoassay
  • the monoclonal antibody of the present invention can be used for immunostaining such as tissue or cell staining, immunoprecipitation, immunoblotting, immunoassay, such as competitive or noncompetitive immunoassay, radioimmunoassay, ELISA, latex agglutination method, protein It can be used for purification, affinity column, etc.
  • immunoassay includes all methods utilizing immunological reactions such as immunohistological examination, immunoblotting, and immunoprecipitation.
  • the present invention also provides a hyperidoma cell line, an immunoassay and a test kit. Furthermore, the present invention provides a monoclonal antibody that specifically recognizes megsin protein, an immunoassay for the detection and quantification of megsin characterized by using this antibody, and a test kit for performing this immunoassay. I will provide a.
  • the monoclonal antibody obtained by the present invention is very useful for detection and quantification of megsin having high specificity for megsin.
  • the present invention also provides a method for measuring megsin protein comprising the following steps.
  • an anti-megsin polyclonal antibody that binds to or binds to a solid phase or a fragment that includes an antigen-binding region thereof, an anti-megsin monoclonal antibody that binds a labeled molecule, or a fragment that includes an antigen-binding region thereof And contacting the biological sample
  • ii) a step of detecting the labeled molecule bound to the megsin protein via the anti-megsin monoclonal antibody bound to the labeled molecule.
  • the origin and preparation method of the antibody necessary for the immunological measurement method of mengsin protein is not limited as long as it can recognize the megsin protein to be detected. Therefore, a polyclonal antibody, a monoclonal antibody, or a mixture thereof can be used.
  • the antibody used in the present invention includes, for example, an antibody against a protein having the amino acid sequence set forth in SEQ ID NO: 1.
  • An antibody against megsin protein or a partial amino acid sequence thereof (for example, a polyclonal antibody or a monoclonal antibody) or antiserum is an oligopeptide containing megsin protein or a partial amino acid sequence thereof.
  • a fusion protein such as a protein as an antigen
  • it can be produced according to a method for producing an antibody or antiserum known per se.
  • a monoclonal antibody can be produced according to the method described above.
  • a synthetic peptide having a partial amino acid sequence is used as an immunogen, it is generally advantageous to use an amino acid sequence that is as specific as possible to megsin protein and has a high hydrophilicity.
  • the megsin protein of the present invention or a synthetic peptide having a partial amino acid sequence of the megsin protein of the present invention is administered to a warm-blooded animal itself or together with a carrier and a diluent.
  • Synthetic peptides are used as immunogens that are bound to carrier proteins such as cythyroglobulin or keyhole limpets and mocyan.
  • carrier proteins such as cythyroglobulin or keyhole limpets and mocyan.
  • it can be administered with complete Freund's adjuvant or incomplete Freund's adjuvant. Administration is usually once every 1 to 6 weeks, 2 to 10 times in total.
  • warm-blooded animals examples include monkeys, rabbits, dogs, guinea pigs, mice, rats, hidges, goats, and chickens. Usagi is preferably used when the monoclonal antibody is a labeled antibody.
  • a monoclonal antibody or a polyclonal antibody that recognizes megsin protein is used in the method for measuring megsin protein of the present invention.
  • the sandwich complex formed by reacting megsin protein with an antibody bound to an insoluble carrier and a labeled antibody bound to a labeled molecule is detected.
  • the labeled human urine-derived megsin protein and the human urine-derived megsin protein in the sample are reacted competitively with the antibody, and the amount of labeled antigen reacted with the antibody determines the human urine-derived megsin protein in the sample. It is possible to measure human urine-derived megsin protein in a specimen using a competitive method for measuring urine.
  • a preferred assembly system in the present invention is a sandwich method.
  • the immobilized antibody is reacted with human urine-derived megsin protein, then unreacted substances are completely removed by washing, and the labeled antibody is removed.
  • a one-step method that sometimes mixes can be used.
  • insoluble carriers used in the measurement include polystyrene, polyethylene, polypropylene, polyvinyl chloride, polyester, polyacrylic ester, nylon, polyacetal, synthetic resins such as fluorinated resin, polysaccharides such as cellulose and agarose, Examples include glass and metal.
  • 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, and a test tube can be used.
  • the carrier on which the antibody is adsorbed is stored in a cool place where appropriate in the presence of a preservative such as sodium azide.
  • Examples of the chemical bonding method include a method using dartalaldehyde, N-succinimidyl-
  • maleimide method using 4- (N-maleimidomethyl) cyclohexane-1-carboxylate and N-succinimidyl-2-maleimide acetate, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride And the carbodiimide method using the above.
  • Other examples include the maleimidobenzoyl-N-hydroxysuccinimide ester method, the N-succimidyl-3- (2-pyridyldithio) propionic acid method, the bis-diazobenzidine method, and the dipalmityllysine method.
  • a complex formed by reacting two types of antibodies different from the substance to be detected and the epitope may be immobilized by immobilizing the third antibody against the antibody by the above method. Is also possible.
  • the monoclonal antibody of the present invention can be used as a labeled antibody for use in immunoassay.
  • enzymes, enzyme substrates, coenzymes, enzyme precursors, apoenzymes, fluorescent materials, dye materials, chemiluminescent compounds, luminescent materials, chromogenic materials, magnetic materials, metal particles, radioactive materials, etc. can be used.
  • a reaction between a thiol group and a maleimide group a reaction between a pyridyl disulfide group and a thiol group, a reaction between an amino group and an aldehyde group can be used.
  • Preferred labeling enzymes include, for example, peroxidase, alkaline phosphatase, ⁇ -D-galactosidase, malate dehydrogenase, staphylococcal nuclease, delta-
  • 5-steroid isomerase ⁇ -glycerol phosphate dehydrogenase, triosephosphate isomerase, horseradish peroxidase, wasparaginase, darcosoxidase, ribonuclease, urease, catalase, glucose-6-phosphine Examples thereof include ate dehydrogenase, darcoamylase, and acetylcholinesterase.
  • fluorescent substances include, for example, fluorescein isothiocyanate, phycobylprotein, rhodamine, phycoerythrin, phycocyanin, aloficocyanin, and orthophthalaldehyde.
  • the luminescent substance include isoluminol, lucigenin, luminol, aromatic atheridi-um ester, imidazole, atalidum salt and its modified ester, luciferin, luciferase, and equorin.
  • Preferred radioactive materials include 125 I, I, m I, “C, 3 H, 32 P, or 35 S.
  • the direct labeling is generally a method in which an antibody or antibody fragment and a label are covalently covalently bound with a crosslinking agent.
  • Cross-linking agents include ⁇ , ⁇ '-orthophenol-di-maleimide, 4- ( ⁇ -maleimidomethyl) cyclohexanoic acid ⁇ -succinimide ester, 6-maleimidohexanoic acid ⁇ -succinimide ester, 4,4 '-Dithiopyridine and other known crosslinking agents can be used.
  • cross-linking agents with enzymes and antibodies may be carried out 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, dinitrophenol, pyridoxal, or fluoresamine is bound to the antibody and indirectly labeled with a binding component that recognizes it can also be employed.
  • Avidin and streptavidin are used as recognition ligands for piotin.
  • dinitrophenol, pyridoxal or fluoresamine antibodies that recognize these haptens are 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 antibodies by the periodate method.
  • an antibody is used as an antibody, for example, Fab ′, Fab, F (ab ′).
  • Polyclonal antibodies and monoclonal antibodies are used as an antibody, for example, Fab ′, Fab, F (ab ′).
  • an enzyme label can be obtained by the same treatment. If the enzyme label obtained using the above-mentioned crosslinking agent is purified by a known method such as affinity chromatography, a more sensitive immunoassay system can be obtained. Purified enzyme-labeled antibodies include thimerosal as a preservative and glycerin as a stabilizer. Add and save. The labeled antibody can be stored for a longer period by lyophilization and storage in a cool and dark place.
  • the labeling agent is an enzyme
  • a substrate and, if necessary, a coloring agent are used to measure the activity.
  • H 0 is used as the substrate solution
  • 2,2′-azino-di- [3-ethylbenzthiazolinesulfonic acid] a is used as the color former.
  • ammonium salt ABTS
  • 5-aminosalicylic acid 2-aminosalicylic acid
  • ortho-phendiamine 4-aminoantipyrine
  • 3,3 ′ 5,5′-tetramethylbenzidine and the like
  • alkaline phosphatase is used as the enzyme
  • ortho-trifluorophosphate para-trifluorophosphate, or the like
  • fluorescein-di- j8-D-galatatopyranoside
  • 4-methylumbelliferyl-iS-D-galactopyranoside etc.
  • the present invention also provides a reagent for immunoassay of megsin protein by labeling the above-mentioned monoclonal antibody and immobilizing a polyclonal antibody, and further, an indicator for detecting the label, a control sample, etc. It also includes those that are kitty.
  • the immunoassay using the monoclonal antibody of the present invention can use any form of solution, colloid solution, non-fluid sample, or the like as a specimen or sample.
  • biological samples specifically blood, plasma, joint fluid, cerebrospinal fluid, saliva, amniotic fluid, urine, other body fluids, cell culture fluid, tissue culture fluid, tissue homogenate, biopsy sample, cell, tissue.
  • Examples include brain tissue, brain-derived cell line, nerve cell line, nerve-derived cell line, breast-derived cell line, breast tissue, ovarian-derived cell line, ovarian tissue, cancer cell line, and cancer tissue.
  • a particularly preferred sample is urine.
  • the present invention by using a polyclonal antibody as a solid-phase antibody and a monoclonal antibody as a labeled antibody, it was possible to measure megsin in concentrated urine.
  • the monoclonal antibody according to the present invention produced by Hypridoma Msl2a is used as a labeled antibody, megsin can be measured with high sensitivity using unconcentrated urine as a sample.
  • the present invention provides a method for measuring megsin protein contained in a biological sample, comprising the following steps. i) An anti-megsin polyclonal antibody that binds to or binds to a solid phase, or a fragment that includes an antigen-binding region thereof, an anti-megsin monoclonal antibody that binds a labeled molecule, or an antigen-binding region thereof A step of contacting the fragment and the biological sample; and ii) detecting the labeled molecule bound to the megsin protein through the anti-megsin monoclonal antibody bound to the labeled molecule, and detecting the megsin concentration of the biological sample. Determine the process
  • the labeled molecule detected in the step ii) is associated with the megsin concentration based on the detected amount of the labeled molecule detected using a standard sample with a known megsin concentration in advance.
  • the detected amount of the labeled molecule and the megsin concentration can be determined using, for example, a calibration curve.
  • a calibration curve is created by plotting the detected amount of labeled molecule obtained by measuring a dilution series of megsin.
  • a calibration curve is also called a standard curve.
  • Creathun correction is a technique for correcting the influence of dilution (or concentration) of the measurement target component due to fluctuations in urine volume based on the Creatun concentration. Based on the fact that the amount of creatine excreted in urine per day is constant, the percentage of total urine excretion in one day is calculated from the concentration of creatine and measured from the same urine.
  • the concentration of the target component can be converted into total excretion per day.
  • Weight correction is a technique for calculating the amount of blood components based on the estimated body volume of the blood sample.
  • the megsin measurement method of the present invention it is possible to diagnose a disease accompanied by a change in megsin concentration in a biological sample. Specifically, a significant increase in urinary megsin concentration was confirmed in diseases such as chronic glomerulonephritis including IgA nephropathy, rapidly progressive glomerulonephritis, diabetic nephropathy, and chronic renal failure.
  • diseases such as chronic glomerulonephritis including IgA nephropathy, rapidly progressive glomerulonephritis, diabetic nephropathy, and chronic renal failure.
  • the present invention provides a method for diagnosing a disease accompanied by a change in megsin concentration in a biological sample including the following steps.
  • 0An anti-megsin polyclonal antibody that binds to or binds to a solid phase or a fragment that includes an antigen-binding region thereof, an anti-megsin monoclonal antibody that binds a labeled molecule, or a fragment that includes an antigen-binding region thereof And ii) detecting the labeled molecule bound to the megsin protein via the anti-megsin monoclonal antibody bound to the labeled molecule, and determining the concentration of megsin contained in the biological sample. Associating process; and
  • the eluate was concentrated by centrifugation using Centricon 10 (Millipore) (3000 g), and the buffer was exchanged with Dulbecco's PBS (-) buffer (Nissui Pharmaceutical). Centrifugation and buffer exchange were repeated three times to obtain purified recombinant megsin.
  • the chromatographic apparatus used was AKTA explorer 10s (Amersham Bioscience), and all operations were performed at 4 ° C.
  • mice (6-week-old male: CLEA Japan) were acclimated for 1 week, then 50 g of purified megsin per mouse was dissolved in 250 ⁇ 1 of PBS and 250 ⁇ 1 Freund's complete adjuvant (Diffco) The mixture was mixed well and prepared into an abdominal cavity. Two weeks later, the same concentration of megsin dissolved in PBS and 250 ⁇ l Freund's incomplete adjuvant (Diffco) were mixed well to prepare an emulsion and administered intraperitoneally. After repeating this three times, the mouse was opened and the spleen was removed.
  • Diffco Freund's complete adjuvant
  • Hybridomas were prepared using Clonacell-HY Hybridoma cloning Kit (manufactured by Stem Cell Technology).
  • the spleen cells were removed from the spleen that had been opened and washed, and then washed with a fusion medium.
  • cultured SP2 cells were washed with a fusion medium, 20 million cells were taken and added to a centrifuge tube containing spleen cells. This was mixed well and fused using polyethylene glycol.
  • the fused cells were cultured in a 75 cm 2 culture flask for 1 day. Cells were collected after 1 day of culture, suspended in 100 ml of selective medium warmed to 37 ° C, and placed in a 10 cm dish. When the cells grew and colony formation became visible, they were blotted using a chip and transferred to a 96-well plate containing growth medium. 3-4 days after transplantation Remove culture supernatant and screen Used for
  • Hypridoma screening ELISA method (direct method)
  • Purified megsin was adjusted to 1 ⁇ g / ml with PBS and adsorbed overnight at 4 ° C. on an ELISA plate at 100 ml / well. After washing 3 times with a washing solution (PBS containing 0.05% Tween 20), blocking was performed with Block Ace diluted 4-fold with PBS. Thereafter, the cells were washed, and each 100 I / well of high-pridoma culture supernatant was added and reacted at room temperature for 2 hours.
  • a washing solution PBS containing 0.05% Tween 20
  • peroxidase-labeled mouse IgG antibody (Chemicon) diluted 5000 times with antibody diluent (Block Ace diluted 10-fold with PBS) was added at 100 / zl / well and reacted at room temperature for 2 hours.
  • Dissolve 0-phenolamine in a substrate reaction solution (0.2 1 / ml citrate-phosphate buffer solution containing hydrogen peroxide / hydrogen peroxide, pH 5.0) to a concentration of 0.4 mg / ml. was prepared.
  • the plate was washed 5 times with the washing solution, and the substrate solution was added to 100 I / well. After reacting the substrate solution for 30 minutes, 2N sulfuric acid was stopped at 100 ⁇ l / well and the absorbance at 490 nm was measured.
  • Msl2a had an OD490 of 0.524, which was higher than other clones and showed a value. Cloning was performed to obtain a single clone cell line.
  • Hyperidoma producing antibody that binds to megsin protein Repeated cloning by limiting dilution method three times to produce antibody that specifically binds to megsin protein, and has stable growth ability
  • the dormer Msl2a cell line was obtained.
  • Purified megsin was adjusted to 40 ng / lane with PBS, mixed with an equal volume of 2 X loading buffer and heated in a boiling bath for 5 minutes to obtain a sample solution.
  • the sample solution was electrophoresed on 10-20% polyacrylamide gel using an electrophoresis apparatus and Tris-glycine buffer.
  • the polyvinylidene difluororide membrane was immersed in methanol and then immersed in purified water. Transfer of protein to PVDF membrane is accomplished by removing the gel from the device after electrophoresis. Take out the filter paper from the anode side on the blotter, place two filter papers soaked in buffer A1 of the electroblot buffer, one filter paper soaked in buffer A2, PVDF membrane, gel and three filter papers soaked in buffer C in this order. Transferred with 80 mA I gel for 1.5 hours. After transfer, the PVDF membrane was blocked by shaking with Block Ace for 1 hour at room temperature. The membrane was then reacted overnight at 4 ° C with the hybridoma culture supernatant. After washing with a washing solution, alkaline phosphatase-labeled anti-mouse IgG antibody was added and reacted at room temperature for 2 hours. After washing with a washing solution, color was developed with an NBT-BCIP solution.
  • Msl2a showed reactivity with megsin protein under both SDS-PAGE 2ME +, 2ME- and native-PAGE conditions.
  • Purified megsin g / ml 200 1 was bound to CM5 chip by amino coupling method. Free active groups were blocked with ethanolamine. The Ms 12a monoclonal antibody obtained by screening was bound and the amount bound was measured.
  • Msl2a showed sufficient binding to megsin protein even in the case of Biacore, which has a three-dimensional structure compared to other methods.
  • Balb / c mice (8-week-old females) were intraperitoneally administered 0.5 ml / pristane, and 10 days later, about 10 7 cells of the high-pridoma Msl2a cell line obtained in the above c. A few / 0.5ml / animal was injected into the abdomen. About 10 days later, as the force was also found to be abdominal hypertrophy of the mouse, the abdomen was opened and ascites collected. The collected ascites was centrifuged at lOOOOrpm and 4 ° C for 10 minutes, and the supernatant was allowed to stand at 37 ° C for 30 minutes and then left at 4 ° C.
  • the resulting supernatant was purified using a protein column Protein A Sepharose (Amersham Bioscience) to purify the monoclonal antibody (Msl2a).
  • the absorbance of this antibody solution at 260, 280 and 320 nm was measured, and the antibody concentration was measured by the Werbulg-Christian method.
  • the obtained antibody was used as a detection antibody for ELISA.
  • reactivity with human urine protein was examined by Western blotting (Fig. 4).
  • Normal human and patient urine were prepared so as to have a protein amount of 10 g / well, megsin was added thereto at 5 ng / well, and electrophoresis was performed by SDS-PAGE.
  • an alkaline phosphatase-labeled anti-mouse IgG (H + L) antibody (Chemicon) diluted 10,000 times was used.
  • an alkaline phosphatase-labeled anti-mouse IgG (H + L) antibody (Chemicon) diluted 10,000 times was used.
  • Msl2a has a higher affinity than other monoclonal antibodies.
  • the amount of binding to megsin was similar to that of Msl2a, but dissociation started immediately after binding, whereas Msl2a had a lower tendency. Because of this, we investigated a sandwich method in which Usagi polyclonal anti-megsin antibody (individual number: 1503) was combined with Msl2a as a solid phase antibody.
  • An ELISA was performed using a purified antigen of a rabbit polyclonal anti-megsin antibody (1 ⁇ g / ml) in a 96-well microplate (NUNC Code442404). Based on the standard stock solution (CHO-megsin Lot. 200205-2: 20 g / ml), 0.078-5 ng / ml serial dilution was used as a standard, and added at 100 1 / well. Protein and non-adsorbed tubes (manufactured by Sumitomo Bakelite) were used for all dilutions of standards and urine samples. The urine sample was stirred well with a vortex mixer, diluted 4 times with the sediment, and added at 100 1 / well.
  • the megsin concentration was examined in the range of 0.002 to 5 ng / ml, and the minimum detection sensitivity was 0.04 ng / ml.
  • Megsin was measured using 89 samples of patient urine supplemented with megsin, and the recovery rate of megsin was measured. Samples with measured values falling within ⁇ 20% of the theoretical value vary slightly from about 70%, but statistical processing suggests that they are not due to inhibition or non-specific reactions. Therefore, it is considered that megsin in the specimen was specifically detected and measured specifically by Msl2a.
  • Megsin (final concentration: lng / ml) supplemented to 8 patient urine samples is neutralized 100% by Msl2a supplement, and is considered effective for recognition of megsin as a detection antibody.
  • the effects of coexisting substances are: ammonium magnesium phosphate, calcium oxalate, calcium carbonate, transferrin, albumin, urea, IgG, IgA, IgM, ascorbic acid, dalcose, pyrilbin (free form), pyrilrubine (conjugation) Type), hemolyzed hemoglobin, milk and THP.
  • Ascorbic acid was strongly inhibited at a concentration of 250 mg / dl, which is thought to be due to a decrease in pH (in this case, pH 4 or less) due to addition at this concentration. When the pH lowered after the addition of ascorbic acid was measured by returning it to neutral, it was not inhibited even at a concentration of 500 mg / dl.
  • the ratio of the average value of megsin in normal human urine + 2SD (0.88 / z g / gCr), that is, statistically significantly higher than the amount of megsin in normal human urine was as follows.
  • megsin protein in a biological sample without concentrating the biological sample It became possible to measure the quantity of quality.
  • the measurement method becomes simple, and the present invention is effective in that a large amount of sample can be measured at a time.
  • megsin concentration in urine samples is useful as a diagnostic marker for various renal diseases. Therefore, based on the present invention, it is possible to measure megsin contained in a urine sample that has been concentrated and used as a diagnostic material for various renal diseases.

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Abstract

L’invention concerne un anticorps monoclonal réagissant à une protéine megsine. L’ELISA utilisant l’anticorps monoclonal peut détecter une megsine dans un échantillon biologique avec une grande sensibilité. Selon le procédé de détermination, un échantillon biologique n’ayant pas subi de prétraitement (par exemple, une concentration) peut être déterminé. Il devient possible de déterminer une megsine dans un échantillon d’urine non concentré et d’utiliser le résultat en tant que mesure pour le diagnostic de diverses néphropathies.
PCT/JP2006/309326 2005-05-09 2006-05-09 Procede de determination d’une megsine dans un echantillon biologique Ceased WO2006121047A1 (fr)

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* Cited by examiner, † Cited by third party
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WO2009147999A1 (fr) * 2008-06-02 2009-12-10 エーザイ・アール・アンド・ディー・マネジメント株式会社 MÉTHODE ET TROUSSE DE DÉTECTION POUR NÉPHROPATHIE À IgA

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WO2000057189A1 (fr) * 1999-03-19 2000-09-28 Kurokawa, Kiyoshi Procede de detection de proteine megsine et son utilisation
WO2001048019A1 (fr) * 1999-12-28 2001-07-05 Kurokawa, Kiyoshi Methode permettant de diagnostiquer la nephrite proliferative a cellules mesangiales chez le rat
WO2003084998A1 (fr) * 2002-04-11 2003-10-16 Fuso Pharmaceutical Industries, Ltd. Anticorps monoclonal neutralisant la megsine
JP2005261244A (ja) * 2004-03-17 2005-09-29 Tokai Univ 腎糸球体上皮細胞局在タンパク質特異性抗体
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WO1999015652A1 (fr) * 1997-09-22 1999-04-01 Kurokawa, Kiyoshi Proteine megsine
JP2006075170A (ja) * 1997-09-22 2006-03-23 Toshio Miyata メグシンタンパク質
WO2000057189A1 (fr) * 1999-03-19 2000-09-28 Kurokawa, Kiyoshi Procede de detection de proteine megsine et son utilisation
WO2001048019A1 (fr) * 1999-12-28 2001-07-05 Kurokawa, Kiyoshi Methode permettant de diagnostiquer la nephrite proliferative a cellules mesangiales chez le rat
WO2003084998A1 (fr) * 2002-04-11 2003-10-16 Fuso Pharmaceutical Industries, Ltd. Anticorps monoclonal neutralisant la megsine
JP2005261244A (ja) * 2004-03-17 2005-09-29 Tokai Univ 腎糸球体上皮細胞局在タンパク質特異性抗体

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INAGI R. ET AL.: "Specific tissue distribution of megsin, a novel serpin, in the glomerulus and its up-regulation in IgA nephropathy", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 286, no. 5, 2001, pages 1098 - 1106, XP002975419 *
TOMINAGA N. ET AL.: "Jinzo Tokuiteki Idenshi Megsin to Post Genome Kenkyu. (Post-genome research on megsin, a kidney-specific serpin gene)", JAPANESE JOURNAL OF CLINICAL MEDICINE, vol. 64, no. SPECIAL EXTRA ISSUE 2, 28 February 2006 (2006-02-28), pages 371 - 374, XP003004689 *
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009147999A1 (fr) * 2008-06-02 2009-12-10 エーザイ・アール・アンド・ディー・マネジメント株式会社 MÉTHODE ET TROUSSE DE DÉTECTION POUR NÉPHROPATHIE À IgA

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