WO2006121047A1 - Method for determination of megsin in biological sample - Google Patents

Abstract

Provided is a monoclonal antibody which reacts with megsin protein. The ELISA utilizing the monoclonal antibody can detect megsin in a biological sample with high sensitivity. According to the determination method, a biological sample which has not undergone any pre-treatment (e.g., concentration) can be determined. It becomes possible to determine megsin in an unconcentrated urine sample and use the result as a measure for the diagnosis of various renal diseases.

Description

 Specification

 Method for measuring megsin in biological samples

 Technical field

 [0001] 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.

 Background art

 [0002] 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.

 [0003] 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. In Japan, however, 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.

[0004] Currently, there are an estimated 210,000 dialysis patients with end-stage renal failure in Japan, and the number of patients per population is the world's highest. The average cost of treatment per person is about 6 million yen per year, which is one of the major causes of pressure on the medical insurance system. In addition, the social burden on the patient himself is large because he / she is restrained for dialysis treatment for 2-3 days a week, 4-6 hours a day.

 [0005] Furthermore, the age of dialysis patients is also increasing with the recent aging of the population. For this reason, there is a recognized need for diagnostic agents and drugs to diagnose and treat kidney disease early and prevent progression to renal failure. However, in the field of renal diseases, there are currently no effective diagnostics or pharmaceuticals that have a poor information research base such as target molecules for drug discovery.

[0006] 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.

 [0007] In addition, the proliferation of mesangial cells themselves during glomerular injury is associated with an increase in the extracellular matrix that is secreted by the mesangial cell force, suggesting that the cells are deeply involved in the onset and progression of the disease. Yes. From these facts, it is considered indispensable to elucidate the biological characteristics of mesangial cells in order to elucidate the molecular mechanism of renal diseases. However, the gene-level specificity for mesangial cells has not been clarified.

 [0008] There are about 60 trillion cells in the human body, and these have the same genomic DNA, but each individual cell, and thus organ, has different biological properties. It is thought to be due to genes that are specifically expressed in cells and organs.

 [0009] 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.

 [0010] 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).

 [0011] As a result, 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.

[0012] Further, amino acid homology search by FASTA program was performed using SwissProt amino acid sequence database. In addition, the 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.

 [0013] That is, 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).

 [0014] In addition, when IgA nephropathy patients, diabetic nephropathy patients, and healthy individuals, the expression level of megsin in the kidney tissue was compared, IgA nephropathy patients and diabetic nephropathy patients expressed megsin expression. The amount is significantly large (Non-Patent Documents 7, 8, and 11). Further, in the experimental mesangial proliferative glomerulonephritis model (Thy-1 nephritis model) using rats, a similar increase in megsin expression was observed (Non-patent Document 9). From this, it was revealed that megsin-expressing casangium cells change with abnormal function and are deeply involved in the onset and progression of the disease.

[0015] To further understand the role of megsin in the function of mesangium, the inventors overexpressed human megsin cDNA in the mouse genome. Two strains of megsin transgenic mice were obtained, which showed progressive mesangial matrix expansion, mesangial cell proliferation, and increased immune complex deposits (Non-Patent Document 10, Patent Document 2). .

 [0016] These findings indicate that megsin has a biologically important effect on the function of mesangyum. Interestingly, single genetic manipulation of megsin can generate early mesangial lesions present in experimental and human glomerulonephritis. Thus, megsin has also been reported to be involved in the development of mesangial proliferative glomerulonephritis in animal individuals.

[0017] On the other hand, in renal disorders, since no significant subjective symptoms appear until the end stage, ie, near renal failure, the kidney has already suffered irreparable damage at the time of its onset and oversight. There are many cases. Therefore, it is important to detect kidney damage as early as possible before observing the onset of subjective symptoms in order to prevent the transition to renal failure. It is also important to avoid insurance financial pressure from treatment.

 [0018] 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

01)

 Disclosure of the invention

 Problems to be solved by the invention

[0019] An object of the present invention is to provide a megsin protein measurement method useful for diagnosis of renal dysfunction.

 Means for solving the problem

[0020] 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.

 [0021] 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.

 [0022] That is, when the megsin protein in the urine was measured by ELISA using the monoclonal antibody of the present invention as a labeled antibody, the probability that megsin was detected from the urine of patients with various kidney diseases was normal and the urine of patients other than kidney disease. It turned out to be a high rate compared to. Furthermore, IgA nephropathy, which is nephritis associated with glomerular injury, was found to have a higher positive rate of megsin detection than other renal diseases in acute progressive glomerulonephritis. In diabetic nephropathy with glomerular mesangial disorder, a group of patients with higher urinary megsin protein content was found compared to other renal diseases.

 From these results, it became possible to detect megsin protein in a biological sample that was not concentrated by the measurement method of the present invention. .

 That is, 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.

 [1] Hybridoma Msl2a deposited as FERM BP-10598.

 [2] A monoclonal antibody produced by the hybridoma Msl2a deposited as FERM BP-10598, or a fragment containing the antigen-binding region thereof.

 [3] 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.

 [4] A method for measuring megsin protein contained in a biological sample, comprising the following steps.

0An anti-megsin polyclonal antibody bound to or capable of binding to the solid phase, or a fragment containing the antigen-binding region thereof, an anti-megsin monoclonal antibody bound to a labeled molecule A step of contacting a body or a fragment containing the antigen-binding region thereof and a biological sample ii) a step of detecting the labeled molecule bound to the megsin protein via an anti-megsin monoclonal antibody bound to the labeled molecule

 [5] The measurement method according to [4], comprising a step of contacting an anti-megsin polyclonal antibody and a biological sample, and then contacting an anti-megsin monoclonal antibody.

 [6] The measurement method according to [5], wherein the biological sample is urine.

 [7] The measurement method according to [6], which is a monoclonal antibody produced from the hybridoma Msl2a deposited as anti-megsin monoclonal antibody force FERM BP-10598.

 [8] The measurement method according to [7], wherein the anti-megsin polyclonal antibody is derived from Usagi.

 The invention's effect

 [0023] 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.

 Brief Description of Drawings

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.

 BEST MODE FOR CARRYING OUT THE INVENTION

[0025] 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.

[0026] 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.

[0027] Hybridoma Msl2a is 1st chome, Tsukuba Sagahito, Ibaraki, Japan, February 9, 2005

Deposited under the accession number FERM BP-10598 to the Patent Biological Depositary Center in the National Institute of Advanced Industrial Science and Technology, located in the sixth center of No.1.

 The contents specifying the deposit are described below.

 (a) Depositor's name

 Name: National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center

 Address: Japan Tsukuba Sakai Higashi 1-chome 1 1 Central 1 (zip code 305-8566)

(b) Deposit date: February 9, 2005 (February 9, 2005)

 (c) Accession number: FERM BP-10598

 [0028] 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).

 In the present invention, the “fragment containing an antigen-binding region” means a fragment having a partial force containing the antigen-binding region of a monoclonal antibody. Specifically, F (ab '), Fab', Fab, Fv (v

 2

ariable fragment of antibody), sFv, scFv, dsFv (disulphide stabilised Fv)! Antibody fragments such as /, ί or dA b (single domain antibody) are included in the “fragment containing the antigen-binding region”. The

 [0030] Here, "F (ab ')" and "Fab'" refer to immunoglobulin (monoclonal antibody), protein component

 2

 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. For example, when IgG is treated with papain, V (L chain variable region) and C (L chain constant region) are cleaved upstream of the disulfide bond existing between the two H chains in the hinge region.

 Region) force L chain and V (H chain variable region) binding y 1 (γ 1 region in H chain constant region)

 Η Η

 It is possible to produce two homologous antibody fragments in which the long chain fragments are linked by a disulfide bond at the C-terminal region. These two homologous antibody fragments are each called Fab '. In addition, when IgG is treated with pepsin, an antibody fragment is produced that is cleaved downstream of the disulfide bond existing between the two H chains in the hinge region and is slightly larger than the two Fab's connected in the hinge region. be able to. This antibody fragment is F (ab ')

 Called 2.

 [0031] A fragment containing an antigen-binding region can also be obtained by expressing DNA encoding the fragment. For example, 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.

[0032] 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.

 Furthermore, 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. . These labeled antibody fragments and fusion proteins are included in the monoclonal antibody of the present invention.

 [0033] 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.

 [0034] 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.

[0035] 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. Examples of promoters that can be used in eukaryotes include viral promoters, promoters for alcohol dehydrogenase, and promoters for glycolytic enzymes. Etc. Commercially available 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.

 [0036] As described above, 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.

 [0037] Further, 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.

 [0038] 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.

[0039] For example, 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.

 [0040] As an immunized animal, 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.

 [0041] 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.

 [0042] 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.

 [0043] 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. For immunization, animals such as mice such as Balb / c mice and FI mice can be used.

 [0044] As described above, 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. For example, 3-4 days after the final immunization, 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 After centrifugation under the conditions described above, remove the supernatant, treat with Tris-HCl buffer (PH7.65) for 1-2 minutes to remove erythrocytes, and then wash 3 times with MEM medium for cell fusion. Spleen cells obtain.

 [0045] Prior to cell fusion, it is necessary to first prepare the tumor cell line to be used. The tumor cell line used prior to cell fusion can be selected, for example, from cell lines that do not produce immunoglobulins. For the permanently proliferating cells to be fused, the ability to use any cell having permanent proliferation is generally used. Myeloma cells (myeloma cells) are generally used. Permanently proliferating cells are preferably derived from the same animal species as the antibody-producing cells.

 For example, in the case of 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))

P3 / NS1 / 1— Ag4— 1 (NS— 1) (Eur. J. Immunol., 6: 511 (1976))

 SP2 / 0-Agl4 (SP-2) (Nature, 276: 269 (1978))

 P3- X63- Ag8653 (653) (J. Immunol, 123: 1548 (1979))

 P3- X63- Ag8 (X63) (Nature, 256: 495 (1975))

These immortalized cell lines, glutamine, 8 Azaguanin medium (RPMI-1640 medium (1.5 mM), 2-mercaptoethanol ⁽⁵ X 10- 5 M), gentamicin (10 μ g / mL) and © shea fetal Subculture with normal medium supplemented with serum (FCS, CLS) (10%) and medium with 8-azaguanine (15 g / mL), and normal medium 3-4 days before cell fusion And secure a cell count of 2 X 10 ⁷ or more on the day of fusion.

[0047] 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. l After centrifugation at 200 rpm for 5 minutes, remove the supernatant, disaggregate the precipitated cells, and maintain at 37 ° C with stirring, and use polyethylene glycol-1000 (PEG-1000 as a cell fusion agent) ) Add 1 to 4 g, 1 to 4 mL of MEM medium and 0.5 to 1.0 mL of dimethyl sulfoxide as a cell fusion promoter, and add 0.1 to 1.0 mL / 10 ⁸ cells to cause cell fusion.

[0048] Thereafter, add 3 mL of MEM medium several times every 10 minutes, add MEM medium to a total volume of 50 mL and dilute to stop cell fusion. Next, centrifuge (1,500 rpm x 5 minutes) After removing the cells and gently lysing the cells, add 100 ml of normal medium (RPMI-1640 medium, 10% FCS) and gently suspend the cells by pipetting with a pipette.

[0049] Dispense 100 μL / well of this suspension into a 96-well culture plate and add 5% CO ink.

2 Incubate at 37 ° C for 3-5 days in an incubator. HAT medium 100 μ 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.

 2

 Incubate for about 2 weeks.

 [0050] For the wells in which the fused cells are observed to grow in colonies, remove half of the supernatant, and add the same amount of HT medium (with HAT medium power minus aminopterin). Incubate for a day. A part of the culture supernatant is collected and the antibody titer against megsin protein is measured by the above-mentioned ELISA.

 [0051] More specifically, for example, 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. As the anti-immunoglobulin antibody, when the cell used for cell fusion is a mouse, an anti-mouse immunoglobulin antibody is used. In addition, 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. Furthermore, 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.

 [0052] Anti-megsin protein monoclonal antibody-producing high-pridoma that showed stable antibody titer of megsin protein by repeating cloning 4 times by limiting dilution method for wells in which production of antibody reacting with megsin protein was observed Select as stock.

[0053] 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. . 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.

[0054] When cultured in vivo, 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. For example, 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. In 2 to 3 weeks, ascites containing a high concentration of monoclonal antibody accumulates in the abdominal cavity of the mouse, and the abdomen becomes enlarged. Collect the mouse force ascites and centrifuge (3,000 rpm x 5 min) to remove solids and purify IgG.

 [0055] 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.

 [0056] For 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).

 [0057] In order to obtain a large amount of monoclonal antibody, ascitic fluidization of hybridoma can be used. In this case, 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.

[0058] 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.

 [0059] 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. When using the ELISA method, a sandwich type assembly is preferable. In addition, the immunoassay includes all methods utilizing immunological reactions such as immunohistological examination, immunoblotting, and immunoprecipitation.

 [0060] 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.

 [0061] The present invention also provides a method for measuring megsin protein comprising 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 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.

[0062] 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. Go-peptide or c-myc- (His) -Tag-megsin protein or MBP-megsin tamper

 6

 Using 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. For example, a monoclonal antibody can be produced according to the method described above. When 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.

 [0063] 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. In order to enhance the ability to produce antibodies upon administration, 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. Examples of warm-blooded animals that can be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, hidges, goats, and chickens. Usagi is preferably used when the monoclonal antibody is a labeled antibody.

 [0064] A monoclonal antibody or a polyclonal antibody that recognizes megsin protein is used in the method for measuring megsin protein of the present invention. As a method for measuring megsin protein using these antibodies, 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. In addition, 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. In the measurement of human urine-derived megsin protein by the sandwich method, first, 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. Add the fixed antibody to the human urine-derived megsin protein labeled antibody to form a two-step method, or use the fixed antibody, labeled antibody and human urine-derived megsin protein together. A one-step method that sometimes mixes can be used.

 [0065] Examples of 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. As the 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.

 [0066] For the immobilization of the antibody, a known chemical bonding method or physical adsorption method can be used.

 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. Alternatively, 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.

 [0067] The monoclonal antibody of the present invention can be used as a labeled antibody for use in immunoassay. For labeling antibodies, 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. For labeling, 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, β-D-galactosidase, malate dehydrogenase, staphylococcal nuclease, delta-

5-steroid isomerase, α-glycerol phosphate dehydrogenase, triosephosphate isomerase, horseradish peroxidase, wasparaginase, darcosoxidase, ribonuclease, urease, catalase, glucose-6-phosphine Examples thereof include ate dehydrogenase, darcoamylase, and acetylcholinesterase. Preferable fluorescent substances include, for example, fluorescein isothiocyanate, phycobylprotein, rhodamine, phycoerythrin, phycocyanin, aloficocyanin, and orthophthalaldehyde. Preferable examples of 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.

 [0068] Techniques for binding the labeling substance to an antibody are known. Specifically, direct and indirect signs can be used. 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 Ν, Ν'-orthophenol-di-maleimide, 4- (Ν-maleimidomethyl) cyclohexanoic acidΝΝ-succinimide ester, 6-maleimidohexanoic acidΝΝ-succinimide ester, 4,4 '-Dithiopyridine and other known crosslinking agents can be used. The reaction of these 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. In addition, 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. On the other hand, for dinitrophenol, pyridoxal or fluoresamine, antibodies that recognize these haptens are labeled.

[0069] When labeling an antibody, 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. In some cases, such an antibody is used as an antibody, for example, Fab ′, Fab, F (ab ′). Polyclonal antibodies and monoclonal antibodies

 2

Regardless of the local antibody, 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.

 [0070] When the labeling agent is an enzyme, a substrate and, if necessary, a coloring agent are used to measure the activity. When peroxidase is used as the enzyme, H 0 is used as the substrate solution, and 2,2′-azino-di- [3-ethylbenzthiazolinesulfonic acid] a as the color former.

twenty two

 For example, ammonium salt (ABTS), 5-aminosalicylic acid, ortho-phendiamine, 4-aminoantipyrine, 3,3 ′, 5,5′-tetramethylbenzidine and the like can be used. When alkaline phosphatase is used as the enzyme, ortho-trifluorophosphate, para-trifluorophosphate, or the like can be used as a substrate. When / 3-D-galactosidase is used as the enzyme, fluorescein-di- (j8-D-galatatopyranoside), 4-methylumbelliferyl-iS-D-galactopyranoside, etc. can be used as a substrate. 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.

 [0071] 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. For example, 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.

 [0072] In particular, in 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. In particular, when 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.

That is, 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

 In the present invention, 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.

[0074] When determining the amount of megsin protein using urine as a sample, for example, if the amount of urine is measured by pooling urine for one day, the amount of megsin protein per day in urine may be clarified. it can. Alternatively, even when urine is used as a sample at any time, it is possible to estimate a value that is similar to the amount by using Creathun correction. 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. For blood, it is possible to estimate the amount by applying numerical corrections generally used in renal function diagnosis such as weight correction. Weight correction is a technique for calculating the amount of blood components based on the estimated body volume of the blood sample.

[0075] In addition, by observing changes in the megsin protein concentration in a biological sample derived from a certain individual, changes in renal function can be followed over time without conversion to an amount. Alternatively, a normal value of the megsin protein concentration of a body fluid sample in a specific species or population such as a race is set in advance, and the megsin protein concentration (or amount) of a specific individual is set. ), It is possible to know the presence or absence of abnormal renal function.

 [0076] By 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.

 That is, 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

 iii) A step of determining chronic glomerulonephritis, rapidly progressive glomerulonephritis, diabetic nephropathy, or chronic renal failure when the megsin concentration in the biological sample is higher than that of a normal subject. All prior art documents cited in are incorporated herein by reference.

 Example

 [Example 1] Preparation of antibody

 a. Preparation of immunogen (recombinant megsin)

 In accordance with a known method [Inagi, R. et al .: Biochem. Biophys. Res. Commun., 286: 1098-1106, 2 001], a Chinese memstar egg nest (CHO ) Recombinant cell culture supernatant was also obtained to obtain human megsin.

[0078] 100 ml of 1 M sodium acetate was added to the culture supernatant 21 and adjusted to pH 4.5, and then 50 mM sodium acetate was added to dilute the mixture twice. The diluted solution was subjected to ion exchange chromatography (HiPrepl6 / 10 SP XL: Amersham Bioscience) (elution condition: 50 mM sodium acetate (pH 4.5), NaCl 0 to 1 M linear gradient; elution volume: 20 X Column bed volume). The eluate was subjected to buffer exchange by gel filtration (HiPrep 26/10 Desalting, 20 mM potassium phosphate). (PH 6.8)), followed by hydroxyapatite chromatography (HT-1: manufactured by Bio-Rad) (elution conditions: 20 mM potassium phosphate (pH 6.8), potassium phosphate 20-400 mM linear gradient) Elution volume: 30 X column bed volume). The eluate was buffer exchanged again by gel filtration (HiPrep 26/10 Desalting, 50 mM MES (pH 5.5), 50 mM NaCl), ion exchange chromatography (Mono S HR 5/5: manufactured by Amersham Biosciences) (Elution conditions: 50 mM MES (pH 5.5), 50 mM NaCl, NaCl 50-100 mM linear gradient; elution volume: 40 X column bed volume). 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.

[0079] b. Immunity

 Balb / c and Ajcl mice (6-week-old male: CLEA Japan) were acclimated for 1 week, then 50 g of purified megsin per mouse was dissolved in 250 μ1 of PBS and 250 μ1 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 μl 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.

[0080] c. Cell fusion

 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. On the other hand, 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

 [0081] d. Hypridoma screening: ELISA method (direct method)

 Purified megsin was adjusted to 1 μg / 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. After washing the plate, 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 μl / well and the absorbance at 490 nm was measured.

 ELISA results 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.

 [0082] e. Closing of Noble Ibridoma

 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.

 [0083] f. Monoclonal antibody typing

 As a result of typing 0.5 ml of the culture supernatant of the cell line obtained in e. Above using Mouse Antibody Isotyping Kit (Roche), the isotype was IgGl.

 [0084] g. Western blotting

 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.

[0085] On the other hand, during electrophoresis, 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.

[0086] h. Biacore study of binding

 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.

 As a result, Msl2a showed sufficient binding to megsin protein even in the case of Biacore, which has a three-dimensional structure compared to other methods.

[0087] i. Preparation and purification of monoclonal antibody (Msl2a) IgG

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. After centrifugation at 12000 rpm and 4 ° C for 10 minutes, 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.

[Example 2] Specificity of Msl2a

 a. Reactivity with various serpins

In addition to megsin, some serpin (α 2-anti Cross-reactivity with plasmin, oc 1-antitrypsin, antithrombin III, PAI-Kplasminogen activator inhibitor 1), MBP PAI-2, maltose biding protein fusion plasminogen activator inhibitor 2)) was examined (Fig. 1). Only a slight reaction to MBP PAI-2 was observed.

[0089] b. Reactivity with various megsins (CHO-expressed megsins, GST-fused human megsins, GST-fused rat megsins, GST-fused mouse megsins) and reactivity with megsin / protease complexes CHO-expressed megsins were used for monoclonal antibody screening. The reactivity of other megsins was also examined by Western blotting (Fig. 2).

 It reacted well against two types of GST-fused human megsins. On the other hand, very little reaction was observed against GST-fused rat megsins and GST-fused mouse megsins with different species differences. We also recognized a complex of CHO-expressed megsin and plasmin, which is considered to be one of the megsin ligands (Fig. 3).

[0090] cReactivity with human urinary protein (normal, patient)

 Similarly, 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. As a secondary antibody, an alkaline phosphatase-labeled anti-mouse IgG (H + L) antibody (Chemicon) diluted 10,000 times was used.

 As a result, the reaction was only with megsin, and no nonspecific reaction with human urinary protein was observed by the Western blotting method.

[0091] d. Reactivity with human plasma (normal, patient)

 Similarly, the reactivity with human plasma was examined by Western blotting (Fig. 5). Plasma was added to normal subjects and patients to 0.15 / ζ ΐΑνθ11 and megsin to 5 ng / well, and electrophoresis was performed by SDS-PAGE. As a secondary antibody, an alkaline phosphatase-labeled anti-mouse IgG (H + L) antibody (Chemicon) diluted 10,000 times was used.

 As a result, except for the reaction derived from the secondary antibody, only the reaction with megsin, and the non-specific reaction with human plasma was not seen in Western blotting.

[0092] [Example 3] Sandwich ELISA method for measuring urinary megsin using Msl2a as detection antibody a. Basic study data

1) Examination of binding properties as detection antibodies using Biacore As a result, Msl2a has a higher affinity than other monoclonal antibodies. In the case of other 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.

[0093] 2) ELISA method

 An ELISA was performed using a purified antigen of a rabbit polyclonal anti-megsin antibody (1 μg / 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. When the addition was completed, the reaction was allowed to proceed at room temperature for 2 hours. After completion of the reaction, the plate was washed with a plate washer 500 1'5 times. Peroxidase-labeled Msl2a (Lot. TT-861) diluted 10,000 times with antibody dilution buffer was added at 100 μl / well and reacted at room temperature for 2 hours. After completion of the reaction, the plate was washed with 500 / ζ 1 · 5 times using a plate washer. MB (MOSS) was added at 100 μl / well and reacted at room temperature for 30 minutes. After 30 minutes 2N- H SO 100 μ 1

 twenty four

/ well Stop the reaction. Measurement was performed at 450 nm (samp) to 650 nm (reel) with V-MAX (Molecular Devices). Using the same urine sample, the creatinine concentration was determined using a commercially available reagent for measuring creatinine (manufactured by Dia Reagent, “Dia” Crea, an automatic analyzer measuring reagent), and the amount of megsin protein was corrected as the creatine ratio.

[0094] 3) Calibration curve (sensitivity, measurement range, linearity)

Regarding the calibration curve (log-log graph: Fig. 6), 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. The appropriate measurement range is 0.078-5 ng / ml. In this case, it is almost in the range of r ² = 0.995-1.

The dilution linearity of a urine specimen, if the sample were added serially diluted megsin in normal persons urine, r ² = 0.994, which was satisfactory in the range of 0.3~32ng / ml.

[0095] 4) Conditions when using Msl2a (buffer composition)

Selection of buffer (PBS or Tris) and optimum pH for antigen-Msl2a antibody reaction! (Figure 7). As a result of changing the pH from Tris buffer to 6.0 to 8.0 in 0.5 increments, there was not much change except pH 8.0 showing a slightly lower OD. However, in comparison with PBS, TBS showed a decrease in OD of at least 17%, so PBS was considered to be more sensitive, so PBS was adopted.

[0096] 5) Reproducibility

 Day difference reproducibility (measured 5 times) was examined for samples supplemented with PBS and urine so that the megsin concentration was 0.1, 0.5, 2 ng / ml. The variation in OD was about 10%, and the variation in measured values was about 20%.

 [0097] 6) Additive recovery test

 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.

 [0098] 7) Neutralization test

 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.

 [0099] 8) Influence of coexisting substances

 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. Other than ascorbic acid, it is thought that there is almost no influence. 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.

[0100] Regarding the effect of blood supplementation, the recovery rate was determined by adding megsin to hemolyzed plasma or serum added to normal human urine. Ivy [0101] b. Urine megsin measurement data

 1) Normal human urine measurement results (Figure 8)

 As for normal human urine, a total of 101 samples including 24 samples of early morning urine and 78 samples of urine at any time were measured. Of these, 2 specimens (2%) exceeded the measurement limit. All other specimens were negative.

[0102] 2) Patient urine measurement results (Figure 8)

 Out of all 2290 patient urine samples (diabetic nephropathy 69%, IgA nephropathy 6%, chronic glomerulonephritis 5%, chronic renal failure 3%, other 15%), positive samples (above the measurement limit in ELISA: 0.31ng / ml) was 242 samples (positive rate 11.2%). The percentage of positive specimens by disease was as follows.

 Diabetic nephropathy 132 specimens (12%)

 IgA nephropathy 27 specimens (24%)

 Chronic glomerulonephritis other than IgA nephropathy 37 specimens (16%)

 Chronic renal failure 14 specimens (19%)

 Rapidly progressive glomerulonephritis 7 specimens (39%)

 Non-renal disease 25 specimens (3%)

 [0103] This includes urine collection specimens (58 specimens) from renal biopsy patients, but 20 specimens (34%) were positive, of which high activity, including lupus nephritis, and glomerulonephritis in urine. Megsin tended to be high.

 In addition, 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.

 Diabetic nephropathy 71 specimens (7%)

 IgA nephropathy 13 specimens (12%)

 Chronic glomerulonephritis other than IgA nephropathy 12 specimens (5%)

 Chronic renal failure 8 specimens (11%)

 Rapidly progressive glomerulonephritis 7 specimens (39%)

 Non-renal disease 3 specimens (0.4%)

 Industrial applicability

[0104] By the measurement method of the present invention, megsin protein in a biological sample without concentrating the biological sample It became possible to measure the quantity of quality. By not pre-treating the biological sample, the measurement method becomes simple, and the present invention is effective in that a large amount of sample can be measured at a time. For example, 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.

Claims

The scope of the claims

 [1] Hybridoma Msl2a deposited as FERM BP-10598.

 [2] A monoclonal antibody produced by the hybridoma Msl2a deposited as FERM BP-10598, or a fragment containing the antigen-binding region thereof.

[3] A method for producing a monoclonal antibody or a fragment containing an antigen-binding region thereof, comprising culturing the hybridoma Msl2a deposited as FERM BP-10598 and recovering the immunoglobulin contained in the culture.

 [4] A method for measuring megsin protein contained in a biological sample, comprising 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 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.

 [5] The measurement method according to claim 4, comprising a step of contacting the anti-megsin polyclonal antibody with the biological sample and then contacting the anti-megsin monoclonal antibody.

 6. The measurement method according to claim 5, wherein the biological sample is urine.

 [7] The method according to claim 6, which is a monoclonal antibody produced from the hybridoma M sl2a deposited as anti-megsin monoclonal antibody strength FERM BP-10598.

8. The measurement method according to claim 7, wherein the anti-megsin polyclonal antibody is derived from a rabbit.