JP2005261244A - Antibody specific to protein localized in glomerular epithelial cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a monoclonal antibody reacting with a megsin protein which is expressed in a glomerular epithelial cell, and to provide a use of the same. <P>SOLUTION: This monoclonal antibody reacting with the megsin protein which is expressed in the glomerular epithelial cell and the use of the same are provided, respectively. It is confirmed that, for example, expression of the megsin protein which is localized in the glomerular epithelial cell is exalted in nephropathy caused by abnormality of a glomerular epithelial cell line, such as membranous nephropathy and minimal change nephrosis, by an immune structure dyeing method using the monoclonal antibody. It is found that, on the other hand, the expression of the megsin protein in glomerular epithelium is depressed in IgA nephropathy, etc. Thus, classification of types of nephric diseases is realized by comparing dye patterns of the megsin protein in the glomerular epithelial cell. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a specific serpin (serine protease inhibitor), that is, a monoclonal antibody that selectively binds to a specific epitope of Megsin, a method for producing the same, and a method for diagnosing renal injury using the monoclonal antibody, and It relates to a composition for diagnosis.

  Renal failure is a condition that ultimately leads to patients with kidney disease. The cause and history are not uniform, and many cases of renal failure occur due to lesions other than the original kidney such as drug addiction, infection, malignant tumor, diabetes, systemic lupus erythematosus (SLE), etc. It is done.

  Renal transplantation is the only treatment for end-stage renal failure where the blood filtration and detoxification of the kidneys do not function at all. However, in Japan, it is difficult to say that the system for supplying transplanted kidneys is sufficiently established. In addition, social recognition 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.

  At present, the number of dialysis patients with end-stage renal disease in Japan is estimated to be approximately 210,000, and the number of patients per population is the top in the world. The average treatment cost per person is about 6 million yen per year, which is one of the major causes of pressure on the medical insurance system. Moreover, since the patient is restrained for dialysis treatment for 2 to 3 days a week and 4 to 6 hours a day, it is difficult for the patient to return to society.

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

  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.

  In addition, the proliferation of mesangial cells themselves and an increase in extracellular matrix secreted from mesangial cells are observed during glomerular injury, and it is thus presumed that the cells are deeply involved in the onset and progression of diseases. From these facts, in order to elucidate the molecular mechanism of renal disease, it is essential to first elucidate the biological characteristics of mesangial cells. However, the gene-level specificity for mesangial cells has not been revealed.

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

  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 renal disease state, to find clues to elucidate the molecular mechanism of renal disease, and to develop new diagnostic and therapeutic methods for renal disease 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.

  First, for the purpose of quantitatively analyzing genes expressed in mesangial cells, the present inventors extracted mRNA from cultured human mesangial cells to prepare a 3′-directed cDNA library. Then, large-scale DNA sequencing and database analysis of gene fragments inserted into the clones were performed (Non-patent Document 1).

  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. The inventors succeeded in isolating and obtaining megsin protein, which is a novel protein consisting of 380 amino acids encoded by the full-length cDNA clone of megsin.

  Furthermore, amino acid homology search by FASTA program was performed using SwissProt amino acid sequence database. And the consensus sequence in the reactive loop region (reactive loop site) important as a bioactive central site of the serine protease inhibitor (SERPIN) superfamily (Non-patent Documents 2 to 6) in the amino acid sequence of megsin protein ( It was found that a sequence (EEGTEATAAT / SEQ ID NO: 3) similar to EEGTEAAAAT / SEQ ID NO: 2) exists.

  That is, megsin has the structural features of serpin and has a reactive loop region (P17-P5 ′: EEGTEATAATGSNIVEKQLPQS / SEQ ID NO: 4) that is an active site like other serpins (Non-patent Document 7). . From these, it was clarified that human megsin protein is a protein belonging to serpin (Non-patent Document 7). A patent application was filed for these findings (Patent Document 1).

  In addition, when comparing the expression level of megsin in kidney tissues between IgA nephropathy patients and diabetic nephropathy patients and healthy individuals, the expression level of megsin in IgA nephropathy patients and diabetic nephropathy patients is significantly higher (non- Patent Documents 7 and 8). 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). This indicates that megsin expression changes with dysfunction of mesangial cells and is deeply involved in the onset and progression of the disease.

  To better understand the role of megsin in mesangial function, we 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).

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

  On the other hand, in renal disorder, since no significant subjective symptoms appear until the end stage, that is, near renal failure, its occurrence is easily overlooked, and at the time of onset, the kidney may have already been damaged beyond recovery. Many. Therefore, it is important to find kidney damage at the earliest possible stage before observing the manifestation of subjective symptoms, in order to prevent the transition to renal failure and to avoid insurance financial pressure from dialysis treatment. .

The present inventors considered that it is necessary to measure a specific protein closely related to the pathological condition in order to determine the definite diagnosis and severity of 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 Document 3). ).
International Publication No.99 / 15652 International Publication No. 01/24628 International Publication No. 00/57189 Yasuda, Y. et al., Kidney Int., 53: 154-158 (1998) Carrell, RW et al., Trends Biochem. Sci., 10: 20 (1985) Carrell, RW et al., Cold Spring Harbor Symp. Quant. Biol., 52: 527 (1987) Kruithof, EKO et al., Blood, 86: 4007 (1995) Potempa, J. et al., J. Biol. Chem., 269: 15957 (1994) Remold-O'Donnell. E., FEBS Lett., 315: 105 (1993) Miyata, T. et al., J. Clin. Invest., 102: 828-836 (1998) Suzuki, D. et al., J. Am. Soc. Nephrol., 10: 2606 (1999) Nangaku, M. et al., Kidney Int., 60: 641 (2001) Miyata, T. et al., J. Clin. Invest., 109: 585 (2002)

  As described above, the method for diagnosing kidney damage using megsin protein antibody is an extremely useful means for early diagnosis of kidney damage and determination of the degree of progression of the disease state. An object of the present invention is to elucidate the binding epitope of the megsin protein antibody and provide a disease state-specific diagnostic agent.

  The present inventors analyzed various tissues and cells by Northern blot and reverse transcription polymerase chain reaction, and megsin was weakly expressed in human fibroblasts, smooth muscle cells, endothelial cells, keratinocytes, and particularly in mesangial cells. It was found that it was strongly expressed. These findings were further obtained in situ hybridization (Miyata, T. et al., J. Clin. Invest., 102: 828 (1998), Suzuki, D. et al., J. Am. Soc. Nephrol., 10 : 2606 (1999)) and immunohistochemistry using megsin antibody (Inagi, R. et al., Biochem. Biophys. Res. Commun., 286: 1098 (2001)).

  Therefore, finding and specifying an antibody (monoclonal antibody) with excellent specificity for megsin protein can be made by elucidating the biological properties of kidney damage, investigating the cause of kidney disease, and eventually treating kidney disease, It is effective for diagnosis.

  The inventors have obtained many antibodies that recognize megsin protein using recombinant megsin protein as an immunogen. Furthermore, the relationship between the specificity for the kidney tissue and the binding epitope was investigated. Then, the present inventors have found a monoclonal antibody 4F3 that reacts with a megsin protein localized in renal glomerular epithelial cells at the time of renal injury caused by glomerular epithelial cell line abnormality, and its epitope (246-260: LSEIENKLTFQNLME / SEQ ID NO: 1) was determined. Furthermore, it was clarified that the localization pattern of megsin protein localized in glomerular epithelial cells varies depending on the disease type, and the present invention was completed.

  That is, when the staining patterns were compared using the monoclonal antibody 4F3 of the present invention, in renal disorders derived from mesangial cell disorders such as IgA nephropathy and diabetic nephropathy, meg localizing in glomerular epithelial cells. On the other hand, the localization of the megsin protein in the glomerular epithelial cells in the case of renal disorders caused by abnormalities in the glomerular epithelial cell lineage such as membranous nephropathy and minimal change nephrosis is reduced. I found out that my presence was increasing. Therefore, for example, by measuring the staining intensity of megsin protein localized in glomerular epithelial cells using means such as immunohistochemical staining using the monoclonal antibody of the present invention, it is possible to diagnose the type of renal disorder. It becomes.

Monoclonal antibody against megsin peptide (WO 00/57189) and antibody that specifically binds to an epitope in the reactive loop region (P17-P5 ': EEGTEATAATGSNIVEKQLPQS / SEQ ID NO: 4) of megsin protein (WO 03/084998 ) Is known. However, none of them is known to be related to the disease type. Furthermore, the knowledge about the reactivity with respect to the megsin protein localized in a glomerular epithelial cell at the time of renal failure resulting from abnormality of a glomerular epithelial cell system was not confirmed. The monoclonal antibody according to the present invention is capable of reacting with megsin protein localized in renal glomerular epithelial cells in renal injury caused by glomerular epithelial cell line abnormality. That is, the present invention relates to the following monoclonal antibodies and uses thereof.
(1) A monoclonal antibody that binds to megsin protein localized in renal glomerular epithelial cells.
(2) A monoclonal antibody that recognizes an epitope constituted by a peptide having the amino acid sequence set forth in SEQ ID NO: 1.
(3) A hybridoma cell line that produces the monoclonal antibody according to (1) or (2).
(4) Hybridoma 4F3 deposited as FERM P-19734.
(5) A monoclonal antibody produced by hybridoma 4F3 deposited as FERM P-19734.
(6) A method for producing a monoclonal antibody, comprising culturing a hybridoma 4F3 deposited as FERM P-19734 and recovering an immunoglobulin contained in the culture.
(7) A method for diagnosing renal injury, comprising a step of detecting a monoclonal antibody that binds to megsin protein localized in renal glomerular epithelial cells.

  The monoclonal antibody 4F3 of the present invention reacts with a megsin protein localized in renal glomerular epithelial cells at the time of renal injury caused by glomerular epithelial cell line abnormality. Therefore, by comparing the staining patterns of renal tissue using the antibody by means such as immunohistochemical staining, it becomes possible to diagnose the disease type of renal disease. In other words, in renal disorders derived from mesangial cell disorders such as IgA nephropathy and diabetic nephropathy, the localization of megsin protein in glomerular epithelial cells is reduced, whereas membranous nephropathy and microvariable type In the case of renal disorders caused by glomerular epithelial cell line abnormalities such as nephrosis, the localization of megsin protein in renal glomerular epithelial cells is enhanced. Thus, it became possible to classify disease types by comparing staining patterns of megsin protein in glomerular epithelial cells.

  The present inventors have found that a monoclonal antibody that recognizes a specific region of megsin protein reacts with megsin protein localized in renal glomerular epithelial cells. That is, the present invention relates to a monoclonal antibody that recognizes a specific region of megsin protein. The region recognized by the monoclonal antibody of the present invention refers to a region consisting of the amino acid sequence shown in SEQ ID NO: 1, particularly in the amino acid sequence of megsin protein. Therefore, the monoclonal antibody of the present invention is a monoclonal antibody that recognizes an epitope constituted by a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1.

  It can be confirmed by epitope mapping that the monoclonal antibody recognizes an epitope composed of a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1. For example, in Examples 3 and 4, epitopes recognized by monoclonal antibodies are specified using peptides having various amino acid sequences.

  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. The monoclonal antibody of the present invention can be obtained by selecting the obtained monoclonal antibody based on the evaluation method as described above.

  As a hybridoma producing the monoclonal antibody of the present invention, for example, a hybridoma 4F3 cell can be shown. The hybridoma 4F3 is a hybridoma that produces a monoclonal antibody that recognizes 246-260 (LSEIENKLTFQNLME / SEQ ID NO: 1) in the amino acid sequence constituting the megsin protein. Monoclonal antibody 4F3 produced by hybridoma 4F3 reacts with megsin protein localized in renal glomerular epithelial cells.

Hybridoma 4F3 is registered with the Patent Biological Depositary Center of the National Institute of Advanced Industrial Science and Technology (AIST) located at 1-1-1, Higashi 1-1-1, Tsukuba, Ibaraki, Japan, on March 16, 2004. -Deposited as 19734.
The contents specifying the deposit are described below.
(a) Name and address of depositary institution Name: National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center Address: Central 1st 1-1, Higashi 1-1-1 Tsukuba City, Ibaraki, Japan (Zip 305-8566)
(b) Date of deposit: March 16, 2004
(c) Accession number: FERM P-19734

  The isotype of the monoclonal antibody produced by this hybridoma 4F3 cell line was IgG1 for the H chain and κ for the L chain. The present invention also encompasses class switch variants of the above antibodies, such as variants belonging to isotype IgG3, IgG1, IgG2b, IgG2a and other immunoglobulin subclasses, and such variants are described in the Martin et al. (Martin, C. et al .: J. Immunol. Methods., 145: 1118, 1991).

  Production of a monoclonal antibody against megsin protein requires megsin protein that can be used as an immunogenic antigen. The megsin protein as an antigen can be obtained using a cultured cell, for example, a megsin protein-producing cell. Examples of megsin protein-producing cells include human kidney-derived cells. This megsin protein-producing cell is cultured using a medium or culture method known in the art or substantially the same, and the megsin protein produced in the culture supernatant is subjected to, for example, ion exchange chromatography. And / or can be purified by affinity chromatography using 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, the transformed host is cultured to obtain the amino acid sequence of megsin protein. In which the polypeptide is produced and the polypeptide is used as an immunogen. A recombinant vector containing megsin cDNA is prepared by a conventional gene recombination technique, for example, by inserting it into a plasmid vector. As a vector, viruses such as vaccinia virus and baculovirus can be used in addition to plasmids and phages.

  As the host, for example, prokaryotes such as Escherichia coli, Bacillus subtilis, actinomycetes and the like, 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 may be used. it can. Examples of promoters that can be used in prokaryotes include tryptophan synthase operon and lactose operon. Examples of promoters that can be used in eukaryotes include viral promoters, promoters for alcohol dehydrogenase, promoters for glycolytic enzymes, and the like. A commercially available vector or plasmid having a multicloning site, a promoter, a resistance gene, a replication origin, a terminator, a liposome binding site, and the like can also be used. Resistance genes include those for tetracycline, ampicillin, neomycin and the like. The megsin protein thus prepared may be further used as an immunogenic conjugate, but can be used as it is for immunization of an animal by mixing it with an appropriate adjuvant.

  As described above, antigens are obtained from various raw materials such as cultured cells, cultured tissues, transformed cells, and other conventionally known methods, for example, salting out such as ammonium sulfate precipitation, gel filtration chromatography using Sephadex, It can be obtained by purification by ion exchange chromatography, hydrophobic chromatography, dye gel chromatography, electrophoresis, dialysis, ultrafiltration, affinity chromatography, high performance liquid chromatography and the like.

  Furthermore, the megsin protein is a fragmented one, or a sequence region selected based on the amino acid sequence deduced from the cloned and sequenced cDNA sequence, and a polypeptide is designed and chemically synthesized. The resulting polypeptide fragment may be combined with various carrier proteins via an appropriate condensing agent to form a hapten-protein immunoconjugate that can be used to recognize specific sequences only. It is also possible to design monoclonal antibodies that can be used. A cysteine residue or the like can be added in advance to the designed polypeptide to facilitate the preparation of an immunogenic conjugate.

  The present invention provides at least one monoclonal antibody that specifically binds to a megsin protein localized in renal glomerular epithelial cells. The monoclonal antibody according to the present invention, after immunizing an animal with recombinant megsin as an immunogen, cell fusion between myeloma cells and antibody-producing cells, selection and monocloning of hybridomas, production of monoclonal antibodies, and ascites as needed It can be produced by a process such as

  The animal is immunized as follows, for example. A human megsin protein purified according to a known method (Miyata, T. et al., J. Clin. Invest., 120: 828 (1998)) is immunized to mammals such as rats and mice. It is preferable to use an animal of the same strain as the partner's permanent proliferating cell for cell fusion. The animal age is preferably 8 to 10 weeks for mice, for example. Sex may be either male or female.

  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 an antibody having the desired reactivity by using a transgenic animal in which an immunoglobulin gene is recombined with a human gene is known. The immunoglobulins that can be obtained in this way are fully human immunoglobulin molecules, although they are obtained from animals.

  The immunization method consists of mixing purified human megsin protein with an appropriate adjuvant (for example, Freund's complete adjuvant or aluminum hydroxide gel-pertussis vaccine, etc.) to make an emulsion, and then subcutaneously, intraperitoneally, intravenously, etc. Administer. Thereafter, this immunization is performed 2 to 5 times at intervals of 1 to 2 weeks. The final immunization is performed by administering 0.5 to 2 μg of human megsin protein intraperitoneally.

  Polyclonal antibodies are obtained from the body fluids of animals immunized in this way. 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 is sufficiently increased, antibody or antibody-producing cells are collected. The antibody titer against megsin can be measured by a technique such as ELISA. ELISA for measuring antibody titer can be performed by adding serum to a plate coated with megsin and further adding a labeled antibody against IgG of the immunized animal.

  Examples of the adjuvant used together with the antigen include Freund's complete adjuvant, livi adjuvant, pertussis vaccine, BCG, liposome, aluminum hydroxide, silica gel and the like. For immunization, animals such as mice such as Balb / c mice and FI mice can be used.

  Antibody-producing cells are collected from animals immunized with human megsin protein as described above. 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 Essential Medium (MEM) medium (manufactured by Nissui Pharmaceutical), disassembled with tweezers, and subjected to conditions of 1,200 rpm x 5 minutes. After centrifugation, remove the supernatant, treat with Tris-HCl buffer (pH7.65) for 1-2 minutes to remove erythrocytes, and then wash with MEM medium 3 times to obtain spleen cells for cell fusion .

  Before cell fusion, it is necessary to prepare the tumor cell line to be used first. The tumor cell line used prior to cell fusion can be selected from, for example, cell lines that do not produce immunoglobulins. As the permanent proliferating cell to be fused, any cell having permanent proliferation can be used, but in general, myeloma cells (myeloma) are 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-U1 (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-Ag14 (SP-2) (Nature, 276: 269 (1978))
P3-X63-Ag8653 (653) (J. Immunol., 123: 1548 (1979))
P3-X63-Ag8 (X63) (Nature, 256: 495 (1975))
These permanently proliferating cell lines consist of 8-azaguanine medium (RPMI-1640 medium with glutamine (1.5 mM), 2-mercaptoethanol (5 × 10 ⁻⁵ M), gentamicin (10 μg / mL) and fetal calf serum (FCS). (Manufactured by CLS) (10%), subcultured with 8-azaguanine (15 μg / mL), and subcultured to normal medium 3-4 days before cell fusion, Secure 2 × 10 ⁷ cells or more on the day of fusion.

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. After centrifuging at 1,200 rpm for 5 minutes, remove the supernatant, thoroughly dissolve the precipitated cells, and maintain at 37 ° C. with stirring, and 1 to 4 g of polyethylene glycol-1000 (PEG-1000) as a cell fusion agent Then, 1 to 4 mL of MEM medium and a mixed solution of 0.5 to 1.0 mL of dimethyl sulfoxide as a cell fusion promoter 0.1 to 1.0 mL / 10 ⁸ cells are added to cause cell fusion.

  Thereafter, 3 mL of MEM medium is added several times every 10 minutes, and MEM medium is added and diluted to a total volume of 50 mL to stop cell fusion. Next, centrifuge (1,500 rpm x 5 minutes) to remove the supernatant, gently disintegrate the cells, add 100 mL of normal medium (RPMI-1640 medium, 10% FCS), and pipet with a pipette. Gently suspend the cells.

This suspension is dispensed at 100 μL / well into a 96-well culture plate and cultured at 37 ° C. for 3-5 days in a 5% CO ₂ incubator. Add 100 μL / well HAT medium (culture medium with hypoxanthine (10 ^-4 M), thymidine (1.5 x 10 ^-5 M) and aminopterin (4 x 10 ^-7 M) to normal medium) to the culture plate, Incubate for an additional 3 days. After that, remove half the volume of the culture supernatant every 3 days, add the same amount of HAT medium, and incubate in a 5% CO ₂ incubator at 37 ° C for about 2 weeks.

  For wells in which fused cells are observed to grow in colonies, remove half the volume of the supernatant, add the same amount of HT medium (HAT medium minus aminopterin), and culture for 4 days. A part of the culture supernatant is collected, and the antibody titer against megsin protein is measured by the aforementioned ELISA.

  More specifically, for example, a hybridoma 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 to measure the label. By doing so, the antibody titer can be measured. 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, instead of the labeled antibody, protein A can be added to detect the anti-megsin protein monoclonal antibody bound to the solid phase. Furthermore, the antibody titer can also be measured by adding the hybridoma culture supernatant to a solid phase on which an anti-immunoglobulin antibody or protein A is adsorbed and adding megsin protein labeled with a radioactive substance or enzyme.

  For wells in which production of antibodies reacting with megsin protein was observed, cloning was repeated four times by limiting dilution, and those showing stable megsin protein antibody titers were selected as anti-megsin protein monoclonal antibody-producing hybridoma strains. .

  A monoclonal antibody is produced by culturing the hybridoma obtained as described above in vitro and in vivo. The desired monoclonal antibody can be cultured in an appropriate medium such as FCS-containing MEM medium or RPMI-1640 medium, and obtained from the culture supernatant. The in vitro culture of the hybridoma is preferably performed in a serum-free medium, and an optimal amount of antibody is given to the supernatant.

When cultured in vivo, hybridomas are transplanted into any animal. 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-pristan-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. Ascites is collected from the mouse, centrifuged (3,000 rpm × 5 minutes) to remove solids, and IgG is purified.

  Ascites fluid and culture supernatant are salted out with 50% ammonium sulfate and dialyzed against PBS for 1-2 weeks. This 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 megsin protein localized in renal glomerular epithelial cells.

  For the antibody isotype, use a commercially available kit (Gibco BRL, Mouse Antibody Isotyping Kit, etc.), or octalony (double immunodiffusion) method (Introduction to immunology experiments, Biochemistry experiment method 15, published by Academic Publishing Center, 74 Page, 1981). The amount of protein is calculated by the foreign method and absorbance at 280 nm (1.4 (OD280) immunoglobulin 1 mg / mL).

  Hybridoma ascites can be used to obtain large amounts of monoclonal antibodies. In this case, monoclonal antibodies produced in the ascites by transplanting each hybridoma into the abdominal cavity of an animal having the same tissue compatibility as that of an animal derived from myeloma cells, and proliferating, or transplanting each hybridoma into a nude mouse or the like. Can be obtained.

  The animal can be administered intraperitoneally with a mineral oil such as pristane prior to transplanting the hybridoma. Ascites fluid can be purified as it is or by a conventional method. For example, it can be purified by salting out such as ammonium sulfate precipitation, gel filtration using Sephadex, ion exchange chromatography, electrophoresis, dialysis, ultrafiltration, affinity chromatography, high performance liquid chromatography, or the like. The characteristics of the monoclonal antibody obtained as described above can be clarified by, for example, an enzyme immunoassay (ELISA method) or the like.

  The monoclonal antibody of the present invention can specifically bind to the 246-260 site of human megsin protein localized in the glomerular epithelial cells of the kidney. In addition, megsin protein localized in renal glomerular epithelial cells is highly localized in renal damage caused by glomerular epithelial cell line disorders. For this reason, the monoclonal antibody of the present invention is considered to be effective for definitive diagnosis in the diagnosis of renal damage.

  In the present invention, “the megsin protein is localized” means that the megsin protein is present at a high concentration. The term “localized in the glomerular epithelial cells” means not only megsin protein directly expressed from the glomerular epithelial cells but also megsin expressed in cells other than the glomerular epithelial cells such as mesangial cells. This includes the case where proteins are secreted extracellularly and accumulated in glomerular epithelial cells.

  The monoclonal antibody of the present invention is used for immunostaining such as tissue or cell staining, immunoprecipitation, immunoblot, immunoassay such as competitive or non-competitive immunoassay, radioimmunoassay, ELISA, latex agglutination method, protein purification, affinity column, etc. can do. When using the ELISA method, a sandwich type assay is preferable. The immunoassay includes all methods utilizing an immune reaction such as immunohistological examination, immunoblot, and immunoprecipitation.

  The monoclonal antibody of the present invention can be a labeled antibody for use in an immunoassay. Enzymes, enzyme substrates, coenzymes, enzyme precursors, apoenzymes, fluorescent substances, dye substances, chemiluminescent compounds, luminescent substances, chromogenic substances, magnetic substances, metal particles, radioactive substances, etc. are used for labeling antibodies. be able to. 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.

  The immunoassay using the monoclonal antibody of the present invention can use any form of solution, colloidal solution, non-fluid sample, etc. 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, ovary-derived cell line, ovarian tissue, cancer cell line, and cancer tissue.

  Accordingly, the present invention also provides such hybridoma cell lines, immunoassays and test kits. Furthermore, the present invention provides a monoclonal antibody that specifically recognizes megsin protein localized in glomerular epithelial cells, an immunoassay for detecting and quantifying megsin characterized by using this antibody, and carrying out this immunoassay To provide a test kit. In addition, the monoclonal antibody obtained by the present invention has high specificity for megsin and is very useful in the detection and quantification of megsin.

The disease type diagnosis of renal disorder based on the present invention is performed, for example, by the following steps. That is, the present invention relates to a method for examining renal injury including the following steps.
(1) a step of measuring the expression intensity of megsin protein in glomerular epithelial cells in a kidney tissue sample collected from a living body, and
(2) a step of detecting renal injury caused by glomerular epithelial cell system abnormality when the localization of megsin protein is increased as compared with normal kidney tissue. Renal disorders resulting from abnormalities in the spherical epithelial cell line include membranous nephropathy and microvariable nephrosis. On the other hand, in the renal tissues of patients with renal disorders derived from mesangial cell disorders such as IgA nephropathy and diabetic nephropathy, the increase in the expression intensity of megsin protein in glomerular epithelial cells is similar to that in healthy subjects. can not see.

Living body renal tissue can be collected by renal biopsy. On the other hand, the expression intensity of megsin protein in glomerular epithelial cells of a tissue specimen can be detected by an antibody that recognizes megsin protein localized in epithelial cells. A preferred antibody in the test method of the present invention is an antibody that recognizes the amino acid sequence shown in SEQ ID NO: 1 as an epitope. An example of such an antibody is a monoclonal antibody produced by a hybridoma deposited as FERM P-19734.
The antibody used in the test method of the present invention is useful as a test reagent for renal damage. The antibody can be labeled for easy detection. For example, a method for labeling and detecting an antibody with a fluorescent molecule, a luminescent molecule, an enzyme molecule, or a binding ligand is known. Alternatively, antibody binding can also be detected by combining a second antibody that recognizes and binds to an antibody that binds to the tissue. When a second antibody is combined, it is the second antibody that is labeled.
The antibody can be combined with a component necessary for detection of a label, a blocking agent useful for blocking a tissue sample, a positive sample, a negative sample, or the like to form a kit for testing a renal disorder.

Hereinafter, the present invention will be described more specifically based on examples.
[Example 1] Preparation of monoclonal antibody
a. Preparation of immunogen (recombinant megsin protein)
According to known methods (Inagi, R. et al., Biochem. Biophys. Res. Commun., 286: 1098-1106 (2001)), Chinese hamster ovary (CHO) cells transfected with human megsin cDNA Recombinant human megsin protein was obtained from the culture supernatant.

  100 mL of 1 M sodium acetate was added to 2 L of the culture supernatant to adjust the pH to 4.5, and then 50 mM sodium acetate was added to dilute it twice. The diluted solution was subjected to ion exchange chromatography (HiPrep 16/10 SP XL: Amersham Bioscience) (elution condition: 50 mM sodium acetate (pH 4.5), NaCl 0 to 1 M linear gradient; elution volume: 20 × column Bed volume). The eluate was subjected to buffer exchange by gel filtration (HiPrep 26/10 Desalting, 20 mM potassium phosphate (pH 6.8)), and then subjected to hydroxyapatite chromatography (HT-1: 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 is again subjected to buffer exchange by gel filtration (HiPrep 26/10 Desalting, 50 mM MES (pH 5.5), 50 mM NaCl), and subjected to ion exchange chromatography (Mono S HR 5/5: Amersham Biosciences). (Elution conditions: 50 mM MES (pH 5.5), 50 mM NaCl, NaCl 50-100 mM linear gradient; elution volume: 40 × column bed volume). The eluate was concentrated by centrifugation (3,000 g) using Centricon 10 (Millipore), and the buffer was exchanged with Dulbecco's PBS (-) buffer (Nissui Pharmaceutical). Centrifugal concentration and buffer exchange were repeated three times to obtain purified recombinant megsin protein. As the chromatography apparatus, AKTAexplorer 10s (manufactured by Amersham Bioscience) was used, and all operations were performed at 4 ° C. The purified human recombinant megsin protein 10 μg (fluid volume 50 μL) thus obtained was used as an immunogen per mouse.

b. Immunization
In a 1.5 mL tube, use 50 μL of Freund's complete adjuvant (SIGMA) and purified megsin protein solution (10 μg / 50 μL) using a 1 mL syringe (Terumo: SS-01T) and a 21 gauge needle (Terumo: NN-2138S) The mixture was mixed until completely emulsified to obtain an antigen stock solution for immunization. Mice (Balb / c) were immunized by injecting the immunogen solution into the peritoneal cavity using a 26 gauge injection needle (Terumo: NN-2613S). Immunization was performed once every week for a total of 4 times. In order to check the antibody titer during the fourth immunization, 50-100 μL of blood was collected from the inferior vein of the mouse and the antibody titer was confirmed by ELISA.

c. Primary screening (ELISA method)
Purified megsin protein at a concentration of 2 μg / mL in 0.1 M carbonate buffer pH 9.0, dispense 50 μL per well of a 96-well microtiter plate (Nunc), and leave it at 25 ° C. for 3 hours. I was sensitized. After removing the solution, 200 μL of 1% BSA (Bovine Serum Albumin) solution (0.1 M carbonate buffer pH 9.0) and 200 μL were dispensed and allowed to stand at 25 ° C. for 1 hour for blocking. After removing the blocking solution, 50 μL of the hybridoma supernatant to be screened or mouse serum solution is dispensed into each well and left at 25 ° C. for 1 hour (primary reaction). After removing the solution, it is washed with 50 mM phosphate buffer pH 7.2 (hereinafter: PBS) containing 0.13 M NaCl. Dispense 50 μL of Horseradish Peroxidase (HRP) labeled goat anti-mouse IgG (H + L) antibody (manufactured by MBL) into each well and leave it at 25 ° C. for 1 hour (secondary reaction). After removing the solution, it is washed with 50 mM phosphate buffer pH 7.2 containing 0.13 M NaCl. Add 50 μL of 0.8 mM TMB (tetramethylbenzidine: 3,3 ′, 5,5′Tetramethylbenzidine: manufactured by Dojindo Laboratories) solution per well, and develop color at 30 ° C. for 5-20 minutes. The color reaction was stopped by adding 50 μL of 1.5NH ₃ PO ₄ per well, and the absorbance at 450 nm was measured using a microtiter plate reader (Wallack: ARVO-SX).

d. Preparation of Monoclonal Hybridoma After confirming sufficient antibody titer, lymphocytes prepared from the extracted mouse spleen were fused with mouse myeloma cells (P3U1) using polyethylene glycol (manufactured by Wako Pure Chemical Industries). Hybridomas were selected by selecting HAT (hypoxanthine, aminopterin, thymidine: hypoxanthine, thymidine, SIGMA H-0137 aminopterin, SIGMA A-5159). A clone having high reactivity to the immunizing antigen was screened by ELISA using a hybridoma culture supernatant. Hybridoma 4F3, which produces an antibody that specifically binds to megsin protein and has a stable growth ability by repeating cloning by limiting dilution method three times for hybridoma that produces an antibody that binds to megsin protein A cell line was obtained. Hybridoma 4F3 cells were transferred to the National Institute of Advanced Industrial Science and Technology patent biological depository center located at 1-1-1 Higashi 1-1-1, Tsukuba, Ibaraki, Japan on March 16, 2004. Deposited as P-19734.
The contents specifying the deposit are described below.
(a) Name and address of depositary institution Name: National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center Address: Central 1st 1-1, Higashi 1-1-1 Tsukuba City, Ibaraki, Japan (Zip 305-8566)
(b) Date of deposit: March 16, 2004
(c) Accession number: FERM P-19734

e. Typing of monoclonal antibody Isotype was investigated using IsoStrip kit (manufactured by Roche) using 0.5 mL of the culture supernatant of the hybridoma 4F3 cell line obtained above diluted 150-fold with PBS. The isotype of the monoclonal antibody produced by the hybridoma 4F3 cell line was IgG1 (FIG. 1).

f. Preparation and purification of monoclonal antibody (4F3)
8 weeks old female Balb / c mice were intraperitoneally administered with 0.5 ml / pristane, and 10 days later, the hybridoma 4F3 cell line obtained by the cloning of d above was about 1 × 10 ⁷ cells / mouse. 0.5 mL / animal was injected intraperitoneally. Since about 10 days later, abdominal hypertrophy of the mice was observed, and ascites was collected after laparotomy. The collected ascites was centrifuged at 1000 rpm and 4 ° C. for 10 minutes, and the supernatant was allowed to stand at 37 ° C. for 30 minutes and then allowed to stand overnight at 4 ° C. After centrifugation at 12000 rpm and 4 ° C. for 10 minutes, the resulting supernatant was purified from a monoclonal antibody (4F3) using an affinity column (Sepharose Protein A: Amersham Biosciences). 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.

g. Immunoprecipitation
Add 500 μL of hybridoma culture supernatant to 25 μL of rmp Protein A Sepharose Fast Flow (hereinafter referred to as gel; manufactured by Amersham Biosciences) in a 1.5 mL Eppendorf tube, and gently stir at room temperature for 1 hour. After removing the culture supernatant, the gel is washed with PBS, 50 μL / tube of 10 μg / mL megsin protein PBS solution is added, and left at room temperature for 1 hour. After washing the gel with PBS, add 30 μL of SDS-PAGE Sample Buffer (Lemuri method) and boil for 3 minutes. 20 μL of the sample is subjected to SDS-PAGE in the same manner as in Example 2 below, followed by Western blotting. For detection of protein, anti-megsin rabbit polyclonal antibody was used as the primary antibody, and horseradish peroxidase (HRP) -labeled goat anti-mouse IgG (H + L) antibody (manufactured by MBL) was used as the secondary antibody. .

[Example 2] Examination of reactivity of monoclonal antibody by Western blotting A 0.1 μg protein solution of megsin protein prepared by the same method as that for the antigen for immunization is electrophoresed on 12.5% SDS-PAGE according to a conventional method. Using a semi-dry transfer device (manufactured by Biocraft), the migrated protein is transferred to an Immobilon-P transfer membrane (manufactured by Millipore). The membrane after transfer is blocked with Block Ace (Dainippon Pharmaceutical Co., Ltd.) (25 ° C, 1 hour), and then reacted with the hybridoma culture supernatant at 25 ° C for 1 hour. After washing the membrane with PBS, the horseradish peroxidase (HRP) -labeled goat anti-mouse IgG (H + L) antibody (manufactured by MBL) is reacted with the membrane at 25 ° C. for 1 hour. After washing the membrane with PBS, chemiluminescence was performed using an ECL Western Blotting kit (Amersham Bioscience), which was baked into Hyperfilm ECL (Amersham Bioscience) and detected.

  The antigen specificity of monoclonal antibody 4F3 was verified by Western blotting. As a result, monoclonal antibody 4F3 reacted with human megsin protein expressed in CHO cells (FIG. 2).

  Similarly, Western blot analysis was performed to examine the reactivity of monoclonal antibody 4F3 against megsin proteins with different species differences. E. coli recombinant mouse megsin and monoclonal antibody 4F3 reacted but did not react with E. coli recombinant rat megsin (FIG. 3).

  Similarly, Western blot analysis was performed to examine cross-reactivity to serpins other than megsin. As a result, the monoclonal antibody 4F3 did not react with α2-antiplasmin, α1-antitrypsin, antithrombin, plasminogen activator inhibitor 1 and E. coli-derived MBP fusion plasminogen activator inhibitor 2, and these serpins No crossing was observed (Fig. 4).

  Furthermore, the reactivity to the serine protease / serpin complex was verified by Western blot analysis. Monoclonal antibody 4F3 recognized a complex with plasmin, which is considered to be one of the megsin ligands (FIG. 5).

[Example 3] Epitope mapping of 4F3 antibody (1) (examination by Western blotting)
Trypsinized fragmented megsin protein Trypsin solution is added to megsin protein solution (megsin 0.5 mg / mL, trypsin 7800 U / mL), and then mixed with an equal volume of 2X loading buffer (Daiichi Kagaku). The sample solution was heated for 5 minutes in a boiling bath. The sample solution was electrophoresed on a 15-25% polyacrylamide gel (Daiichi Kagaku) using an SDS electrophoresis apparatus (Daiichi Kagaku) and Tris-Glycine buffer (Daiichi Kagaku). During electrophoresis, 2 sheets of 3MM filter paper (Whattman) for buffer A (Daiichi Kagaku), 1 sheet for buffer B (Daiichi Kagaku), 3 for buffer C (Daiichi Kagaku) for blotting I immersed it. A polyvinylidene difluoride membrane (PVDF membrane, manufactured by Millipore) was immersed in methanol and then immersed in purified water.

  Transfer the protein to the PVDF membrane. After the electrophoresis, remove the gel from the device, blotter (manufactured by Pharmacia), two filter papers soaked in buffer A from the anode side, one filter paper soaked in buffer B, PVDF The membrane, gel, and three filter papers soaked in buffer C were placed in the order listed and run at 80 mA / gel for 1.5 hours. After the transfer, a part of the PVDF membrane was stained with CBB. Moreover, a part of PVDF membrane was blocked by permeating with Block Ace (manufactured by Snow Brand Milk Products) at room temperature for 1 hour.

  After blocking, the membrane was reacted overnight at 4 ° C. with monoclonal antibody 4F3 diluted with antibody diluent. Thereafter, alkaline phosphatase-labeled anti-mouse IgG antibody was added, reacted for 1 hour at room temperature, and then developed with an NBT-BGIP solution. In this result, an amino acid sequence was performed on the band reacted with the monoclonal antibody 4F3 and confirmed by CBB staining. As a result, it was confirmed that this band was a megsin protein fragment having an N-terminal corresponding to amino acid sequence 204 of the megsin protein.

b. E. coli expressed fragmented megsin protein The following E. coli expressed fragmented megsin protein was prepared.
Total length: 1-380
1-6: 1-100
2-7: 101-168
3-8: 169-266
4-9: 267-321
5-10: 322-380
2-10: 101-380
3-10: 169-380
4-10: 267-380
Using these Escherichia coli-expressed fragmented megsins, the Western blotting method was performed in the same manner as in a. Above, and a band reacting with the monoclonal antibody 4F3 was confirmed. The results are shown in Table 1. From these results, it was confirmed that the site of the megsin protein that reacts with the monoclonal antibody 4F3 is in the amino acid sequence 204-266.

[Table 1]
---------------------------------------------
E. coli expressed megsin protein peptide fragment reactivity with 4F3 antibody
---------------------------------------------
1-380 ○
101-380 ○
169-380 ○
267-380 ×
1-100 x
169-266 ○
267-321 ×
322-380 ×
Vector ×
---------------------------------------------

[Example 4] 4F3 antibody epitope mapping (2)
a. ELISA method using Kobalink plate Based on the results of the fragmented E. coli expressed megsin protein obtained in Example 3, peptides of 10 to 20 residues were synthesized by a conventional method. Synthetic peptide is solid-phased on a Kobalink plate by the amine coupling method, washed twice with a washing solution, and blocked by adding Block Ace (manufactured by Snow Brand Milk Products) diluted 4 times with PBS. Add 100 μL / well of diluted monoclonal antibody 4F3 to the plate, and react at room temperature for 2 hours. After washing 4 times with the washing solution, 100 μL / well of POD-labeled anti-mouse IgG antibody is added and reacted at room temperature for 2 hours. After washing 4 times with the washing solution, 100 μL / well of TMB was added and reacted at room temperature for 30 minutes, and the absorbance (OD450) was measured (Tables 2 to 4).

[Table 2]
--------------------------------------------
Megsin protein peptide fragment OD450
--------------------------------------------
None 0.097
204-223 0.102
216-231 0.099
224-243 0.104
236-253 0.099
245-260 0.276
253-269 0.099
--------------------------------------------

[Table 3]
--------------------------------------------
Megsin protein peptide fragment OD450
--------------------------------------------
None 0.036
245-259 0.032
245-258 0.033
245-256 0.033
245-255 0.031
245-254 0.033
245-260 0.602
--------------------------------------------

[Table 4]
--------------------------------------------
Megsin protein peptide fragment OD450
--------------------------------------------
None 0.024
251-260 0.032
250-260 0.030
249-260 0.032
248-260 0.033
247-260 0.030
246-260 0.684
245-260 0.639
--------------------------------------------

b. Measurement of specificity by the biacore method Each megsin protein peptide was immobilized on the sensor chip CM5 (Biacore) by the amine coupling method, and the reactivity with the monoclonal antibody 4F3 was measured using the biacore method. The results are shown in Table 5.
[Table 5]
------------------------------------------------
Megsin protein peptide fragment Response (RU)
------------------------------------------------
236-253 8.2
246-260 207.7
253-269 1.9
------------------------------------------------

  As described above, from the results of Examples 3 to 4, it was found that the epitope recognized by the monoclonal antibody 4F3 is 246-260 (LSEIENKLTFQNLME / SEQ ID NO: 1).

Example 4 Comparison of Localization of Various Antibodies by Double Staining / Fluorescent Antibody Method Kidney tissue OCT-embedded frozen specimens were sectioned into 4 μm, air-dried, and ethanol / ether (1: 1) for 10 minutes with ethanol Fixed for 10 minutes. Incubation was performed at room temperature for 20 minutes in methanol supplemented with 0.3% aqueous hydrogen peroxide to block endogenous peroxidase. After washing 3 times with PBS containing 0.25% Tween20, the sections were blocked with PBS containing 10% goat serum at room temperature for 2 hours, then megsin monoclonal antibody 4F3, Cy3-labeled anti-vimentin antibody (manufactured by SIGMA), anti-von Willerbrand Factor antibody (Manufactured by DAKO), anti-megsin peptide polyclonal antibody (antibody against the 72-86 peptide fragment of megsin protein: P2 antibody), and PBS containing 2% bovine serum albumin at 4 ° C. overnight. As secondary antibodies, FITC-labeled anti-mouse antibody and Rhodamine anti-rabbit antibody were used to compare the localization of various antibodies.

  The results are shown in FIGS. The staining patterns of the megsin monoclonal antibody 4F3 and the glomerular epithelial cell marker (vimentin) almost coincided (FIG. 6-1). On the other hand, it did not coincide with the staining pattern of the endothelial cell marker (von Willerbrand Factor) (FIG. 6-2). Moreover, since the anti-megsin peptide polyclonal antibody (P2 antibody) reacted with the megsin protein in mesangial cells, the staining pattern was significantly different from that of the monoclonal antibody 4F3 (FIG. 6-3). From the above, it was confirmed that the monoclonal antibody 4F3 reacts with the megsin protein localized in the glomerular epithelial cells.

[Example 5] Comparative examination of stained images of various diseases by immunohistochemical staining using monoclonal antibody 4F3 In order to perform immunohistochemical analysis, the kidney was excised, fixed with methyl carnoir, and then a paraffin-embedded block was prepared. . The paraffin-embedded block was sliced into 4 μm with a microtome to obtain paraffin sections. Deparaffinized in xylene for 5 minutes 3 times, treated with 100% ethanol 3 times 5 minutes, 90% ethanol 1 time, 70% ethanol 1 time, hydrated with PBS, 0.05% Pronase (DAKO ) For 15 minutes to activate the antigen. After washing with PBS, endogenous peroxidase was blocked using Peroxidase blocking reagent (DAKO), and endogenous biotin was blocked using Biotin blocking system (DAKO). After washing with PBS, the sections were blocked in a humidified chamber (37 ° C.) with Block Ace (manufactured by Snow Brand Milk Products) for 1 hour, and then the monoclonal antibody 4F3 was added as a primary antibody and incubated at 4 ° C. overnight. After washing with PBS three times, the section was incubated for 1 hour at 37 ° C. in a humidified chamber using a biotin-labeled horse anti-mouse IgG antibody (manufactured by VECTOR) diluted 400 times. Tissue sections were washed 3 times with PBS, incubated with peroxidase avidin D (VECTOR) diluted 1: 1000 in a humidified chamber for 30 minutes at room temperature, and the tissue sections were washed 3 times with PBS. For detection of peroxidase, a 3,3′-diaminobenzidine (DAB) solution containing 0.03% hydrogen peroxide was used, and then nuclear staining was performed with hematoxylin.

  As a result, the megsin localization in the glomerular epithelium was enhanced in membranous nephropathy (Fig. 7-2) and minute change nephrosis (Fig. 7-3), compared with normal renal glomeruli (Fig. 7-1). There was a tendency to On the other hand, in IgA nephropathy (FIGS. 7-4), the localization of megsin in the glomerular epithelium was decreased. These results show that the staining intensity varies depending on the type of renal disease in immunohistochemical staining using monoclonal antibody 4F3, which preferentially detects megsin protein in glomerular epithelial cells. Usefulness as a diagnostic antibody was confirmed.

  According to the monoclonal antibody of the present invention and the diagnostic method using the same, diseases caused by abnormalities of renal epithelial cells (membrane nephropathy and minute change nephrosis) and diseases caused by abnormal mesangial cells (IgA nephropathy and diabetes) (Nephropathy) can be differentiated.

It is a photograph which shows the isotype of the monoclonal antibody 4F3 using IsoStrip kit made from Roche. It is a photograph showing the results of Western blotting showing the reactivity between the monoclonal antibody 4F3 of the present invention and human megsin protein. It is a photograph showing the results of Western blotting showing the reactivity between the monoclonal antibody 4F3 of the present invention and megsin proteins having different species differences. It is a photograph showing the result of Western blotting showing the cross-reactivity between the monoclonal antibody 4F3 of the present invention and serpin other than megsin protein. 2 is a photograph showing the results of Western blotting showing the reactivity between the monoclonal antibody 4F3 of the present invention and a serine protease / serpin complex. It is a photograph showing the localization of the monoclonal antibody 4F3 of the present invention and the epithelial cell marker (vimentin) using double staining / fluorescence antibody method. It is a photograph which shows the localization of the monoclonal antibody 4F3 of this invention and endothelial cell marker (von Willerbrand Factor) using the double dyeing | staining and the fluorescent antibody method. It is a photograph showing the localization of monoclonal antibody 4F3 of the present invention and anti-megsin peptide polyclonal antibody using double staining / fluorescence antibody method. 2 is a photograph showing a stained image of normal kidney glomeruli by immunohistochemical staining with the monoclonal antibody 4F3 of the present invention. 2 is a photograph showing a stained image by immunohistochemical staining of glomeruli with membranous nephropathy using the monoclonal antibody 4F3 of the present invention. FIG. 3 is a photograph showing a stained image by immunohistochemical staining of glomeruli of minute change nephrosis with the monoclonal antibody 4F3 of the present invention. 2 is a photograph showing a stained image of an IgA nephropathy glomerulus by immunohistochemical staining with the monoclonal antibody 4F3 of the present invention.

Claims (7)

Monoclonal antibody that binds to megsin protein localized in renal glomerular epithelial cells. A monoclonal antibody that recognizes an epitope constituted by a peptide having the amino acid sequence set forth in SEQ ID NO: 1. A hybridoma cell line producing the monoclonal antibody according to claim 1 or 2. Hybridoma 4F3 deposited as FERM P-19734. Monoclonal antibody produced by hybridoma 4F3 deposited as FERM P-19734. A method for producing a monoclonal antibody, comprising culturing a hybridoma 4F3 deposited as FERM P-19734 and recovering an immunoglobulin contained in the culture. A method for diagnosing renal injury, comprising a step of detecting a monoclonal antibody that binds to a megsin protein localized in glomerular epithelial cells.