WO2007074864A1 - Antibody capable of recognizing asymmetric dimethylarginine, method for production of the antibody, and method for detection of protein having posttranslationally modified amino acid - Google Patents

Abstract

Disclosed are: a novel antibody capable of reacting specifically with asymmetric dimethylarginine (ADMA) or a fragment of the antibody; a hybridoma capable of producing the antibody; a method for production of the antibody; and a method for analysis of an ADMA-containing basic protein using the antibody. The antibody can bind specifically to ADMA and shows an increased reactivity to a acylated protein. A protein containing ADMA has not been identified yet, but it now becomes possible to detect the protein by utilizing the combination of the antibody and the acetylation of a protein.

Description

 Specification

 Antibody recognizing asymmetric dimethylarginine, production method thereof, and detection method of post-translationally modified amino acid-containing protein

 Technical field

 [0001] The present invention relates to a novel antibody or antibody fragment that specifically reacts with asymmetric dimethylarginine, a hybridoma that produces the antibody, a method for producing the antibody, and asymmetric dimethylarginine using the antibody. The present invention relates to a method for detecting a contained protein.

 Background art

 [0002] It is known that in vivo proteins undergo various post-translational modifications after being translated from mRNA, without functioning as they are. For example, protein phosphates play an important role as signal cascades in transmitting extracellular signals to the nucleus or as regulators for normal cell cycle progression. Histone acetylenes, which are involved in the formation of nuclear chromatin structures, are important for efficient transcription. It is also known that when a protein is ubiquitinated, it is transported to the proteanome and decomposes to lose its activity. On the other hand, many proteins become active when the signal peptide is cleaved off by a signal peptidase present in the endoplasmic reticulum membrane. In this way, after being translated from mRNA, the protein is subjected to various modifications, thereby exhibiting each function at an appropriate place at an appropriate time. On the other hand, this post-translational modification abnormality is considered to cause various diseases.

[0003] Among such post-translational modifications of proteins, methylation of arginine residues in proteins has recently attracted attention. Methyl arginine includes symmetric dimethyl arginine (hereinafter sometimes referred to as SDMA), asymmetric dimethyl arginine (hereinafter sometimes referred to as ADMA), and an intermediate from arginine to SDMA or ADMA. There is a certain monomethylarginine (hereinafter sometimes referred to as MMA). Of these, ADMA in blood is thought to affect NO metabolism in vascular tissues and vascular endothelial function, and is considered to be related to arteriosclerotic lesions. [0004] In order to investigate the post-translational modification of proteins and the functional expression of proteins, or the relationship with diseases, including ADMA, it is important to first examine what proteins have undergone post-translational modifications. As a means for detecting a protein subjected to post-translational modification, a method using a probe molecule specific for each post-translational modification can be considered. For example, as a probe that recognizes phosphorylated tyrosine, many antibodies that react specifically with phosphorylated tyrosine are commercially available and widely used. However, useful antibodies that can detect ADMA efficiently are not generally available. Only about 20 types of proteins containing ADMA have been identified.

 [0005] Under such circumstances, Boisvert et al. Formed a peptide represented by SEQ ID NO: 1 (KG—ADMA—G—ADMA—G—ADMA—G—) in which ADMA and glycine form a cluster! ADMA-G-P-P-P-P-P-ADMA-G-ADMA-G-ADMA-G-ADMA-G (hereinafter referred to as ADMA-G rich peptide) An antibody (ASYM24) was obtained. The reason why they used this peptide is that the main enzymes that methylate arginine, PRMTl (typel) and PRMT5 (typell) forces Arginine and glycine form a clustered region (hereinafter RG-rich) This is because it was known to selectively methylate arginine (called a cluster). They found that the protein containing ADMA was isolated by immunoprecipitation using this antibody, and the amino acid sequence of the protein was determined to have most of the protein strength RG-rich cluster. Therefore, it is presumed that the ASYM2 4 antibody, which is an antibody against ADMA, recognizes ADMA present in the RG rich cluster. (Non-patent document 1).

 [0006] Non-Patent Document 1: “Molecular & Cellular Proteomics” (USA), 2003, No. 2, No. 12, p. 1319-1330 Disclosure of the Invention

 Problems to be solved by the invention

[0007] However, the present inventors thought that asymmetric dimethylarginine exists in addition to the region of the RG rich cluster of the protein. Conventional antibodies were thought to detect proteins with ADMA present in RG-rich clusters. It was difficult to analyze proteins with ADMA existing in other regions. As a result of earnest research on antibodies capable of detecting a novel protein having ADMA, the present inventors have immunized animals with a complex in which a peptide having a structure different from that of a conventional immunogen is bound to a carrier protein. It has been found that an antibody exhibiting reactivity different from that of a known anti-ADMA antibody can be prepared. Furthermore, the antibody of the present invention can detect ADMA present in proteins with a small positive charge. Further, the present inventors have found a method for detecting a basic protein containing ADMA, which has not been identified in the past, by combining the antibody of the present invention with a chemical modification of a protein, in particular, acetylene cocoon. The present invention is based on these findings.

Means for solving the problem

 The above-mentioned problem is obtained by chemically modifying a protein side chain on a protein containing asymmetric dimethylarginine and reacting specifically with asymmetric dimethylarginine. This can be solved by an antibody that reacts specifically with the metric dimethylarginine-containing protein. In a preferred embodiment of the antibody of the present invention, it specifically binds to asymmetric dimethylarginine and has the formula (4):

 Gly— ADMA— Lys— Cys— BSA (4)

 [ADMA is asymmetric dimethylarginine, and BSA is ushi serum albumin].

Gly-ADMA-AcLys-Cys-BSA (8)

[ADMA is asymmetric dimethylarginine, AcLys is acetylated lysine, and BSA is ushi serum albumin]. In another preferred embodiment of the antibody of the present invention, it is a mouse monoclonal antibody, in particular, an antibody secreted and produced by a hybridoma having the accession number FERM BP-10458. The present invention also relates to an antibody fragment, which is a fragment of the antibody, comprising an antigen-binding site that specifically reacts with asymmetric dimethylarginine. The present invention also relates to a hybridoma that produces the above-mentioned antibody, and in particular, a high number having the accession number FERM BP-10458. About Pridoma.

 [0009] Further, the present invention provides a general formula (9):

 X-ADMA-Z-Cys (9)

 [Wherein X is a peptide fragment consisting of the same or different amino acid residues 1 to 5 other than Cys, ADMA is asymmetric dimethylarginine, and Z is the same or different amino acid residues 1 to 5 other than Cys. The present invention also relates to a method for producing an anti-asymmetric dimethylarginine antibody, characterized in that a peptide represented by the sequence of [5 is a peptide fragment consisting of 5] is bound to a carrier protein to immunize an animal. In a preferred embodiment of the method for producing an antibody of the present invention, the peptide is represented by the general formula (9), wherein X is Gly and Z is ε-aminocaproic acid. In another preferred embodiment of the method for producing an antibody of the present invention, the animal is an autoimmune disease mouse, particularly an MRL lprZlpr mouse.

 [0010] Furthermore, the present invention includes (1) a step of chemically modifying a protein side chain on a test sample that may contain a post-translationally modified amino acid-containing protein, and (2) a protein side chain. A step of contacting a probe that specifically reacts with a post-translationally modified amino acid-containing protein obtained by chemical modification of the sample and the chemically modified test sample; and (3) formed by the contacting step. The present invention also relates to a method for detecting the post-translationally modified amino acid-containing protein, comprising a step of detecting a binding (complex) between the chemically modified post-translationally modified amino acid-containing protein and the probe. A preferred embodiment of the method for detecting a post-translationally modified amino acid-containing protein of the present invention is a protein containing a post-translationally modified amino acid-containing protein strength asymmetric dimethylarginine. In another preferred embodiment of the method for detecting a post-translationally modified amino acid-containing protein of the present invention, the chemical modification of the protein side chain is acylation or alkylation. In another preferred embodiment of the method for detecting a post-translationally modified amino acid-containing protein of the present invention, the antibody or antibody fragment specifically reacts with the probe power ADMA for the modified amino acid. In another preferred embodiment of the method for detecting a post-translationally modified amino acid-containing protein of the present invention, the method further comprises a step of fractionating the protein by isoelectric point, molecular weight, or a combination of isoelectric point and molecular weight.

[0011] Furthermore, the present invention provides (1) a test that may contain a post-translationally modified amino acid-containing protein. A process for changing the charge of the protein on the sample; (2) a process for separating the test sample subjected to the charge change by isoelectric point of the protein; and (3) after the separation. A step of contacting a test sample with a probe that specifically reacts with a post-translationally modified amino acid, and (4) binding (complex) of the fractionated protein formed by the contact step and the probe. The present invention also relates to a method for detecting a post-translationally modified amino acid-containing protein, comprising a step of detecting. In a preferred embodiment of the method for detecting a post-translationally modified amino acid-containing protein of the present invention, fractionation based on the molecular weight of a protein is performed before or after the fractionation step. In another preferred embodiment of the method for detecting a post-translationally modified amino acid-containing protein of the present invention, the probe is an antibody against methyllysine.

 The antibody of the present invention is useful for searching for and identifying a protein containing asymmetric dimethylarginine. In addition, the post-translationally modified amino acid-containing protein detection method combining the antibody and protein chemical modification of the present invention has made it possible to identify a powerful protein containing ADMA that could not be identified conventionally. Using the antibody and detection method of the present invention, it becomes possible to identify an ADMA-containing protein as a target molecule associated with a disease. Furthermore, the antibody of the present invention can be used for therapy by controlling the function of a target molecule associated with the identified disease and for diagnosis by detecting the target molecule.

 Brief Description of Drawings

[0013] [Fig. 1] Antibody titer of immunized mice Shows the antibody titer in blood by the ELISA method of BalbZc and MRL-lprZlpr mice 3 days after the final immunization with RME-GaR _£ AC-KLH complex.

[Figure 2] Monoclonal antibody reactivity (ELISA by antigen) Established mouse monoclonal antibody DMA2—2G11, ADMA2—2H5, ADMA2—2H7, ADMA2—4E6, A DMA2—5E10, or ADMA2—3C10 (sub) Clones ADMA2—3C10A and A DMA2-3C10B) ADMA-containing peptides RME-LaRC-BSA (A), RME-GaRKC—BSA (B), RME—GaR ε AC—BSA (C), and ADMA-free peptides The reactivity with a troll peptide is shown. As antibody controls, the reactivity of monoclonal antibody Clone 7E6 and polyclonal antibody ASYM24 is shown. [Figure 3] Reactivity of monoclonal antibodies (ELISA: by antibody) Established mouse monoclonal antibodies ADMA2-5E10, ADMA2-3C10, and ADMA2-2H5 ADMA-containing peptides RME—GaR ε AC—BSA RME—GaRKC—BSA ゝ RME—LaRC—BSA, a peptide containing SDMA RME—LsRC—BSA, acetylated! The reaction with LRGRGRKC-BSA, AcK-BSA (complex of acetylated lysine and BSA) and MeK-BSA (complex of methyllysine and BSA), which are peptides containing arginine, is shown. As antibody controls, the reactivity of polyclonal antibody ASYM24, monoclonal antibody Clone 7E6 and SDMA to polyclonal antibody SYM11 is shown.

 [Fig. 4] Inhibition of monoclonal antibody reaction by amino acids (ELISA) Inhibition of mouse monoclonal antibodies ADMA2-5E10, ADMA2-3C10, and ADMA2-2H5 by ADMA, MMA, or arginine (Arg). Show.

[Fig. 5] Effect of sulfo NHS acetate (ELISA) By treating RME-GaRKC-BSA (10 μg / mL) and LRGRGRKC—BSA with sulfo NHS acetate, the mouse monoclonal antibodies ADMA2-5E10, ADMA2-3C10 3 shows changes in reactivity of ADMA2-2H5, polyclonal antibody ASYM24, and monoclonal antibody Clone 7E6.

 [FIG. 6] Effect of sulfo NHS acetate (Western) The reactivity of mouse monoclonal antibody ADM A2-2H5 and polyclonal antibody ASYM24 to the protein of MOLT-4F cells treated or not treated with sulfo NHS acetate is shown. ACK2F12 is a monoclonal antibody against acetyl lysine, indicating that the protein lysine is acetylated.

 [FIG. 7] Western blotting of mouse organs Western blotting using mouse monoclonal antibody ADMA2-2H5 for the extracted tissue protein.

FIG. 8 shows the results of Western blotting using mouse monoclonal antibody ADMA2-2H5 after 2D electrophoresis of 2D- Western HepG2 cell protein of HepG2 lysate. [Fig. 9] Detection of ADMA-containing protein by sulfo NHS acetate treatment Western blotting using mouse monoclonal antibody ADMA2-2H5 after two-dimensional electrophoresis of protein of HepG2 cells with sulfo NHS acetate treatment or untreated. Show fruit.

 [Fig. 10] Detection of methyllysine-containing protein by treatment with sulfo NHS acetate Shows the results of Western blotting using mouse monoclonal antibody MEK3D7 after 2D electrophoresis of proteins in Hep G2 cells treated with sulfo NHS acetate or untreated .

BEST MODE FOR CARRYING OUT THE INVENTION

 The antibody of the present invention specifically reacts with asymmetric dimethylarginine (ADMA) and does not react with symmetric dimethylarginine (SDMA). ADMA recognized by the antibody of the present invention is represented by the general formula (1):

[Chemical 1]

An arginine in which two hydrogen atoms in the side chain of an arginine represented by the formula are substituted with a methyl group. SDMA to which the antibody of the present invention does not react is a general formula (2):

[Chemical 2]

The arginine substituted by the hydrogen atom of the amino group in the side chain of arginine and the hydrogen atom cation group of the imino group. The antibodies of the present invention include monomethylarginine and arginine. It is an antibody that reacts specifically with ADMA and does not react with ginin. That is, specifically reacting with ADMA means reacting with ADMA but not with SDMA, monomethylarginine, and arginine.

[0015] The antibody of the present invention specifically reacts with a complex of the following three types of peptides containing ADMA and BSA.

 General formula (3):

 Leu-ADMA-Cys-BSA (3)

 (BSA is ushi serum albumin: hereinafter sometimes referred to as RME-LaRC-BSA) General formula (4):

 Gly-ADMA-Ly s-Cys-BSA (4)

 (Hereafter referred to as RME—GaRKC—BSA)

 General formula (5):

 Gly-ADMA-Acp-Cys-BSA (5)

 (Acp is ε-aminocaproic acid: hereafter referred to as RME-GaR ε AC— BSA)

 [0016] On the other hand, the antibody of the present invention does not react with the following peptide-BSA complex containing SDMA and methylated! /, NA! /, Arginine.

 General formula (6):

 Leu-SDMA- Cys-BSA (6)

 (Hereafter, it may be called RME—LsRC—BSA)

 General formula (7):

 Leu-Arg-Gly-Arg-Gly-Arg-Lys-Cys-BSA (7) (hereinafter sometimes referred to as LRGRGR KC BSA)

[0017] The reaction of the antibody of the present invention against RME-GaRεAC-BSA, which is a complex containing ADMA, is inhibited in a dose-dependent manner by adding free ADMA to the reaction system. The addition of arginine and arginine was not inhibited. These reactivities are those in which the antibody of the present invention specifically recognizes one asymmetric dimethylarginine, and an antibody that recognizes ADMA and its surrounding amino acids. It shows no.

 [0018] Further, the antibody of the present invention is a complex of RSA-GaRKC-BSA lysine acetylated peptide and BSA.

 General formula (8):

 Gly-ADMA-AcLys-Cys-BSA (8)

 (Hereinafter sometimes referred to as RME—GaRacKC—BSA),

 On the other hand, it shows a stronger reaction than that for RME-GaRKC-BSA. The antibody of the present invention does not react with LRGRGRKC- BSA lysine acetylated complex, and therefore reacts with ADMA rather than reacting with peptide or BSA acetylated lysine. . Furthermore, the antibody of the present invention becomes highly reactive to proteins by acetylating lysine contained in proteins in cells. Therefore, by acetylating the protein, it becomes possible to detect a protein containing ADMA, which has been impossible to detect conventionally.

 [0019] On the other hand, as shown in Example 6 described later, Clone 7E6, which is a commercially available monoclonal antibody against ADMA, does not react with RME-GaRKC-BSA or RME-GaRacKC BSA. . In addition, the ASYM24 antibody, which is a polyclonal antibody obtained using an ADMA-G rich peptide as an immunogen, is more reactive to RME-GaRKC-BSA than RME-GaRacKC-BSA, in contrast to the antibody of the present invention. To do. Furthermore, the ASYM24 antibody becomes less reactive to the protein by acetylating lysine contained in the protein in the cell.

[0020] The antibody of the present invention is characterized in that the binding force of the antibody to RME-GaRacK C BSA is stronger than the binding force to RME-GaRKC-BSA. In this specification, the strong binding force means that the affinity constant is high, the dissociation constant is low, or the binding amount of the antibody is increased. The binding force of the antibody of the present invention to RME—GaRKC BS A or RME—GaRacKC—BSA can be measured by solid-phase enzyme immunoassay (ELISA), Western blotting, radioimmunoassay, etc. is there. For example, when examining the binding force by ELISA, (a) RME—Ga RKC-BSA (10 g / mL) is immobilized on an ELISA plate. (b) Sulfo NHS carbonate in PBS 0 Dissolve in mM (untreated), ImM, or lOmM, and treat for 1 hour at 25 ° (50). (c) Block each well of the plate with 1% BSA-PBST for 1 hour. (d) Antibody to be measured Dilute to 1000 ngZmL, 333 ng / mL, ll lng / mL, 37 ng / mL, 12.3 ng / mL, 4. lng / mL, or 1.4 ng / mL with PBST, add 50 / z L, and 30 minutes at room temperature (E) Add 50 μL · (l μg / mL) of HRP-labeled anti-mouse IgG antibody and allow to react for 30 minutes at room temperature (f) OPD substrate solution [20 mM—o-phenylenediamine, 0 0.1% citrate phosphate buffer solution (pH 5.0) containing 05% hydrogen peroxide solution] Add 100 L and react for 10 minutes at 25 ° C. (G) Measure absorbance at 492 nm. In the antibody of the invention, the binding force of the antibody to RME-GaRacKC-BSA is stronger in any of the above-mentioned antibody concentrations by ImM treatment or 10 mM treatment than OmM treatment (untreated) of sulfo NHS acetate. Absorbance power at 492 nm Specifically, the strong binding force of an antibody indicates that the absorbance (A) of sulfo NHS acetate in ImM treatment or 10 mM treatment is the absorbance (B) of sulfo NHS acetate in OmM treatment. (X) Force means greater than 1, preferably 1.01 or more, more preferably 1.1 or more, and even more preferably 1.5 or more.

 An increase in binding force can also be expressed by an increase in association constant (Association constant, Ka) or a decrease in dissociation constant (Dissociation constant, Kd). The reaction in which an antigen and an antibody are combined to form an antigen-antibody conjugate is a reversible reaction. When the reaction reaches equilibrium, the concentrations of unbound antigen, unbound antibody, and antigen-antibody conjugate are respectively Ag], [Ab], [Ag'Ab]

Ka = [Ag'Ab], [Ag] [Ab]

 Kd = [Ag] [Ab] Z [Ag'Ab], which can be obtained by analysis such as Scatchard plot using radiolabeled antigen. An approximate value can also be obtained by ELISA. For example,

 [Ag] + [Ab] <"~ [Ag'Ab]

 Kd = [Ag] [Ab] / [Ag-Ab]

It becomes. If the total amount (concentration) of bound antibody is Ab,

 tot

[Ag'Ab] = Ab-[Ag] / (Kd + [Ag]) It becomes. When [Ag] when 50% of Ab is bound to Ag is Ag,

 1/2

 [Ag-Ab] = l / 2-Abtot

 So, if you substitute these,

 Kd = Ag

 1/2

 It becomes. Therefore, Kd can be estimated by assuming that [Ag] is hardly affected by the binding of Ag in the liquid phase and is proportional to the signal strength [Ag 'Ad] after washing in the ELISA method. And a decrease in the value of Kd represents an increase in binding force. This estimation method is particularly effective when the concentration of the adsorbed antibody is sufficiently smaller than the Kd value. Specifically, strong antibody binding means that Ka in sulfo NHS acetate ImM treatment or 10 mM treatment is greater than the value of 1 when divided by Ka in sulfo NHS acetate OmM treatment, Preferably it is 1.01 or more, More preferably, it is 1.1 or more, More preferably, it is 1.5 or more. Alternatively, strong antibody binding means that the Kd in OmM treatment of sulfo NHS acetate divided by Kd in ImM treatment or 10 mM treatment of sulfo NHS acetate is greater than 1. Preferably it is 1.01 or more, More preferably, it is 1.1 or more, More preferably, it is 1.5 or more.

[0022] The antibody of the present invention includes a polyclonal antibody and a monoclonal antibody, more preferably a monoclonal antibody. The antibody fragment of the present invention is a fragment of the antibody of the present invention, and is an antibody fragment that has the same reaction specificity as the original antibody. That is, the antibody fragment of the present invention specifically reacts with ADMA and does not react with SDM A, monomethylarginine, or unmethylated arginine. In addition, RME-GaRacKC-BSA, which is a peptide in which lysine continuous with ADMA is acetylated, exhibits a stronger response than RME-GaRKC-BSA, and acetylates lysine contained in proteins in cells. Increases the reactivity to proteins. Examples of the antibody fragment of the present invention include Fab, Fab \ F (ab '), or Fv

 2 etc. are included. These fragments can be obtained, for example, by digesting the monoclonal antibody of the present invention with a proteolytic enzyme according to a conventional method, and subsequently following a conventional method for protein separation and purification.

[0023] The antibody of the present invention has the general formula (9): X-ADMA-Z-Cys (9)

 [Wherein X is a peptide fragment consisting of the same or different amino acid residues 1 to 5 other than Cys, ADMA is asymmetric dimethylarginine, and Z is the same or different amino acid residues 1 to 5 other than Cys. It is a peptide fragment consisting of 5]

 The peptide represented by the sequence can be prepared by binding to a carrier protein and immunizing an animal.

 In the general formula (9), X is any amino acid other than cysteine. X is not particularly limited as long as it is an amino acid other than Cys, but amino acids that do not affect ADMA on the C-terminal side are preferred.For example, amino acids having nonpolar side chains and uncharged polar side chains. Is preferred. Specific amino acids include glycine, alanine, parin, serine and the like, with glycine being particularly preferred. Further, the continuous length of the amino acid of X is not particularly limited, but is preferably an integer of 1 to 5, and more preferably 1.

 Z is also any amino acid other than cysteine. Z is not particularly limited as long as it is an amino acid other than Cys, but preferably an amino acid that does not affect ADMA on the N-terminal side, for example, an amino acid whose side chain has a nonpolar side chain and an uncharged polar side chain. preferable. Specific examples of amino acids include glycine, alanine, Norin, serine, and ε-aminocaproic acid (6-aminocaproic acid), and ε-aminocaproic acid (6-aminocaproic acid) is particularly preferable. Further, the continuous length of the wrinkles is not particularly limited, but is preferably an integer of 1 to 5, more preferably 1. The peptide represented by the general formula (9) is bound to a carrier protein using the thiol group (SH group) of Cys on the C-terminal side. For this reason, when Cys is contained in X or Z, Cys other than the C-terminal side may bind to the carrier protein and ADMA may be hidden, which is not preferable in terms of the structure of the immunogen.

 [0025] The peptide of the general formula (9) can be prepared by chemical synthesis, for example, Fmoc solid phase synthesis method or Boc solid phase synthesis method. The synthesized peptide can be purified by a known method such as HPL C. The peptide is bound to a carrier protein using the SH group of Cys of the peptide of the general formula (9).

In the present specification, the “carrier protein” is not particularly limited as long as it is a protein capable of binding to the peptide to form a complex and exhibiting immunogenicity. More than 10,000, preferably 40,000 to 1 million proteins are preferred. Specific examples include sushi serum albumin, immunoglobulin, ovalbumin, mosquito hemocyanin (KLH), and the like. The peptide and carrier can be bound using the SH group of cysteine and the functional group of the carrier protein. The functional group of the carrier protein is not limited as long as it is a functional group that binds to the SH group, and includes a thiol group or an amino group. The binding method can be carried out according to a conventionally known method. For example, maleimide, carbodiimide, glutaraldehyde, sulfo GMBS, or GMBS can be used as a cross-linking agent between a peptide and a carrier protein. it can.

 [0027] The antibodies of the present invention include animal polyclonal antibodies and human mouse monoclonal antibodies. Methods for immunizing animals to obtain antibodies and methods for obtaining hyperprideomas producing monoclonal antibodies It can be carried out by a known method except that a complex of the peptide of the general formula (9) and a carrier protein is used as an immunogen. For example, it can be performed according to the method described in the Second Biochemistry Experiment Course (Japan Biochemical Society) or the Immunobiological Research Method (Japan Biochemical Society).

 [0028] The antibody of the present invention can be obtained by immunizing an animal using the complex of the peptide of the general formula (9) and a carrier protein as an immunogen. The animal used for immunization is not particularly limited, and hidge, goat, rabbit, mouse, rat, guinea pig, bird, horse, horse and the like can be used. However, since asymmetric dimethylarginine is an amino acid that is naturally present in the body of a normal animal, an antibody against ADMA may be difficult to produce in an animal with normal immune function. Therefore, the animal used for immunization is preferably an animal with an autoimmune disease that easily produces autoantibodies. In particular, when obtaining the antibody of the present invention in a mouse, it is preferable to use an MRL-lprZlpr mouse, which is an autoimmune disease mouse.

[0029] The immunization method is not particularly limited as long as a known method is used. For example, the complex is emulsified and mixed with an equal amount of Freund's complete adjuvant or Titer-Max gold (Titer Max), and a rabbit is used. Administered subcutaneously or intraperitoneally in mice. Thereafter, immunize several times with the same procedure at 1-2 week intervals. The antibody of the present invention can be prepared by collecting the blood of the immunized animal to obtain serum or plasma. [0030] The hyperidoma of the present invention that produces the monoclonal antibody of the present invention can be obtained from an animal that has undergone the above-described immunization. For example, two weeks after the immunization of mice, the peptide dissolved in phosphate buffered saline (PBS) or the like is inoculated from the tail vein. Two to three days later, the spleen containing lymphocytes that produce mouse antibody is aseptically removed. This lymphocyte can be established as a hybridoma that produces a monoclonal antibody by cell fusion with myeloma cells in the presence of polyethylene glycol, for example.

 [0031] When cell fusion is performed, for example, lymphocytes and myeloma cells are fused in the presence of polyethylene glycol. As the myeloma cell, various known cells can be used, and examples thereof include cells such as p3-NS-l / l-Ag4.1 or SP2Z0-Agl4. Fused cells are selected by killing non-fused cells using a selective medium such as HAT medium. Next, screening is performed for the presence or absence of antibody production in the culture supernatant of the growing hyperidoma. Screening can be performed by measuring the production of specific antibodies against asymmetric dimethylarginine by solid phase enzyme immunoassay (ELISA). Thus, the hybridoma cell line ADMA2-2H5 [Accession No. FERM BP-10458] producing the representative monoclonal antibody of the present invention can be selected. As of November 30, 2005, it was deposited internationally at the National Institute of Advanced Industrial Science and Technology patent biological deposit center (address: 1st, 1st, 1st, 1st, 6th, Tsukuba, Higashi 305-8566, Japan). It was.

 [0032] The hyperidoma of the present invention can be subcultured in any known medium, for example, RPMI1640. The monoclonal antibody of the present invention can be prepared by culturing this hyperidoma, for example, by adding 10% fetal bovine serum to RPMI1640 medium and culturing at 37 ° C in the presence of 5% CO. Antibody produced in the culture supernatant

 2

It is. In addition, antibodies can be produced in ascites by inoculating Hypridoma into the abdominal cavity of mice and collecting ascites. The antibody of the present invention can be purified by a known method. For example, a purification method using Protein G, a method using a affinity column to which ADMA is bound, or a method using ion exchange column chromatography. It can be purified by methods.

 [0033] As described above, the antibody of the present invention reacts more strongly with RME-GaRacKC-BSA in which lysine of RME-GaRKC-BSA is acetylated than with RME-GaRKC-BSA. Furthermore, by reacting cell protein lysine with acetylene, reactivity to many proteins is enhanced. By utilizing this property of the antibody of the present invention, it becomes possible to detect a protein containing ADMA, which could not be detected until now. In other words, since the binding between the antibody of the present invention and ADMA in the protein is strengthened by acetylating the protein, it can be bound to a protein having strong ADMA that could not be identified conventionally. Clone 7E6, a conventionally known antibody, cannot bind to acetylated protein. In addition, since the ASYM24 antibody recognizes RG-rich cluster ADMA and becomes less reactive to acetylated protein, it cannot be effectively used in the method for detecting a post-translationally modified amino acid-containing protein of the present invention.

 [0034] << Action >>

 The reason why the antibody of the present invention reacts more strongly with RME-GaRacKC-BSA than with RME-GaRKC-BSA, and the reason why protein lysine enhances the reactivity to protein. Is not fully elucidated, but can be inferred as follows. However, the present invention is not limited by the following description. First, lysine of RME-GaRKC-BSA is a basic amino acid. The acetyl group replaces the hydrogen atom of the side chain of the lysine with the acetyl group. Therefore, it is considered that the positive charge of lysine disappears. The antibody of the present invention is capable of recognizing ADMA. When a basic amino acid is present in the vicinity of ADMA, binding to ADMA is considered to be inhibited under the influence of positive charge. However, when lysine is acetylated, the positive charge around ADMA is weakened, and the binding of the antibody of the present invention to ADMA is thought to increase. In the case of intracellular proteins, as in the case of the aforementioned peptides, the positive charge around ADMA is neutralized by the acetylation of the lysine amino group present around ADMA recognized by the antibody of the present invention. It may be considered that the antibodies of the present invention can be combined and bind!

[0035] On the other hand, ASY is a known antibody obtained using an ADMA-G rich peptide as an immunogen. In contrast to the antibody of the present invention, M24 has the ability to weaken the reactivity to the protein by acetylating the lysine of the protein of the cell. ADMA-G-rich peptide is an antibody with a strong positive charge. It is thought that it binds to peptides and proteins with strong positive charges in the vicinity.

 Next, a method for detecting two post-translationally modified amino acid-containing proteins according to the present invention will be described. In the first method for detecting a post-translationally modified amino acid-containing protein of the present invention, (1) a protein side chain is chemically modified on a test sample that may contain a post-translationally modified amino acid-containing protein. (2) contacting a probe that specifically reacts with a post-translationally modified amino acid-containing protein obtained by chemical modification of the protein side chain with the chemically modified test sample; and (3) And a step of detecting the binding between the chemically modified post-translationally modified amino acid-containing protein formed by the contacting step and the probe [hereinafter referred to as a chemical modification detection method]. The second method for detecting a post-translationally modified amino acid-containing protein of the present invention comprises (1) a treatment for changing a protein charge on a test sample that may contain a post-translationally modified amino acid-containing protein. Steps to be performed, (2) Steps for performing separation by isoelectric point of proteins for test samples subjected to charge change treatment, (3) Specific reaction to post-sorting test samples and post-translationally modified amino acids And (4) detecting the binding between the separated protein and the probe formed by the contacting step. [Hereinafter referred to as charge change detection method] In the chemical modification detection method and the charge change detection method of the present invention, the above steps are usually performed in this order.

 [0037] The test sample used in the chemical modification detection method and the charge change detection method of the present invention is not particularly limited as long as it may contain a post-translationally modified amino acid-containing protein. , Cultured cells, culture supernatants of cultured cells, plant tissues, biological samples of animals, particularly tissues, blood, plasma, serum, urine and the like.

[0038] The post-translationally modified amino acid-containing protein detected by the chemical modification detection method and the charge change detection method of the present invention is not limited as long as it contains a post-translationally modified amino acid. The post-translationally modified amino acid recognized by the probe in the chemical modification detection method and the charge change detection method of the present invention is not limited as long as it is an amino acid modified after translation in vivo. However, examples include amino acids that are phosphorylated, ubiquitinated, acetylated, methylated, farnesylated, sulfated, carboxylated, glycosylated, or lipid modified, and specifically, asymmetric dimethylarginine. , Methyllysine, phosphorylated tyrosine, phosphoric acid serine, or phosphorylated threonine. In particular, the chemical modification detection method is preferably asymmetric dimethylargin, and the charge change detection method is methyllysine.

 [0039] The chemical modification carried out by the chemical modification detection method of the present invention is not particularly limited, but alkylation, acylation, acetylation, amido, glycosylation, succination, phosphorylation, sulfation, Forces such as reboylation, force rubymilation, or methyl cocoon Acidic proteins that favor chemical modification that changes the isoelectric point of the protein when combined with the separation of proteins by isoelectric point In the case of, a chemical modification that neutralizes the negative charge is preferable, and in the case of a basic protein, a chemical modification that neutralizes the positive charge is preferable. In particular, in the case of a basic protein, it is preferably acylic acid, and it is not limited as long as it replaces an amine group or a hydroxyl group with an isyl group, and examples thereof include acetylation and benzoylation. Acetyl cocoon is preferable. The acetylene is not limited as long as the amino acid in the protein is acetylylated. As the amino acid to be acetylated, the positive charge disappears when acetylene is used. The acetylating reagent is not particularly limited, and examples thereof include acetic anhydride, N-acetylsuccinimide, N-hydroxysuccinimide acetate (NHS-acetate), N-acetylenomidazole, and sulfo-NHS acetate. . In addition, in the chemical modification, in the relationship with the probe that binds to the post-translationally modified amino acid, the chemical modification that inhibits the binding to the probe by chemical modification is not preferable, and the chemical bond is stronger, so that the probe binding becomes stronger. Chemical modification is preferred. For example, in the case of a probe that does not easily bind to an acidic protein, chemical modification that neutralizes negative charges is preferable. In addition, in the case of a probe that does not easily bind to a basic protein, chemical modification that neutralizes positive charge is preferred. For example, lysine acetylene is preferred.

[0040] The probe used in the chemical modification detection method of the present invention may be a probe capable of recognizing a post-translationally modified amino acid present in a protein subjected to chemical modification of a protein side chain. For example, for antibodies that bind to modified amino acids, such as phosphorylated, ubiquitinated, acetylated, methylated, farnesylated, sulfated, carboxylated, glycosylated, or lipid modified amino acids An antibody is mentioned, More preferably, it is an antibody with respect to ADM A. In the chemical modification detection method of the present invention, the post-translationally modified amino acid-containing protein is chemically modified. Therefore, the post-translationally modified amino acid-containing protein is chemically modified to prevent the antibody from reacting with the protein. Cannot be used. Therefore, the antibody used in the detection method of the present invention can bind to a chemically modified protein, and preferably, the reactivity with a post-translationally modified amino acid is reduced by chemically modifying the protein. More preferably, the antibody is more reactive.

 [0041] Further, in the chemical modification detection method of the present invention, a step of separating the post-translationally modified amino acid-containing protein can be further performed. The method for fractionation is not particularly limited as long as it is a method for fractionating proteins. For example, force using a known method can be used, for example, electrophoresis using talylamide gel or capillary. The fractionation step may be performed before or after any step of the detection method, but is preferably before the step (2) of contacting with a probe that recognizes a post-translationally modified amino acid. This is because the isoelectric point of the molecular weight may change due to the binding between the probe and the protein, which may make the analysis difficult. Methods for separating proteins include a method using molecular weight, a method using isoelectric point, and a method using other principles. These methods can be performed alone or in combination. To separate many proteins, a combination of multiple separation principles is preferred. For example, isoelectric point Two-dimensional electrophoresis combined with fractionation by molecular weight.

[0042] Since proteins are ampholytes having an amino group and a carboxyl group, the charge varies depending on the type and configuration of the amino acid. Therefore, when electrophoresis is performed in a pH gradient, it can be separated by the difference in isoelectric point. In the present specification, the “basic protein” means a protein having an isoelectric point on the basic side, for example, a protein having 7.0 or more, preferably 8.0 or more, more preferably 9.0 or more. is there. In the detection method of the chemically modified detection method and the charge change detection method of the present invention, a method for detecting all proteins in a specimen The force that can be used as it is. Preferably, only acidic protein or basic protein is separated and used in the detection method. As a method for separating acidic protein or basic protein, known protein separation methods can be used. For example, an ion exchange chromatography column can be used.

The charge change process carried out by the charge change detection method of the present invention is not particularly limited as long as it is a process that changes the charge of the protein !, but changes the charge using a covalent bond or a non-covalent bond. Can be combined. In the case of non-covalent bonds, ionic bonds and hydrophobic bonds can be used. When a covalent bond is used, the charge of the protein can be changed, for example, by chemically modifying the amino acid side chain of the protein. The chemical modification is not particularly limited, but alkylation, acylation, acetylation, amidation, glycosylation, succination, phosphorylation, sulfation and reboylation, force ruamylation, methylation, phosphorylation, or sulfuric acid In the case of acidic proteins, chemical modifications that neutralize negative charges are preferred, and in the case of basic proteins, chemical modifications that neutralize positive charges are preferred. In particular, in the case of a basic protein, it is preferably an acyl group, and any amine group or hydroxyl group may be substituted with an isyl group, and examples thereof include acetylation and benzoylation, and more preferably. Acetylation. Acetylation is not limited as long as an amino acid in a protein is acetylated. As the amino acid to be acetylated, lysine can be cited as the acetylated amino acid, thereby eliminating the positive charge. The reagent for acetylation is not particularly limited, but acetic anhydride, N-acetylsuccinimide, N-hydroxysuccinimide acetate (NHS-acetate), N-acetylimidazole, or sulfo NHS acetate (sulfo-NHS acetate) Are listed. In addition, the charge mutation treatment is not preferable in terms of the relationship with the probe that binds to the post-translationally modified amino acid, and charge mutation treatment that inhibits binding to the probe by charge mutation treatment is not preferable. Is preferable. For example, in the case of a probe that does not easily bind to an acidic protein, charge mutation treatment that neutralizes negative charges is preferable. In the case of a probe that does not easily bind to a basic protein, charge mutation treatment that neutralizes positive charge is preferred. For example, lysine acetylation by chemical modification is preferred. [0044] The probe used in the charge change detection method of the present invention is not particularly limited as long as it is a probe capable of recognizing a post-translationally modified amino acid present in a protein subjected to charge change treatment of a protein side chain. Phosphorylated, ubiquitinated, acetylated, methylated, farnesylated, sulfated, carboxylated, glycosylated, and lipid modified amino acids, which are antibodies that bind to glycine, and preferably antibodies against methyllysine . In the charge change detection method of the present invention, the post-translationally modified amino acid-containing protein is subjected to charge change treatment, and therefore an antibody that does not react with the protein by the charge change treatment of the post-translationally modified amino acid-containing protein cannot be used. Therefore, the antibody used in the detection method of the present invention can bind to a protein subjected to charge change treatment. Preferably, the reactivity of the protein to a post-translationally modified amino acid is reduced by subjecting the protein to charge change treatment. More preferably, the antibody is more reactive.

 [0045] "Fractionation by isoelectric point" to be performed in step (2) of the charge change detection method of the present invention is not particularly limited as long as it is a method of separating proteins by isoelectric point. An example is point electrophoresis. Furthermore, in order to separate many proteins, it is preferable to use a separation method that combines other separation principles in addition to separation by isoelectric point. For example, two-dimensional electrophoresis that combines separation by isoelectric point and molecular weight is preferred. Can be mentioned.

 [0046] The probe bound to the post-translationally modified amino acid can be detected by a known method. For example, the probe can be detected by labeling the probe with a labeling substance. An enzyme (eg, peroxidase or alkaline phosphatase), a fluorescent dye [eg, fluorescein isothiocyanate (FITC)], a luminescent substance, or a radioactive substance can be used. For example, these labeling substances can be detected by a known color development method, luminescence method, fluorescence method or the like.

 Example

 [0047] Hereinafter, the ability to specifically explain the present invention by way of examples. These do not limit the scope of the present invention.

<Example 1> Preparation of antigen

 (A) Peptide synthesis

Peptides with the following sequences were synthesized by the Fmoc solid phase method. Gly—ADMA—Acp—Cys (shown as SEQ ID NO: 2; hereinafter referred to as RME—GaR ε AC)

 Leu—ADMA—Cys (hereinafter referred to as RME—LaRC)

 Gly—ADMA—Lys—Cys (shown as SEQ ID NO: 3; hereinafter referred to as RME—GaRKC) Leu—SDMA—Cys (hereinafter referred to as RME—LsRC)

 Leu Arg— Gly Arg— Gly Arg— Lys Cys (shown as SEQ ID NO: 4; hereinafter referred to as LR GRGRKC)

 In the peptides of the above sequences, Gly is glycine, ADMA is asymmetric dimethyl arginine, Acp is epsilon aminocaproic acid, Cys is cysteine, Leu is leucine, SDMA is symmetric dimethylarginine, Arg Means arginine.

 [0049] (B) Binding of peptide to mussel guanine (KLH) or BSA

 The RME-GaRεAC peptide was bound to KLH, which is a carrier protein, through C-terminal Cys. That is, KLH (Nacalai Testa Co.) or BSA (Sigma) lOmg was dissolved in PBS lmL containing 5 mM EDTA, and then the crosslinking agent GMBS (N-γ-maleimidobutyryloxysulfosuccinimide ester (Ν (γ Maleim laobutyryloxy) sulfosuccinimide ester: Concentration 20mg / mL, Dojinki) 匕 3.5 μL was added and allowed to react at 30 ° C for 30 minutes with stirring, and then a PD-10 column (Farmasia Biotech) was added. Then, unreacted GMBS was removed by gel filtration, and then lmg peptide [100 L, dissolved in lOmgZmL in PBS] was added to KLHIOmg or BSAlOmg with GMBS attached. However, after reacting at 30 ° C for 3 hours, 2 L of 2-mercaptoethanol was added, and further reacted at room temperature for 30 minutes, and then unreacted peptide was filtered by gel filtration using a PD-10 column. The resulting RME—GaR ε AC peptide and KLH Complexes, and RME- GaR ε AC- KLH, a complex of the BSA R ME - called GaR ε AC- BSA.

[0050] RME-LaRC, RME—GaRKC® RME—: LPERC and LRGRG RKC are used as peptides, and the complex of each peptide and BS A is prepared by the same procedure as that for the RME GaR ε AC and BSA. Prepared. Obtained RME—LaRC, RME-GaRKC, RM The complex of E—LsRC and LRGRGRKC peptide and BSA is referred to as RME—La RC—BSA RME—GaRKC—BSA RME—: LsRC—BSA ゝ and LRGRGRKC BSA, respectively.

<Example 2> Establishment of a monoclonal antibody-producing hyperpridoma

 (A) Immunity

The RME—GaR ε AC KLH complex antigen solution (2 mg / mL) obtained in Example 1 was mixed with an equal amount of Titer—Max Gold (Titer Max USA) until emulsified, and the mixed solution 0. ImL was added. Immunization was carried out by intraperitoneal administration of three 6-week-old female BalbZc mice or 3 6-week-old female MRL-lprZlpr mice (MRLZMpJUmmCrjlprZlpr mice). (First immunization). Twice every 2 weeks, 0. ImL of the mixed solution prepared in the same manner as in the previous period was administered intraperitoneally (second and third immunizations). Two weeks after the second and third immunizations, mouse force was collected and used for the antibody titer measurement in (B) below. For mice with increased antibody titers, RME-GaRεAC-KLH complex antigen solution (2 mgZmL) was diluted with an equal volume of PBS, and 0.1 mL of the diluted solution was administered to the abdominal cavity of the mice. On the next day of administration, RME-GaR ε Yoji-10 ^ 1 complex antigen solution (2111 ₈ 7111 was diluted with an equal volume of PBS, and 0.0 ImL of the diluted solution was administered intravenously to mice. (Final immunization) After 3 days from the final immunization, the mouse-powered spleen was aseptically removed and used in the following cell fusion step (C).

 [0052] (B) Measurement of antibody titer by ELISA

50 μL each of the RME-GaRKC-BS A complex (10 μg / mL) prepared in Example 1 was dispensed into a 96-well ELISA plate (Nunc) and left at 4 ° C. overnight. Next, each well of the plate was blocked with phosphate buffered saline (PBS) containing 1% BSA and 0.05% Tween 20 (hereinafter referred to as 1% BSA-PBST) for 30 minutes. After removing this supernatant, the serum obtained in the above step (A) was diluted from 30 times to 7290 times with PBST, and 50 / z L was added. After standing at room temperature for 30 minutes, it was washed 3 times with 0.5% Tween20ZPBS (hereinafter referred to as PBST). Subsequently, horseradish peroxidase (HRP) -labeled anti-mouse IgG antibody (goat) 50 L (1 g / mL) was added, allowed to stand at room temperature for 2 hours, and washed again with PBS T three times. OPD Substrate Solution [20mM—o-Phenylenediamine, 0.05% peracid 0.1 M citrate phosphate buffer containing hydrogen water (pH 5.0)] 100 L was added to each well, reacted at 25 ° C. for 10 minutes, and the absorbance of each well was measured at 492 nm. The results are shown in Figure 1.

 [0053] The antibody titer against RME-GaRK C-BSA was increased in both BalbZc mice and MRL-lprZlpr mice. However, compared with BalbZc mice, the antibody titer of MRLlpZlpr mice was particularly high, and one mouse (MRL-1) showed a very high antibody titer.

 [0054] (C) Cell fusion

 The spleen of the aseptically extracted MRL-1 mouse was placed in a petri dish containing 8 mL of sterile PBS. After spleen cells were drained, the spleen cell suspension was passed through a nylon mesh, collected in a 15 mL centrifuge tube, and centrifuged at 380 X g for 3 minutes. After performing this operation twice, the suspension was suspended in 8 mL of RPMI medium and centrifuged at 380 X g for 3 minutes. This operation was performed twice. The cell pellet thus obtained was resuspended in 8 mL of RPMI 1640 medium, and the number of splenocytes was measured.

[0055] On the other hand, mouse myeloma cells (myeloma cells) cultured in advance in 50 mL tubes were spalled into the spleen cells (approximately 1 × 10 ⁷ cells) S P2Z0—Agl4 5 × 10 ⁷ ), mixed well in RPMI1640 medium, and centrifuged (380 × g, 5 minutes) o Aspirate the supernatant, thaw the pellet well, and incubate at 37 ° C 1 mL of the 40% polyethylene glycol (PEG) 4000 solution was dropped, and the centrifuge tube was gently rotated by hand for 1 minute to mix the PEG solution and the cell pellet. Next, 10 mL of RPMI 1640 medium kept at 37 ° C was added, and the tube was gently rotated. Then, after centrifugation (170 X g, 5 minutes) and removing the supernatant, the cell pellet contains 10% urine fetal serum and 5% Blyclone (human IL-6, Dainippon Pharmaceutical). HAT medium (RPMI1 640 medium 〖this aminopterin 4 X 10 ^_7 M, thymidine 1. 6 X 10 ^_5 M, and hypoxanthine 1 X 10 ^_4 those that have been添Ka卩such that M) was suspended in 50 mL. 100 μL of this cell suspension was dispensed into each well of a 96-well cell culture plate, and culture was started in a 37 ° C carbon dioxide incubator containing 5% carbon dioxide. During culture, remove about 100 L of each well medium at intervals of 2-3 days, and grow in HAT medium by adding 100 L of the above HAT medium. Selected High Pridoma. On the 10th day, the following high-pridoma screening was performed: o

 [0056] (D) Screening of hybridoma

 The high-pridoma was screened in the same manner as the antibody titer measurement by the ELISA method in the above step (ii), except that 50 μL of the high-pridoma culture supernatant was used instead of the serum-powered sample. Each hyperidoma in the well where antibody production was observed was cloned by the limiting dilution method. Ten days later, a hybridoma clone producing the monoclonal antibody of the present invention was screened by the same ELISA method. as a result

ADMA2-2G11, ADMA2-2H5, ADMA2-2H7, ADMA2-4E6, ADM A2-5E10, ADMA2-3C10, six clones of high-pridoma strains were established. Hybridoma cell line ADMA2-2—2H5 (Accession No. FERM BP—10458), dated November 30, 2005, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (address: 305-8566 Japan) It was deposited internationally at Tsukuba Sakai Higashi 1-chome 1 Ibaraki Pref. 1 Central 6).

[Example 4] Preparation of isolated monoclonal antibody

 (A) Preparation of monoclonal antibody with strong culture supernatant

 The established mouse hyperidoma was cultured in a serum-free medium (Hybridoma—SFM, GIBCO) at 37 ° C. in a 5% carbon dioxide atmosphere for 72 to 96 hours. The culture solution was applied to a protein G column (Amersham Biosciences). The antibody was eluted with a buffer solution of pH 3.5 to obtain a purified monoclonal antibody of the present invention. About 20 mg of antibody was obtained from 5 OOmL of the culture solution. In the present specification, the name of each hybridoma is also used as the name of the monoclonal antibody produced by the hyperidoma force.

 (B) Preparation of monoclonal antibody from ascites

Hypridoma ADMA2-2H5 grown in the abdominal cavity of 6-week-old BalbZc-nuZnu mice was inoculated at 5 × 10 ⁶ cells per mouse. About 15 mL of ascites was obtained from 5 mice, and about 10 mg of antibody was obtained from 2 mL of ascites. Purification of the monoclonal antibody in the ascites was performed in the same manner as the purification from the culture supernatant. [0058] (C) HRP labeling of antibody

 The antibody was labeled with HRP using Pierce's EZ-Link Plus Activated Peroxidase. This method is a method in which HRP into which an aldehyde group is introduced is bound to an amino group in an antibody molecule. Labeling was performed according to the protocol provided by the manufacturer. The HRP-labeled antibody obtained from ADMA2-2H5 is referred to as HRP-ADMA2-2H5.

 [Example 5] Examination of reactivity of established monoclonal antibody

 (A) Reactivity of established monoclonal antibodies to ADMA-containing peptides

 50 μL each of RME—GaR ε AC—BSA, RME—LaRC—BSA, RME—GaRKC—BSA, and control peptide (10 g / mL) prepared in Example 1 on a 96-well ELISA plate Poured and left overnight at 4 ° C. Each well on the plate was then blocked with 1% BSA-PBST for 30 minutes. After removing this supernatant, each antibody obtained in the step (B) of Example 5 and ASYM24 (upstate), which is a polyclonal antibody against ADMA, were diluted with PBST as a control and added in 50 L portions. . After standing at room temperature for 30 minutes, it was washed 3 times with PBST. Next, add 50 L (1 g / mL) of HRP-labeled anti-mouse IgG antibody or 50 L (1 g / mL) of HRP-labeled anti-rabbit IgG antibody, leave it at room temperature for 1 hour, and then again with PBST three times. Washed. 100 L of OPD substrate solution was added to each well, reacted at 25 ° C for 30 minutes, and the absorbance of each tool at 492 nm was measured. The result is shown in figure 2. Each monoclonal antibody reacted with RME-GaRεAC-BSA, RME-LaRC-BSA, and RME-GaRKC-BSA, but did not react with the control peptide.

 [0060] (B) Specificity of established monoclonal antibodies for ADMA antigen

RME — LsRC— BSA, LRGRGRKC— BSA ゝ AcK— BSA (complex of acetylated lysine and BSA) and MeK— BSA (methyllysine and BSA) are antigens that are immobilized on the plate. The same procedure as in (A) was performed except that Clone 7E6 (Abeam) and SYM11 (upstate) were measured as control antibodies. Clone 7E6 is commercially available! Monoclonal antibody against ADMA and SYM11 is a polyclonal antibody against symmetric dimethylarginine. The results are shown in Figure 3. [0061] ADMA2—5E10, ADMA2—3C10, ADMA2—2H5, and ASYM24 do not react to RME—LsR C—BSA, which is a peptide containing SDMA that reacts with peptides containing AD MA, and LRGRGRKC— Neither BSA, AcK-BSA nor MeK-BSA reacted. Clone 7E6 reacted with RME GaR ε AC -BSA and RME-LaRC-BSA, which are peptides containing ADMA, but showed a slightly non-specific reaction even with peptides that did not contain ADMA. SYM11 reacted with RME-LsRC-BSA, a peptide containing SDMA, but showed a strong non-specific reaction with RME-LsR C BSA, a peptide containing ADMA.

 [0062] (C) Amino acid competition of established monoclonal antibody reaction

ADMA2-5E10, ADMA2-3C10, and ADMA2-2H5 were tested for inhibition by ADMA to confirm their specificity. 50 μL each of RME GaR ε AC BSA (10 μg / mL) prepared in Example 1 was dispensed into a 96-well ELISA plate and left overnight at 4 ° C. Next, each well of the plate was blocked with 1% BSA-PBST for 30 minutes. Monoclonal antibody and ADMA, monomethylarginine (MMA), or algin (Arg) were mixed and incubated at 25 ° C for 30 minutes. The final concentrations during incubation are 10 ng / mL for AD MA2-2H5, 37 ng / mL for ADMA2-5E10, 2.34 ng / mL for ADMA2-3C10, and each amino acid has a final concentration of 2 ° to ²⁵ mM. Within range, diluted with PBST. After removing 1% BSA-PBST from the plate, 50 L each of the antibody and amino acid mixture was added. After standing at room temperature for 30 minutes, it was washed 3 times with PBST. Subsequently, 50 μL (lOOngZmL) of an HRP-labeled anti-mouse IgG antibody was added, left at room temperature for 30 minutes, and washed again with PBST three times. 100 L of OPD substrate solution was added to each well, reacted at 25 ° C for 10 minutes, and the absorbance at 492 nm of each well was measured. The results are shown in Fig. 4. Neither antibody competes with ADMA, but competes with MMA and Arg.

 Example 6 Enhancement of Monoclonal Antibody Reactivity by Treatment with Sulfo-NHS Acetate (ELISA)

Dispense 50 μL each of RME—GaRKC—BSA (10 μg / mL) and LRGRGRKC—BSA (10 μg / mL) prepared in Example 2 to a 96-well ELISA plate at 4 ° C. one Left at night. Next, PBS or a solution of sulfo NHS acetate dissolved in ImM or 10 mM in PBS was added 50 L at a time and treated at 25 ° C. for 1 hour. Next, each well of the plate was blocked with 1% BSA-PBST for 1 hour. After removing the supernatant, ADMA2-5E10, ADMA2-3C10, ADMA2-2H5, ASYM24 and Clone 7E6 were diluted with P BST and added in 50 L portions. After standing at room temperature for 30 minutes, it was washed 3 times with PBST. Subsequently, 50 L (1 g / mL) of an HRP-labeled anti-mouse IgG antibody (goat) was added, allowed to stand at room temperature for 30 minutes, and then washed again with PBST three times. 100 L of OPD substrate solution was added to each well, reacted at 25 ° C for 10 minutes, and the absorbance at 492 nm of each well was measured. The results are shown in FIG. The ADMA2-5E10, ADMA2-3C10, and ADMA2-2H5 antibodies became more reactive to RME-GaRK C-BSA as the concentration of the treatment with sulfo NHS acetate increased. In addition, as in the case of each monoclonal antibody, the serum of MRL 1 mice obtained in step (2) of Example 2 also became more reactive to RME GaRKC BS A as the concentration of treatment with sulfo NHS acetate increased. (Not shown in the figure). In contrast, ASYM24 became less reactive to RME-GaRKC-BSA as the concentration of treatment with sulfo NHS acetate increased. In addition, Clone 7E6 did not react with RME—GaRKC—BSA, but it did not increase its reactivity with force sulfo NHS phosphate treatment.

Example 7 Enhancement of monoclonal antibody reactivity by treatment with sulfo-NHS acetate (Western blot)

MOLT—4F cells (approximately 5 × 10 ⁶ cells) were added to Lysis buffer (25 mM Tris—HCl (pH 8.0), 120 mM NaCl, 0.5% NP-40, ImM CaCl, protease inhibitor potency.

 2

It was suspended and dissolved in kutel (protease inhibitor cocktail: Roche). The cell lysate was crushed with ultrasonic waves. The disrupted cell lysate was centrifuged (10,000 X g, 15 minutes), and the supernatant was collected. Each supernatant 10 / ^ (10 / ^ protein amount) 12% 303? After electrophoresis on an AGE gel, the protein in the gel was electrically transferred to a polypyridene difluoride (Polyvinylidene difluoride, Bio-rad: hereinafter referred to as PVDF) membrane. The membrane was treated with 10 mM sulfo NHS acetate for 1 hour to acetylate the protein. The control membrane is 10 mM sulfo NHS No cassette treatment was performed. Blocking was performed by immersing in 10 mM Tris-HC1 buffer (pH 7.5) containing 3% BSA, 3% polybutylpyrrolidone K30 (PVP, Wako) and 0.15M-NaCl for 1 hour at 25 ° C. As a primary antibody, the monoclonal antibody ADMA2-2H5 antibody (1 μgZmL) against ADMA obtained in Example 2 was reacted at 25 ° C. for 1 hour. As controls, Usagi polyclonal antibody against ADMA, ASY M24 (0.11 μg / mL), and ACK 2F12 (1 g / mL), a monoclonal antibody against acetylyllysine, were reacted at 25 ° C. for 30 minutes.

 [0065] After washing the membrane three times with 0.05% Tween—20 and 0.15M—NaCl-containing 10 mM Tris—HC1 buffer (pH 7.5), HRP-labeled mouse anti-mouse IgG ( KPL) or HRP-labeled anti-rabbit IgG (KPL) is diluted 20000 times with 10 mM Tris-HC1 buffer (pH 7.5) containing 0.5% skim milk and 0.15M-NaCl. The mixture was reacted at C for 30 minutes. After the membrane was washed by the same method as described above, a luminol luminescence reaction catalyzed by peroxidase and hydrogen peroxide was performed using an ECL Western protein detection kit (Amersham Co., Ltd.). This luminescence was detected with LAS-1000 (Fujifilm). The result is shown in FIG.

 [0066] The ADMA2-2H5 antibody, which is a monoclonal antibody against ADMA, was enhanced in response to some proteins by treatment with sulfo N HS acetate, and there was almost no protein that weakened the reaction. In contrast, ASYM24 antibody attenuated the reactivity of MOLT-4F to most proteins (Figure 6). The reactivity of ACK2F12, a monoclonal antibody to acetylyllysine, was enhanced by sulfo NHS acetate treatment, indicating that the lysine of the protein in the cell was acetylated.

 [0067] << Example 8 >> Analysis of mouse organ lysate using ADMA2-2H2 monoclonal antibody (Western blot)

MOLT-4F force Example 7 except that the extracted protein is used in place of the extracted protein and the membrane to which the protein has been transferred is not treated with sulfo NHS acetate. The mouse organ protein was analyzed with the ADMA2-2H5 monoclonal antibody in the same manner as described above. Tissue protein before The Lysis Buffer was prepared, homogenized and used. The results are shown in FIG. Forces that could detect proteins with ADMA in most tissues In the heart and skeletal muscle, proteins with ADM A were less powerful.

[0068] << Example 9 >> Analysis of HepG2 cell lysate using ADMA2-2H2 monoclonal antibody (two-dimensional electrophoresis)

 HepG2 cells were suspended in 2D Lysis buffer [9.8Murea® 0.5% CHAPS® lOmMdithiothreitol (DTT)] and disrupted with ultrasound. The disrupted cell lysate was centrifuged (10,000 X g, 5 minutes), and the supernatant was collected.

 [0069] Dissolve 20 μg of tannic acid in 9.8 M urea, 0.5% CHAPS, lOmMDTT, 0.2% Biolytes, 0.001% bromphenol blue 125 L, and use this protein solution. An IPG strip (7 cm) in the pH range 3-10 was swollen. Set the strip after swelling in an isoelectric focusing device (BioRad), Stepl: 250V for 1 hour, Step2: 250-4000V for 2 hours (increase with linear gradient), Step3: 4000V for 10 hours, Isoelectric focusing was performed. After stripping, the strip is reduced in 0.375M Tris—HCl (pH8), 6M urea, 2% SDS, 20% glycerol, and 130mMDTT (15 minutes), and further 0.375M Tris—HCl (pH8), Alkylation (15 min) in 6M urea, 2% SDS, 20% glycerol, and 135 mM odoacetamide. The strip after the reductive alkylation treatment was placed on the SDS polyacrylamide midenore and developed in the second dimension.

 [0070] Proteins in the gel subjected to two-dimensional electrophoresis were electrically transferred to a PVDF membrane.

 This membrane was soaked in 10 mM Tris-HC1 buffer (pH 7.5) containing 3% BSA, 3% PVP and 0.15M-NaCl for 1 hour at 25 ° C. for blocking. The HRP-ADMA2-2H5 antibody (0.2 / z gZmL) prepared in the step (C) of Example 4 was reacted at 25 ° C for 30 minutes. Wash the membrane 3 times with 10 mM Tris-HC1 buffer (pH 7.5) containing 0.5% Tween-20 and 0.15M NaCl, then use ECL Western Blotting Detection Kit (Amersham Co., Ltd.). Used to carry out a luminescent reaction. This luminescence was detected with LAS-1000 (Fuji Film). The results are shown in FIG.

 [Example 10] Detection of basic protein by anti-ADMA antibody

(A) Basic protein acetylene HepG2 cells were suspended in the Lysis buffer and sonicated. The disrupted cell lysate was centrifuged (10, OOO X g, 15 minutes), and the supernatant was collected. The collected culture supernatant is equilibrated buffer (25 mM Tris-HCl (pH 8.0), 0.5% NP-40, ImM CaCl)

 Diluted 10-fold with 2. The culture supernatant diluted with Q-Sepharose equilibrated with the equilibration buffer was applied, and the flow-through fraction was collected. The passing fraction was acetone-precipitated and dried by a centrifugal evaporator. The pellet was dissolved in 2D Lysis buffer [9.8Murea, 0.5% CHAPS, lOmMdithiothreitol (DTT)] and solubilized. Sulfo NHS acetate dissolved in DMSO was added to 50 g of protein to a final concentration of 10 mM. After reacting at 25 ° C for 1 hour, 1/10 volume of 1M Tris-HCl (pH 8.0) was added to stop the reaction. The control sample was not treated with sulfo NHS acetate. The protein in the reaction solution was precipitated with acetone and again dissolved in 2D lysis buffer to prepare a sample for two-dimensional electrophoresis.

 [0072] (B) Analysis of intracellular ADMA-containing protein by two-dimensional electrophoresis

 Dissolve 20 L / zg of tannin in 9.8 M urea, 0.5% CHAPS, lOmMDTT, 0.2% Biolytes, 0.001% bromophenol blue 125 L, and use this protein solution to adjust the pH range. — Ten IPG strips (7 cm) were swollen. Set the strip after swelling in an isoelectric focusing device (BioRad), Stepl: 250V for 1 hour, Step2: 250-4000V for 2 hours (increase with linear gradient), Step3: 4000V for 10 hours, Isoelectric focusing was performed. After stripping, the strip is reduced in 0.375M Tris—HCl (pH8), 6M urea, 2% SDS, 20% glycerol, and 130mMDTT (15 minutes), and further 0.375M Tris—HCl (pH8), Alkylation (15 min) in 6M urea, 2% SDS, 20% glycerol, and 135 mM odoacetamide. The strip after the reductive alkylation treatment was placed on the SDS polyacrylamide midenore and developed in the second dimension.

 [0073] (C) Detection with monoclonal antibody ADMA2-2H5

Proteins in the gel subjected to two-dimensional electrophoresis were electrically transferred to a PVDF membrane. This membrane was soaked in 10 mM Tris-HC1 buffer (pH 7.5) containing 3% BSA, 3% PVP and 0.15M-NaCl for 1 hour at 25 ° C. for blocking. The HRP-ADMA2-2H5 antibody (0.2 / z gZmL) prepared in step (C) of Example 4 was reacted at 25 ° C for 30 minutes. Let After washing the membrane 3 times with 05% Tween-20 and 0.15M-lOmM Tris-HC1 buffer (pH 7.5), using ECL Western Blotting Detection Kit (Amersham Co., Ltd.) The luminescence reaction was performed. This luminescence was detected with LAS-1000 (Fuji Film). The results are shown in FIG. When the sulfo NHS acetate treatment is not performed, the basic protein strength is concentrated in the vicinity of H10, so that it is difficult to separate the spots of the basic protein. However, when the sulfo NHS acetate treatment is performed, the protein spots are separated and can be analyzed. When ASYM24 and Clone 7E6 were used, most spots where the reaction to the acetylated protein was weak could not be detected.

 [Example 11] Detection of basic protein with anti-methyllysine antibody

 A basic protein was detected in the same manner as in Example 10 except that MEK3 D7 (Japanese Patent Application No. 2003-403313), which is an antibody against methyllysine, was used instead of the HRP-ADMA2-2H5 antibody. The result is shown in FIG. When the sulfo NHS acetate treatment is not performed, the basic protein strength is concentrated in the vicinity of H10, so that it is difficult to separate the basic protein spots. However, when treated with sulfo NHS acetate, protein spots are separated and can be analyzed.

 Industrial applicability

[0075] The antibody of the present invention has increased reactivity with respect to acetylated protein, and thus can be used for searching and identifying a protein containing asymmetric dimethylarginine, which could not be detected conventionally. Further, the post-translationally modified amino acid-containing protein detection method combining the antibody of the present invention and the protein acetylene can be used for the search and identification of basic proteins including ADMA that cannot be identified conventionally. By examining the correlation with a disease using the antibody of the present invention and the detection method, it becomes possible to identify an ADMA-containing protein that is a target molecule associated with the disease. Furthermore, the antibody of the present invention can be used for diagnosis that detects a target molecule associated with the identified disease, or for therapeutic treatment by controlling the function of the target molecule.

 As mentioned above, although this invention was demonstrated along the specific aspect, the deformation | transformation and improvement obvious to those skilled in the art are included in the scope of the present invention.

Claims

 Claims An antibody that reacts specifically with a chemically-modified asymmetric dimethylarginine-containing protein obtained by subjecting a protein containing ginin to chemical modification of the protein side chain.

 [2] Binds specifically to asymmetric dimethylarginine, with formula (4):

 Gly— ADMA— Lys— Cys— BSA (4)

 [ADMA is asymmetric dimethylarginine, and BSA is ushi serum albumin].

 Gly-ADMA-AcLys-Cys-BSA (8)

 An antibody characterized in that its binding power to a peptide represented by [ADMA is asymmetric dimethylarginine, AcLys is acetylated lysine, and BSA is ushi serum albumin] increases.

[3] The antibody according to claim 1 or 2, which is a mouse monoclonal antibody.

[4] The mouse monoclonal antibody according to claim 3, which is an antibody that also produces a hyperpridoma force of accession number FERM BP-10458.

[5] An antibody fragment according to any one of claims 1 to 4, comprising an antigen-binding site that specifically reacts with asymmetric dimethylarginine.

 [6] A hybridoma that produces the antibody according to any one of claims 1 to 4.

 7. The hybridoma according to claim 6, wherein the hybridoma power accession number is FERM BP-10458.

[8] General formula (9):

 X— ADMA— Z— Cys (9)

[Wherein X is a peptide fragment consisting of the same or different amino acid residues 1 to 5 other than Cys, ADMA is asymmetric dimethylarginine, and Z is the same or different amino acid residues 1 to 5 other than Cys. A peptide fragment consisting of 5], which binds a peptide represented by the sequence to a carrier protein and immunizes an animal. A method for producing a dimethyl arginine antibody.

 [9] The anti-asymmetric dimethylarginine according to claim 8, wherein the peptide represented by the general formula (9) of the general formula (1) is an X force Gly and Z is ε-aminocaproic acid. Antibody manufacturing method.

 10. The method for producing an anti-asymmetric dimethylarginine antibody according to claim 8 or 9, wherein the animal is an autoimmune disease mouse.

[11] The method for producing an anti-asymmetric dimethylarginine antibody according to [10], wherein the autoimmune disease mouse is an MRL-lprZlpr mouse.

[12] (1) A step of chemically modifying a protein side chain on a test sample that may contain a post-translationally modified amino acid-containing protein,

 (2) contacting a probe that specifically reacts with a protein containing a post-translationally modified amino acid obtained by chemical modification of a protein side chain with the chemically modified test sample; and (3) the contact A step of detecting a complex of a chemically modified post-translationally modified amino acid-containing protein and the probe formed by the step

 The method for detecting a post-translationally modified amino acid-containing protein, comprising:

13. The method for detecting a post-translationally modified amino acid-containing protein according to claim 12, wherein the post-translationally modified amino acid-containing protein is a protein containing asymmetric dimethylarginine.

 [14] The method for detecting a post-translationally modified amino acid-containing protein according to [12] or [13], wherein the chemical modification of the protein side chain is ashirui.

[15] Probe power for the modified amino acid Detection of the post-translationally modified amino acid-containing protein according to any one of claims 12 to 14, which is the antibody or antibody fragment according to any one of claims 1 to 5. Method.

[16] The post-translationally modified amino acid-containing protein according to any one of claims 12 to 15, further comprising a step of fractionating the protein by isoelectric point, molecular weight, or a combination of isoelectric point and molecular weight. Detection method.

[17] (1) A step of subjecting a test sample possibly containing a post-translationally modified amino acid-containing protein to a process of changing the charge of the protein, (2) A step of performing separation based on the isoelectric point of the protein on the test sample subjected to charge change treatment,

 (3) contacting the test sample after separation with a probe that specifically reacts with the post-translationally modified amino acid; and

 (4) A step of detecting a complex between the separated protein and the probe formed by the contact step

 The method for detecting a post-translationally modified amino acid-containing protein, comprising:

18. The method for detecting a post-translationally modified amino acid-containing protein according to claim 17, wherein the fractionation according to the molecular weight of the protein is performed before or after the fractionation step.

[19] The method for detecting a post-translationally modified amino acid-containing protein according to claim 17 or 18, wherein the probe is an antibody against methyllysine.