JP4012722B2 - Antibodies against carboxymethylated peptides - Google Patents

Description

【００６７】
表１から判るように、本発明のモノクローナル抗体NF-1Gを用いた場合には、測定感度に優れるため、臨床検体に含まれるCML量を十分に測定することができた。
【００６８】
【発明の効果】
本発明により、CM化タンパク質、CM化ペプチド、CM化アミノ酸に特異的に反応するモノクローナル抗体が提供される。また、本発明により、糖尿病の検出方法、及び前記抗体の用途が提供される。
CM化タンパク質、CM化ペプチド、CM化アミノ酸は、糖尿病合併症にみられる組織障害の発症と進展に深く関連していることから、医学研究や臨床検査の領域で、糖尿病合併症などの臨床マーカーとして使用可能である。
【００６９】
また、本発明の抗体の使用により、CM化タンパク質におけるCM化アミノ酸量を好感度に測定することができる。これにより、試料中に含まれるCM化タンパク質におけるCML量を定量的に検出することができ、糖尿病又は老化現象とCM化タンパク質等との関連について、より詳細に検討することができる。
【図面の簡単な説明】
【図１】各種モノクローナル抗体とCM化HSA及びCE化HSAとの反応性を示すELISA法による特性図であり、(a)はモノクローナル抗体6D12のCM化HSAに対する反応性を示し、(b)はモノクローナル抗体CMS-10のCM化HSAに対する反応性を示し、(c)はモノクローナル抗体KNH-30のCM化HSAに対する反応性を示し、(d)はモノクローナル抗体NF-1GのCM化HSAに対する反応性を示し、(e)はモノクローナル抗体6D12のCE化HSAに対する反応性を示し、(f)はモノクローナル抗体CMS-10のCE化HSAに対する反応性を示し、(g)はモノクローナル抗体KNH-30のCE化HSAに対する反応性を示し、(h)はモノクローナル抗体NF-1GのCE化HSAに対する反応性を示している。
【図２】各種モノクローナル抗体とCML遊離体及びCEL遊離体との反応性を示す競合ELISA法による特性図であり、（ａ）はモノクローナル抗体6D12、（ｂ）はモノクローナル抗体CMS-10また（ｃ）はモノクローナル抗体NF-1Gを用いた場合を示している。
【図３】モノクローナル抗体NF-1Gを用いた競合法ELISAにおいて検出感度を検討した結果を示す特性図である。
【図４】各種モノクローナル抗体とCML-HSAとの反応性に対するEDTAの影響をELISA法で検討した結果を示す特性図であり、（ａ）はモノクローナル抗体6D12、（ｂ）はモノクローナル抗体CMS-10また（ｃ）はモノクローナル抗体NF-1Gを用いた場合を示している。
【図５】モノクローナル抗体NF-1Gを用いた臨床検体に含まれるCML量を測定する際に用いた標準曲線を示す特性図である。標準物質として、酵素処理したCM化HSAを用いた。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an antibody that reacts with a carboxymethylated amino acid present in a protein or peptide, a method for evaluating diabetic complications and the progress of aging, and use of the antibody.
[0002]
[Prior art]
Proteins in the body are saccharified by reacting with reducing sugars non-enzymatically. This reaction is generally called the Maillard reaction, and is composed of an early stage reaction and a late stage reaction.
The first stage of the Maillard reaction is that the amino acid side chain amino group or N-terminal amino group of the protein reacts with the carbonyl group of the sugar to produce an Amadori rearrangement product via a Schiff base. As products of this reaction, hemoglobin A1C and glycated albumin are known, and these products are widely used as clinical markers of diabetes. Basically, all proteins in the living body are considered to be glycated at present, and many protein dysfunctions as a result have been reported.
[0003]
The late stage of the Maillard reaction is characterized by the fact that the Amadori rearrangement product produced by the early stage undergoes complex irreversible reactions such as dehydration, oxidation, and condensation, resulting in fluorescence, browning or intramolecular / intermolecular crosslink formation. This is the stage that yields the final product of the reaction. The final product in the late stage is called AGE (Advanced Glycation End products).
[0004]
AGE is considered to be an assembly of a plurality of structures, and its quantification is performed by measuring fluorescence intensity, instrumental analysis, antigen-antibody reaction, and the like. To date, pyrazine, pentosidine, croslin, carboxymethyllysine (CML), etc. have been proposed as AGE structures (Biochemistry vol.35, No.24, 8075-8083, 1996, etc.). The significance of the existence of the body is not yet detailed.
[0005]
In addition to the above physicochemical characteristics, AGE has a biological characteristic that it is specifically recognized by the cell membrane receptor of macrophages, and has received much attention. In addition, small vascular disorders (nephropathy, retinopathy, peripheral neuropathy, etc.) observed in diabetes and aging, arteriosclerosis, cataracts, sclerosis and thickening of connective tissues of skin, blood vessels, and joints, etc. (Horiuchi S. et al., Nephrol. Dial. Transplant. 11 (5) (1996)). Therefore, AGE is considered to be deeply involved in the onset of these tissue disorders, and the importance of AGE in the fields of medical research and clinical examination is increasing.
[0006]
By the way, carboxymethyl lysine (CML), which is a representative product by oxidative cleavage of the glucose-derived Amadori rearrangement product, was identified in 1986 (Ahmed MU et al., J. Biol. Chem. 261, 4889-94 (1986)) has attracted attention in relation to diabetes and aging as described above. In the tissues, including reports that CML levels in human skin collagen are significantly higher in diabetic patients (Dyer DG. Et al., J. Clin. Invest, 91 (6), 2463-9 (1993)) There have been many reports that the CML concentration of urine increases significantly with the progression of diabetes and aging. Studies on epitopes of antibodies against AGE have revealed that CML is the main structure of AGE (Ikeda K. et al., Biochem. 35, 8075-8083 (1996)).
[0007]
On the other hand, recently, carboxyethyl lysine (CEL) with a structure very similar to CML was found as a new structure of AGE, and the concentration in human lens protein increased with aging by gas chromatography / mass spectrometry. (Ahmed MU. Et al., Biochem Journal, 324, 565-570 (1997)). CEL is considered that the side chain of lysine in a protein is carboxyethylated (CE-ized) by methylglyoxal (MGO). MGO exists as a degradation product of triose phosphate and acetol metabolite in glycolysis and is reported to increase in the blood of diabetic patients (Mclellan AC et al., Clinical Science 87, 21-29 ( 1994)). Moreover, even in vitro, it is known that when erythrocytes are cultured in a medium containing a high concentration of sugar, the MGO concentration in the cells increases.
As mentioned above, CML and CEL are being shown to be important as AGE structures. Thus, the present inventors have been able to obtain an antibody that reacts with CML, which is important as an AGE structure, and does not react with CEL (see JP 2000-219700 A).
[0008]
However, the antibody disclosed in the above publication can distinguish between carboxymethylated protein and carboxyethylated protein by specifically reacting with carboxymethylated protein. In addition, for example, it hardly reacted to a carboxymethylated peptide consisting of several amino acids as well as a carboxymethylated free amino acid. Therefore, the antibody disclosed in the above publication cannot detect carboxymethylated peptides and carboxymethylated amino acids after enzymatic treatment or hydrolysis with acid.
[0009]
[Problems to be solved by the invention]
Therefore, the present invention provides an amino acid having a carboxymethylated side chain amino group after enzymatic treatment or acid hydrolysis of a carboxymethylated protein, an antibody capable of reacting with a peptide containing the amino acid, and diabetes using the antibody. An object is to provide a method for evaluating the degree of complications and aging, and use of the antibody.
[0010]
[Means for Solving the Problems]
As a result of intensive studies to solve the above problems, the present inventor uses a protein or peptide in which the amino group of the amino acid side chain is carboxymethylated as an antigen, and uses the carboxymethylated protein or peptide for screening. Thus, the present invention was completed by successfully producing an antibody that reacts with an amino acid or peptide in which a side chain amino group is carboxymethylated in a protein or peptide.
That is, the present invention is an antibody that reacts with an amino acid or peptide in which the side chain amino group is carboxymethylated.
[0011]
Examples of the carboxymethylated amino acid include N-ε-carboxymethyllysine. Moreover, as a peptide, what consists of two or more amino acids can be mentioned. Furthermore, the antibody may be a polyclonal antibody or a monoclonal antibody. Examples of monoclonal antibodies include those produced by a hybridoma whose accession number is FERM P-18589.
[0012]
Furthermore, the present invention is a method for measuring a carboxymethylated amino acid in a protein, comprising reacting the antibody with an amino acid having a side chain amino group carboxymethylated.
Furthermore, the present invention is a method for evaluating diabetic complications, which comprises reacting the antibody with the amino acid. Examples of diabetic complications include diabetic fine blood vessel complications such as diabetic nephropathy, retinopathy, and neuropathy, or diabetic macrovascular complications typified by arteriosclerosis.
[0013]
Furthermore, the present invention is a method for evaluating the progress of aging, comprising reacting the antibody with the amino acid.
Furthermore, the present invention is a reagent for detecting an amino acid containing a carboxymethylated side chain amino group, comprising the antibody.
Furthermore, the present invention is a reagent for evaluating diabetic complications, a reagent for evaluating the degree of aging, or a reagent for staining an immune tissue, comprising the antibody.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
[0015]
The present invention relates to an amino acid whose side chain amino group is carboxymethylated (hereinafter referred to as “carboxymethylated amino acid” or “CM amino acid”), a peptide containing a carboxymethylated amino acid (hereinafter referred to as “carboxymethylated peptide”). Alternatively, an antibody that reacts with a protein containing a carboxymethylated amino acid (hereinafter referred to as “carboxymethylated protein” or “CMylated protein”) is provided.
[0016]
Examples of the CM-modified amino acid and the CM-modified peptide to which the present antibody reacts include those obtained by enzymatic treatment of a CM-protein with a protease or hydrolysis with an acid.
Here, CM protein is a variety of microangiopathies (nephropathy, retinopathy, peripheral neuropathy, etc.) observed in diabetes and aging, arteriosclerosis, cataracts, hardening and thickening of connective tissues of skin, blood vessels, and joints. It is one of the AGE structures (Horiuchi S. et al., Nephrol. Dial. Transplant. 11 (5) (1996)) present at the site of tissue damage. Therefore, if the CM protein is quantitatively detected from the various tissues, the progress of the tissue damage can be grasped. That is, in the fields of medical research and clinical examination, the importance of quantitatively detecting CM-modified proteins in the various tissues is increasing.
[0017]
However, the carboxymethylation site in the CM protein is not constant depending on the type and structure of the protein. Therefore, conventional antibodies cannot react depending on the type of CM protein, and it has been difficult to quantify the CM protein contained in the various tissues. On the other hand, the antibody according to the present invention reacts with a CM-modified amino acid and a CM-modified peptide obtained by, for example, protease treatment or acid, so that the carboxymethylation in the CM-protein contained in the above various tissues is performed. It reacts with all the sites, and the CM protein contained in the various tissues can be quantified.
[0018]
One of the other AGE structures different from the CM protein is an amino acid in which the amino group of the side chain is carboxyethylated (CE). The structures of CM amino acids and CE amino acids are very similar. The antibody according to the present invention reacts only with CM-converted amino acid residues and does not react with CE-converted amino acid residues. Therefore, by using the antibody according to the present invention, the CE protein and the CM protein contained in the various tissues can be separated or identified, and only the CM protein can be quantified.
[0019]
Here, “carboxymethylated amino acid” or “CM amino acid” means an amino acid having an amino group in the side chain, wherein the amino group is carboxymethylated. The type of amino acid having an amino group in the side chain is not particularly limited, and examples include lysine, arginine, asparagine, and glutamine. Lysine (for example, N-ε-carboxymethyllysine) is preferable.
[0020]
“Carboxymethylated peptide” or “CM peptide” means a structure comprising a plurality of amino acids formed by peptide bonding of the above CM amino acid and other amino acids. The other amino acids are not particularly limited, and are meant to include any amino acids and their derivatives and modifications. Further, the “carboxymethylated peptide” or “CM peptide” may include a plurality of CM amino acids adjacent to or separated from each other. In addition, the “carboxymethylated peptide” or “CM peptide” only needs to contain at least one or more CM amino acids, and includes other amino acids in which the side chain amino group is not CM-ized. Also good. Furthermore, the “carboxymethylated peptide” or “CM peptide” only needs to contain at least one CM amino acid, and may contain a CE amino acid. The “CE peptide” is a peptide containing an amino acid in which at least some of the side chain amino groups are carboxyethylated, and does not contain a carboxymethylated amino acid (“carboxyethyl”). Also referred to as a “modified peptide”).
[0021]
In addition, “carboxymethylated peptide” or “CM peptide” may contain two or more amino acids, but when there are a plurality of CM amino acids per protein molecule, this antibody is used after hydrolysis. Therefore, measurement can be performed with high detection sensitivity.
In this case, the antibody according to the present invention can react with amino acids in which the amino group of the side chain is converted to CM among the amino acids present in the peptide.
[0022]
1. Preparation of CM protein, CM peptide and CM amino acid
The protein to be commercialized is not particularly limited, and may be any protein such as complex protein, simple protein, glycoprotein, and lipoprotein. Examples of these proteins include albumin (such as BSA), hemoglobin, keyhole limpet hemocyanin (KLH), human serum albumin (HSA), ribonuclease (RNase), β ₂ microglobulin, histone, collagen, blood cell membrane protein, or Examples thereof include low density or high density lipoprotein. Moreover, it is not limited to proteins, and peptides such as oligopeptides and polypeptides may be used, and those synthesized by modification or degradation from proteins can also be used.
[0023]
In order to convert these proteins or peptides into CM, several tens mg / ml (for example, 5 to 50 mg / ml) of protein such as BSA and tens of mg / ml (for example, 10 to 100 mg / ml) of glyoxylic acid are phosphoric acid. For example, a method of reacting at room temperature for about 24 hours in a buffer solution (containing NaCNBH _{3 in} a molar amount 5 times that of glyoxylic acid, pH 7.4) is employed.
The CM protein obtained by the above method is purified by dialysis, liquid column chromatography or the like, and then used for production of a polyclonal antibody or a monoclonal antibody.
[0024]
The CM peptide and the CM amino acid can be obtained by subjecting the CM protein obtained as described above to a hydrolysis treatment with a protease such as trypsin, pepsin, papain, aprotinin, or leupeptin, or an acid such as hydrochloric acid or sulfuric acid. For example, several tens of mg / ml (for example, 5 to 70 mg / ml) of the above CM-modified protein tens of μL (for example, 10 to 50 μL) and several tens of mg / ml (for example, 10 to 80 mg / ml) of trypsin solution of several tens of μL ( For example, 20 to 50 μL) is mixed and reacted in a 37 ° C. water bath for 3 hours. The reaction is stopped with 5 mM AEBSF, whereby the CM-modified protein can be enzymatically treated, and a CM-modified peptide and a CM-modified amino acid can be obtained. The trypsin hydrolysis reaction can be stopped by adding a trypsin inhibitor (for example, 4- (2-Aminoethyl) benzenesulfonylfluoride, HCL (AEBSF)) to the mixed solution.
[0025]
2. Antibody to CM protein In the present invention, the term “antibody” refers to an entire antibody molecule or a fragment thereof (for example, Fab or F (ab ′)) that can bind to the CM protein, CM peptide and CM amino acid that are antigens. ₂ fragment), which may be a polyclonal antibody or a monoclonal antibody.
Antibodies (polyclonal antibodies and monoclonal antibodies) can be produced by any of a variety of methods. Methods for producing such antibodies are well known in the art [see, eg, Sambrook, J et al., Molecular Cloning, Cold Spring Harbor Laboratory Press (1989)].
[0026]
(1) Production of monoclonal antibody against CM protein
(i) Collection of antibody-producing cells CM-produced protein or CM-produced peptide prepared as described above is administered as an antigen to mammals such as rats, mice, rabbits and the like. The dose of the antigen per animal is 0.1 to 100 mg when no adjuvant is used, and 1 to 100 μg when an adjuvant is used. Examples of adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant. Immunization is performed mainly by injecting intravenously, subcutaneously or intraperitoneally. The immunization interval is not particularly limited, and immunization is performed 1 to 10 times, preferably 2 to 5 times at intervals of several days to several weeks, preferably 2 to 5 weeks. Then, antibody-producing cells are collected 1 to 60 days after the last immunization day, preferably 1 to 14 days later. Examples of antibody-producing cells include spleen cells, lymph node cells, peripheral blood cells, etc., but spleen cells or local lymph node cells are preferred.
[0027]
(ii) Cell fusion A cell fusion between an antibody-producing cell and a myeloma cell is performed to obtain a hybridoma. As myeloma cells to be fused with antibody-producing cells, generally available cell lines of animals such as mice can be used. The cell line to be used has drug selectivity and cannot survive in a HAT selection medium (including hypoxanthine, aminopterin, and thymidine) in an unfused state, but can survive only in a state fused with antibody-producing cells. Those having the following are preferred. Examples of myeloma cells include mouse myeloma cell lines such as P3X63-Ag.8.U1 (P3U1) and NS-I.
[0028]
Next, the myeloma cell and the antibody-producing cell are fused. Cell fusion is performed in animal cell culture media such as serum-free DMEM and RPMI-1640 media, and 1 × 10 ⁶ to 1 × 10 ⁷ antibody-producing cells and 2 × 10 ⁵ to 2 × 10 ^6. Per ml of myeloma cells (cell ratio of antibody-producing cells to myeloma cells is preferably 5: 1), and a fusion reaction is performed in the presence of a cell fusion promoter. As the cell fusion promoter, polyethylene glycol having an average molecular weight of 1000 to 6000 daltons can be used. Alternatively, antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device utilizing electrical stimulation (for example, electroporation).
[0029]
(iii) Selection and cloning of hybridoma The target hybridoma is selected from the cells after cell fusion treatment. As a method, the cell suspension is appropriately diluted with, for example, fetal bovine serum-containing RPMI-1640 medium, then plated on a microtiter plate at about 3 × 10 ⁵ cells / well, a selective medium is added to each well, and thereafter appropriate. The culture is carried out with the selective medium changed. As a result, cells that grow from about 14 days after the start of culture in the selective medium can be obtained as hybridomas.
[0030]
Next, the culture supernatant of the hybridoma that has grown is screened for the presence of antibodies that react with the CMized protein. Hybridoma screening is not particularly limited, and may be performed according to ordinary methods. For example, a part of the culture supernatant contained in a well grown as a hybridoma can be collected and screened by enzyme immunoassay, radioimmunoassay or the like.
[0031]
Specifically, cells that react with multiple types of CM proteins by ELISA, etc., and produce monoclonal antibodies that react with the former and not with the latter, using multiple types of non-CM proteins as antigens. Establish a hybridoma.
Cloning of fused cells is performed by the limiting dilution method or the like. Finally, a hybridoma that is a cell producing a monoclonal antibody that reacts with a plurality of types of CM proteins but does not react with non-CM proteins is established.
[0032]
In the present invention, a hybridoma NF-1G (referred to as “Mouse-Mouse hybridoma NF-1G”) that produces the monoclonal antibody was obtained. This Mouse-Mouse hybridoma NF-1G was deposited as FERM P-18589 on November 6, 2001 at the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center (1-1 1-1 Higashi, Tsukuba City, Ibaraki Prefecture) Has been.
[0033]
(iv) Collection of monoclonal antibody As a method for collecting a monoclonal antibody from the established hybridoma, a normal cell culture method or ascites formation method can be employed.
In the cell culture method, the obtained hybridoma is cultured in an animal cell culture medium such as RPMI-1640 medium, MEM medium or serum-free medium containing 10% fetal bovine serum under normal culture conditions (for example, 37 ° C., 5% CO ₂ (Concentration) for 7 to 14 days, and antibodies are obtained from the culture supernatant.
[0034]
In the case of the ascites formation method, about 1 × 10 ⁷ hybridomas are administered into the abdominal cavity of a mammal derived from a myeloma cell and the same strain, and the hybridomas are proliferated in large quantities. And ascitic fluid is collected after 1-2 weeks.
When antibody purification is required in the antibody collection method, known methods such as ammonium sulfate salting-out method, ion exchange chromatography, gel filtration, affinity chromatography are appropriately selected, or a combination thereof is used. Can be purified.
[0035]
(2) Preparation of polyclonal antibody against CM protein The CM protein or peptide is used as an antigen and administered to mammals such as rats, mice, rabbits and the like. The dose of the antigen per animal is 0.1 to 100 mg when no adjuvant is used, and 1 to 100 μg when an adjuvant is used. Examples of adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant. Immunization is performed mainly by injecting intravenously, subcutaneously, intraperitoneally, or the like. The immunization interval is not particularly limited, and immunization is performed 1 to 10 times, preferably 2 to 5 times at intervals of several days to several weeks, preferably 2 to 5 weeks. And 6 to 60 days after the last immunization, antibody titer is measured by enzyme immunoassay (ELISA (enzyme-linked immunosorbent assay) or EIA (enzyme immunoassay)), radioimmunoassay (RIA), etc. On the day when the maximum antibody titer is shown, blood is collected to obtain antiserum.
[0036]
Thereafter, CM and CE proteins (such as BSA) are used, and the reactivity of polyclonal antibodies in the antiserum against these proteins is measured by ELISA or the like. By collecting fractions that react with a certain type of CM protein and do not react with a non-CM protein, a polyclonal antibody that reacts only with the CM protein can be obtained.
[0037]
3. Method for Measuring CM-Containing Amino Acid In the present invention, the above-mentioned antibody can be used to measure or quantify CM-containing amino acids, CM-containing peptides, and CM-containing proteins. For example, by reacting the monoclonal antibody or polyclonal antibody of the present invention with a CM-modified amino acid, a CM-modified peptide and a CM-modified protein, and using an anti-mouse IgG antibody labeled with horseradish peroxidase (HRP) or the like, a CM-modified amino acid, CMized peptides and CMized proteins can be quantified.
[0038]
4). Method for evaluating the degree of diabetic complications and aging Progression of the diabetic complications and aging can be evaluated by reacting the antibody of the present invention with a CM amino acid, a CM peptide and a CM protein in a biological sample. it can. Examples of diabetic complications include diabetic fine blood vessel complications such as diabetic nephropathy, retinopathy, and neuropathy, or diabetic macrovascular complications typified by arteriosclerosis. In the present invention, these diseases for detecting complications may be one type or a combination of two or more types.
[0039]
Blood, urine, tissue, etc. are collected from patients with diabetic complications, for example, patients suspected of having diabetic nephropathy, diabetic retinopathy, etc., and subjected to appropriate pretreatment to prepare a measurement sample. The measurement sample can be used as a clinical marker for diabetes. Next, the measurement sample and the antibody are reacted. After the reaction, a CM amino acid, a CM peptide and a CM protein are detected and quantified by a conventional method using an anti-mouse IgG antibody labeled with horseradish peroxidase (HRP) or the like.
When the detection result is positive or the quantitative value is high, it can be used as a judgment material for evaluating the presence or absence of diabetic complications and the progress of aging.
[0040]
Examples of the pretreatment include enzyme treatment with protease and the like, and hydrolysis treatment with acid and the like. By applying an enzyme treatment and a hydrolysis treatment, the CM protein can be decomposed into CM amino acids and CM peptides. Since this antibody has particularly excellent reactivity with CM-modified amino acids and CM-ized peptides, by performing enzyme treatment and hydrolysis treatment as the above pretreatment, the CMization site in the CMized protein contained in the measurement sample Can be measured quantitatively. Therefore, by using this antibody, diabetic complications and the progress of aging can be evaluated in detail.
[0041]
5). Reagents Containing the Antibody of the Present Invention In the present invention, antibodies against CM-modified amino acids, CM-ized peptides and CM-modified proteins can be used as various reagents. For example, in the case of using as a CM peptide or a CM amino acid detection reagent, the above 3. When the detection is performed using the measurement method described in 1. and used as a reagent for detecting diabetes, 4. Detection is performed by the method described in 1.
[0042]
When the antibody of the present invention is used as a reagent for immunohistochemical staining, detection is performed according to a normal immunohistological staining method.
For example, various tissue sections obtained from a biopsy of a diabetic patient are prepared by a conventional method, and the antibody of the present invention is bound thereto. Horseradish peroxidase (HRP) -labeled anti-mouse IgG antibody is bound to the antibody of the present invention as a secondary antibody, treated with 3,3′-diaminobenzidine, and then stained. Microscopic observation is performed after staining, and it can be determined that the area stained brown has undergone commercialization.
[0043]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.
[0044]
[Example 1] Preparation of monoclonal antibody
(1) Preparation of antigen
(i) Carboxymethylated protein In this example, carboxymethylated human serum albumin (hereinafter referred to as “CM HSA”) was used as the carboxymethylated protein.
5 mg of protein (HSA) was reacted in 0.2 M phosphate buffer (pH 7.8) containing 1,100 mM glyoxylic acid and 0.45 M NaCNBH ₃ at 37 ° C. for 24 hours, dialyzed against PBS, CM Protein was obtained (Reddy S et al., Biochemistry 1995, 34, 10872-10878). The resulting CM protein was confirmed to be CM HSA from the results of amino acid analysis.
(ii) Carboxyethylated protein
5 mg of protein (HSA) was reacted in PBS containing 0.2 M pyruvate and 0.3 M NaCNBH ₃ for 24 hours, and then dialyzed with PBS to obtain CE protein (Ahmed MU et al., Biochem J 1997, 324, 565-570). From the results of amino acid analysis of the generated CE protein, it was confirmed that it was CE-converted HSA (referred to as “CE-converted HSA”).
[0045]
(2) Animal immunity
1 mg / ml antigen (CM HSA) was mixed with an equal amount of Freund's adjuvant to prepare an emulsion, and 200 μl / mouse was immunized into the back skin of BALB / c mice.
Booster immunization was performed every 2 weeks, and after immunization 4 times from the initial immunization, blood was collected from the tail vein to confirm the antibody titer.
[0046]
(3) Measurement of antibody titer The antibody titer was measured using ELISA method. That is, CM HSA as an antigen was coated on a plate at a concentration of 5 μg / ml at 50 μl / well at 4 ° C. overnight. After washing three times with PBS containing 0.05% Tween 20 (PBS-T), blocking was performed for 1 hour with a phosphate buffer solution (pH 7.4) containing 0.5% gelatin. After washing, mouse serum diluted serially with PBS-T was added at 50 μl / well and allowed to stand for 1 hour. After washing, a secondary antibody (horseradish peroxidase (HRP) -labeled anti-mouse IgG) diluted 2500 times with PBS-T was added in an amount of 50 μl / well and allowed to stand for 1 hour. After washing, citrate buffer (pH 5.0) containing 0.5 mg / ml OPD 1,2-phenylenediamine dihydrochloride (OPD) was added at 100 μl / well and allowed to react for 10 minutes. . After the reaction, 2N sulfuric acid was added to stop the reaction, and the absorbance at 490 nm was measured. As a result, the antiserum showed significant reactivity with the antigen in a 10,000-fold diluted solution.
[0047]
(4) Cell fusion and cloning of antibody-producing hybridoma Mouse spleen cells and myeloma cells P3U1 confirmed to have increased antibody titers were fused at a ratio of 5: 1 by the polyethylene glycol method, and the hybridomas were cultured in a HAT selective medium. Selective culture was performed. The hybridoma culture supernatant was collected on the 10th day of cell fusion, and ELISA was performed in the same manner as the method for measuring antibody titer (see (3) above), and the reactivity with CM HSA and CM KLH was confirmed. A strain that was positive and negative for HSA reactivity was screened.
[0048]
The spleen cells of the mice in which the increase in antibody titer was confirmed were collected by magnetic beads (PIERCE) bound with streptavidin. That is, magnetic beads labeled with streptavidin and CM-modified HSA labeled with biotin are mixed in the spleen cell suspension, and then the magnetic beads are recovered by applying a magnet. The streptavidin bound to the collected magnetic beads is bound to biotin of CM HSA, and as a result, spleen cells bound to CM HSA by antigen-antibody reaction are captured by the magnetic beads.
[0049]
After measuring the number of hybridoma cells that were positive in the above screening, the cells were seeded (subcloning) into a 96-well plate at 2 × 10 ⁵ cells / well, and after 10 days, only single colony wells were screened again. Similarly, the subcloning was performed again for the hybridoma that became positive in the screening, and the screening operation was continued until the properties were 100% identical.
[0050]
As a result, a monoclonal antibody-producing cell line (NF-1G) that had positive reactivity with CM-ized HSA and CM-type KLH and did not react with HSA was obtained.
The hybridoma NF-1G (name: “Mouse-Mouse hybridoma NF-1G”) was established by the National Institute of Advanced Industrial Science and Technology, Patent Deposit Center (1-1-1, East 1-chome, Tsukuba City, Ibaraki Prefecture) Deposited as FERM P-18589 on May 6th.
[0051]
[Example 2] Examination of properties (CML and CEL discrimination) of monoclonal antibodies of the present invention 1
In this example, the difference in reactivity to CML and CEL was examined for the monoclonal antibody-producing cell line NF-1G specific for CML.
Antigens with different CML contents per molecule were prepared. That is, 6-fold dilution series was prepared 3 times each of 1,100 mM glyoxylic acid, and 1 ml of 5 mg / ml HSA was added to each concentration of 100 μl. The mixture was allowed to stand at room temperature for 2 hours, and 50 μl of NaCNBH ₃ was added and mixed. After standing overnight at room temperature, dialyzed with PBS to obtain CM protein. As a result of amino acid analysis of the generated CM protein, it was confirmed that it was CM HSA. Antigens with different CEL contents were prepared in the same manner using pyruvic acid instead of glyoxylic acid.
[0052]
Differences in the reactivity of the monoclonal antibody-producing cell line NF-1G to CML and CEL were examined by the following method. That is, CM HSA and CE HSA prepared by the above method were dissolved in a phosphate buffer solution (pH 7.4) at a concentration of 5 μg / ml, respectively, and coated on an ELISA plate by 50 μl / well. Thereafter, blocking was performed with 0.5% gelatin (200 μl / well) dissolved in carbonate buffer (pH 9.5). 1 μg / ml NF-1G purified antibody was added at 50 μl / well and reacted for 1 hour, and then HRP-labeled anti-mouse IgG antibody was added at 50 μl / well and reacted for 1 hour. Next, 1,2-phenylenediamine dihydrochloride (OPD) was added to perform a color reaction, and the absorbance at 490 nm was measured with an ELISA reader.
[0053]
At this time, as controls, 6D12 (manufactured by Transgenic Co., Ltd.), an existing anti-CML monoclonal antibody, CMS-10 (manufactured by Transgenic Co., Ltd.), and KNH-30 (Kumamoto University, an anti-CEL monoclonal antibody). The more purified antibody was used.
As a result, it was confirmed that the monoclonal antibody (NF-1G) of the present invention reacts with CM-ized HSA but does not show reactivity with CE-ized HSA. 6D12 reacted almost the same as CM HSA and CE HSA, and it was revealed that the two could not be distinguished. Furthermore, it was confirmed that KNH-30 reacts with CE-modified HSA but does not react with CM-ized HSA. On the other hand, CMS-10 specifically reacted with CM-ized HSA, but it was confirmed that the reactivity with CM-ized HSA was lower than that with NF-1G.
[0054]
Furthermore, since the reactivity increases according to the number of CML, it can be seen that it reacts with CML.
Therefore, it was found that the monoclonal antibody (NF-1G) of the present invention clearly shows a superior reactivity specifically to CML and does not react with CEL, unlike the existing anti-AGE monoclonal antibody ( Figure 1).
The monoclonal antibody subtype of the present invention was confirmed to be IgG _2a by a commercially available typing kit.
[0055]
[Example 3] Examination of properties of monoclonal antibody of the present invention (discriminability of CML free form) 2
In this example, the discriminability of CML educts was examined for 6D12, CMS-10, and NF-1G in which reactivity to CMized HSA was observed in Example 1. CML educts were synthesized by chemical methods.
[0056]
A competitive ELISA was performed using CMLHSA as a solid-phased antigen, and the reactivity of 6D12, CMS-10, and NF-1G to CML educts was examined. In the competitive ELISA, CML-HSA as an antigen was coated on a plate at a concentration of 2.5 μg / ml at 50 μl / well overnight at 4 ° C. After washing three times with PBS containing 0.05% Tween 20 (PBS-T), blocking was performed with a phosphate buffer solution (pH 7.4) containing 0.5% gelatin for 1 hour. After washing, CML educt and 0.1 μg / ml 6D12, CMS-10 and NF-1G reacted in advance at room temperature for 1 hour were added at 50 μl / well and allowed to stand for 1 hour. After washing, a secondary antibody (horseradish peroxidase (HRP) -labeled anti-mouse IgG (γ)) diluted to 2500 times was added at 50 μl / well and allowed to stand for 1 hour. After washing, a citrate buffer solution (pH 5.0) containing 0.5 mg / ml 1,2-phenylenediamine dihydrochloride (OPD) was added 100 μl / well and allowed to react for 10 minutes. After the reaction, 2N sulfuric acid was added to stop the reaction, and the absorbance at 490 nm was measured.
[0057]
Similarly, competition method ELISA using chemically synthesized CEL free form as an antigen was performed. The results are shown in FIGS. 2 (a), (b) and (c). From FIG. 2 (a), it was confirmed that 6D12 did not show reactivity with both CML and CEL educts. Further, from FIG. 2 (b), it was confirmed that CMS-10 also showed no reactivity with both the CML free form and the CEL free form. On the other hand, it was confirmed from FIG. 2 (c) that NF-1G showed reactivity with CML educts but not CEL educts.
[0058]
Therefore, according to the present example, it was found that the monoclonal antibody (NF-1G) of the present invention clearly differs from the existing anti-AGE monoclonal antibody and can specifically react with the CML educt.
In addition, in the competitive ELISA using NF-1G, the measurement sensitivity was examined by adjusting the concentration of CML free form. The results are shown in FIG. From FIG. 3, it was found that in the competitive ELISA using NF-1G, the CML educt contained in the sample can be detected even when the CML educt is a low concentration of about 10 μM. Since the detection sensitivity of CML free form by high performance liquid chromatography is 1-100 μM, there should be no difference in detection sensitivity between competitive method ELISA using NF-1G and high performance liquid chromatography. Can do.
[0059]
[Example 4] Examination of properties of monoclonal antibody of the present invention (effect of anticoagulant) 3
In this example, the influence of reactivity by EDTA was examined on 6D12, CMS-10, and NF-1G in which reactivity to CM HSA was observed in Example 1. In this example, CM-modified HSA prepared in the same manner as in Example 1 was used as a solid-phased antigen, and the effect of EDTA on reactivity with various antibodies was examined.
[0060]
The measurement was performed by ELISA using CM HSA as a solid phase antigen. That is, CML-HSA as an antigen was coated on a plate at a concentration of 2.5 μg / ml at 50 μl / well overnight at 4 ° C. After washing three times with PBS containing 0.05% Tween 20 (PBS-T), blocking was performed with a carbonate buffer solution (pH 9.5) containing 0.5% gelatin for 1 hour. After washing, 50 μl of an antibody solution containing 10,5,2.5,1.25,0.625,0.3125 mM EDTA was added and allowed to react at room temperature for 1 hour, then placed in a plate and allowed to react for 1 hour. After washing, a secondary antibody (horseradish peroxidase (HRP) -labeled anti-mouse IgG (γ)) diluted to 2500 times was added at 50 μl / well and allowed to stand for 1 hour. After washing, a citrate buffer solution (pH 5.0) containing 0.5 mg / ml 1,2-phenylenediamine dihydrochloride (OPD) was added 100 μl / well and allowed to react for 10 minutes. After the reaction, 2N sulfuric acid was added to stop the reaction, and the absorbance at 490 nm was measured.
[0061]
The results are shown in FIGS. 4 (a), (b) and (c). As shown in FIG. 4 (b), in CMS-10 that specifically reacts with CMLHSA, the reactivity with CML-HSA decreased as the EDTA concentration increased. In contrast, as shown in FIGS. 4 (a) and (c), in 6D12 and NF-1G, the reactivity to CML-HSA was constant regardless of the EDTA concentration.
Therefore, according to this example, the monoclonal antibody (NF-1G) of the present invention can be accurately detected without being affected by EDTA contained in the sample, for example, when detecting or quantifying CML-HSA contained in the sample. Can be detected or quantified.
[0062]
[Example 5] Clinical specimen measurement using the monoclonal antibody of the present invention In this example, the monoclonal antibody (NF-1G) of the present invention was used to measure the amount of CML contained in a clinical specimen, whereby NF- We examined the possibility of using 1G for clinical specimen measurement.
[0063]
In this example, a standard curve as shown in FIG. 5 was prepared using a CMized peptide obtained by treating CMized HSA with trypsin. The CMized peptide was prepared by adding 40 μL of a 70 mg / ml trypsin solution to 40 μL of the solution containing CML-HSA prepared in Example 1, reacting in a 37 ° C. water bath for 1 hour, and adding 80 μL of 5 mM AEBSF. Obtained by stopping the reaction. By this operation, the CM HSA is cleaved at the C-terminal of the lysine residue and the arginine residue, and becomes a plurality of CM-converted peptides whose N-terminal is lysine or arginine.
[0064]
For clinical specimens, blood was collected from the cubital veins of diabetic patients, allowed to stand at room temperature for 20-30 minutes, and then centrifuged at 3,000 rpm for 10-15 minutes to separate serum. 40 μl of a 70 mg / ml trypsin solution was added to 40 μl of the patient serum thus obtained, and the mixture was reacted for 1 hour in a 37 ° C. water bath, and the reaction was stopped by adding 80 μl of 5 mM ABESF.
[0065]
The amount of CML contained in the thus prepared clinical specimen was measured as follows.
Using CML-HSA treated with trypsin as STANDARD, a total of 8 concentrations were prepared in 2-fold serial dilutions from 80 μM, and a calibration curve was prepared by competitive ELISA. The specimen pretreated as described above was diluted 2 times, 4 times, and 8 times, and the CML measurement value was obtained from the prepared calibration curve.
The results are shown in Table 1.
[0066]
[Table 1]

[0067]
As can be seen from Table 1, when the monoclonal antibody NF-1G of the present invention was used, the measurement sensitivity was excellent, so that the amount of CML contained in the clinical specimen could be sufficiently measured.
[0068]
【The invention's effect】
According to the present invention, a monoclonal antibody that specifically reacts with a CM protein, CM peptide, or CM amino acid is provided. The present invention also provides a method for detecting diabetes and uses of the antibody.
CM proteins, CM peptides, and CM amino acids are closely related to the onset and progression of tissue damage in diabetic complications, so in the field of medical research and clinical examination, clinical markers such as diabetic complications Can be used as
[0069]
Further, by using the antibody of the present invention, the amount of CM amino acid in the CM protein can be measured with good sensitivity. Thereby, the amount of CML in the CM protein contained in the sample can be quantitatively detected, and the relationship between diabetes or aging phenomenon and the CM protein can be examined in more detail.
[Brief description of the drawings]
FIG. 1 is a characteristic diagram by ELISA showing the reactivity of various monoclonal antibodies with CM HSA and CE HSA, (a) shows the reactivity of monoclonal antibody 6D12 to CM HSA, (b) The reactivity of monoclonal antibody CMS-10 to CM HSA, (c) shows the reactivity of monoclonal antibody KNH-30 to CM HSA, (d) shows the reactivity of monoclonal antibody NF-1G to CM HSA (E) shows the reactivity of monoclonal antibody 6D12 to CE-modified HSA, (f) shows the reactivity of monoclonal antibody CMS-10 to CE-ized HSA, and (g) shows the CE of monoclonal antibody KNH-30. (H) shows the reactivity of monoclonal antibody NF-1G to CE-modified HSA.
FIG. 2 is a characteristic diagram by competitive ELISA showing the reactivity of various monoclonal antibodies with CML free form and CEL free form, (a) is monoclonal antibody 6D12, (b) is monoclonal antibody CMS-10 or (c ) Shows the case where the monoclonal antibody NF-1G was used.
FIG. 3 is a characteristic diagram showing the results of examination of detection sensitivity in a competitive ELISA using monoclonal antibody NF-1G.
FIG. 4 is a characteristic diagram showing the results of examination of the influence of EDTA on the reactivity of various monoclonal antibodies with CML-HSA by ELISA. (A) is monoclonal antibody 6D12, (b) is monoclonal antibody CMS-10. (C) shows the case where the monoclonal antibody NF-1G was used.
FIG. 5 is a characteristic diagram showing a standard curve used when measuring the amount of CML contained in a clinical specimen using monoclonal antibody NF-1G. Enzyme-treated CM HSA was used as a standard substance.

Claims (10)

  A monoclonal antibody produced by a hybridoma whose accession number is FERM P-18589, which reacts with an amino acid whose side chain amino group is carboxymethylated. A method for measuring an amino acid, comprising reacting the antibody according to claim 1 with a carboxymethylated amino acid in a biological sample . A method for evaluating diabetic complications, which comprises reacting the antibody according to claim 1 with a carboxymethylated amino acid in a biological sample . The evaluation method according to claim 3 , wherein the diabetic complication is diabetic microvascular complication and / or diabetic macrovascular complication. A method for evaluating the progress of aging, comprising reacting the antibody according to claim 1 with a carboxymethylated amino acid in a biological sample . A reagent for detecting a carboxymethylated amino acid, comprising the antibody according to claim 1 . A reagent for evaluating diabetic complications, comprising the antibody according to claim 1 . The reagent according to claim 7, wherein the diabetic complication is diabetic microvascular complication and / or diabetic macrovascular complication. A reagent for evaluating the progress of aging, comprising the antibody according to claim 1 . A reagent for immunohistochemical staining comprising the antibody according to claim 1 .

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