JP2003160599A - Antibody against carboxymethylated peptide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antibody against a carboxymethylated amino acid. <P>SOLUTION: The antibody reacts with the amino acid of which the amino group in the side chain is carboxymethylated. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antibody that reacts with a carboxymethylated amino acid present in a protein or peptide, a method for evaluating the progress of diabetic complications and aging using the antibody, and the antibody. Regarding the use of.

[0002]

BACKGROUND OF THE INVENTION Proteins in the living body are non-enzymatically reacted with reducing sugars to be saccharified. This reaction is generally called the Maillard reaction and is composed of early and late stage reactions. The first stage of the Maillard reaction is that the side chain amino group and the N-terminal amino group of the amino acids that make up the protein react with the carbonyl group of the sugar to form the Amadori rearrangement product via the Schiff base. Hemoglobin A1C and glycated albumin are known as products of this reaction, and these products are widely used as clinical markers of diabetes. Basically, at present, it is considered that all the proteins in the living body are glycated, and many dysfunctions of the proteins as a result have been reported.

In the latter stage of the Maillard reaction, the Amadori rearrangement product produced in the first stage undergoes complicated irreversible reactions such as dehydration, oxidation and condensation, and is characterized by fluorescence, browning or intramolecular / intermolecular crosslink formation. Is a step of producing the final product of the Maillard reaction. And the final product of the latter stage is AGE (Advanced Glycation End produ
cts).

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, the structures of AGE include pyraline, pentosidine, crosulin,
Carboxymethyllysine (CML) has been proposed (B
iochemistry vol.35, No.24, 8075-8083, 1996 and others)
The details of the reaction pathway and the significance of the existence of the structure have not yet been clarified.

In addition to the above-mentioned physicochemical characteristics, AGE has a biological characteristic that it is specifically recognized by the cell membrane receptor of macrophages, and has received a great deal of attention. In addition, AGEs in various tissue lesions such as microangiopathy (nephropathy, retinopathy, peripheral neuropathy, etc.) seen in diabetes and aging, arteriosclerosis, cataract, hardening / thickening of connective tissue of skin / vessel / joint Has been confirmed (Horiuch
i S. et al., Nephrol. Dial. Transplant. 11 (5) (199
6)). Therefore, AGE is considered to be deeply involved in the development of these tissue disorders, and AGE is becoming more important in the fields of medical research and clinical examination.

By the way, carboxymethyllysine (CML), which is a typical product of oxidative cleavage of glucose-derived Amadori rearrangement product, has been identified since 1986 (Ahm
ed MU et al., J. Biol. Chem. 261, 4889-94 (1986)),
It has attracted attention in connection with the above-mentioned diabetes and aging phenomenon. CML concentration in human skin collagen is significantly higher in diabetic patients (Dyer DG. Et al., J. Clin. In
vest, 91 (6), 2463-9 (1993)) and other CML in the organization.
There have been many reports that the concentration significantly increases with the progress of diabetes and aging. In addition, studies on the epitope of antibodies to AGE have revealed that CML is a major structure of AGE (Ikeda K. et al., B.
iochem. 35, 8075-8083 (1996)).

On the other hand, in recent years, as a new structure of AGE, C
Carboxyethyllysine (CEL) with a structure very similar to ML was found, and it was revealed by gas chromatography / mass spectrometry that the concentration in human lens protein increases with aging (Ahmed MU.et. al., Biochem Journal, 32
4, 565-570 (1997)). In CEL, the side chain of lysine in a protein is considered to be carboxyethylated (CE) by methylglyoxal (MGO). It has been reported that MGO exists as a degradation product of triosephosphate in the glycolysis system and a metabolite of acetol and is increased in blood of diabetic patients (Mclellan AC et al., Clinical
Science 87, 21-29 (1994)). Even in vitro, when erythrocytes were cultured in a medium containing a high concentration of sugar, MGO in the cells was
It is known that the concentration increases. As mentioned above,
CML and CEL are being shown to be important as AGE structures. Therefore, the present inventors were 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 is capable of discriminating between carboxymethylated protein and carboxyethylated protein by specifically reacting with carboxymethylated protein. The detection sensitivity was not sufficient, and there was almost no reaction with, for example, a carboxymethylated peptide consisting of several amino acids and a carboxymethylated free amino acid. Therefore, the antibody disclosed in the above publication could not detect the carboxymethylated peptide and the carboxymethylated amino acid after enzymatic treatment or hydrolysis with an acid.

[0009]

Therefore, the present invention is directed to the reaction with amino acids whose side chain amino groups are carboxymethylated after carboxymethylated proteins are treated with an enzyme or hydrolyzed with an acid, and peptides containing the amino acids. It is an object of the present invention to provide a possible antibody, a method for evaluating the degree of progression of diabetic complications and aging using the antibody, and the use of the antibody.

[0010]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventor has found that a protein or peptide in which the amino group of the amino acid side chain is carboxymethylated is used as an antigen and carboxymethylated. By using such a protein or peptide for screening, an antibody that reacts with an amino acid or peptide having a side chain amino group present in the protein or peptide that is carboxymethylated was successfully prepared, and the present invention was completed. It was That is, the present invention is an antibody that reacts with an amino acid or peptide in which the amino group of the side chain is carboxymethylated.

Examples of carboxymethylated amino acids include N-ε-carboxymethyllysine. Moreover, as the peptide, those composed of two or more amino acids can be mentioned. Further, the antibody may be a polyclonal antibody or a monoclonal antibody. As the monoclonal antibody, for example, the accession number is FERM P-1
8589 produced by the hybridoma.

Furthermore, the present invention is a method for measuring a carboxymethylated amino acid in a protein, which comprises reacting the above antibody with an amino acid whose side chain amino group is 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 nephropathy, retinopathy, neuropathy and other diabetic microvascular complications, and diabetic macrovascular complications represented by arteriosclerosis.

Further, the present invention is a method for evaluating the degree of aging, which comprises reacting the antibody with the amino acid. Furthermore, the present invention is a reagent for detecting an amino acid in which a side chain amino group is carboxymethylated, which comprises the above antibody. Furthermore, the present invention comprises the above antibody,
It is a reagent for evaluating diabetic complications, a reagent for evaluating the progress of aging, or a reagent for immunohistological staining.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.

The present invention is directed to amino acids whose side chain amino groups are carboxymethylated (hereinafter referred to as "carboxymethylated amino acids" or "CM amino acids") and peptides containing carboxymethylated amino acids (hereinafter referred to as "carboxymethylated"). And a protein containing a carboxymethylated amino acid (hereinafter referred to as "carboxymethylated protein" or "CM-modified protein").

The CM-modified amino acid and CM-modified peptide to which the present antibody reacts include, for example, those obtained by enzymatically treating a CM-modified protein with a protease or hydrolyzing it with an acid. Here, the CM protein is
AGEs present in various tissue lesions such as microangiopathy (nephropathy, retinopathy, peripheral neuropathy, etc.) observed in diabetes and aging phenomena, arteriosclerosis, cataract, hardening / thickening of connective tissue of skin / vessel / joint Structure (Horiuchi S. et al., Nephrol.
Dial. Transplant. 11 (5) (1996)). Therefore, if the CM protein is quantitatively detected from the various tissues, the progress of the tissue disorder can be grasped. That is, in the fields of medical research and clinical examinations, the importance of quantitatively detecting the CM-ized protein in the above various tissues is increasing.

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-ized protein, and it has been difficult to quantify the CM-ized protein contained in the various tissues described above. On the other hand, the antibody according to the present invention reacts with, for example, a CM-ized protein and a CM-modified amino acid obtained by treatment with a protease or an acid, so that carboxymethylation of the CM-modified protein contained in the various tissues described above. Since it reacts with all the sites, the CM-ized protein contained in the above various tissues can be quantified.

Further, other AGE different from the CM protein
One of the structures 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-modified amino acid residues and does not react with CE-modified amino acid residues. Therefore, by using the antibody according to the present invention, the CE-modified protein and the CM-modified protein contained in the above various tissues can be separated or discriminated and only the CM-modified protein can be quantified.

Here, "carboxymethylated amino acid"
Alternatively, the “CM-modified amino acid” means an amino acid having a side chain having an amino group, in which the amino group is carboxymethylated. The type of amino acid having an amino group in the side chain is not particularly limited, and examples thereof include lysine, arginine, asparagine, and glutamine, and lysine (for example, N-ε-carboxymethyllysine) is preferable.

“Carboxymethylated peptide” or “CM
The “modified peptide” means a structure including a plurality of amino acids, which is formed by peptide-bonding the above CM-modified amino acid and other amino acids. Other amino acids include:
There is no particular limitation and it is meant to include any amino acid and its derivatives and modified forms. Further, the “carboxymethylated peptide” or “CM-modified peptide” may include a plurality of CM-modified amino acids adjacent to or separated from each other. Further, the "carboxymethylated peptide" or "CM-modified peptide" need only include at least one or more CM-modified amino acids, and may include other amino acids whose side chain amino groups are not CM-modified. Is also good. further,
"Carboxymethylated peptide" or "CM peptide"
As long as it contains at least one or more CM-modified amino acids, it may contain CE-modified amino acids.
The term "CE-modified peptide" refers to a peptide containing an amino acid in which at least some of the side chain amino groups are carboxyethylated, and does not include a carboxymethylated amino acid ("carboxyethylated"). (Also referred to as "modified peptide").

The "carboxymethylated peptide" or "CM-modified peptide" may include two or more amino acids, but when there are a plurality of CM-modified amino acids per protein molecule, the present antibody after hydrolysis is used. By using, it is possible to measure with high detection sensitivity. In this case, the antibody according to the present invention can react with an amino acid having a side chain amino group converted to CM among the amino acids existing in the peptide.

1. CM protein, CM peptide and CM
Preparation of modified amino acid The protein to be CM-converted is not particularly limited, and may be any of complex protein, simple protein, glycoprotein, lipoprotein and the like. Examples of these proteins include albumin (BSA etc.),
Hemoglobin, keyhole limpet hemocyanin (KL
H), human serum albumin (HSA), ribonuclease (RNase), β ₂ microglobulin, histone, collagen, blood cell membrane protein, low density or high density lipoprotein, and the like. Further, not only a protein but also a peptide such as an oligopeptide or a polypeptide may be used, and those synthesized by modification or decomposition from a protein can be used.

To convert these proteins or peptides into CM, several tens of mg / ml of protein such as BSA (for example, 5 to 50 m
g / ml) and several tens of mg / ml (eg 10-100 mg / ml) of glyoxylic acid in phosphate buffer (containing 5 times the molar amount of NaCNBH ₃ of glyoxylic acid, pH 7.4) at room temperature. The method of reacting for about 24 hours is adopted. The CM-modified protein obtained by the above method is purified by dialysis, liquid column chromatography, etc., and then used for producing a polyclonal antibody or a monoclonal antibody.

The CM-modified peptide and the CM-modified amino acid can be obtained by hydrolyzing the CM-modified protein obtained as described above with a protease such as trypsin, pepsin, papain, aprotinin, leupeptin or an acid such as hydrochloric acid or sulfuric acid. You can For example, several tens of mg / ml (for example, 5 to 70 mg
/ ml) tens of microliters of CM protein (for example, 10 to 50 μl)
L) and several tens of mg / ml (for example, 10 to 80 mg / ml) of trypsin solution (tens of μL, for example, 20 to 50 μL) are mixed and allowed to react in a 37 ° C. water bath for 3 hours. The reaction is stopped with 5 mM AEBSF, whereby the CM-ized protein can be enzymatically treated, and a CM-modified peptide and a CM-modified amino acid can be obtained. In addition, trypsin inhibitor (for example, 4-
(2-Aminoethyl) benzenesulfonylfluoride, HCL (AEBS
The hydrolysis reaction by trypsin can be stopped by adding F)).

2. Antibodies to CM-ized proteins In the present invention, the term "antibody" means the entire CM-ized protein that is an antigen, the CM-ized peptide and the CM-ized amino acid, or a whole fragment thereof (for example, Fab or F (ab ')
₂ fragment) and may be a polyclonal antibody or a monoclonal antibody. Antibodies (polyclonal and monoclonal antibodies) can be produced by any of various methods. Methods for producing such antibodies are well known in the art [eg Sambrook, J et al.,
Molecular Cloning, Cold Spring Harbor Laboratory
Press (1989)].

(1) Preparation of monoclonal antibody against CM-ized protein (i) Collection of antibody-producing cells Using the CM-ized protein or CM-formed peptide prepared as described above as an antigen, mammals such as rat, mouse,
Administer to rabbits. The dose of the antigen per animal is 0.1 to 100 mg when no adjuvant is used,
When an adjuvant is used, it is 1 to 100 μg. As the adjuvant, Freund's complete adjuvant (FCA),
Examples include Freund's incomplete adjuvant (FIA) and aluminum hydroxide adjuvant. Immunization is mainly performed by injecting intravenously, subcutaneously, or intraperitoneally. Further, the interval of immunization is not particularly limited, and is from several days to several weeks, preferably at intervals of 2 to 5 weeks, 1 to 10 times, preferably
Immunize 2-5 times. Then, antibody-producing cells are collected 1 to 60 days, preferably 1 to 14 days after the final immunization day.
Examples of antibody-producing cells include spleen cells, lymph node cells, peripheral blood cells and the like, but spleen cells or local lymph node cells are preferred.

(Ii) Cell fusion To obtain a hybridoma, cell fusion between antibody-producing cells and myeloma cells is performed. As the myeloma cells to be fused with the antibody-producing cells, commonly available cell lines of animals such as mice can be used. The cell line used has drug selectivity and cannot survive in HAT selective medium (including hypoxanthine, aminopterin, and thymidine) in the unfused state, and can survive only in a state fused with antibody-producing cells. Those having are preferred. Examples of myeloma cells include mouse myeloma cell lines such as P3X63-Ag.8.U1 (P3U1) and NS-I.

Next, the myeloma cells and antibody-producing cells are fused with each other. Cell fusion is serum-free DME
1 × in M, RPMI-1640 medium or other animal cell culture medium
10 ⁶ to 1 × 10 ⁷ cells / ml of antibody-producing cells and 2 × 10 ⁵ to 2 × 10 ⁶
Individual / ml myeloma cells are mixed (a cell ratio of antibody-producing cells and 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. Also electrical stimulation
The antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device utilizing (for example, electroporation).

(Iii) Selection and cloning of hybridomas The desired hybridomas are selected from the cells after the cell fusion treatment. As a method, after appropriately diluting the cell suspension with, for example, RPMI-1640 medium containing fetal bovine serum, spread 3 × 10 ⁵ cells / well on a microtiter plate, add a selective medium to each well, and then add an appropriate solution. The selective medium is exchanged and the culture is performed. As a result, cells that start to grow about 14 days after the start of culture in the selective medium can be obtained as hybridomas.

Next, it is screened whether or not the antibody that reacts with the CM protein is present in the culture supernatant of the grown hybridoma. The screening of hybridomas may be performed according to a usual method and is not particularly limited. For example, a part of the culture supernatant contained in the wells grown as hybridomas can be collected and screened by enzyme immunoassay, radioimmunoassay or the like.

[0031] Specifically, a plurality of types of C are prepared by the ELISA method or the like.
A hybridoma, which is a cell that produces a monoclonal antibody that reacts with the protein that has been converted to M and that does not react with the latter and that does not react with the latter, is established using multiple types of proteins that have not been converted to CM as antigens. Cloning of fused cells
Perform by the limiting dilution method or the like. And finally,
A hybridoma, which is a cell that produces a monoclonal antibody that reacts with a CM-modified protein but not with a protein that is not CM-modified, is established.

In the present invention, the hybridoma NF-1G (“Mouse-Mouse
hybridoma NF-1G ”) was obtained. This Mouse-
Mouse hybridoma NF-1G is a patent biological deposit center of National Institute of Advanced Industrial Science and Technology (1-1, Higashi 1-chome, Tsukuba City, Ibaraki Prefecture).
No. 1) was deposited as FERM P-18589 on November 6, 2001.

(Iv) Collection of Monoclonal Antibody As a method for collecting the monoclonal antibody from the established hybridoma, an ordinary cell culture method, ascites formation method or the like can be adopted. In the cell culture method, the obtained hybridomas are cultured under normal culture conditions (eg, 37 ° C., 5% CO _{2 in} an animal cell culture medium such as RPMI-1640 medium containing 10% fetal bovine serum, MEM medium or serum-free medium). (Concentration) 7 to 1
Culture for 4 days, and obtain the antibody from the culture supernatant.

In the case of the ascites formation method, approximately 1 × 10 ⁷ hybridomas are intraperitoneally administered to a mammal derived from a myeloma cell and an animal of the same species to proliferate the hybridomas in a large amount. Then, after 1 to 2 weeks, ascites is collected. When purification of the antibody is required in the above method of collecting the antibody,
The purification can be performed by appropriately selecting a known method such as an ammonium sulfate salting-out method, ion exchange chromatography, gel filtration, affinity chromatography, or a combination thereof.

(2) Preparation of polyclonal antibody against CM-ized protein Using the CM-modified protein or CM-modified peptide as an antigen, this is administered to mammals such as rats, mice, and rabbits. 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 the adjuvant include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), aluminum hydroxide adjuvant and the like. Immunity is mainly intravenous, subcutaneous,
It is performed by injecting into the abdominal cavity 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. Then, 6 to 60 days after the final immunization date, an enzyme-linked immunosorbent assay (ELISA)
Or EIA (enzyme immunoassay)), radioimmunoassay (RI
The antibody titer is measured by A; radioimmuno assay, etc., and blood is collected on the day when the maximum antibody titer is shown, to obtain antiserum.

After that, the CM-modified and CE-modified proteins (BS
Reactivity of polyclonal antibodies in antiserum to these proteins is measured by ELISA method or the like. Then, by collecting the fractions that react with a certain CM-modified protein and do not react with the non-CM-modified protein, a polyclonal antibody that reacts only with the CM-modified protein can be obtained.

3. In the present invention, a method for measuring CM-ized amino acids, etc.
Peptide and CM protein can be measured or quantified. For example, a monoclonal antibody or a polyclonal antibody of the present invention and CM-ized amino acids, CM-modified peptide and CM-ized protein reacted, by using an anti-mouse IgG antibody labeled with horseradish peroxidase (HRP), CM-ized amino acid, It is possible to quantify CM peptides and CM proteins.

4. Method for assessing diabetic complications and aging progression By reacting the antibody of the present invention with CM amino acid, CM peptide and CM protein in a biological sample, it is possible to evaluate diabetic complications and aging progression. it can. Examples of diabetic complications include diabetic nephropathy, retinopathy, neuropathy and other diabetic microvascular complications, and diabetic macrovascular complications represented by arteriosclerosis. In the present invention, the diseases for detecting these complications may be one kind or two or more kinds may occur together.

Patients with diabetic complications, such as diabetic nephropathy,
Blood from patients suspected of having diabetic retinopathy,
Urine, tissue, etc. are collected and subjected to appropriate pretreatment to prepare a measurement sample. The measurement sample can be used as a clinical marker for diabetes. Then, the measurement sample is reacted with the antibody. After the reaction, CM-modified amino acids, CM-modified peptides and CM-modified proteins 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 when the quantitative value is high, it can be used as judgment data for evaluating the presence or absence of diabetic complications and the progress of aging.

Examples of the pretreatment include enzyme treatment with protease and the like and hydrolysis treatment with acid and the like. The CM-modified protein can be decomposed into CM-modified amino acids and CM-modified peptides by performing enzymatic treatment and hydrolysis treatment. This antibody is especially useful for CM
Since it has excellent reactivity to modified amino acids and commercialized peptides, it is possible to quantitatively measure the commercialized site in the commercialized protein contained in the measurement sample by performing enzymatic treatment and hydrolysis treatment as the pretreatment. You can Therefore, by using this antibody, the progress of diabetic complications and aging can be evaluated in detail.

5. Reagents Containing Antibodies of the Present Invention In the present invention, CM amino acids, CM peptides and CM
Antibodies to the modified protein can be used as various reagents. For example, when used as a reagent for detecting a CM-modified peptide and a CM-modified amino acid, the above 3. When the detection is performed using the measuring method described in 4 above, and the reagent is used as a reagent for detecting diabetes, 4. Detection is performed by the method described in 1.

When the antibody of the present invention is used as a reagent for immunohistological staining, detection is carried out according to a usual 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)
A labeled anti-mouse IgG antibody was bound to the antibody of the present invention as a secondary antibody to give 3,3′-diaminobenzidine (3,3′-diamin
Stain with obenzidine) treatment. After dyeing, microscopic observation can be performed, and it can be determined that the brown-stained region has undergone CMization.

[0043]

EXAMPLES The present invention will be described in more detail below with reference to examples. However, the technical scope of the present invention is not limited to these examples.

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”) was used as the carboxymethylated protein.
HSA ”) was used. 5 mg of protein (HSA), 1,
0.2 with 100 mM glyoxylic acid and 0.45 M NaCNBH ₃
After reacting in M phosphate buffer (pH 7.8) at 37 ° C for 24 hours,
It was dialyzed against PBS to obtain a CM protein (Reddy S et a
L., Biochemistry 1995, 34, 10872-10878). Generated C
From the result of amino acid analysis of the M-protein, it was confirmed that the protein was CM-HSA. (ii) Carboxyethylated protein 5 mg protein (HSA) was added to 0.2 M pyruvic acid and 0.3 M
After reacting for 24 hours in PBS containing M NaCNBH ₃ , it was dialyzed against PBS to obtain CE-modified protein (Ahmed MU et al., Bioc
hem J 1997, 324, 565-570). From the result of amino acid analysis of the produced CE-modified protein, it was confirmed to be CE-modified HSA (referred to as “CE-modified HSA”).

(2) Immunization of animal 1 mg / ml of antigen (CM HSA) was mixed with an equal amount of Freund's adjuvant to prepare an emulsion, and then 200 μl /
BALB / c mice were immunized intradermally in the back. Booster immunization was performed every 2 weeks, and blood was collected from the tail vein after immunizing four times from the initial immunization to confirm the antibody titer.

(3) Measurement of antibody titer The antibody titer was measured by the ELISA method. That is, CM HSA was used as an antigen at a concentration of 5 μg / ml in 50 μl / well each for 4 nights.
Plates were coated at ° C. After washing three times with PBS containing 0.05% Tween 20 (PBS-T), blocking was carried out for 1 hour with a phosphate buffer (pH 7.4) containing 0.5% gelatin. After washing, 50 μl / well of mouse serum serially diluted with PBS-T was added and left standing for 1 hour. After washing, 50 μl / well of a secondary antibody (horseradish peroxidase (HRP) -labeled anti-mouse IgG) diluted 2500 times with PBS-T was added and left still for 1 hour. After washing, 0.5 mg / ml OPD 1,2-phenylenediamine
Dihydrochloride (1,2-phenylenediamine dihydroch
100 μl / w of citrate buffer (pH 5.0) containing loride, OPD)
Each ell was added and reacted 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, a 10,000-fold diluted solution of antiserum showed significant reactivity with the antigen.

(4) Cell Fusion and Cloning of Antibody-Producing Hybridoma Mouse spleen cells confirmed to have an increased antibody titer and myeloma cells P3U1 were cell-fused at a ratio of 5: 1 by the polyethylene glycol method, and a HAT selection medium was prepared. Then, selective culture of hybridoma was performed. On the 10th day of cell fusion, the hybridoma culture supernatant was collected and the antibody titer was measured (see above).
(3)) ELISA is performed in the same manner as described in CM for HSA and CM.
A strain having a positive reactivity with KLH and a negative reactivity with HSA was screened.

The mouse spleen cells in which the increase in antibody titer was confirmed were collected by streptavidin-bound magnetic beads (PIERCE). That is, magnetic beads labeled with streptavidin and biotin
CM-containing HSA is mixed into the spleen cell suspension, and then,
The magnetic beads are collected by operating a magnet. The streptavidin bound to the collected magnetic beads will be bound with biotin of CM-ized HSA, and as a result, the spleen cells bound by CM-HSA by the antigen-antibody reaction will be captured by the magnetic beads.

After measuring the number of hybridoma cells that became positive in the above screening, the cells were seeded in a 96-well plate (subcloning) at 2 × 10 ⁵ cells / well, 1
After 0 days, only single colony wells were screened again. Similarly, the hybridomas that became positive in the screening were subcloned again, and the screening operation was continued until the properties matched 100%.

As a result, a monoclonal antibody producing cell line (NF-1G) having a positive reactivity with CM-modified HSA and CM-modified KLH and not reacting with HSA was obtained. Hybridoma NF-1G (name: "Mouse-Mouse hybridoma NF-1G")
On November 6, 2001 at the National Institute of Advanced Industrial Science and Technology Patent Depository Center (1-1-1, Higashi, Tsukuba, Ibaraki)
Deposited as FERM P-18589 on date.

[Example 2] Examination of properties of the monoclonal antibody of the present invention (discrimination between CML and CEL) 1 In this Example, the reaction of CML-specific monoclonal antibody-producing cell line NF-1G to CML and CEL was performed. The sex difference was examined. Antigens with different CML contents per molecule were prepared. That is, 1100 mM glyoxylic acid was tripled, and 6-step dilution series were prepared.
1 mg of HSA of 5 mg / ml was added to each. Let stand at room temperature for 2 hours,
Add 50 μl of NaCNBH ₃ and mix, and leave at room temperature overnight,
It dialyzed with PBS and CM-ized protein was obtained. As a result of amino acid analysis of the produced CM protein, it was commercialized.
It was confirmed to be HSA. For the preparation of antigens having different CEL contents, pyruvic acid was used instead of glyoxylic acid, and the same method was used.

Regarding the monoclonal antibody producing cell line NF-1G, the difference in reactivity to CML and CEL was examined by the following method. That is, CMized HSA and CE produced by the above method
Phosphate HSA at a concentration of 5 μg / ml each (pH 7.4)
It was dissolved in 50 μl / well and coated on an ELISA plate by 50 μl / well. Then 0.5% dissolved in carbonate buffer (pH 9.5)
Blocked with gelatin (200 μl / well). 1 μg / ml
50 μl / well of NF-1G purified antibody was added 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 dihydrochlo
ride, OPD) was added to carry out color reaction, and the absorbance at 490 nm was measured by an ELISA reader.

At this time, as controls, existing anti-CML monoclonal antibodies 6D12 (manufactured by Transgenic Co., Ltd.), CMS-10 (manufactured by Transgenic Co., Ltd.), anti-CEL were used.
KNH-30, a monoclonal antibody (provided by Kumamoto University)
The purified antibody was used. As a result, it was confirmed that the monoclonal antibody (NF-1G) of the present invention reacts with CM HSA but does not show reactivity with CE HSA. Also, 6D12 is CM
It became clear that they reacted in the same manner as the modified HSA and CE modified HSA, and that they could not be distinguished. Furthermore, KNH-30 is CE
It was confirmed that it reacts with HSA but does not react with CM HSA. On the other hand, it was confirmed that CMS-10 specifically reacts with CM-HSA, but has lower reactivity with CM-HSA than NF-1G.

Furthermore, since the reactivity increases with the number of CMLs, it can be seen that CML is reacting. Therefore, the monoclonal antibody (NF-1G) of the present invention,
Clearly, unlike existing anti-AGE monoclonal antibodies, C
It was found that it showed excellent reactivity specifically to ML and did not react with CEL (Fig. 1). Incidentally, the subtype of the monoclonal antibodies of the present invention was confirmed to be IgG _2a with a commercially available typing kit.

[Example 3] Examination of properties of the monoclonal antibody of the present invention (discrimination of CML free form) 2 In this example, 6D12 and CMS-10, which had a reactivity with CM HSA in Example 1, were observed. For NF-1G and NF-1G, the discriminability of CML educts was examined. The CML educt was synthesized by a chemical method.

A competitive ELISA was carried out using CMLHSA as a solid-phased antigen, and the reactivity of 6D12, CMS-10 and NF-1G to the CML free form was examined. In the competitive ELISA, CML-HSA was used as the antigen at a concentration of 2.5 μg / ml and 50 μl / well.
The plates were coated overnight at 4 ° C. 0.05% Tw
After washing 3 times with PBS containing een20 (PBS-T), blocking was performed with a phosphate buffer (pH 7.4) containing 0.5% gelatin for 1 hour.
After washing, in advance with CML educt and 0.1 μg / ml 6D12, CMS-10 and
NF-1G was reacted at room temperature for 1 hour, 50 μl / well was added to each, and the mixture was allowed to stand for 1 hour. After washing, a secondary antibody (horseradish peroxidase (HRP) -labeled anti-mouse IgG (γ)) diluted 2500 times was added at 50 μl / well and left standing for 1 hour. After washing, 0.5 mg / ml 1,2-phenylenediamine dihydrochlori
de, OPD) containing citrate buffer (pH 5.0) was added at 100 μl / well and reacted for 10 minutes. After the reaction, 2N sulfuric acid was added to stop the reaction, and the absorbance at 490 nm was measured.

Similarly, a competitive ELISA was performed using the chemically synthesized CEL educt as an antigen. The result is shown in Figure 2.
Shown in (a), (b) and (c). From FIG. 2 (a), it was confirmed that 6D12 did not show reactivity with both the CML educt and the CEL educt. In addition, from FIG. 2 (b), CMS-10 also shows CML educts and
It was confirmed that neither CEL educts show reactivity.
On the other hand, from FIG. 2 (c), it was confirmed that NF-1G showed reactivity to the CML educt and not the CEL educt.

Therefore, according to the present example, it was revealed that the monoclonal antibody (NF-1G) of the present invention is capable of specifically reacting with a CML free form, unlike the existing anti-AGE monoclonal antibody. 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. 3. From FIG. 3, it was found that the competitive ELISA using NF-1G can detect the CML educt contained in the sample even at a low concentration of about 10 μM. CM by high performance liquid chromatography
Since the detection sensitivity of L educts is 1 to 100 μM, NF-1G
It can be said that there is no difference in detection sensitivity between the competitive method ELISA using and the high performance liquid chromatography.

Example 4 Examination of Properties of Monoclonal Antibody of the Present Invention (Effect of Anticoagulant) 3 In this example, 6D12, CMS-10 and 6D12, which showed reactivity to CM HSA in Example 1, were obtained. About NF-1G, EDTA
The influence of the reactivity was investigated. In this example, CM-HSA prepared in the same manner as in Example 1 was used as a solid-phased antigen, and the effect of EDTA on the reactivity with various antibodies was examined.

The measurement is carried out by an ELISA using CM HSA as a solid phase antigen.
I went by law. That is, 2.5 μg / ml of CML-HSA as an antigen
Plates were coated with 50 μl / well at a concentration of ml overnight at 4 ° C. After washing three times with PBS containing 0.05% Tween 20 (PBS-T), it was blocked with a carbonate buffer (pH 9.5) containing 0.5% gelatin for 1 hour. After washing 10,5,2.5,1.25,0.625,0.31
After 50 μl of an antibody solution containing 25 mM EDTA was added and reacted at room temperature for 1 hour, the mixture was placed on a plate and allowed to react for 1 hour. After washing, 50 μl / well of secondary antibody (horseradish peroxidase (HRP) -labeled anti-mouse IgG (γ)) diluted 2500 times
Each was placed and left for 1 hour. After washing, 0.5 mg / ml 1,2-phenylenediamine dihydrochloride (1,2-phenylene
Citrate buffer containing diamine dihydrochloride (OPD)
(pH 5.0) was added at 100 μl / well and reacted for 10 minutes. After the reaction, 2N sulfuric acid was added to stop the reaction, and the absorbance at 490 nm was measured.

The results are shown in FIGS. 4 (a), (b) and (c). Figure 4
As shown in (b), in CMS-10, which specifically reacts with CMLHSA, the reactivity with CML-HSA decreased with increasing EDTA concentration. On the other hand, as shown in FIGS. 4 (a) and 4 (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 of the present invention (NF-1G)
For example, when CML-HSA contained in a sample is detected or quantified, it can be accurately detected or quantified without being affected by EDTA contained in the sample.

Example 5 Measurement of Clinical Specimen Using Monoclonal Antibody of the Present Invention In this Example, the amount of CML contained in a clinical sample was measured using the monoclonal antibody (NF-1G) of the present invention. , N
We investigated the applicability of F-1G for clinical sample measurement.

In this example, a standard curve as shown in FIG. 5 was prepared using CM-modified peptides obtained by treating CM-modified HSA with trypsin. The CM-modified peptide was prepared in Example 1.
To a 40 μL solution containing CML-HSA, add 70 mg / ml trypsin solution 4
Add 0 μL, react for 1 hour in a 37 ° C water bath, and add 5 mM AEB.
The reaction was stopped by adding 80 μL of SF.
By this operation, the CM-modified HSA is cleaved at the C-terminals of the lysine residue and the arginine residue, resulting in a plurality of CM-modified peptides having N-terminal lysine or arginine.

[0064] For the clinical specimen (details on specimen collection and pretreatment, etc., below), blood was collected from the elbow vein of a diabetic patient, left at room temperature for 20 to 30 minutes, and then 3,000 r
Serum was separated by centrifugation at pm for 10-15 minutes. 40 μl of patient serum thus obtained, 40 μl of 70 mg / ml trypsin solution
Was added and reacted in a water bath at 37 ° C for 1 hour, and 5 mM ABESF was added.
The reaction was stopped by adding 80 μl.

CM contained in clinical specimens prepared in this way
The amount of L was measured as follows. Using CML-HSA treated with trypsin as STANDARD, a total of 8 concentrations were prepared by serial dilution from 80 μM in 2-fold increments, and a calibration curve was prepared by a competitive ELISA. The sample pretreated as described above was diluted 2-fold, 4-fold, and 8-fold, and the CML measurement value was obtained from the prepared calibration curve.
The results are shown in Table 1.

[0066]

[Table 1]

As can be seen from Table 1, when the monoclonal antibody NF-1G of the present invention was used, the measurement sensitivity was excellent, and therefore the amount of CML contained in the clinical sample could be sufficiently measured.

[0068]

INDUSTRIAL APPLICABILITY The present invention provides a monoclonal antibody that specifically reacts with a CM-ized protein, a CM-modified peptide, and a CM-ized amino acid. The present invention also provides a method for detecting diabetes and uses of the antibody. Since CM protein, CM peptide, and CM amino acid are closely related to the onset and progression of tissue damage in diabetic complications, clinical markers such as diabetic complications in the field of medical research and clinical examination. Can be used as.

Further, by using the antibody of the present invention, the amount of CM amino acid in the CM protein can be measured with high sensitivity. As a result, the CML amount in the CM-modified protein contained in the sample can be quantitatively detected, and the relationship between the diabetes or aging phenomenon and the CM-modified protein or the like can be examined in more detail.

[Brief description of drawings]

[Figure 1] Various monoclonal antibodies and CM HSA and CE HSA
Is a characteristic diagram by ELISA method showing the reactivity with, (a) shows the reactivity to the CM-ized HSA of the monoclonal antibody 6D12, (b) shows the reactivity to the CM-ized HSA of the monoclonal antibody CMS-10, (c) CM of monoclonal antibody KNH-30 H
Shows reactivity to SA, (d) is monoclonal antibody NF-
1G shows the reactivity to CM HSA, (e) shows the reactivity of monoclonal antibody 6D12 to CE HSA, (f) shows the reactivity of monoclonal antibody CMS-10 to CE HSA, (g) Shows the reactivity of the monoclonal antibody KNH-30 with CE-labeled HSA, and (h) shows the CE-labeled HS of the monoclonal antibody NF-1G.
It shows reactivity to A.

FIG. 2 is a characteristic diagram by a competitive ELISA method showing the reactivity of various monoclonal antibodies with a CML educt and a CEL educt, where (a) is the monoclonal antibody 6D12, (b) is the monoclonal antibody CMS-10 and (c). ) Is the monoclonal antibody NF
The case where -1G is used is shown.

[Fig. 3] Competitive ELISA using monoclonal antibody NF-1G
FIG. 6 is a characteristic diagram showing the results of examination of detection sensitivity in FIG.

FIG. 4 is a characteristic diagram showing the results of examining the effect of EDTA on the reactivity of various monoclonal antibodies with CML-HSA by the ELISA method. (A) is the monoclonal antibody 6D12, (b)
Shows the case of using the monoclonal antibody CMS-10, and (c) shows the case of using the monoclonal antibody NF-1G.

FIG. 5 is a characteristic diagram showing a standard curve used when measuring the amount of CML contained in a clinical sample using the monoclonal antibody NF-1G. As the standard substance, enzyme-treated CM HSA was used.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] April 5, 2002 (2002.4.5)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0064

[Correction method] Change

[Correction content]

As a clinical sample, blood was collected from the elbow vein of a diabetic patient, left at room temperature for 20 to 30 minutes, and then 10 to 3,000 rpm.
The serum was separated by centrifugation for 15 minutes. To 40 μl of patient serum thus obtained, add 40 μl of 70 mg / ml trypsin solution,
It was obtained by reacting in a water bath at 7 ° C for 1 hour and stopping the reaction by adding 80 µl of 5 mM ABESF.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Ryuji Nagai             Kumamoto City, Kumamoto City             Takahashi 303 (72) Inventor Tamami Sakamoto             1-13-8 Rendaiji Temple, Kumamoto City, Kumamoto Prefecture F-term (reference) 4B064 AG26 AG27 CA10 CA20 CC24                       DA01 DA13                 4H045 AA11 AA30 CA41 DA75 DA76                       EA20 EA50 FA72

Claims (15)

[Claims] 1. An antibody in which an amino group of a side chain reacts with a carboxymethylated amino acid. 2. The antibody according to claim 1, wherein the amino acid is present in a peptide. 3. The antibody according to claim 2, wherein the peptide consists of 2 or more amino acids. 4. The carboxymethylated amino acid is N-
The antibody according to claim 1, which is ε-carboxymethyllysine. 5. The antibody according to claim 1, which is a polyclonal antibody or a monoclonal antibody. 6. A monoclonal antibody produced by a hybridoma having an accession number of FERM P-18589, which comprises:
A monoclonal antibody in which the amino group of the side chain reacts with a carboxymethylated amino acid. 7. A method for measuring an amino acid, which comprises reacting the antibody according to any one of claims 1 to 6 with a carboxymethylated amino acid. 8. A method for evaluating diabetic complications, which comprises reacting the antibody according to claim 1 with a carboxymethylated amino acid. 9. The evaluation method according to claim 8, wherein the diabetic complication is diabetic microvascular complication and / or diabetic macrovascular complication. 10. A method for evaluating the degree of aging, which comprises reacting the antibody according to any one of claims 1 to 6 with a carboxymethylated amino acid. 11. A reagent for detecting a carboxymethylated amino acid, which comprises the antibody according to any one of claims 1 to 6. 12. A reagent for evaluating diabetic complications, which comprises the antibody according to any one of claims 1 to 6. 13. The reagent according to claim 11, wherein the diabetic complication is diabetic microvascular complication and / or diabetic macrovascular complication. 14. A reagent for evaluating the degree of aging, which comprises the antibody according to any one of claims 1 to 6. 15. A reagent for immunohistochemical staining, which comprises the antibody according to any one of claims 1 to 6.