JP3542227B2 - Immunoreagent - Google Patents

Description

【００５１】
実施例２ 糖尿病合併症患者由来血液中のＣＭ化物の測定
実施例１で糖尿病患者由来血液の代わりに、糖尿病の他に腎症または網膜症を併発している糖尿病合併症患者由来血液を被検体としたこと以外は、実施例１の方法に従って被検体中のＣＭ化物の測定を行った。該患者の平均年齢は６０．４歳であった。測定結果を表２に示す。
【００５２】
【表２】

【００５３】
比較例１
実施例１で糖尿病患者由来血液の代わりに、糖尿病ではない健常者由来の血液を被検体としたこと以外は、実施例１の方法に従って被検体中のＣＭ化物の測定を行った。該健常者の平均年齢は６１．２歳であった。測定結果を表３に示す。
【００５４】
【表３】

【００５５】
健常者群と実施例１で測定した糖尿病患者群の発光強度の間には、統計学的に差があることを検証した。即ち、「差がない」と考えられる確率を計算し、その確率が非常に小さいから「差がある」という手順を取り、統計学的に検証した（Ｍａｃｉｎｔｏｓｈ統計マニュアル、長田 理、１９９５年２月１０日発行、真興交易医書出版部）。該検証にはウェルチのｔ検定を用いた。その結果、「差がない」と考えられる確率は５％未満で、糖尿病患者群の発光強度と健常者群の発光強度の間には有意に差があった。このことは、糖尿病患者由来の血液には、健常者由来の血液よりも、有意にＣＭ化物が多いことを意味する。
【００５６】
また、健常者群と実施例２で測定した糖尿病合併症患者群の発光強度の間に差があるか否かをウェルチのｔ検定にて検証したところ、「差がない」と考えられる確率は５％未満で、糖尿病合併症患者群の発光強度と健常者群の発光強度の間には有意に差があった。このことは、糖尿病合併症患者由来の血液には、健常者由来の血液よりも、有意にＣＭ化物が多いことを意味する。
【図面の簡単な説明】
【図１】作製したＣＭ化物に対する抗体の抗原特異性を競合法ＥＬＩＳＡにて調べた結果を示す図である（縦軸が４０５ｎｍの吸光度、横軸が各阻害剤の添加量）。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a diagnostic reagent for diabetes and diabetic complications, comprising an antibody against N-carboxymethyllysine (hereinafter abbreviated as CML) or an antibody against a peptide or protein having an N-carboxymethyllysine residue (hereinafter abbreviated as CM). Or a drug for evaluating the efficacy of a drug for treating diabetes or a complication for treating diabetes complications.
[0002]
[Prior art]
It is known that proteins in blood are saccharified by non-enzymatically reacting with glucose to become glycated proteins. The saccharification reaction is called a Maillard reaction and is divided into two reactions, an early stage and a late stage. The reaction in the first stage is said to be until the side chain amino group or N-terminal amino group of the protein reacts with the carbonyl group of the sugar to generate an Amadori rearrangement compound via a Schiff base. As the first-stage reaction products, for example, hemoglobin A1C and glycated albumin are known, and it is a well-known fact that they are used as clinical markers of diabetes.
[0003]
It is known that after the early stage reaction, it changes in two directions. One is the oxidative cleavage of an Amadori rearrangement compound involving oxygen and a transition metal, and the other is the late product of the Maillard reaction involving fluorescence, browning, or intra- and intermolecular cross-linking (hereinafter AGE). Abbreviated). The AGE is considered to be an aggregate of a plurality of compounds, and the qualitative and quantitative determination is performed by measuring the fluorescence intensity or the antigen-antibody reaction.
[0004]
Recently, it has been reported that the late-stage reaction occurs in vivo and is involved in the development of vascular complications (Monnier, VM, et al, New England Journal of Medicine, vol 314, p403, 1986). . In particular, in diabetic patients, AGE generated in the late-stage reaction has been attracting attention as being involved in the development and progression of diabetic complications. At present, 2-furoyl-4 (5)-(2-furanyl) -1H-imidazole, pyralin, pentosidine, and crosslin A & B have been proposed as one of the structures of AGE, but the basic structure of AGE is still unknown. There are many points at present.
[0005]
On the other hand, CML has been identified as a result of oxidative cleavage of the Amadori rearrangement compound (Ahmed, MU, et al, Journal of Biological Chemistry, vol 261, p4889, 1986), lens protein of elderly and diabetic patients, skin It has been reported that it is present in collagen and the like (Dunn, JA, et al, Biochemistry, vol 30, p1205, 1991). In addition, the modified protein in which the hydrogen of the side chain amino group of lysine constituting bovine serum albumin (hereinafter abbreviated as BSA) is replaced with a carboxymethyl group (hereinafter sometimes abbreviated as a CM group) is a modified protein in the lens protein. There is also a report that the reaction between AGE and anti-AGE antibody is strongly inhibited (Reddy, S., et al, Biochemistry, vol 34, p10872, 1995). However, these methods have a drawback in that a subject such as a lens protein and skin collagen that is difficult to use in a clinical test is used as a subject. Furthermore, since these were not directly measured immunologically with antibodies against CML or BSA substituted with a CM group, the operation was complicated and the measurement took a long time. Furthermore, the relationship between CML and CM compounds and diabetes and diabetic complications was unknown.
[0006]
[Problems to be solved by the invention]
Accordingly, an object of the present invention is to provide a method for directly and easily measuring CML or a CM compound using a body fluid, such as blood, serum, or urine, which is frequently used as a subject in a clinical test. It is to provide an immunological reagent for complications.
[0007]
[Means for Solving the Problems]
The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, by using an antibody against CML or an antibody against a CM compound (hereinafter, both of them are also referred to as an anti-CM antibody), they are generally used for clinical tests. It has been found that CML or CM-compounds can be easily quantified using the biological component to be tested as a subject, and that CML or CM-compounds are associated with diabetes or diabetic complications. .
[0008]
That is, the present invention An antibody against hemoglobin having an N-carboxymethyllysine residue was used. It is an immunoreagent for diabetes or diabetic complications.
[0009]
Hereinafter, the immunoreagent of the present invention will be described in detail, but is not limited to the following substances and production methods.
[0010]
The N-carboxymethyl lysine of the present invention includes lysine in which hydrogen of the side chain amino group of lysine is substituted with a carboxymethyl group, lysine in which an α-amino group is modified with a t-butoxycarbonyl group, a tosyl group, or the like. It refers to a lysine derivative such as lysine in which the α-carboxyl group is modified with methyl ester, ethyl ester, or the like, in which the hydrogen of the side chain amino group is substituted with a carboxymethyl group.
[0011]
For substituting the hydrogen of the side chain amino group with a carboxymethyl group, a known method is used without any limitation. For example, after protecting the α-amino group of lysine with a protecting group such as a t-butoxycarbonyl group or a benzyloxycarbonyl group, the lysine is treated in the presence of a hydride reducing agent such as sodium borohydride or sodium cyanoborohydride. Lysine and glyoxylic acid may be reacted at pH 7 or higher.
[0012]
The antibody against CML used in the present invention specifically binds to lysine in which the hydrogen of the side chain amino group is substituted with a carboxymethyl group or lysine derivative in which the hydrogen of the side chain amino group is substituted with a carboxymethyl group. Refers to an antibody. The antigen for producing the antibody, that is, the immunogen may be lysine or a lysine derivative in which the hydrogen of the side chain amino group is substituted with a carboxymethyl group, and the lysine or lysine derivative has an ability to induce an immune response. Is very weak, it is more preferable to use CML bound to a conventionally known carrier protein such as hemocyanin, bovine serum albumin, β-galactosidase or the like as an immunogen in preparing an antibody. For the binding of the lysine or lysine derivative to the carrier protein, the method usually used for the purpose is used without any limitation.
[0013]
The peptide or protein having an N-carboxymethyllysine residue of the present invention refers to a peptide or protein having an N-carboxymethyllysine residue. The peptide may be a natural peptide or a synthetic peptide, as long as it has lysine. Even if it is an oligopeptide having 2 to 9 amino acids, it is a polypeptide having 10 or more amino acids. May be. The protein may be a simple protein such as albumin, globulin, prolamin, or a complex protein such as a glycoprotein, a phosphoprotein, a lipoprotein, or a metal protein, as long as it has lysine. In order to prepare the CM product, the hydrogen of the amino group on the lysine side chain of the peptide or protein having lysine may be replaced with a carboxymethyl group. Known methods are used for the substitution without any limitation. For example, as in the reductive alkylation method described in “New Chemistry Experiment Course 1, Protein 4” (edited by the Biochemical Society of Japan, pp. 13-16, Tokyo Kagaku Dojin, published on March 20, 1991), glyoxylic acid is used. And a protein having lysine is dissolved in an aqueous solution such as a borate buffer or a phosphate buffer, and reacted at pH 7 to 10 in the presence of a hydride reducing agent such as sodium borohydride or sodium cyanoborohydride. Good. If the pH is lower than this, the hydride reducing agent becomes unstable, and if the pH is higher than this, the peptide or protein may be denatured.
[0014]
The antibody against the above-mentioned CM product used in the present invention means an antibody that specifically binds to a peptide or protein having an N-carboxymethyllysine residue. An antigen for producing the antibody, that is, an immunogen may be a peptide or protein having an N-carboxymethyllysine residue.
[0015]
Antibodies produced using such antigens are not particularly limited in their origin, and antiserum, ascites fluid, etc. obtained by immunizing host animals such as rabbits, goats, mice, and guinea pigs with the antigens can be used as they are. Alternatively, it can be purified as a conventionally known method by salting-out method, gel filtration method, ion exchange chromatography, affinity chromatography, electrophoresis and the like and used as a polyclonal antibody. Furthermore, a monoclonal antibody prepared from a hybridoma obtained by fusing antibody-producing cells such as mammalian spleen cells and lymph node cells sensitized with an antigen with myeloma cells can be used as such or by a salting-out method known in the art. , Gel filtration, ion exchange chromatography, affinity chromatography, electrophoresis and the like.
[0016]
The antibody thus obtained may use the antibody molecule itself as an anti-CM antibody, or the activity of an antibody such as Fab, Fab ', F (ab') 2 obtained by treating these antibodies with an enzyme. Fragments (portions containing the antigen recognition site of the antibody) may be used as anti-CM antibodies.
[0017]
The immunoreagent of the present invention refers to an antigen-antibody reaction that occurs when an anti-CM antibody or an antibody to a biological component or an antigen in a sample is carried on an insoluble carrier and is brought into contact with the antigen or the anti-CM antibody in the sample. It refers to a labeled immunoassay reagent that detects using luminescence, fluorescence, and the like, and an immunoagglutination reagent that detects using, for example, aggregation of an insoluble carrier.
[0018]
Examples of the qualitative reagent include a latex agglutinating reagent and a microtiter reagent, and examples of the quantitative reagent include a radioimmunoassay reagent, an enzyme immunoassay reagent, a fluorescent immunoassay reagent, and a latex quantitative reagent.
[0019]
The shape of the anti-CM antibody or the antibody to the biological component, or the shape of the insoluble carrier that carries the antigen in the sample may be appropriately selected depending on the purpose of use. For example, beads, test plates, spheres, disks Shape, tube shape, filter shape and the like. In addition, as the material thereof, those used as ordinary immunoassay carriers, for example, glass, polysaccharides or derivatives thereof, silica gel, porous ceramics, metal oxides, red blood cells, propylene, styrene, acrylamide, acrylonitrile, etc. Synthetic resins and those obtained by introducing a reactive functional group such as a sulfone group and an amino group into these by a known method are exemplified.
[0020]
As a method for immobilizing an anti-CM antibody or an antibody against a biological component on an insoluble carrier, or an antigen in a specimen, known methods such as a physical adsorption method, a covalent bonding method, an ionic bonding method, and a crosslinking method can be used without any limitation. .
[0021]
As the antibody carried on the insoluble carrier, an anti-CM antibody or an antibody against a biological component is used. Examples of the antibody against the biological component include an antibody against albumin, an antibody against hemoglobin, an antibody against γ-globulin, an antibody against low-density lipoprotein, an antibody against transferrin, and an antibody against erythrocyte membrane protein. As the antibody against such a biological component, an antibody against only one biological component may be used, or an antibody against two or more biological components may be used. When an antibody against a biological component is carried on an insoluble carrier, the antigen in the sample may be brought into contact with the anti-CM antibody. When the anti-CM antibody is carried on the insoluble carrier, the anti-CM antibody may be contacted again after bringing the antigen in the sample into contact.
[0022]
The basic operation of measuring CML or CM compound in a labeled immunoassay reagent can be performed according to a usual assay method, for example, a radioimmunoassay (RIA) method, an enzyme immunoassay (EIA) method, or the like. The operation, procedure, and the like in each of these assay methods are not particularly different from those generally employed, and can be in accordance with known non-competitive methods, competitive methods, sandwich methods, and the like.
[0023]
For example, an anti-CM antibody or an antibody to a biological component, or an antigen in a sample is added to an insoluble carrier at 0.01 to 1000 μg / cm. ² , And contact with 0.001 to 1000 μg of the antigen in the sample or the anti-CM antibody for measurement. When an anti-CM antibody or an antibody against a biological component is carried on an insoluble carrier, as described above, after the antigen in the sample is brought into contact, the anti-CM antibody is further brought into contact. The anti-CM antibody not supported on the insoluble carrier is preferably labeled with a labeling agent.
[0024]
Examples of the labeling agent include radioactive substances such as radioactive iodine and radioactive carbon, fluorescent substances such as fluorescein isothiocyanate and tetramethylrhodamine, and enzymes such as alkaline phosphatase and peroxidase. The antigen-antibody reaction product obtained by such a method is detected using colorimetry, fluorescence, luminescence and the like.
[0025]
The basic operation of measuring CML or a CM compound in an immunoagglutination reagent can be performed according to a usual assay method, for example, a hemagglutination method, a passive agglutination method, an immunoassay method, an immunoturbidimetry method, or the like. The operations, procedures, and the like in each of these assay methods can be based on those generally employed.
[0026]
For example, particles containing 0.001 to 100 mg of anti-CM antibody per gram of particulate insoluble carrier (hereinafter abbreviated as sensitizing particles) in an aqueous medium may be used in an amount of 0.001 to 15% by weight. And used as an active ingredient of an immunoreagent. The particle size of the insoluble carrier carrying the antibody is preferably an insoluble carrier having an average particle size of 0.05 to 10 μm from the viewpoint of the ease of aggregation after the antigen-antibody reaction and the ease of discrimination of aggregation. It is. The sensitized particles prepared by such a method are brought into contact with the antigen in the sample, and the degree of aggregation of the sensitized particles is measured. Conventional methods such as visual observation and optical measurement can be used for the degree of particle aggregation without any limitation.
[0027]
Utilizing the above various immunoassays, the immunological reagent for diabetes or diabetic complications of the present invention is useful as a diagnostic reagent for diabetes, diabetic complications, or a drug for evaluating the efficacy of drugs for treating diabetic complications. Used for
[0028]
When used as a diagnostic reagent, an anti-CM antibody is used to measure the antigen in the sample, that is, the ratio of CML or CM compound contained in a specific biological component. Examples of the biological component include blood, urine, lymph fluid, amniotic fluid, peritoneal fluid, body fluids such as saliva, skin collagen, lens protein, arteries, tissues such as kidneys, and the like. It is more preferable to use a bodily fluid.
[0029]
When used as a drug efficacy evaluation reagent, the degree of decrease in the ratio of CML or CM compound contained in a specific biological component by administration of a therapeutic agent for diabetes or a therapeutic agent for diabetes complications is measured.
[0030]
【The invention's effect】
Antibodies to CML or CM conjugate were obtained by the usual method of immunizing rabbits with human serum albumin containing CML or CM conjugate. This antibody reacted significantly with various glycated proteins produced in vitro, but did not react with the unmodified product. Furthermore, it was revealed that the antibody also reacted with human-derived hemoglobin, which was significantly stronger in diabetic patients and diabetic complication patients than in healthy subjects. Further, the antibody against CML or the CM compound and AGE showed immunological cross-reactivity. Various studies have shown that diabetic patients are exposed to higher oxygen stress than healthy subjects, and that CML or CM formed by saccharification and oxidative reactions may cause diabetes complications. It is highly useful as a new index for diagnosing progression.
[0031]
By using the immunoreagent of the present invention using an antibody against CML or a CM compound, it has become possible to easily diagnose diabetes and diabetic complications using body fluids and tissues as subjects. Further, the diagnostic reagent completed according to the present invention can be used for evaluating the efficacy of drugs for treating diabetes and complications for diabetes.
[0032]
【Example】
EXAMPLES Examples will be shown below for more specifically describing the present invention, but the present invention is not limited to these examples.
[0033]
Example 1
(1) Preparation of CM product
In order to prepare a CM product, the hydrogen of the amino group in the side chain of the protein was replaced with a carboxymethyl group (hereinafter, this operation is also referred to as carboxymethylation).
[0034]
1 ml of 1 mg / ml human serum albumin (Fraction V, manufactured by Sigma) adjusted to pH 9 and 1 ml of 0.25 M glyoxylic acid (manufactured by Sigma) containing 1 mg of sodium cyanoborohydride adjusted to pH 9 were mixed. At 0 ° C. for 1 hour. As a control, human serum albumin was treated in the same manner except that glyoxylic acid was not added.
[0035]
In order to confirm the replacement of the amino group hydrogen with a carboxymethyl group, the unreacted amino group of the prepared human serum albumin in which the hydrogen of the amino group was replaced with a carboxymethyl group (hereinafter abbreviated as substituted human serum albumin) was used. Quantification was performed using nitrobenzenesulfonic acid (hereinafter abbreviated as TNBS). In addition, amino groups of human serum albumin not treated with glyoxylic acid as a control were quantified. 0.5 ml of the sample was added to 0.5 ml of a 0.1 M aqueous sodium hydroxide solution containing 0.1 M sodium tetraborate. Next, 20 μl of 1.1 M TNBS which had been recrystallized and washed with diluted hydrochloric acid was added thereto, followed by stirring. Thirty minutes later, 2 ml of 98.5 mM sodium dihydrogen phosphate containing 1.5 mM sodium sulfite was added to stop the reaction, and the absorbance at 420 nm was measured. In addition, a reaction with TNBS was performed in the same manner except that the prepared substituted human serum albumin or human serum albumin not treated with glyoxylic acid was not included, and the absorbance was measured. It is 0.03, that of human serum albumin without glyoxylate treatment is 1.25, that of the sample without the substituted human serum albumin and human serum albumin without glyoxylate treatment is 0.03, The carboxymethylation rate of the substituted human serum albumin was 100%.
[0036]
Substituted human serum albumin produced by such a method is dialyzed against a 20 mM phosphate buffer (pH 7.4) at 4 ° C. for 2 days to remove unreacted glyoxylic acid and sodium cyanoborohydride, and then a CM product For the production of antibodies to
[0037]
(2) Preparation of antibodies against CM
Rabbits weighing 2 kg or more were immunized using the prepared CM compound as an antigen in the following manner.
[0038]
0.5 ml of the antigen solution adjusted to 2 mg / ml and 0.5 ml of Freund's complete adjuvant were added to the ear vein of the rabbit. Thereafter, every two weeks, 0.25 ml of the 2 mg / ml antigen and 0.25 ml of Freund's incomplete adjuvant were boosted. During this period, rabbits were partially bled once every two weeks from the peripheral ear vein in order to confirm whether antibodies against the CM compound were produced. Six weeks later, it was confirmed by an enzyme immunoassay (ELISA) method that an antibody against the CM compound was produced, and the whole blood was collected.
[0039]
(3) Preparation of affinity purification column
After washing 25 ml of Affigel 15 (manufactured by Bio-Rad) with 75 ml of 10 mM acetate buffer (pH 4.5), 62.5 ml of a 10 mg / ml human serum albumin solution was added, followed by gentle stirring at room temperature for 1 hour. Next, unreacted human serum albumin was removed by filtration, 30 ml of 1 M ethanolamine was added, and the mixture was gently stirred at room temperature to block unreacted N-hydroxysuccinimide ester. The support on which the human serum albumin was immobilized was packed in a column, and washed with ion-exchanged water until the absorbance at 280 nm became zero. Further, the column was equilibrated with a 20 mM phosphate buffer (pH 7.4) containing 0.15 M sodium chloride.
[0040]
(4) Affinity purification of antibody against CM compound
The prepared antibody to the CM product was diluted with a 20 mM phosphate buffer (pH 7.4) containing 0.15 M sodium chloride so as to have a concentration of 1 mg / ml. I applied. Then, the phosphate buffer was flowed at a flow rate of 0.5 ml / min until the absorbance at 280 nm became 0. The antibody that did not bind to the column was recovered as an antibody against the CM compound. When the absorbance at 280 nm became 0, the phosphate buffer was changed to 0.1 M glycine buffer (pH 3.0), and the antibody bound to the column was eluted. The column was equilibrated with a phosphate buffer (pH 7.4), the recovered antibody was applied to the column again, and the antibody not bound to the column was recovered. This operation was repeated once more, and the resultant was used as an antibody for biotin labeling.
[0041]
(5) Biotin labeling of antibody to CM compound
Biotin labeling of the purified antibody was performed using a protein biotilation system (manufactured by Gibco).
[0042]
A solution prepared by diluting or concentrating an antibody against the purified CM compound to a concentration of 1.5 mg / ml with a 20 mM phosphate buffer (pH 7.4) containing 0.15 M sodium chloride to a concentration of 0.05 M was added. Sodium carbonate buffer (pH 9.0) was added. Next, 26 μl of a 50 mg / ml CAB-NHS ester solution prepared according to the instructions was added to 6.7 ml of the antibody solution, and the mixture was gently stirred at room temperature for 1 hour, and ammonium chloride was added to a concentration of 0.11 M for reaction. Was stopped. Thereafter, the antibody solution was desalted using a column attached to this kit. Further, when the number of moles of the introduced biotin was calculated using Avidin / HABA included in the kit, 14 moles of biotin were bound to 1 mole of the antibody to the CM compound.
[0043]
(6) Antigen specificity of antibody to CM compound
The antigen specificity of the antibody to the biotin-labeled CM product was confirmed by competition ELISA.
[0044]
Each of the prepared CM products was added to an antibody against CM products diluted with 10 mM phosphate buffer (pH 7.4) (hereinafter abbreviated as PBS) containing 0.15 M sodium chloride so as to be 1 μg / ml. , 1, 10, 100 μg / ml. This solution was left at 37 ° C. for 1 hour, and used as an antibody solution inhibited by a CM compound.
[0045]
Carboxymethyl tosyl-lys (hereinafter abbreviated as CM-tosyl-lys) prepared from α-tosyl-lysine methyl ester (hereinafter abbreviated as tosyl-lys) (manufactured by Sigma) in the same manner as in the preparation of the CM compound. Prepared. The CM-tosyl-lys was added to a 1 μg / ml antibody solution against a CM compound at a concentration of 0.1, 1, 10, 100 μg / ml, respectively. This solution was left at 37 ° C. for 1 hour and used as an antibody solution inhibited by CM-tosyl-lys.
[0046]
In performing the competitive ELISA, the prepared CM product was diluted with PBS to 1 μg / ml. Next, the diluted CM compound solution was applied to a 96-well immunoplate (manufactured by NUNC) at 100 μl per well and left at 37 ° C. for 1 hour to fix the CM compound to the immunoplate. After 1 hour, the CM solution was removed, 100 μl of PBS containing 0.5% gelatin was applied per well, and the mixture was allowed to stand at 37 ° C. for 1 hour to block a portion where the CM product was not fixed. One hour later, the gelatin solution was removed, and the well was washed three times with PBS. Then, the antibody solution inhibited by the CM compound at the above concentration or the antibody solution inhibited by CM-tosyl-lys at the above concentration was added per well. 100 μl was applied and left at 37 ° C. for 1 hour. Thereafter, the plate was washed three times with PBS, 100 µl of an anti-rabbit IgG antibody solution (manufactured by Cosmo Bio) labeled with 1 µg / ml of alkaline phosphatase was applied per well, and left at 37 ° C for 1 hour. Further, the plate was washed three times with PBS, and 100 μl of a substrate solution prepared per well using an alkaline phosphatase substrate kit (manufactured by Bio-Rad) according to the instructions was applied. After standing at room temperature for 5 minutes, a 0.4 M sodium hydroxide solution was added at 100 μl per well to stop the alkaline phosphatase reaction, and the absorbance at 405 nm was measured. The results are shown in FIG. From these results, it can be seen that the prepared antibody against the CM compound shows reactivity not only with the CM compound but also with CM-tosyl-lys, since the antigen-antibody reaction of the CM compound and the antibody to the CM compound was inhibited. It was suggested.
[0047]
(7) Measurement of CM compounds in blood from diabetic patients
50 μl of blood collected from 10 diabetic patients without complications using a vacuum blood collection tube containing EDTA-2K, washed once with 250 μl of physiological saline, and lysed by adding 1 ml of purified water Was used as a subject. The average age of the patient was 61.3 years.
[0048]
The measurement of the CM compound in the subject was performed by the dot blotting method. After measuring the hemoglobin concentration by the cyanmethemoglobin method, the hemoglobin was adsorbed to a PVDF membrane (manufactured by Bio-Rad) using a dot blotting apparatus (manufactured by Bio-Rad) so that the hemoglobin became 500 ng. The membrane was immersed in a 20 mM phosphate buffer (pH 7.4) containing 0.2% Tween 20 for 1 hour at room temperature. The membrane was taken out, and 5 ml of the biotin-labeled antibody solution against 1 μg / ml CM was added. added. After 1 hour incubation at room temperature, the membrane was washed three times with 50 ml of 20 mM phosphate buffer (pH 7.4) containing 0.05% Tween20. Next, 5 ml of an avidin-peroxidase-labeled biotin complex solution (Vectastain ABC kit: manufactured by Funakoshi) was added to the membrane, and the mixture was incubated at room temperature for 1 hour. After washing the membrane three times with 50 ml of a 20 mM phosphate buffer (pH 7.4) containing 0.05% Tween 20, 2 ml of an ECL western blotting detection reagent (manufactured by Amersham) was added. The emission intensity of the film was detected using a Bio-Rad GS-363 molecular imager.
[0049]
A commercial hemoglobin (manufactured by Sigma) was used as a subject, and a CM compound was measured in the same manner as described above. With the luminescence intensity of the commercially available hemoglobin as 100, the luminescence intensity of hemoglobin derived from the diabetic patient was indicated. Table 1 shows the measurement results.
[0050]
[Table 1]

[0051]
Example 2 Measurement of CM Compound in Blood from Patients with Diabetic Complications
The method of Example 1 was repeated except that blood from a diabetic complication patient who had nephropathy or retinopathy in addition to diabetes was used instead of blood from the diabetic patient in Example 1. Was measured. The average age of the patient was 60.4 years. Table 2 shows the measurement results.
[0052]
[Table 2]

[0053]
Comparative Example 1
A CM compound in a subject was measured according to the method of Example 1 except that blood from a healthy subject without diabetes was used as the subject instead of blood from a diabetic patient in Example 1. The average age of the healthy subjects was 61.2 years. Table 3 shows the measurement results.
[0054]
[Table 3]

[0055]
It was verified that there was a statistical difference between the luminescence intensities of the healthy group and the diabetic patient group measured in Example 1. That is, the probability of “no difference” was calculated, and since the probability was very small, the procedure of “there was a difference” was taken and statistically verified (Macintosh Statistical Manual, Osamu Nagata, February 1995) Published on 10th, Shinko Trading Medical Book Publishing Department). For the verification, Welch's t-test was used. As a result, the probability of being considered “no difference” was less than 5%, and there was a significant difference between the luminescence intensity of the diabetic patient group and the luminescence intensity of the healthy subject group. This means that blood from a diabetic patient has significantly more CM compounds than blood from a healthy person.
[0056]
Further, when the difference between the luminescence intensities of the group of healthy subjects and the group of diabetic complications measured in Example 2 was verified by Welch's t-test, the probability of “no difference” was found. At less than 5%, there was a significant difference between the luminescence intensity of the diabetic complication patient group and the luminescence intensity of the healthy group. This means that blood from patients with diabetic complications has significantly more CM compounds than blood from healthy persons.
[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a diagram showing the results of examining the antigen specificity of an antibody against a produced CM product by a competition ELISA (the vertical axis indicates absorbance at 405 nm, and the horizontal axis indicates the amount of each inhibitor added).

Claims (1)

An immunological reagent for diabetes or diabetic complications using an antibody against hemoglobin having an N-carboxymethyllysine residue .

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