JP6553491B2 - Method for predicting morbidity or risk of diseases associated with final glycation products (AGEs) - Google Patents

Description

  The present invention relates to a method for predicting the onset or risk of diseases associated with final glycation products (AGEs), and more specifically, methylglyoxal-hydroimidazolone (MG-H1), which is one of the final glycation products (AGEs). Using a monoclonal anti-AGEs antibody that specifically binds to), and measuring MG-H1 in a biological sample collected from a subject to predict the morbidity or risk of diseases associated with advanced glycation end products (AGEs) Regarding the method.

  A non-enzymatic reaction between an amino group present in a protein and an aldehyde group of a reducing sugar such as glucose forms a glycated product. This reaction is referred to as a non-enzymatic saccharification reaction or Maillard reaction. Non-enzymatic saccharification occurs also in vivo. The non-enzymatic glycation reaction is considered to occur in two steps, the early reaction in which an Amadori rearrangement product is formed via a Schiff base and the late reaction in which complex cleavage, condensation, etc. occur, and in the late reaction, the final glycated product Various non-single substances called (= AGEs: Advanced Glycation End Products) are generated.

  About AGEs, the relationship with various symptoms, diseases, etc. accompanying diabetes and aging has been reported. For example, AGEs are related to the accumulation of AGEs in renal disorders and retinopathy in diabetes, and to various tissue disorders recognized with age, for example, hardening of connective tissues such as skin, blood vessel wall, joints, etc. In addition, it has been reported that skin thickening, wrinkle formation, arteriosclerosis, arthritis and the like can be caused, and that AGEs are involved in accumulation in brain of β-amyloid peptide in Alzheimer's disease. In addition, in the reduction of bone strength such as osteoporosis, it has been reported that glycation (AGEs formation) of collagen in bone is one of the causes. Furthermore, it has been suggested that AGEs are involved in cancer diseases such as their proliferation and metastasis.

In order to investigate the relationship between AGEs and various diseases, and to develop their prevention and treatment methods, a technique for detecting or quantifying specific AGEs focused on from various AGEs is required. Methods for detecting or quantifying AGEs include high performance liquid chromatography (HPLC), gas chromatography mass spectrometry (GC / MS), liquid chromatography mass spectrometry (LC-MS / MS), and specific AGEs. There is known a method using an anti-AGE antibody that is recognized by the human body. Among these, the method using an anti-AGE antibody is excellent in that it is not necessary to use a large and expensive device.
As a method using an anti-AGE antibody, for example, Patent Document 1 discloses an antibody specifically reacting with AGEs derived from 3,4-dioxyglucosone-3-ene (DGE) and AGEs using the same. A detection method is described, and Patent Document 2 describes a monoclonal antibody specific for AGEs derived from guanidino groups in a biological sample and a method for detecting AGEs using the same. Patent Document 3 describes an antibody against AGE derived from fructose and a method of measuring AGE derived from fructose using the same.

  By the way, MG-H1 is known as a type of AGEs, and the association of this MG-H1 with symptoms and diseases associated with diabetes and aging is also reported. For example, Patent Document 4 describes the use of levels of two or more biomarkers generated from plasma as an indicator of the risk or rate of developing diabetic nephropathy, eye disease, or cardiovascular complications. The use of MG-H1 as one of the biomarkers is described. Non-patent document 1 relates to methylglyoxal-hydroimidazolone in soluble human lens protein and measures the concentration of two structural isomers (MG-H1, MG-H2) and compares it with the concentrations of other AGEs. It is clarified that there is a relationship between MG-H1 or MG-H2 and aging or cataract.

However, regarding MG-H1, there are few documents that mention detection and quantification using an anti-AGE antibody, and they are not described in Patent Documents 1 to 3 described above. Patent Document 4 described above also describes that the level of a biomarker is determined by analyzing a biological sample obtained from an individual using liquid chromatography / quadrupole mass spectrometry (LC-MS / MS). On the other hand, analysis using an anti-AGE antibody is not described. Further, Non-Patent Document 1 does not describe that methylglyoxal (MG) imidazolone (MG-HI, MG-H2) is analyzed using an antibody that specifically recognizes them.
On the other hand, Non-Patent Document 2 discloses that an antibody obtained by immunizing a mouse with a protein modified with methylglyoxal (MG) includes an antibody whose epitope is imidazolone. However, Non-Patent Document 2 does not describe that imidazolone that is not bound to a protein is synthesized and then bound to the protein and used as an immunogen. Monoclonal antibodies with high binding ability to MG-H1 are not described.

  Patent Document 5 describes cardiovascular diseases such as arteriosclerosis, Alzheimer's disease, and the like in which glycated proteins formed by hyperglycemic state characteristic of diabetes are associated with advanced glycation end products (AGEs). It has been described that it can be an important cause of complications such as proliferation and metastasis, inflammatory reaction, and such glycated proteins can be detected using the binding activity of lectins called galectins to sugar chains. Have been described. However, as a disease associated with final glycation products (AGEs), in the prediction of osteoporosis, anemia, arthritis, cataract, hypertension, gastroesophageal reflux disease and hyperlipidemia or the risk thereof, as final glycation products (AGEs) MG-H1 is not known to be a useful biomarker.

JP 2006-31621 A Special Table 2002-517224 JP 2004-323515 A JP2013-257328A JP 2012-225762 A

Naila Abmed et al., "Methylglyoxal-derived hydroimidazole advanced glycation end-products of human lens proteins.", Invest Ophthalmol Vis Sci, 2003; Vol. 44, No. 12: P. 5287-5292 B.K.kilhovd et al., "Increased serum levels of the specific AGE-compound methylglyoxal-derived hydroimidazolone in patients with type 2 diabetes.", Metabolism, 2003; Vol.52, No2: P.163-167

  An object of the present invention is to provide a method useful for predicting the morbidity or risk of a disease associated with final glycation end products (AGEs).

  As a result of intensive studies to solve the above problems, the inventor of the present invention measured MG-H1 in a biological sample collected from a subject using a monoclonal antibody having high specificity and binding ability to MG-H1, Among the diseases related to the final glycation products (AGEs), the present inventors have found that it is useful for predicting the incidence or risk of osteoporosis, anemia, arthritis, cataract, hypertension, gastroesophageal reflux disease and hyperlipidemia.

The present invention has been made on the basis of the above findings, and a method for predicting the onset or risk of a disease associated with the following final glycation products (AGEs) of (1) to (6), and (7) final glycation Methods for providing data to predict the onset or risk of diseases associated with products (AGEs), and (8) Kits used in these methods.
(1) A method for predicting morbidity or risk of diseases associated with final glycation products (AGEs), which is specific to methylglyoxal-hydroimidazolone (MG-H1) in biological samples collected from subjects One wherein the disease is selected from osteoporosis, anemia, arthritis, cataract, hypertension, gastroesophageal reflux disease and hyperlipidemia, comprising measuring using a monoclonal anti-MG-H1 antibody that binds selectively And a method characterized by the above-mentioned diseases.
(2) The method according to (1), wherein the measurement method is an ELISA method.
(3) The amount of MG-H1 in a biological sample collected from a subject is compared with the amount of MG-H1 in a healthy subject's biological sample. The method according to (1) or (2).
(4) The method according to any one of (1) to (3), wherein the biological sample is serum or plasma.
(5) Monoclonal anti-MG-H1 antibody was obtained by synthesizing free MG-H1 not bound to protein and immunizing an animal with the adduct obtained by binding it to protein as an antigen. The method according to any one of (1) to (4), wherein the carboxymethylarginine and carboxymethyllysine, which are known AGEs, have no cross-reactivity.
(6) The monoclonal anti-MG-H1 antibody is an antibody produced by a hybridoma deposited as NITE P-1898 (OYC111), NITE P-1899 (OYC112) or NITE P-2064 (OYC113). Method described.
(7) The amount of MG-H1 in a biological sample collected from a subject is measured using a monoclonal anti-MG-H1 antibody that specifically binds to this, and the obtained measurement value and the biological sample of a healthy person A method of providing data for predicting the onset or risk of diseases associated with final glycation products (AGEs), including determining whether it is significantly higher or lower compared to the value of MG-H1 in .
(8) A kit for use in the method according to any one of (1) to (7), which comprises a monoclonal anti-MG-H1 antibody that specifically binds to MG-H1.

  The method for predicting the morbidity or risk of a disease associated with the final glycated products (AGEs) of the present invention is a method for determining the final glycated products (AGEs) by measuring MG-H1 in a biological sample such as blood collected from a subject Among diseases related to osteoporosis, it is very useful because osteoporosis, anemia, arthritis, cataract, hypertension, gastroesophageal reflux disease and hyperlipidemia can be easily and rapidly predicted. . In addition, the value measured using the monoclonal anti-MG-H1 antibody is significantly higher than the measured value of MG-H1 in the obtained biological sample and the value of MG-H1 in the biological sample of a healthy subject. By determining if it is low or not, data is provided to predict the morbidity or risk of diseases associated with advanced glycation end products (AGEs).

It is a graph of the amount of MG-H1 in the healthy person and each disease patient serum by competitive ELISA method using a monoclonal anti-MG-H1 antibody.

Hereinafter, the present invention will be described based on preferred embodiments (modes).
The monoclonal anti-AGE antibody used in the method for predicting the morbidity or risk of diseases associated with the final glycation products (AGEs) of the present invention is an antibody against the final glycation products (AGEs) and specific to MG-H1 To be bound. In the present invention, “MG-H1” is a non-free product or a free product produced by the reaction of methylglyoxal (MG), which is a precursor of AGEs, with non-free arginine or free arginine such as arginine residues in proteins. MG-H1 of the body, and “specifically binds to MG-H1” means that it reacts by recognizing at least non-free MG-H1 while it is AGE other than MG-H1 Any of carboxymethylarginine (CMA: Nω- (carboxymethyl) arginine), carboxymethyllysine (CML: Nε- (carboxyethyl) lysine) and carboxymethyllysine (CEL) It means no reaction or any reactivity to them is significantly lower than the reactivity to non-free MG-H1.

  The monoclonal anti-AGEs antibody used in the method of the present invention is preferably an antibody having the ability to bind MG-H1 in free form not bound to a protein. An antibody having the binding ability to free MG-H1 can be used, for example, in competitive ELISA. In a competitive inhibition assay, for example, non-free MG-H1 bound protein is immobilized in a well (well) of a microplate, and the binding of the antibody to MG-H1 coexists with the free MG-H1. If it is inhibited, it is judged to have the ability to bind MG-H1 in free form, and when it is not inhibited, it has no ability to bind MG-H1.

  The free form MG-H1 which is not bound to a protein means MG-H1 which is not bound to a protein including a peptide, and is a methylglyoxal (MG) modification of arginine having no peptide bond. That is, the monoclonal anti-AGEs antibody of the present invention preferably has not only the reactivity of MG-H1 bound to the protein but also the reactivity of MG-H1 of the free form not bound to either peptide or protein. .

  The monoclonal anti-AGEs antibody used in the method of the present invention is an immunogen obtained by synthesizing the free MG-H1 which is not bound to a protein and then binding the synthesized MG-H1 to a protein as an immunogen. It is obtained by immunizing an animal. The monoclonal anti-AGEs antibody of the present invention, when prepared in this manner, has a specificity for MG-H1 and / or MG-H1 as compared to when the same animal is immunized with an immunogen obtained by reacting a protein with methylglyoxal. The binding capacity of the free form to MG-H1 is high.

Examples of monoclonal anti-AGE antibodies used in the method of the present invention include antibodies 1 to 3 produced by the following hybridomas. The hybridoma producing antibodies 1 and 2 was deposited on July 15, 2014, and the hybridoma producing antibody 3 was deposited with the Patent Organism Depositary, National Institute of Technology and Evaluation on June 5, 2015. The respective accession numbers are as follows.
Hybridoma producing antibody 1 (OYC111): accession number NITE P-1898
Hybridoma producing antibody 2 (OYC112): accession number NITE P-1899
Hybridoma producing antibody 3 (OYC113): accession number NITE P-2064

Hereinafter, a method for producing a monoclonal anti-AGE antibody suitable for the method of the present invention will be described.
A preferred method for producing the monoclonal anti-AGE antibody comprises the following steps (1) to (3) and the following step (4) which is a conventional method for producing monoclonal antibodies.
(1) Step of synthesizing free MG-H1 not bound to protein (2) Step of binding synthesized MG-H1 to protein using crosslinker (3) Protein bound to MG-H1 Step of immunizing animal as antigen (4) Step of producing a hybridoma that produces monoclonal anti-AGE antibody using antibody-producing cells of the immunized animal

Hereinafter, steps (1) to (4) will be described.
[Step (1): Preparation of free MG-H1]
The step (1) is a step of preparing a free MG-H1, preferably a mixture of methylglyoxal and arginine, and a free MG represented by the following formula (1) by a non-enzymatic saccharification reaction. -H1 is generated.

  More specifically, by mixing methylglyoxal and arginine, adjusting the pH to 10 or more with an alkali such as sodium hydroxide, and incubating it at a temperature of 37 ° C. for 1 hour, the above-mentioned free form is obtained. MG-H1 can be produced.

Methylglyoxal-derived hydroimidazolone obtained by reacting methylglyoxal (MG) with arginine has the following three structural isomers. MG-H1 can be used in a state where other structural isomers are mixed, but it is preferable to use MG-H1 alone from the viewpoint of improvement in production efficiency and specificity of free form to MG-H1. The structure of MG-H1 can be confirmed by, for example, a liquid chromatograph mass spectrometer (LC-MS).
MG-H1: Nδ- (5-hydro-5-methyl-4-imidazol-2-yl) -ornithine
MG-H2: 2-amino-5- (2-amino-5-hydro-5-methyl-4-imidazolon-1-yl) heptanoic acid
MG-H3: 2-amino-5- (2-amino-4-hydro-4-methyl-5-imidazolon-1-yl) heptanoic acid

The synthesized free MG-H1 is purified by an appropriate means.
It is preferable to increase the purity of the synthesized free MG-H1 from the viewpoint of improving the specificity of the obtained monoclonal antibody for MG-H1 and / or the binding ability of the free antibody to MG-H1. Various known methods can be employed as the purification method, and for example, liquid chromatography is preferably used. Examples of liquid chromatography include ion exchange chromatography, adsorption chromatography, partition chromatography, molecular exclusion chromatography, affinity chromatography and the like, and these may be carried out singly or in two or more species. The above may be sequentially performed.
The synthesized free MG-H1 is preferably purified to 99% by mass or more, more preferably 99.9% by mass, and used in the next step. In the present invention, MG-H1 may be a salt such as sodium salt or hydrochloride, or may not be a salt.

[Step (2): Antigen preparation step]
The step (2) is a step of preparing an immunogen, and by itself, free MG-H1 having low immunogenicity (immunity induction ability) is bound to a protein to give an antigen having sufficient immunogenicity. As a protein to which MG-H1 is bound, various known proteins can be used without particular limitation, and examples thereof include albumin such as serum albumin, hemocyanin, myoglobin, and the like. Proteins derived from mammals such as cows, rabbits and humans, shells such as squash shells, birds such as chickens, and the like can be used. Among these, squash hemocyanin (KLH) and bovine serum albumin (BSA) are preferable. These proteins can be used singly or in combination of two or more.
A crosslinker is preferably used for binding of MG-H1 to the carrier protein. As the crosslinker, various known ones can be used. For example, glutaraldehyde, EDC (1-ethy-3- (3-dimethylaminopropyl) carbodiimide hydrochloride), SPDP, DST, DSG and the like can be used. Among these, it is preferable to use glutaraldehyde or EDC. A crosslinker can also be used individually by 1 type or in combination of 2 or more types.

[Step (3): Animal Immunization Step]
The step (3) is a step of immunizing an animal. An animal other than a human is immunized with the protein (adduct) bound to MG-H1 prepared as described above as an antigen, and an antibody is injected into the animal body. Produce producer cells. This step (3) can be performed according to a conventional method for producing a monoclonal antibody, except that a protein (adduct) bound with MG-H1 prepared as described above is used as an antigen.
Although the kind of animal is not specifically limited, A mammal is preferable. Examples of mammals include rodents such as mice, rats, and rabbits. As the mouse, it is preferable to use a BALB / C strain mouse because a cell line derived from myeloma used for producing a hybridoma has been established. Immunization can be performed by various known methods, for example, injection subcutaneously, intracutaneously, intravenously or intraperitoneally in a mammal. Immunization is preferably repeated several times after the initial immunization, and the immunization schedule can be appropriately determined according to the type and strain of the animal to be immunized. In order to enhance the immune response, it is preferable to administer the protein to which MG-H1 has been bound mixed with an adjuvant before or at the time of administration. As the adjuvant, various known ones can be used. In addition, for example, complete Freund's adjuvant (CFA) can be used for the first immunization, and incomplete Freund's adjuvant (IFA) can be used for the second and subsequent times.

[Step (4): Production Step of Hybridoma Producing Monoclonal Anti-AGEs Antibody]
In the step (4), antibody producing cells of the immunized animal are used to produce a hybridoma capable of producing a monoclonal anti-AGE antibody.
The immunized animals are bled at appropriate intervals to confirm that antibodies against MG-H1 are produced. For confirmation of antibody production, known analysis methods such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence immunoassay can be used. After confirmation of antibody production, it is also preferable to perform a boost (boost injection of immunogen). After the final immunization, spleen cells are removed from the immunized animal and fused with cells derived from myeloma. As a method for cell fusion, for example, a known method such as a polyethylene glycol method, a method using Sendai virus, or a method of applying electrical stimulation can be used. The fused cells can be selected using a HAT medium containing hypoxanthine, aminopterin, thymidine, or the like. In addition, from the obtained fusion cells, the ability to produce an antibody having binding ability to MG-H1 can be obtained by known analysis methods such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence immunoassay. Select highly fused cells. Then, using the selected cells, cloning is performed by a known method such as a limiting dilution method or a soft agar method. In this way, a hybridoma that produces a monoclonal anti-AGE antibody having high specificity for MG-H1 and high binding ability to MG-H1 in free form is obtained.

  The hybridoma obtained as described above is cultured and proliferated, and the monoclonal anti-AGE antibody of the present invention is obtained in a large amount by separating and purifying the monoclonal anti-AGE antibody from the culture solution or the like. As a method for culturing hybridomas, various known methods such as a method of injecting into abdominal cavity of a mammal and growing in ascites fluid and a method of culturing using an appropriate medium outside the animal can be adopted. For purification of antibodies obtained from ascites fluid, culture fluid or culture supernatant, known methods such as ion exchange chromatography, affinity chromatography and the like can be used without particular limitation.

  The monoclonal anti-AGEs antibody used in the method of the present invention includes not only the antibody thus obtained but also fragments of the antibody containing the recognition site of MG-H1. In addition, it may be a chimeric antibody or a humanized antibody produced using genetic recombination technology so as to contain the recognition site of MG-H1.

  Since the monoclonal anti-AGEs antibody thus obtained is excellent in the specificity for MG-H1 and / or the binding ability of the free form to MG-H1, it is highly sensitive or sensitive to MG-H1 in various biological samples. It can be measured and / or quantified with high accuracy.

  The method for predicting the morbidity or risk of a disease associated with the final glycation products (AGEs) of the present invention is to measure MG-H1 in a biological sample collected from a subject using the monoclonal anti-MG-H1 antibody described above. And / or quantify. As a method for measuring and / or quantifying MG-H1 in a biological sample collected from a subject using a monoclonal anti-AGE antibody, various known measurement and / or quantification methods using a monoclonal antibody can be employed without particular limitation, Examples include enzyme immunoassay (ELISA), radioimmunoassay (RIA), luminescence immunoassay, precipitation, agglutination, western blot analysis, flow cytometry, etc., with ELISA being preferred. The ELISA method may be either a competitive inhibition ELISA method or a competitive binding ELISA method. What is necessary is just to select suitably the secondary antibody, label | marker, etc. which are used in ELISA method according to a conventional method.

  In the present invention, examples of biological samples collected from recipients include all cells, tissues, and body fluids collected from living organisms, and examples include skin, muscle, bone, adipose tissue, cerebral nervous system, heart, and the like. Circulatory system such as blood vessels, lung, liver, spleen, pancreas, kidney, digestive system, thymus, lymph, blood, whole blood, serum, plasma, lymph, saliva, urine, ascites, sputum, etc., and culture media thereof Among them, serum or plasma is preferable for reasons such as ease of collection. As the biological sample, one subjected to any treatment such as dilution with a liquid such as physiological saline or water, filtration treatment, homogenate treatment, chemical treatment or the like is used as necessary.

In the present invention, the disease associated with final glycation products (AGEs) is one or more selected from osteoporosis, arthritis, other bone-related diseases, anemia, cataract, hypertension, gastroesophageal reflux disease and hyperlipidemia. It is a disease.
Other bone-related diseases other than osteoporosis and arthritis include, for example, osteopenia, juvenile osteoporosis, osteogenesis imperfecta, hypercalcimemia, hyperparathyroidism, osteomalacia, osteocalcification loss , Osteolytic bone disease, osteonecrosis, Paget's disease, rheumatoid arthritis, bone loss due to osteoarthritis, inflammatory arthritis, osteomyelitis, glucocorticoid treatment, metastatic bone disease, periodontal bone loss, Bone loss due to cancer, bone loss due to aging, and other bone loss.
Examples of anemia include iron deficiency anemia, aplastic anemia, ironblastic anemia, hemolytic anemia, megaloblastic anemia, renal anemia, thalassemia and the like. Cataracts include age-related cataracts, atopic cataracts, concomitant cataracts, diabetic cataracts and the like. For hypertension, primary hypertension, secondary hypertension (e.g. aortic constriction, renovascular hypertension, renal parenchymal hypertension, primary aldosteronism, Cushing syndrome, drug-induced hypertension, brainstem vascular compression, hypothyroidism) Disease).
Examples of gastroesophageal reflux disease include non-erosive gastroesophageal reflux disease, reflux esophagitis, Barrett's esophagus, etc. Hyperlipidemia includes hypercholesterolemia, hypertriglyceridemia, familial dyslipidemia, etc. Can be mentioned.

  In the method of the present invention, the amount of MG-H1 in the biological sample collected from the subject is significantly higher than the amount of MG-H1 in the biological sample of a healthy subject by the above-mentioned monoclonal anti-MG-H1 antibody and measurement method. Or if it is low, predict that you have or will be at risk for the disease. Specifically, when the amount of MG-H1 in the subject's biological sample is significantly higher than the amount of MG-H1 in the healthy subject's biological sample, osteoporosis, anemia, arthritis, cataract and hypertension It is judged that the at least one disease of or is at high risk. On the other hand, at least one disease of gastroesophageal reflux disease and hyperlipidemia in which the amount of MG-H1 in the biological sample of the subject is significantly lower than the amount of MG-H1 in the biological sample of a healthy person It is judged that the patient is affected or has a high risk.

  Furthermore, the present invention measures the amount of MG-H1 in a biological sample collected from a subject using a monoclonal anti-MG-H1 antibody that specifically binds to it, and the obtained measurement value and a healthy person The data are used to predict the morbidity or risk of diseases associated with final glycation end products (AGEs), including determining whether they are significantly higher or lower than the value of MG-H1 in biological samples of It is also possible to use the method provided.

  The kit of the present invention is a method for predicting the morbidity or risk of a disease associated with the above-mentioned advanced glycation end products (AGEs), and data for predicting morbidity or risk of a morbidity associated with final glycation end products (AGEs) And a kit comprising a monoclonal anti-MG-H1 antibody that specifically binds to MG-H1 for use in the method of The kit of the present invention preferably comprises at least a monoclonal anti-AGE antibody and a reagent for detecting an antibody bound to MG-H1.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not restrict | limited at all by these Examples.

[Preparation of free MG-H1]
(1) Free MG-H1 was synthesized by the following method.
Mix 49 mL of 1N NaOH and 862 μl of 40% Methyl glyoxal (5.8 M), then mix 871 mg of L-Arginine (MW = 174.2) with 100 mM (final) L-Argine and 100 mM (final) Methylglyoxal. 50 mL of the mixture was prepared. The mixture was incubated at 37 ° C. for 1 hour with shaking and then neutralized with HCl. After that, it was immediately concentrated by an evaporator, redissolved in water and freeze-dried.
(2) Purification of Synthesized MG-H1 The synthesized free form of MG-H1 was purified in two steps by the following method.
Purification 1 (purification by Dowex50 column)
The first purification of the synthesized MG-H1 sample was performed by ion exchange chromatography using a column packed with a cation exchange resin. The column was washed with a resin-filled column (inner diameter 20 mm, resin-filled portion height 20 cm) with 10% pyridine (prepared with ultrapure water), about 5 times the amount of the column, and then 5 times the amount of ion. The column was washed with exchange water, then with column 5 volume of 10% HCl, and then column 5 volume ion exchange water was used.
The synthesized MG-H1 sample was dissolved in a small amount of ultrapure water and applied to the column, and 3 times the amount of ultrapure water was poured. Next, elution was carried out with 10% pyridine, and about 3 ml each was collected. Each fraction was spotted with a capillary 10 times on filter paper and detected with a 0.5% ninhydrin solution. The presence of MG-H1 in the fraction developed with ninhydrin was confirmed by TLC. The TLC was spotted on a silica gel plate, and a pure product was spotted, developed with a developing solvent (chloroform: MeOH: water = 2: 3: 1), dried with a drier, and developed with a ninhydrin reagent. The colored part was collected, evaporated with pyridine by an evaporator, frozen at -80 ° C and lyophilized.
The ion-exchange resin, Dowex ^TM 50 using (Dow Chemical Company), as the ultra pure water using milliQ water. MilliQ water is ultrapure water manufactured by Millipore's ultrapure water production system MilliQ.

Purification 2 (Purification by silica gel open column)
The MG-H1 sample after purification by purification 1 was purified a second time by liquid chromatography using a silica gel column.
The silica gel column is packed with loose cotton padded at the bottom of a glass column with an inner diameter of 20 mm and a length of 50 cm, and then silica gel (“silica gel 60” manufactured by Kanto Chemical Co., Inc.) is injected from the top using a funnel and lightly applied to the floor several times. Prepared by applying air to remove air. The height of the silica gel packed portion was 44 cm.
The silica gel column is loaded with the MG-H1 sample after freeze-drying (after the first purification) dissolved in a small amount (about 1 mL) of ultrapure water, and the container is further washed with 0.5 ml of ultrapure water. I also loaded water. Then, chloroform: MeOH: water = 2: 3: 1 was injected to start elution, and the eluate was collected. The liquid of each fraction was spotted on paper and developed with 0.5% ninhydrin. The presence of MG-H1 in the fraction developed with ninhydrin was confirmed by TLC. In addition, TLC was spotted on a silica gel plate to spot a pure product, developed with a developing solvent (chloroform: MeOH: water = 2: 3: 1), dried with a drier, and developed with a ninhydrin reagent. The fractions in which MG-H1 was identified were collected, concentrated by an evaporator, frozen at -80 ° C and lyophilized. Also in purification 2, milliQ water was used as ultrapure water.

(3) Confirmation of structure The MG-H1 sample purified by purification 1 and purification 2 was analyzed by liquid chromatography / quadrupole mass spectrometry (LC-MS / MS). The conditions are as follows.
Sample injection volume: 10 μl of 1 μg / ml
LC section: Accela Pump + Thermo PAL (Thermo Scientific)
Column: ZIC (R) -HILIC 150 x 2.1 mm, 5 μm (Merck Millipore)
Mobile phase: Gradient with 0.1% aqueous formic acid solution and 0.1% formic acid acetonitrile Flow rate: 200 μl / min
MS part: Triple quadrupole mass spectrometer for high sensitivity quantitative analysis TSQ Vantage (Thermo Scientific)
Ionization method: Positive ESI
Spray voltage: 3500 V
Vaporizer temperature: 200 ° C
Ion transfer tube temperature: 300 ° C
Collision gas, pressure: Argon, 1.2 mTorr
As a result of the above analysis, it was confirmed that the isolated compound was MG-H1.

[Preparation of Antigen]
In order to prepare an antigen (immunogen), MG-H1 binding KLH was prepared by binding the high purity MG-H1 prepared as described above with mussel hemocyanin (KLH) used as a carrier protein. . As a binding method, the EDC method using the EDC described above as a crosslinker was used. The specific method is as follows.
(Preparation of MG-H1 binding KLH)
The following components were mixed sequentially from the above components by the following amounts to prepare a total of 1000 μL of mixed solution.
KLH (10mg / mL) 300μL
PBS 445 μL
MG-H1 (1mg / 100μL) 150μL (1.5mg / 150μL)
NHS (5.4 mg / 50 μL) 5 μL
EDC (100mg / 100μL) 100μL
Details of each component are shown below.
KLH: SIGMA KLH
PBS: Phosphate buffered saline (pH 7.2)
MG-H1: MG-H1 of the synthesized free form (purity 99% or more)
NHS: N-hydroxysuccinimide (condensing agent)
EDC: 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide "PIERCE"
The mixture prepared as described above was incubated with shaking for 1 hour, and then 3 μL of 2-Mercaptoethanol was added to inactivate unreacted NHS. Then, it was dialyzed overnight with 2 L of PBS.
After dialysis, the protein concentration was measured with a BCA protein determination kit (PIERCE (registered trademark) Thermo Fisher Scientific Co., Ltd.).

(Preparation of MG-H1 binding BSA)
MG-H1-bound BSA (MG-BSA) was prepared in the same manner as MG-H1-bound KLH except that bovine serum albumin (BSA) (10 mg / mL) was used instead of KLH (10 mg / mL) .

[Immunization and production of hybridoma by subcutaneous immunization method]
Prepare MG-H1-conjugated KLH prepared as described above and an emulsion each mixed with an adjuvant, and prepare 50-100 μg / mouse / back of 6-8 week old female BALB / c mice (Charles River Japan) in the back. I was immunized at times. The immunization interval was performed once every two weeks for a total of 5 times. As an adjuvant, complete Freund's adjuvant (CFA) was used for the first time, and incomplete Freund's adjuvant (IFA) was used for the second and subsequent times. Blood was collected in the week following the third and fourth immunizations, and a titer check was performed. The titer check was performed using MG-H1 binding BSA as a solid phase. As the final immunization, an antigen diluted with phosphate buffered saline (PBS) (-) was intraperitoneally administered.
Spleen cells removed from the mouse 3 to 4 days after the final immunization were mixed with myeloma cells and cell fusion was performed by the PEG method. RPMI 1640 + 10% FBS + hypoxanthine-aminopterin-thymidine (HAT) + hybridoma seeded in 96-well plate at 0.5-1.0 × 10 ⁵ cells / well as spleen cells
The cells were cultured in cloning supplement (HCS).
About 2 weeks after cell fusion, selective culture was performed in HAT medium. After selecting the fused cells, a part of the culture supernatant was sampled from each well and screened by ELISA in which MG-H1-binding BSA was solid-phased at 1 μg / well. As a secondary screening, ELISA using a solid phase of methylglyoxal-modified BSA was performed, and positive wells were confirmed for each of the immunizing antigen and methylglyoxal-modified BSA.
Cloning was performed by limiting dilution using 10 wells of cells selected based on the results of ELISA. Ten wells forming positive single colonies were selected and finally narrowed to three clones in consideration of their growth potential. Conditioned culture was performed so as to grow in the conditioned medium (RPMI1640 + 10% FBS) from the various additive-containing cultures used in the establishment process.

[Production of monoclonal anti-AGEs antibody by mouse peritoneal method]
The cloned and conditioned hybridoma was cultured in a conditioned medium, and cultured for 2 to 3 passages in a 10 cm dish so that the viable cell ratio increased. While confirming the state of living cells, the culture scale was increased, and the required amount (2 × 10 ⁷ cells) of the logarithmic growth phase hybridoma was secured on the day of transplantation. Hybridomas were recovered from the hybridoma suspension by centrifugation (1000 rpm, 5 min). After washing the cells with PBS (−), the number of cells was measured and adjusted to 4 × 10 ⁶ cells / ml with RPMI 1640 (FBS free). 0.5 ml / animal (2 × 10 ⁶ cells / animal) was implanted into 10 BALB / c male mice intraperitoneally. The degree of ascites fluid was confirmed, and ascites was collected using an 18 G injection needle. The collected ascites fluid was centrifuged (3000 rpm, 5 min) to collect ascites supernatant.
Ascites fluid was diluted 3-fold with equilibration / adsorption buffer (PBS (-), pH 7.4). The protein A column was then washed with 5-10 bed vol equilibration and adsorption buffer and diluted ascites was applied. Thereafter, the protein A column was washed again with 5-10 bed vol equilibration / adsorption buffer. Next, elute the adsorbed protein with 3-5 bed vol elution buffer (100 mM citrate buffer, pH 3.0), collect A280 = 0.1 or more, and neutralize with 2/10 Tris-HCl, pH 9.0. ˜1 / 20 times was added, and the eluted fraction was rapidly adjusted to pH 7.0 to 7.5. The neutralized elution fraction was dialyzed against the final buffer (50 mM Tris-HCl, pH 8.0, 0.15 M NaCl), and then sterilized by filtration through a 0.22 μm filter.
In this way, monoclonal anti-AGEs antibodies that specifically bind to MG-H1 were obtained.

[Measurement of MG-H1 in patient serum by competitive ELISA]
Serum MG-H1 levels were measured in healthy subjects and patients with various diseases.
1. Human sera used Human sera of healthy subjects and patients with 8 diseases (osteoporosis, anemia, arthritis, cataract, gastroesophageal reflux disease, hypertension, glaucoma, hyperlipidemia) were obtained from PROMEDDX. The average age of healthy individuals and patients with each disease is as follows. Healthy person 75.0 years old, osteoporosis 75.5 years old, anemia 72.4 years old, arthritis 76.8 years old, cataract 84.7 years old, gastroesophageal reflux disease 76.0 years old, hypertension 80.5 years old, glaucoma 84 .0 years old, hyperlipidemia 72.0 years old. The number of human serum samples from healthy and diseased patients is as follows. Healthy 10, osteoporosis 10, anemia 7, arthritis 7, cataract 3, gastroesophageal reflux 3, glaucoma 2, hypertension 6, hyperlipidemia 2

2. Method
[Preparation of MG-BSA to be coated on ELISA plate]
(1) Two 50 mL tubes were taken, and Methylglyoxal (MG) and BSA were prepared in 1 × PBS to 5.8 M and 4 mg / mL, respectively.
(2) MG was diluted to 2 mM and BSA to 2 mg / mL with 1 × PBS.
(3) 5 mM each of 2 mM MG and 2 mg / mL BSA were added to a 15 mL tube (1 mM (1 mg / mL) MG-BSA) and incubated at 37 ° C. for 12 hours.
(4) Dialyzed against 1 L of 1 × PBS for 12 hours or more.
(5) The solution after dialysis was collected and passed through a 0.45 μm filter.
(6) The amount of MG-BSA protein recovered by the BCA method was measured.

[Measurement of MG-H1 in patient serum by competitive ELISA]
(1) 1 mM (1 mg / mL) MG-BSA solution incubated for 12 hours with 1 × PBS
After preparing 0.5 μg / mL MG-BSA, 100 μL / well was added to a 96-well ELISA plate, covered with a wrap, and allowed to stand at room temperature for 2 hours.
(2) The liquid in the ELISA plate was removed, the washing liquid (PBS + 0.5% Tween 20) was added at 200 μL / well, and the liquid was removed as it was three times.
(3) 0.5% Gelatin (a solution obtained by adding Gelatin to PBS to 0.5%) was added at 200 μL / well and allowed to stand at room temperature for 1 hour for blocking.
(4) The above operation (2) was performed.
(5) In order to prepare a calibration curve, 2 mM MG-H1 was first prepared with a washing solution, diluted to 1 mM, and then diluted three times in order to prepare 12 steps.
(6) Serum was diluted 4-fold with a washing solution and stirred sufficiently, and then added to a predetermined well at 50 μL / well together with MG-H1 prepared in (6) above.
(7) Anti-MG-H1 antibody diluted to 2 μg / mL with a washing solution was added to each well at 50 μL, followed by reaction at room temperature for 1 hour.
(8) The above operation (2) was performed.
(9) After adding 100 μL / well of Goat anti-Mouse IgG (γ) -HRP antibody prepared to 0.1 μg / mL in the washing solution, the mixture was reacted at room temperature for 1 hour.
(10) The above operation (2) was performed.
(11) 100 μL / well of TMB reagent was added at 5 second intervals per plate, and after confirming color development, 2 N sulfuric acid was added at 100 μL / well at 5 second intervals to stop the reaction.
(12) The absorbance was measured with a microplate reader FLUO star OPTIMA (BMG LABTECH) at an absorbance of 450 nm and a reference of 595 nm.

3. Reagents and equipment used are as follows: Methylglyoxal solution, 40%: Sigma, M0252-25mL
・ BSA: Sigma, A-7030
Dialysis tube: Adias, UC24-32-100 Cellulose tube 24/32
・ 0.45 μm filter: Commercially available 0.45 μm filter ・ BCA reagent: Pierce, 23225 BCA Protein Assay Reagent
96-well ELISA plate: NUNC MAXISORP (NUNC 439454)
・ PBS: 10 × PBS prepared with commercially available reagents, diluted with ultrapure water ・ Tween 20: Wako Pure Chemical Industries, Ltd., Polyoxyethylene (20) sorbitan Monolaurate
・ Gelatin: Nacalai Tesque, Gelatin purified powder 16631-92
MG-H1: MG-H1 of the above-mentioned free form that has been synthesized by the method described above and then purified in two steps
・ Primary antibody (anti-MG-H1 antibody): Antibody 3 (5.2 mg / mL)
Secondary antibody: KPL, 214-1802 Goat Anti-Mouse IgG (gamma) Antibody, F (ab ') 2 fragment, Human Serum Adsorbed and Peroxidase labeled
・ Sulfuric acid: Nacalai Tesque, Cat. 16631-92

4. Results FIG. 1 shows the amount (average value) of MG-H1 in the sera of healthy individuals and patients with various diseases by the competitive ELISA method.
From this result, the amount of MG-H1 in serum can be measured by an immunological assay such as an anti-MG-H1 monoclonal antibody or a competitive ELISA using the same. And for osteoporosis, anemia, arthritis, cataracts and hypertension diseases, MG-H1 levels are higher than in healthy subjects, and for gastroesophageal reflux disease and hyperlipidemia, MG-H1 levels are higher than in healthy subjects. The low value indicates that the presence or absence of these diseases affects the amount of MG-H1. This is because the monoclonal anti-AGE antibody of the present invention and the immunoassay method such as competitive ELISA using the same, diseases such as cataract and osteoporosis, specific symptoms, and MG in biological samples such as serum -It means that the presence or absence of a correlation with the amount of H1 can be examined, and based on the correlation, it can be used to determine the presence or absence of a disease or a specific symptom, and the risk of developing the disease or symptom. Is shown. According to the method of the present invention, osteoporosis, anemia, arthritis, cataract and hypertension can also be measured in a short time by measuring MG-H1 in various diseases and symptoms or their risks.

Claims (7)

A method for predicting morbidity or risk of diseases associated with final glycation products (AGEs), which specifically binds methylglyoxal-hydroimidazolone (MG-H1) in a biological sample collected from a subject. One or more diseases selected from the group consisting of osteoporosis, anemia, arthritis, cataracts, hypertension, gastroesophageal reflux disease and hyperlipidemia, comprising measuring using a monoclonal anti-MG-H1 antibody der is,
Monoclonal anti-MG-H1 antibody was obtained by synthesizing free MG-H1 that is not bound to protein and immunizing animals with the adduct obtained by binding it to protein as an antigen. The method is characterized by having no cross-reactivity with carboxymethylarginine and carboxymethyllysine, which are known AGEs .   The method according to claim 1, wherein the measurement method is an ELISA method.   The amount of MG-H1 in the biological sample collected from the subject is compared with the amount of MG-H1 in the biological sample of a healthy person, and it is predicted that the disease is afflicted or at risk if it is significantly higher or lower Item 3. The method according to Item 1 or 2.   The method according to any one of claims 1 to 3, wherein the biological sample is serum or plasma. Monoclonal anti-MG-H1 antibody, NITE P-1898 (OYC111) , NITE P-1899 (OYC112) or NITE P-2064 (OYC113) deposited as hybridoma is an antibody produced any of claims 1 to 4 Or the method described in paragraph 1 . The amount of MG-H1 in a biological sample collected from a subject is measured using a monoclonal anti-MG-H1 antibody that specifically binds to this, and the measured value obtained and the MG in the biological sample of a healthy person compared with the values of -H1, it sees contains to determine if significantly higher or lower,
Monoclonal anti-MG-H1 antibody was obtained by synthesizing free MG-H1 that is not bound to protein and immunizing animals with the adduct obtained by binding it to protein as an antigen. The known AGEs are characterized by having no cross-reactivity with carboxymethylarginine and carboxymethyllysine,
A method of providing data to predict the morbidity or risk of a disease associated with final glycation end products (AGEs). The kit used for the method according to any one of claims 1 to 6 , comprising the monoclonal anti-MG-H1 antibody that specifically binds to MG-H1.