JP6553491B2 - Method for predicting morbidity or risk of diseases associated with final glycation products (AGEs) - Google Patents
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Description
本発明は、最終糖化産物（AGEs）が関連する疾患の罹患又はそのリスクを予測する方法に関し、より詳細には最終糖化産物（AGEs）の１種であるメチルグリオキサール−ハイドロイミダゾロン（MG-H1）に特異的に結合するモノクローナル抗AGEs抗体を使用し、被験者から採取した生体試料中のMG-H1を測定することを含む最終糖化産物（AGEs）が関連する疾患の罹患又はそのリスクを予測する方法に関する。 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.
タンパク質中に存在するアミノ基とグルコース等の還元糖のアルデヒド基とが、非酵素的に反応すると糖化産物が形成される。この反応は、非酵素的糖化反応又はメイラード反応と称される。非酵素的糖化反応は、生体内においても起きている。非酵素的糖化反応は、シッフ塩基を経てアマドリ転位生成物が形成される前期反応と、複雑な開裂や縮合等が起こる後期反応との２段階で起こると考えられ、後期反応では、最終糖化産物(=AGEs:Advanced Glycation End Products)と呼ばれる、単一ではない様々な物質が生成する。 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.
AGEsについては、糖尿病や老化に伴う各種の症状や疾患等との関連性が報告されている。例えば、AGEsが、糖尿病における腎障害や網膜症にAGEsの蓄積が関係していることや、加齢で認められる種々の組織障害、例えば、皮膚、血管壁、関節等の結合組織の硬化に関連し、皮膚の肥厚、しわの形成、動脈硬化、関節炎等の原因になり得ることや、アルツハイマー病におけるβ−アミロイドペプチドの脳内蓄積にAGEsが関与していること等が報告されている。また、骨粗鬆症等の骨の強度の低下において、骨中のコラーゲンの糖化（AGEs化）が原因の一つであることが報告されている。さらに、AGEsが癌疾患、例えばその増殖や転移に関与していることも示唆されている。 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.
AGEsと各種の疾患との関連性の究明やその予防や治療法の開発には、多様なAGEsのなかから着目した特定のAGEsを検出又は定量する技術が必要である。AGEsを検出又は定量する方法としては、高速液体クロマトグラフィー(HPLC法)やガスクロマトグラフィー質量分析法(GC/MS)法、液体クロマトグラフィー質量分析(LC-MS/MS)法、AGEsを特異的に認識する抗AGEs抗体を用いる方法が知られている。これらの中でも、抗AGEs抗体を用いる方法は大型で高価な機器を使用しなくてもよい点で優れている。
抗AGEs抗体を用いる方法としては、例えば、特許文献１には、３，４−ジオキシグルコソン-3-エン(DGE)に由来するAGEsに特異的に反応する抗体及びそれを用いたAGEsの検出方法が記載され、特許文献２には、生体サンプル内におけるグアニジノ基由来のAGEsに特異的なモノクローナル抗体及びそれを用いたAGEsの検出方法が記載されている。また、特許文献３には、フルクトース由来のAGEに対する抗体及びそれを用いたフルクトース由来のAGEの測定方法が記載されている。
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.
ところで、AGEsの１種としてMG-H1が知られており、このMG-H1についても糖尿病や老化に伴う症状や疾患との関連性が報告されている。例えば、特許文献４には、糖尿病性の腎症、眼疾患、又は心血管合併症を発症するリスク又は割合の指標として、血漿から生成された２つ以上のバイオマーカーのレベルを使用するに当たり、その一つのバイオマーカーとしてMG-H1を使用することが記載されている。また、非特許文献１は、可溶性ヒト水晶体タンパク質中のメチルグリオキサール−ハイドロイミダゾロンに関し、２種の構造異性体(MG-H1,MG-H2)の濃度を測定し、他のAGEsの濃度と比較することによって、MG-H1又はMG-H2と老化や白内障との間に関連性があることを明らかにしている。 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.
しかし、このMG-H1については、抗AGEs抗体を用いて検出や定量等を行うことに言及した文献は少なく、上述した特許文献１〜３にも記載されていない。上述した特許文献４においても、個体から得た生体試料を、液体クロマトグラフィー／四重極質量分析法(LC-MS/MS)を使用した分析によりバイオマーカーのレベルを決定することが記載されている一方、抗AGEs抗体を用いて分析することは記載されていない。また、非特許文献１にも、メチルグリオキサール(MG)イミダゾロン(MG-HI,MG-H2)を、それらを特異的に認識する抗体を用いて分析することは記載されていない。
一方、非特許文献２には、メチルグリオキサール(MG)で修飾したタンパク質をマウスに免疫して得られた抗体に、エピトープがイミダゾロンである抗体が含まれることを開示されている。しかし、非特許文献２には、タンパク質に結合していないイミダゾロンを合成した後、それをタンパク質と結合して免疫原として用いることは記載されておらず、MG-H1に対する特異性や遊離体のMG-H1に対する結合能が高いモノクローナル抗体は記載されていない。
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.
なお、特許文献５には、糖尿病で特徴的な高血糖状態により形成される糖化タンパク質が、最終糖化産物（AGEs）が関連する疾患とされる動脈硬化症等の心血管疾患、アルツハイマー、がんの増殖及び転移、炎症反応などの合併症の重要な原因となり得ることが記載されており、また、そのような糖化タンパク質を、ガレクチンと呼ばれるレクチンの糖鎖に対する結合活性を用いて検出することが記載されている。しかし、最終糖化産物（AGEs）が関連する疾患として、骨粗鬆症、貧血症、関節炎、白内障、高血圧、胃食道逆流症及び高脂血症の罹患又はそのリスクの予測において、最終糖化産物（AGEs）としてMG-H1が有用なバイオマーカーであることは知られていない。 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.
本発明の課題は、最終糖化産物（AGEs）が関連する疾患の罹患又はそのリスクの予測に有用な方法を提供することにある。 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).
本発明者は、上記課題を解決すべく鋭意研究を重ねた結果、MG-H1に対する特異性や結合能の高いモノクローナル抗体により、被験者から採取した生体試料中のMG-H1を測定することが、最終糖化産物（AGEs）が関連する疾患のうち、骨粗鬆症、貧血症、関節炎、白内障、高血圧、胃食道逆流症及び高脂血症の罹患又はそのリスクの予測に有用であることを知見した。 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.
本発明は、上記の知見に基づきなされたもので、下記の（１）〜（６）の最終糖化産物（AGEs）が関連する疾患の罹患又はそのリスクを予測する方法、（７）の最終糖化産物（AGEs）が関連する疾患の罹患又はそのリスクを予測するためにデータを提供する方法、及び（８）これらの方法に使用されるキットを提供するものである。
（１）最終糖化産物（AGEs）が関連する疾患の罹患又はそのリスクを予測する方法であって、被験者から採取した生体試料中のメチルグリオキサール−ハイドロイミダゾロン（MG-H1）を、これに特異的に結合するモノクローナル抗MG-H1抗体を使用して測定することを含み、当該疾患が、骨粗鬆症、貧血症、関節炎、白内障、高血圧、胃食道逆流症及び高脂血症から選択される１つ以上の疾患であること、及びを特徴とする方法。
（２）測定方法が、ＥＬＩＳＡ法である（１）に記載の方法。
（３）被験者から採取した生体試料中のMG−H1の量を、健常者の生体試料中のMG-H1量と比較し、有意に高い又は低い場合に疾患の罹患又はそのリスクがあると予測する、（１）又は（２）に記載の方法。
（４）生体試料が血清または血漿である（１）〜（３）のいずれかに記載の方法。
（５）モノクローナル抗MG-H1抗体が、タンパク質に結合していない遊離体のMG-H1を合成し、それをタンパク質に結合して得られた付加体を抗原として動物に免疫して得られたものであり、既知AGEsである、カルボキシメチルアルギニン及びカルボキシメチルリジンに交叉反応性を有しないことを特徴とする、（１）〜（４）のいずれかに記載の方法。
（６）モノクローナル抗MG-H1抗体が、ＮＩＴＥ Ｐ−１８９８（ＯＹＣ１１１）、ＮＩＴＥ Ｐ−１８９９（ＯＹＣ１１２）又はＮＩＴＥ Ｐ−２０６４（ＯＹＣ１１３）として寄託されたハイブリドーマが産生する抗体である、（５）に記載の方法。
（７）被験者から採取した生体試料中のMG−H1の量を、これに特異的に結合するモノクローナル抗MG-H1抗体を使用して測定し、得られた測定値と、健常者の生体試料中のMG-H1の値と比較し、有意に高い又は低い場合かを判定することを含む、最終糖化産物（AGEs）が関連する疾患の罹患又はそのリスクを予測するためにデータを提供する方法。
（８）MG-H1に特異的に結合するモノクローナル抗MG-H1抗体を含む、（１）〜（７）のいずれかに記載の方法に使用されるキット。
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.
本発明の最終糖化産物（AGEs）が関連する疾患の罹患又はそのリスクを予測する方法は、被験者から採取した血液等の生体試料中のMG-H1を測定することにより、最終糖化産物（AGEs）が関連する疾患のうち、骨粗鬆症、貧血症、関節炎、白内障、高血圧、胃食道逆流症及び高脂血症の罹患又はそのリスクを簡便、且つ迅速に予測することができるため、非常に有用である。また、当該モノクローナル抗MG-H1抗体を使用して測定し、得られた生体試料中のMG−H1の測定値と、健常者の生体試料中のMG-H1の値と比較し、有意に高い又は低い場合かを判定することで、最終糖化産物（AGEs）が関連する疾患の罹患又はそのリスクを予測するためにデータが提供される。 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).
以下、本発明をその好ましい実施形態（態様）に基づいて説明する。
本発明の最終糖化産物（AGEs）が関連する疾患の罹患又はそのリスクを予測する方法において使用するモノクローナル抗AGEs抗体は、最終糖化産物（AGEs）に対する抗体であって、MG-H1に対して特異的に結合するものである。本発明における「MG-H1」は、AGEsの前駆体であるメチルグリオキサール(MG)が、タンパク質中のアルギニン残基等の非遊離のアルギニン又は遊離のアルギニンと反応して生じた非遊離体又は遊離体のMG-H1であり、「MG-H1に対して特異的に結合する」とは、少なくとも非遊離体のMG-H1を認識して反応する一方、MG-H1以外のAGEであって既知のAGEs構造である、カルボキシメチルアルギニン(CMA：Nω-(carboxymethyl)arginine)、カルボキシメチルリジン(CML：Nε-(Carboxymethyl)lysine)及びカルボキシエチルリジン(CEL：Nε-(carboxyethyl)lysine)のいずれとも反応しないか、それらに対する反応性がいずれも非遊離体のMG-H1に対する反応性に対して有意に低いことを意味する。
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.
本発明の方法において使用するモノクローナル抗AGEs抗体は、タンパク質に結合していない遊離体のMG-H1に対する結合能を有する抗体であることが好ましい。遊離体のMG-H1に対する結合能を有する抗体であると、例えば競合ELISAで使用可能となる。競合的阻害アッセイにおいては、例えばマイクロプレートのウェル（穴）に非遊離体のMG-H1が結合した蛋白を固定しておき、そのMG-H1に対する抗体の結合が遊離体のMG-H1を共存させることにより阻害されるか否かを調べ、阻害される場合には遊離体のMG-H1に対する結合能を有し、阻害されない場合にはMG-H1に対する結合能を有しないと判断する。 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.
タンパク質に結合していない遊離体のMG-H1は、ペプチドを含めてタンパク質に結合していないMG-H1を意味し、ペプチド結合を有しないアルギニンのメチルグリオキサール(MG)修飾物である。即ち、本発明のモノクローナル抗AGEs抗体は、好ましくは、タンパク質に結合したMG-H1のみならず、ペプチド及び蛋白の何れにも結合していない遊離体のMG-H1に対する反応性も有している。 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. .
本発明の方法において使用するモノクローナル抗AGEs抗体は、タンパク質に結合していない遊離体のMG-H1を合成した後、その合成したMG-H1をタンパク質に結合して得られる付加体を、免疫原として動物に免疫することにより得られる。本発明のモノクローナル抗AGEs抗体は、斯かる方法で調製することにより、タンパク質をメチルグリオキサールと反応させて得た免疫原で同動物を免疫する場合に比べて、MG-H1に対する特異性及び／又は遊離体のMG-H1に対する結合能が高いものとなる。 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.
本発明の方法において使用するモノクローナル抗AGEs抗体としては、下記のハイブリドーマが産生する抗体１〜３が挙げられる。抗体１及び２を産生するハイブリドーマは、２０１４年７月１５日付け、抗体３を産生するハイブリドーマは、２０１５年６月５日付けで、独立行政法人 製品評価技術基盤機構特許生物寄託センターに寄託されており、それぞれの受託番号は下記の通りである。
抗体１を産生するハイブリドーマ（ＯＹＣ１１１）：受託番号ＮＩＴＥ Ｐ−１８９８
抗体２を産生するハイブリドーマ（ＯＹＣ１１２）：受託番号ＮＩＴＥ Ｐ−１８９９
抗体３を産生するハイブリドーマ（ＯＹＣ１１３）：受託番号ＮＩＴＥ Ｐ−２０６４
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
以下、本発明の方法に好適なモノクローナル抗AGEs抗体の製造方法について説明する。
当該モノクローナル抗AGEs抗体の好ましい製造方法は、下記工程（１）〜（３）を具備するとともに、モノクローナル抗体の製法において常法である下記工程（４）を具備する。
（１）タンパク質に結合していない遊離体のMG-H1を合成する工程
（２）合成したMG-H1を、クロスリンカーを用いてタンパク質に結合する工程
（３）MG-H1が結合したタンパク質を抗原として動物を免疫する工程
（４）免疫した動物の抗体産生細胞を用いて、モノクローナル抗AGEs抗体を産生するハイブリドーマを作製する工程
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
以下、工程（１）〜（４）について説明する。
〔工程（１）：遊離体のMG-H1の調製工程〕
前記工程（１）は、遊離体のMG-H1の調製工程であり、好ましくは、メチルグリオキサールとアルギニンとを混合し、非酵素的糖化反応により下記式（１）で表される遊離体のMG-H1を生成させる。
[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.
より具体的には、メチルグリオキサールとアルギニンとを混合し、例えば水酸化ナトリウム等のアルカリでｐＨを１０以上に調整して、３７℃の温度で、１時間インキュベートすることにより、上記構成の遊離体のMG-H1を生じさせることができる。 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.
メチルグリオキサール(MG)とアルギニンとを反応させて得られる、メチルグリオキサール由来のハイドロイミダゾロンは、以下の３つの構造異性体を有する。MG-H1は、他の構造異性体が混在した状態で用いることもできるが、MG-H1を単独で用いることが、製造効率や遊離体のMG-H1に対する特異性の向上の点から好ましい。MG-H1の構造は、例えば、液体クロマトグラフ質量分析計(LC-MS)により確認することができる。
MG-H1：Ｎδ-(5-ヒドロ-5-メチル-4-イミダゾロン-2-イル)-オルニチン
MG-H2：2-アミノ-5-(2-アミノ-5-ヒドロ-5-メチル-4-イミダゾロン-1-イル)ヘ゜ンタン酸
MG-H3：2-アミノ-5-(2-アミノ-4-ヒドロ-4-メチル-5-イミダゾロン-1-イル)ヘ゜ンタン酸
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
合成した遊離体のMG-H1は、適宜の手段により精製する。
合成した遊離体のMG-H1を高純度のものとすることは、得られるモノクローナル抗体のMG-H1に対する特異性及び／又は遊離体のMG-H1に対する結合能を向上させる観点から好ましい。精製の方法は、各種公知の方法を採用することができ、例えば、液体クロマトグラフィーが好ましく用いられる。液体クロマトグラフィーとしては、イオン交換クロマトグラフィー、吸着クロマトグラフィー、分配クロマトグラフィー、分子排斥クロマトグラフィー、アフィニティークロマトグラフィー等が挙げられ、これらは１種を単独で行っても良いし同種又は異種の２種以上を順次行っても良い。
合成した遊離体のMG-H1は、純度が９９質量％以上、より好ましくは９９．９質量％となるまで精製して次工程に用いることが好ましい。なお、本発明において、MG-H1は、ナトリウム塩、塩酸塩等の塩であっても良いし、塩でなくても良い。
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.
〔工程（２）：抗原の調製工程〕
前記工程（２）は、免疫原の調製工程であり、単独では免疫原性（免疫誘導能）が低い遊離体のMG-H1をタンパク質に結合して充分な免疫原性を有する抗原とする。MG-H1を結合させるタンパク質としては、キャリアタンパク質として公知の各種のものを特に制限なく用いることができ、例えば血清アルブミン等のアルブミンや、ヘモシアニン、ミオグロビン等が挙げられる。また、タンパク質は、ウシ、ウサギ、ヒト等の哺乳動物、スカシ貝等の貝類、鶏等の鳥類等に由来するもの等を用いることができる。これらの中でも、スカシ貝ヘモシアニン(KLH)及びウシ血清アルブミン(BSA)が好ましい。これらのタンパク質は、一種を単独で又は二種以上を組み合わせて用いることもできる。
MG-H1のキャリア蛋白への結合には、クロスリンカーを用いることが好ましい。クロスリンカーとしては、各種公知のものを用いることができ、例えば、グルタルアルデヒド、EDC(1-ethy-3-(3-dimethylaminopropyl) carbodiimide hydrochloride)、SPDP、DST、DSGなどを用いることができる。これらの中でも、グルタルアルデヒド、又はEDCを用いることが好ましい。クロスリンカーは、一種を単独で又は二種以上を組み合わせて用いることもできる。
[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.
〔工程（３）：動物の免疫工程〕
前記工程（３）は、動物を免疫する工程であり、上述のようにして調製した、MG-H1が結合したタンパク質（付加体）を抗原としてヒト以外の動物を免疫し、その動物体内に抗体産生細胞を産生させる。本工程（３）は、上述のようにして調製した、MG-H1が結合したタンパク質（付加体）を抗原として用いる以外は、モノクローナル抗体の製法の常法に従って行うことができる。
動物の種類は、特に限定されないが、哺乳動物が好ましい。哺乳動物としては、例えばマウス、ラット、ウサギ等のげっ歯類が挙げられる。マウスは、ＢＡＬＢ／Ｃ系統のマウスを用いることが、ハイブリドーマの作製に用いる骨髄腫由来の細胞株が確立している点から好ましい。免疫は、各種公知の方法により行うことができ、例えば、哺乳動物の皮下、皮内、静脈、または腹腔内に注射する。免疫は、初回の免疫後に何度か繰り返し行うことが好ましく、免疫のスケジュールは、免疫する動物の種類や系統に応じて適宜に決定することができる。また、免疫応答を増強させるために、MG-H1が結合したタンパク質は、投与前又は投与時にアジュバントと混合して投与することが好ましい。アジュバントとしては、各種公知のものを用いることができる。また、例えば、初回免疫時には完全フロイントアジュバント(CFA:Complete Freund's adjuvant)を用い、２回目以降は不完全フロイントアジュバント(IFA:Incomplete Freund's adjuvant)を用いる等、２種以上のアジュバントを用いることもできる。
[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.
〔工程（４）：モノクローナル抗AGEs抗体を産生するハイブリドーマの作製工程〕
前記工程（４）においては、免疫した動物の抗体産生細胞を用いて、モノクローナル抗AGEs抗体の産生能を有するハイブリドーマを作製する。
上記の免疫後の動物は、適宜の間隔で採血し、MG-H1に対する抗体が産生されていることを確認する。抗体産生の確認には、酵素免疫測定法(ELISA)、放射免疫アッセイ法(RIA)、蛍光免疫測定法等の公知の分析方法を用いることができる。抗体産生の確認後、ブースト（免疫原の追加注射）を行うことも好ましい。最終免疫後、免疫した動物から脾臓細胞を摘出し、骨髄腫由来の細胞と細胞融合させる。細胞融合の方法は、例えば、ポリエチレングリコール法、センダイウイルスを用いる方法、電気刺激を与える方法等の公知の方法を用いることができる。融合細胞は、ヒポキサンチン、アミノプテリン、チミジンを含むＨＡＴ培地等で選択可能である。また、得られた融合細胞から、酵素免疫測定法(ELISA)、放射免疫アッセイ法(RIA)、蛍光免疫測定法等の公知の分析方法により、MG-H1に対する結合能を有する抗体の産生能が高い融合細胞を選択する。そして、選択した細胞を用いて、限界希釈法、軟寒天法等の公知の方法によりクローニングを行う。このようにして、MG-H1に対する特異性や遊離体のMG-H1に対する結合能の高いモノクローナル抗AGEs抗体を産生するハイブリドーマが得られる。
[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.
上記のようにして得られたハイブリドーマは、培養して増殖させ、その培養液等からモノクローナル抗AGEs抗体を分離精製することにより、本発明のモノクローナル抗AGEs抗体が大量に得られる。ハイブリドーマの培養方法としては、哺乳動物の腹腔内に注射し腹水内で増殖させる方法や動物の体外で適切な培地を用いて培養する方法等の各種公知の方法を採用可能である。また、腹水や培養液または培養上清から得た抗体の精製には、例えば、イオン交換クロマトグラフィー、アフィニティークロマトグラフィー等の公知の方法を特に制限なく使用可能である。 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.
なお、本発明の方法に使用するモノクローナル抗AGEs抗体には、このようにして得られた抗体のほか、MG-H1の識別部位を含む該抗体の断片も含まれる。また、MG-H1の識別部位を含むように遺伝子組換え技術を用いて製造したキメラ抗体やヒト化抗体であっても良い。 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.
このようにして得られたモノクローナル抗AGEs抗体は、MG-H1に対する特異性及び／又は遊離体のMG-H1に対する結合能に優れているため、各種の生体試料中のMG-H1を高感度あるいは高精度に測定及び／又は定量可能である。 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.
本発明の最終糖化産物（AGEs）が関連する疾患の罹患又はそのリスクを予測する方法は、被験者から採取した生体試料中のMG-H1を、上述したモノクローナル抗MG-H1抗体を使用して測定及び／又は定量する。モノクローナル抗AGEs抗体を用いて被験者から採取した生体試料中のMG-H1を測定及び／又は定量する方法としては、モノクローナル抗体を用いた各種公知の測定及び／又は定量方法を特に制限なく採用でき、例えば、酵素免疫測定法(ＥＬＩＳＡ法)、放射免疫アッセイ法(RIA)、発光免疫測定法、沈降法、凝集法、ウエスタンブロット分析、フローサイトメトリー等が挙げられるが、ＥＬＩＳＡ法が好ましい。なお、ＥＬＩＳＡ法としては、競合阻害ＥＬＩＳＡ法や競合結合ＥＬＩＳＡ法のいずれでもよい。ＥＬＩＳＡ法において用いる二次抗体、標識等は、常法に従って適宜選択すればよい。 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.
また、本発明において、被権者から採取した生体試料としては、生体から採取されたあらゆる細胞、組織、及び体液等が挙げられ、例えば、皮膚、筋肉、骨、脂肪組織、脳神経系、心臓及び血管等の循環器系、肺、肝臓、脾臓、膵臓、腎臓、消化器系、胸腺、リンパ、血液、全血、血清、血漿、リンパ液、唾液、尿、腹水、喀痰等、並びにそれらの培養液物が挙げられるが、採取のし易さ等の理由から、血清または血漿が好ましい。生体試料は、必要に応じて、生理食塩水や水等の液体による希釈、濾過処理、ホモジネート処理、化学処理等の任意の処理を行ったものを用いる。 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.
本発明において最終糖化産物（AGEs）が関連する疾患とは、骨粗鬆症、関節炎、その他の骨関連疾患、貧血症、白内障、高血圧、胃食道逆流症及び高脂血症から選択される１つ以上の疾患である。
骨粗鬆症や関節炎の以外のその他の骨関連疾患としては、例えば骨量減少症、若年性骨粗鬆症、骨形成不全、高カルシムム血症、上皮小体機能亢進症、骨軟化症、骨石灰化脱失症、骨溶解性骨疾患、骨壊死、パジェット病、関節リウマチ、変形性関節症による骨の低下、炎症性関節炎、骨髄炎、グルチコルチコイド処置、転移性の骨疾患、歯周の骨の喪失、癌による骨の喪失、加齢による骨の喪失、及びその他の骨量減少症が挙げられる。
貧血症としては、例えば鉄欠乏性貧血、再生不良性貧血、鉄芽球性貧血、溶血性貧血、巨赤芽球性貧血、腎性貧血、サラセミア等が挙げられる。白内障としては、加齢白内障、アトピー白内障、併発白内障、糖尿病白内障等が挙げられる。高血圧としては、原発性高血圧、二次性高血圧 (例えば大動脈縮窄症、腎血管性高血圧、腎実質性高血圧、原発性アルドステロン症、Cushing 症候群、薬剤誘発性高血圧、脳幹部血管圧迫、甲状腺機能低下症）が挙げられる。
胃食道逆流症としては、非びらん性胃食道逆流症、逆流性食道炎、バレット食道等が挙げあれ、高脂血症としては、高コレステロール血症、高トリグリセリド血症、家族性脂質異常症等が挙げられる。
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.
本発明の方法において、上記モノクローナル抗MG-H1抗体及び測定方法により、被験者から採取した生体試料中のMG−H1量を、健常者の生体試料中のMG-H1量と比較し、有意に高い又は低い場合に疾患の罹患又はそのリスクがあると予測する。具体的には、被権者の生体試料中のMG−H1の量が、健常者の生体試料中のMG-H1量と比較して有意に高い場合、骨粗鬆症、貧血症、関節炎、白内障及び高血圧の少なくとも１つの疾患の罹患又はそのリスクが高いと判断される。一方、被権者の生体試料中のMG−H1の量が、健常者の生体試料中のMG-H1量と比較して有意に低い、胃食道逆流症及び高脂血症少なくとも１つの疾患の罹患又はそのリスクが高いと判断される。 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.
また、本発明は、被験者から採取した生体試料中のMG−H1の量を、これに特異的に結合するモノクローナル抗MG-H1抗体を使用して測定し、得られた測定値と、健常者の生体試料中のMG-H1の値と比較し、有意に高い又は低い場合かを判定することを含む、最終糖化産物（AGEs）が関連する疾患の罹患又はそのリスクを予測するためにデータを提供する方法しても利用可能である。 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.
本発明のキットは、上述した最終糖化産物（AGEs）が関連する疾患の罹患又はそのリスクを予測する方法及び、最終糖化産物（AGEs）が関連する疾患の罹患又はそのリスクを予測するためにデータを提供する方法に使用するための、MG-H1に特異的に結合するモノクローナル抗MG-H1抗体を含むキットである。本発明のキットは、少なくともモノクローナル抗AGEs抗体と、MG-H1と結合した抗体を検出する試薬を含むことが好ましい。 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.
以下、本発明を実施例により更に詳細に説明するが、本発明はこれらの実施例により何ら制限されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not restrict | limited at all by these Examples.
〔遊離体のMG-H1の調製〕
（１）遊離体のMG-H1を下記方法により合成した。
1N NaOH 49mLと40%Methyl glyoxal(5.8M)862μlとを混合し、次いで、それに871mgのL-Arginine(MW=174.2)を混合して、100mM(final)L-Argineと100mM(final)Methylglyoxalの混合液50mLを調製した。その混合液を振盪させながら、37℃で1時間インキュベートした後、HClで中性にした。その後、即エバポレーターで濃縮後、水に再溶解して凍結乾燥した。
（２）合成したMG-H1の精製
合成した遊離体のMG-H1について、下記方法による２段階の精製を行った。
精製１（Dowex50カラムによる精製)
陽イオン交換樹脂を充填したカラムを用いたイオン交換クロマトグラフィーにより、合成したMG-H1サンプルの１回目の精製を行った。カラムは、樹脂を充填したカラム（内径20mm、樹脂充填部の高さ20cm)を、カラムの約５倍量の10％ピリジン（超純水で調製）で洗浄した後、カラム５倍量のイオン交換水で洗浄し、次いでカラム５倍量の10％HClで洗浄した後、カラム５倍量のイオン交換水で洗浄して用いた。
このカラムに、合成したMG-H1サンプルを少量の超純水に溶かしてアプライし、カラムの３倍量の超純水を流した。次いで、10％ピリジンで溶出し、約3mlずつ分取した。ろ紙に各フラクションを10回ずつキャピラリーでスポットし0.5％ニンヒドリン溶液で検出した。ニンヒドリンで発色したフラクションでのMG-H1の存在をTLCで確認した。TLCは、シリカゲルプレートにスポットし純品もスポットして、展開溶媒（クロロホルム：MeOH:水＝2：3：1）で展開し、ドライヤーで乾燥後、ニンヒドリン試薬で発色させた。発色した部分を集めエバポレーターでピリジンをとばした後-80℃で凍らせ、凍結乾燥した。
イオン交換樹脂としては、DowexTM50(ダウ・ケミカル社)を用い、超純水としてはmilliQ水を用いた。milliQ水は、ミリポア社の超純水製造装置MilliQで製造した超純水である。
[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.
精製２（シリカゲルオープンカラムによる精製)
精製１による精製後のMG-H1サンプルについて、シリカゲルカラムを用いた液体クロマトグラフィーにより２回目の精製を行った。
シリカゲルカラムは、内径20mm、長さ50cmのガラス製カラムの底部に脱脂綿をほぐして詰め、次いで、シリカゲル（関東化学社製「シリカゲル60」）を上端からロートを用いて注入し床に軽く数回当てて空気を抜いて調製した。シリカゲル充填部の高さは44cmであった。
このシリカゲルカラムに、凍結乾燥後（１回目の精製後）のMG-H1サンプルを少量（約1mL）の超純水に溶かしてロードし、更に超純水0.5mlで容器を洗浄し、その洗浄水もロードした。そして、クロロホルム：MeOH:水＝2：3：1を注入して溶出を開始し、溶出液を分取した。各フラクションの液を、紙にスポットし、0.5％ニンヒドリンで発色させた。ニンヒドリンで発色したフラクションでのMG-H1の存在をＴＬＣで確認した。また、ＴＬＣは、シリカゲルプレートにスポットし純品もスポットし、展開溶媒（クロロホルム：MeOH:水＝2：3：1）で展開し、ドライヤーで乾燥後、ニンヒドリン試薬で発色させた。MG-H1が確認されたフラクションを集め、エバポレーターで濃縮後に-80℃で凍らせ凍結乾燥した。精製２においても超純水としては、milliQ水を用いた。
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）構造の確認
精製１及び精製２により精製したMG-H1サンプルについて、液体クロマトグラフィー／四重極質量分析(LC-MS/MS)により分析した。条件は下記の通りである。
試料インジェクション量：1μg/mlを10 μl
LC部：Accela Pump + Thermo PAL (Thermo Scientific)
カラム：ZIC(R)-HILIC 150 x 2.1 mm, 5 μm (Merck Millipore)
移動相：0.1%ギ酸水溶液および0.1%ギ酸アセトニトリルによるグラジュエント
流速： 200 μl/min
MS部：高感度定量分析用トリプル四重極質量分析計 TSQ Vantage (Thermo Scientific)
イオン化法 : Positive ESI
スプレー電圧 : 3500 Ｖ
ベポライザー温度: 200 ℃
イオントランスファーチューブ温度 : 300 ℃
コリジョンガス、圧力 : Argon、1.2 mTorr
上記分析の結果、単離された化合物は、MG-H1であることが確認された。
(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.
〔抗原の調製〕
抗原（免疫原）を調製するために、上記のようにして調製した高純度のMG-H1を、キャリアタンパク質として利用するスカシ貝ヘモシアニン（KLH）と結合させて、MG-H1結合KLHを作製した。結合方法としては、クロスリンカーとして前述したEDCを用いたEDC法を用いた。具体的方法は下記の通りである。
（MG-H1結合KLHの調製）
下記成分を下記の量ずつ上の成分から順に混和して合計1000μLの混合液に調製した。
KLH(10mg/mL) 300μL
PBS 445μL
MG-H1(1mg/100μL) 150μL (1.5mg/150μL)
NHS (5.4mg/50μL) 5μL
EDC (100mg/100μL) 100μL
各成分の詳細を以下に示す。
KLH: SIGMA社製KLH
PBS: リン酸緩衝生理食塩水（ｐH7.2）
MG-H1：合成した遊離体のMG-H1（純度99％以上）
NHS: N-ヒドロキシコハク酸イミド（縮合剤）
EDC: 1-エチル-3-(3-ジメチルアミノプロピル)カルボジイミド「PIERCE」
上記のようにして調製した混合液を、１時間、振盪しながらインキュベートした後、3μLの2-Mercaptoethanolを加え未反応のNHSを不活化した。そして、2LのPBSで一晩透析した。
透析後、蛋白濃度をBCA蛋白定量キット（PIERCE(登録商標）サーモフィッシャーサイエンティフィック株式会社製）で測定した。
[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.).
（MG-H1結合BSAの調製）
KLH(10mg/mL)に代えてウシ血清アルブミン（BSA）(10mg/mL)を用いる以外は、MG-H1結合KLHの調製と同様にして、MG-H1結合BSA（MG-BSA）を調製した。
(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) .
〔皮下免疫法による免疫及びハイブリドーマの作製〕
上述のようにして調製したMG-H1結合KLHを、アジュバントとそれぞれ混合したエマルジョンを用意し6〜8週齢メスのBALB/cマウス(日本チャールス・リバー)の背部皮下に50〜100μg/匹/回で免疫した。免疫間隔は２週間に１回で合計５回実施した。アジュバントとして初回は完全フロイントアジュバント(CFA)、2回目以降は不完全フロイントアジュバント(IFA)を使用した。3回目・4回目免疫の翌週に採血し、タイターチェックを実施した。タイターチェックはMG-H1結合BSAを固相にして行った。最終免疫としてリン酸緩衝生理食塩水（PBS, Phosphate buffered saline ）(-)で希釈した抗原を腹腔内に投与した。
最終免疫3〜4日後にマウスより摘出した脾臓細胞をミエローマと混合してPEG法により
細胞融合を実施した。脾臓細胞数として0.5〜1.0×105 cells/wellで96 well plateに播
種し、RPMI 1640 +10% FBS + hypoxanthine-aminopterin-thymidine (HAT) + hybridoma
cloning supplement (HCS)で培養した。
細胞融合後約2週間HAT培地により選択培養を行った。融合細胞を選択後、各ウェルから一部の培養上清をサンプリングし、MG-H1結合BSAを1μg/wellで固相したELISAによりスクリーニングを行った。二次スクリーニングとしてメチルグリオキサール修飾BSAを固相したELISAを行い、免疫抗原、メチルグリオキサール修飾BSAそれぞれで陽性のウェルを確認した。
ELISAの結果を元に選択された10ウェルの細胞を用いて限界希釈法にてクローニングを行った。陽性のシングルコロニーを形成した10個のウェルを選択し、増殖能を加味して最終的に３クローンに絞った。樹立過程において使用した各種添加剤含有培養から馴化培地（RPMI1640 + 10% FBS）で増殖するよう馴化培養を行った。
[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.
〔マウス腹腔法によるモノクローナル抗AGEs抗体の生産〕
クローニング、馴化したハイブリドーマを馴化培地で培養し、生細胞率が上昇するように10ｃｍディッシュで２〜３継代の培養を行った。生細胞の状態を確認しながら培養スケールをあげ、移植当日に対数増殖期のハイブリドーマを必要量（2x107cells）確保した。ハイブリドーマ懸濁液から遠心操作（1000rpm, 5min）によりハイブリドーマを回収した。PBS(-)で細胞を洗浄後、細胞数を測定し、4x106cells/mlにRPMI 1640(FBS free)にて調製した。0.5ml/匹（2x106cells/匹）で１０匹のBALB/c雄マウス腹腔内に移植した。腹水の貯留具合を確認し、18Gの注射針を用いて腹水を採取した。採取腹水を遠心分離（3000rpm, 5min）し、腹水上清を回収した。
腹水を平衡化・吸着バッファー（PBS(-), pH 7.4）で３倍希釈した。次にプロテインAカラムを５〜１０bed volの平衡化・吸着バッファーで洗浄し、希釈した腹水をアプライした。その後、プロテインAカラムを再度５〜１０bed volの平衡化・吸着バッファーで洗浄した。次に３〜５bed volの溶出バッファー（100mM クエン酸バッファー, pH3.0）で吸着蛋白質を溶出させ、A280=0.1以上を集め、中和液（2M Tris-HCl, pH9.0）を1/10〜1/20倍添加し、溶出画分を素早くpH7.0〜7.5にした。中和した溶出画分を最終バッファー（50mM Tris-HCl, pH8.0, 0.15M NaCl）にて透析し、その後0.22μmフィルターで濾過滅菌した。
このようにして、MG-H1に特異的に結合するモノクローナル抗AGEs抗体を得た。
[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.
〔競合ELISA法による患者血清中MG-H1の測定〕
健常者及び様々な疾患患者の血清中MG-H1量を測定した。
１．使用したヒト血清
今回健常者と８種類の疾患(骨粗鬆症、貧血症、関節炎、白内障、胃食道逆流症、高血圧症、緑内障、高脂血症)患者のヒト血清をPROMEDDX社から入手した。健常者と各疾患患者の平均年齢は次の通りである。健常者７５．０歳、骨粗鬆症７５．５歳、貧血症７２．４歳、関節炎７６．８歳、白内障８４．７歳、胃食道逆流症７６．０歳、高血圧症８０．５歳、緑内障８４．０歳、高脂血症７２．０歳。また、健常者及び疾患患者のヒト血清のサンプル数は以下の通り。健常１０、骨粗鬆症１０、貧血症７、関節炎７、白内障３、胃食道逆流症３、緑内障２、高血圧６、高脂血症２
[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．方法
[ELISAプレートにコーティングするMG-BSAの調製]
(1) 50 mLチューブを2本取り、Methylglyoxal (MG) とBSAを1×PBSでそれぞれ5.8 Mと 4 mg/mLに調製した。
(2) 1×PBSでMGを2 mM、BSAを2 mg/mLに希釈した。
(3) 15 mLチューブに2 mM MGと2 mg/mL BSAを各5 mLずつ加えて(1 mM (1 mg/mL) MG-BSA)、37℃で12時間インキュベートした。
(4) 1 Lの1 ×PBSにて12時間以上透析した。
(5) 透析後の溶液を回収し、0.45 μmのフィルターに通した。
(6) BCA法にて回収したMG-BSAのタンパク量を測定した。
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.
[競合ELISA法による患者血清中MG-H1の測定]
(1) 12時間インキュベートした1mM (1 mg/mL) MG-BSA溶液を1×PBSで
0.5 μg/mL MG-BSAに調製した後、96穴ELISAプレートに100μL/well加え、ラップでふたをして2時間室温で静置した。
(2) ELISAプレート内の液を除去し、洗浄液(PBS+0.5%Tween20)を200μL/well加え、そのまま液を除去する工程を3回実施した。
(3) 0.5%Gelatin(PBSにGelatinを加え0.5%にした溶液)を200μL/well加え、１時間室温に置き、ブロッキングを行った。
(4) 上記(2)の操作を行った。
(5) 検量線作成のため、まず2mM MG-H1を洗浄液にて作製後、1mMに希釈し、その後3倍ずつ順に希釈し、12段階調製した。
(6) 血清を洗浄液で４倍希釈して十分撹拌した後、上記(6)で調製したMG-H1とともに所定のウェルに50μL/wellで加えた。
(7) 洗浄液にて2μg/mLに希釈した抗MG-H1抗体を、ウェルに50μLずつ加えた後、1時間室温で反応させた。
(8) 上記(2)の操作を行った。
(9) 洗浄液にて0.1 μg/mLに調製したGoat anti-Mouse IgG (γ)-HRP抗体を100 μL/well加えた後、1 時間室温で反応させた。
(10) 上記(2)の操作を行った。
(11) TMB試薬をプレート1列ごとに5秒間隔で100 μL/well加え、発色確認後2 Nの硫酸を5 秒間隔で100 μL/well加えて反応を止めた。
(12) マイクロプレートリーダーFLUO star OPTIMA (BMG LABTECH)で450nm、レファレンス595 nmの吸光度で測定した。
[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．使用した試薬・備品は以下の通り
・Methylglyoxal solution, 40%：Sigma, M0252-25mL
・BSA：Sigma, A-7030
・透析チューブ：エーディアス, UC24-32-100 セルロースチューブ24/32
・0.45 μmフィルター：市販品0.45 μmフィルター
・BCA試薬：Pierce, 23225 BCA Protein Assay Reagent
・96穴ELISAプレート：NUNC MAXISORP(NUNC 439454)
・PBS：市販の試薬にて10×PBS作製後、超純水で希釈した
・Tween 20：和光純薬工業（株）, Polyoxyethylene (20) sorbitan Monolaurate
・Gelatin：ナカライテスク（株）,Gelatin精製粉末 16631-92
・MG-H1：前述した方法により、合成した後、２段階の精製を行った前記の遊離体のMG-H1
・一次抗体（抗MG-H1抗体）：抗体３(5.2 mg/mL)
・二次抗体：KPL, 214-1802 Goat Anti-Mouse IgG (gamma) Antibody, F(ab')2 fragment, Human Serum Adsorbed and Peroxidase labeled
・硫酸：ナカライテスク（株）, Cat. 16631-92
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．結果
図１に、競合ELISA法による健常者と各疾患患者血清中のMG-H1量（平均値）を示した。
この結果から、抗MG-H1モノクローナル抗体やそれを用いた競合ELISA等の免疫学的測定法によれば、血清中のMG-H1量を測定することができる。そして骨粗鬆症、貧血症、関節炎、白内障並びに高血圧症疾患については、健常者よりもMG-H1量が高値を示し、胃食道逆流症及び高脂血症については、健常者よりもMG-H1量が低値を示すことから、これらの疾患の有無がMG-H1量に影響することが確認された。このことは、本発明のモノクローナル抗AGEs抗体や、それを用いた競合ELISA等の免疫学的測定法によれば、白内障や骨粗鬆症等の疾患や特定の症状と、血清等の生体試料中のMG-H1量との間の相関関係の有無を調べることができることを意味し、また、その相関関係に基づき、疾患や特定の症状の有無、疾患や症状が生じるリスクの判定に利用可能であることを示している。本発明の方法により、骨粗鬆症、貧血症、関節炎、白内障及び高血圧症もMG-H1を測定することで、各種疾患や症状、又はそれらのリスク等を短時間で判定することが可能となる。
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)
モノクローナル抗MG-H1抗体が、タンパク質に結合していない遊離体のMG-H1を合成し、それをタンパク質に結合して得られた付加体を抗原として動物に免疫して得られたものであり、既知AGEsである、カルボキシメチルアルギニン及びカルボキシメチルリジンに交叉反応性を有しないことを特徴とする方法。 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 .
モノクローナル抗MG-H1抗体が、タンパク質に結合していない遊離体のMG-H1を合成し、それをタンパク質に結合して得られた付加体を抗原として動物に免疫して得られたものであり、既知AGEsである、カルボキシメチルアルギニン及びカルボキシメチルリジンに交叉反応性を有しないことを特徴とする、
最終糖化産物（AGEs）が関連する疾患の罹患又はそのリスクを予測するためにデータを提供する方法。 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).
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