JPH0424554A - Method and kit for detecting calpastatin abnormality disease - Google Patents
Method and kit for detecting calpastatin abnormality diseaseInfo
- Publication number
- JPH0424554A JPH0424554A JP12700390A JP12700390A JPH0424554A JP H0424554 A JPH0424554 A JP H0424554A JP 12700390 A JP12700390 A JP 12700390A JP 12700390 A JP12700390 A JP 12700390A JP H0424554 A JPH0424554 A JP H0424554A
- Authority
- JP
- Japan
- Prior art keywords
- calpastatin
- serum
- kit
- antibody
- amount
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 102100035037 Calpastatin Human genes 0.000 title claims abstract description 78
- 108010044208 calpastatin Proteins 0.000 title claims abstract description 78
- ZXJCOYBPXOBJMU-HSQGJUDPSA-N calpastatin peptide Ac 184-210 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H](CCSC)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC(O)=O)NC(C)=O)[C@@H](C)O)C1=CC=C(O)C=C1 ZXJCOYBPXOBJMU-HSQGJUDPSA-N 0.000 title claims abstract description 78
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title claims abstract description 21
- 201000010099 disease Diseases 0.000 title claims abstract description 20
- 238000000034 method Methods 0.000 title claims description 22
- 230000005856 abnormality Effects 0.000 title abstract description 5
- 210000002966 serum Anatomy 0.000 claims abstract description 27
- 238000004519 manufacturing process Methods 0.000 claims abstract description 16
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- 230000002159 abnormal effect Effects 0.000 claims description 8
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- 238000002835 absorbance Methods 0.000 abstract description 7
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- 238000011534 incubation Methods 0.000 abstract description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 abstract 1
- 229910052760 oxygen Inorganic materials 0.000 abstract 1
- 239000001301 oxygen Substances 0.000 abstract 1
- 238000005259 measurement Methods 0.000 description 14
- 208000010125 myocardial infarction Diseases 0.000 description 14
- 210000004027 cell Anatomy 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 210000003743 erythrocyte Anatomy 0.000 description 7
- 210000000056 organ Anatomy 0.000 description 7
- 102000013415 peroxidase activity proteins Human genes 0.000 description 7
- 108040007629 peroxidase activity proteins Proteins 0.000 description 7
- 238000011891 EIA kit Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 238000003018 immunoassay Methods 0.000 description 6
- 206010008132 Cerebral thrombosis Diseases 0.000 description 5
- 201000001429 Intracranial Thrombosis Diseases 0.000 description 5
- 206010008118 cerebral infarction Diseases 0.000 description 5
- 208000026106 cerebrovascular disease Diseases 0.000 description 5
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 102000007590 Calpain Human genes 0.000 description 4
- 108010032088 Calpain Proteins 0.000 description 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 238000011088 calibration curve Methods 0.000 description 4
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- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 2
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- 238000011161 development Methods 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 238000010904 focused beam reflectance measurement Methods 0.000 description 2
- 206010017758 gastric cancer Diseases 0.000 description 2
- 108091006086 inhibitor proteins Proteins 0.000 description 2
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- 239000012089 stop solution Substances 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- MLIWQXBKMZNZNF-KUHOPJCQSA-N (2e)-2,6-bis[(4-azidophenyl)methylidene]-4-methylcyclohexan-1-one Chemical compound O=C1\C(=C\C=2C=CC(=CC=2)N=[N+]=[N-])CC(C)CC1=CC1=CC=C(N=[N+]=[N-])C=C1 MLIWQXBKMZNZNF-KUHOPJCQSA-N 0.000 description 1
- 208000009746 Adult T-Cell Leukemia-Lymphoma Diseases 0.000 description 1
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- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
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- 102100023174 Methionine aminopeptidase 2 Human genes 0.000 description 1
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- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 1
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- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 1
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Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、ヒト心筋梗塞症、ヒト脳血栓症、ヒト癌に代
表される様なカルパスタチン産生異常症の検出方法及び
その定量用キットに関する。DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for detecting disorders of calpastatin production such as human myocardial infarction, human cerebral thrombosis, and human cancer, and a kit for quantifying the same.
カルパスタチンはカルパイン（ＢＣ３，４，２２゜１７
）を特異的に阻害する内在性インヒビタータンパク質で
あり、１９６８年にドラモント（Ｄｒｕｍｍｏｎｄ）
Ｇ、Ｉ、 ら〔ジャーナル オブ バイオロジカル
ケミストリー（Ｊ、 Ｂｉｏｌ、Ｃｈｅｍ、 ）第２４
１巻、第３０９７頁（１９６８） ）により、ホスホリ
ラーゼｂキナーゼの活性化因子の阻害因子として報告さ
れ、ニジムラ（Ｎｉｓｈｉｍｕｒａ）１、らしジャーナ
ル オブ バイオケミストリー（Ｊ、 Ｂｉｏｃｈｅｍ
、 ） 、第８４巻、第１６５７頁（１９７８）］によ
ってカルパインの特異的インヒビタータンパク質である
と報告された。現在までにそのｃＤＮＡ配列も報告され
ている〔アサダ（Ａｓａｄａ） Ｋ、 ラ、エンザイム
インヒビジョン（Ｂｎｚｙｍｅ Ｉｎｈｉｂ、） 、
第３巻、第４９頁（１９８９）］。Calpastatin is calpain (BC3,4,22゜17
) is an endogenous inhibitor protein that specifically inhibits
G, I, et al [Journal of Biology
Chemistry (J, Biol, Chem, ) No. 24
1, p. 3097 (1968)) as an inhibitor of the activator of phosphorylase b kinase, Nijimura 1, Rashi Journal of Biochemistry (J, Biochem).
), Vol. 84, p. 1657 (1978)] reported that it is a specific inhibitor protein of calpain. To date, its cDNA sequence has also been reported [Asada K, LA, Bnzyme Inhib,
Volume 3, page 49 (1989)].
カルパスタチンの生理的意義についてはカルパインを特
異的に阻害することにより、種々の酵素（プロティンキ
ナーゼＣ，）ランスグルタミナーゼ等）、細胞骨格タン
パク（ＭＡＰ２、ニュロフィラメント、ケラチン等）
レセプタータンパク（ＥＧＦレセプター ステロイド
ホルモンレセプター等）の活性を調節することで、細胞
の刺激応答系の一つとして理解されていた。The physiological significance of calpastatin is that by specifically inhibiting calpain, it inhibits various enzymes (protein kinase C, transglutaminase, etc.), cytoskeletal proteins (MAP2, neurofilament, keratin, etc.)
It was understood as one of the stimulus response systems of cells by regulating the activity of receptor proteins (EGF receptor, steroid hormone receptor, etc.).
一方、カルパスタチンのカルパイン阻害活性を指標とし
てラットの各種組織のカルパスタチン含量が検討され、
単位組織重量当りでは、肝、肺、心筋、肝細胞内に多量
に存在することが報告されている〔ムラチ（Ｍｕｒａｃ
ｈｉ） Ｔ、 ら、バイオケミストリー インターナ
ショナル（８ｉｏ−ｃｈｅｔｒｌＩｎｔ、） 、第２巻
、第６６１頁、 （１９８１）］ 。更にヒト赤血球よ
り精製したカルパスタチンを抗原としてウサギ抗カルパ
スタチンポリクローナル抗体が作製され、酵素免疫測定
法（ＢＩＡ）により、赤血球中のカルパスタチン量を測
定する方法が報告されているしタカノ（Ｔａｋａｎｏ）
Ｂ、 ら、ジャーナル 才ブ アプライド バイオ
ケミストリー（Ｊ、Ａｐｐｌ。Ｂｉｏｃｈｅｍ、） 、
第２巻、第１１７頁（１９８４）］。更にポントレモリ
（Ｐｏｎｔｒｅｍｏ Ｉｉ） Ｓ、 ら、〔バイオ
ケミカル アンド バイオフィジカル リサーチ コミ
ュニケーションズ（Ｂｉｏｃｈｅｍ、 Ｂｉｏｐｈｙ、
Ｒｅｓ、Ｃｏｍｍ、 ）、第１５７巻、第８６７頁（
１９８８）］は赤血球由来のカルパスタチンを抗原とし
、マウス抗カルパスタチンモノクローナル抗体を作製し
、赤血球中のカルパスタチン含量をウェスタンブロッテ
ィング法で測定し、本態性高血圧患者の赤血球中カルパ
スタチン量が健常人に比べ著しく低下していることが報
告されている。On the other hand, the content of calpastatin in various rat tissues was investigated using the calpain inhibitory activity of calpastatin as an index.
It has been reported that it exists in large amounts in the liver, lungs, myocardium, and hepatocytes per unit tissue weight [Murac
hi) T. et al., Biochemistry International (8io-chetrlInt.), Vol. 2, p. 661, (1981)]. Furthermore, a rabbit anti-calpastatin polyclonal antibody was produced using calpastatin purified from human red blood cells as an antigen, and a method for measuring the amount of calpastatin in red blood cells by enzyme immunoassay (BIA) has been reported.
B. et al., Journal of Applied Biochemistry (J, Appl. Biochem,),
Vol. 2, p. 117 (1984)]. Furthermore, Pontremo Ii S, et al. [Biochemical and Biophysical Research Communications (Biochem, Biophy,
Res, Comm, ), Volume 157, Page 867 (
[1988)] used calpastatin derived from red blood cells as an antigen, produced a mouse anti-calpastatin monoclonal antibody, and measured the calpastatin content in red blood cells by Western blotting. It has been reported that this is significantly lower than that of
また、成人Ｔ細胞白血病の原因ウィルスであるＨＴＬＶ
−Ｉが感染した細胞では非感染細胞に比較して、細胞内
カルパスタチン含量が増加すること〔アダナ（Ａｄａｃ
ｈ ｉ） Ｙ、ら、バイオロジカルケミストリー ホッ
プーセイラ−（Ｂ ｉｏ ］、Ｃｈｅｍ。In addition, HTLV, the virus that causes adult T-cell leukemia,
Intracellular calpastatin content increases in cells infected with -I compared to uninfected cells [Adana
h i) Y, et al., Biological Chemistry B io ], Chem.
Ｈｏｐｐｅ−８ａｙｌｅｒ） 、第３６９巻、第２２３
頁（１９８８）］が報告されている。Hoppe-8ayler), Volume 369, No. 223
Page (1988)] has been reported.
しかし、これらの報告はあくまでも赤血球内及び細胞内
・組織内のカルパスタチン含量の変化について述べてい
るにすぎない。また、ヒト疾病と組織・細胞内のカルパ
スタチン量の変化との関連は本態性高血圧症についての
みが報告されているにすぎず、その他の報告は、あくま
でも試験管内又は動物実験での結果である。更に、今ま
でにはヒト血清、血漿又は尿中にカルパスタチンが存在
すること、また、ヒト血清、血漿又は尿中のカルパスタ
チン量と特定の疾病との関連を報告した例はない。更に
、カルパスタチンの測定方法については活性測定法〔ム
ラカミ （Ｍｕｒａｋａｍｉ） Ｔ、ら、ジャーナル
オブ バイオケミストリー、第９０巻、第１８０９頁（
１９８１）〕、ポリクローナル抗体を用いたＥＩＡ、モ
ノクローナル抗体を用いたウェスタンプロティング法、
モノクローナル抗体を用いたＥＩＡ（特願平１−２１１
２５８号明細書記載）が報告されているが、これらの方
法は細胞・組織から抽出、又は精製したカルパスタチン
の測定方法についてのみが記載されてふり、血清、血漿
又は尿中のカルパスタチンとの反応性、更には血清、血
漿又は尿中にカルパスタチンが存在すること、そして血
清、血漿又は尿中のカルパスタチン量と疾病との関連に
ついてはなんら記載されていない。However, these reports merely describe changes in calpastatin content within red blood cells, cells, and tissues. Furthermore, the relationship between human diseases and changes in the amount of calpastatin in tissues and cells has only been reported for essential hypertension; other reports are based solely on in vitro or animal experiments. . Furthermore, there has been no report to date of the presence of calpastatin in human serum, plasma, or urine, or of the relationship between the amount of calpastatin in human serum, plasma, or urine and a specific disease. Furthermore, regarding the method for measuring calpastatin, the activity assay method [Murakami T., et al., Journal
of Biochemistry, Volume 90, Page 1809 (
1981)], EIA using polyclonal antibodies, Western protting method using monoclonal antibodies,
EIA using monoclonal antibodies (Patent application No. 1-211
258 specification), but these methods only describe methods for measuring calpastatin extracted from cells/tissues or purified; There is no mention of reactivity, the presence of calpastatin in serum, plasma or urine, or the relationship between the amount of calpastatin in serum, plasma or urine and disease.
本発明の目的は、カルパスタチンの産生異常を伴う疾病
を血清、血漿又は尿中のカルパスタチン量を指標として
検出する新規な測定方法及び定■用キットを提供するこ
とにある。An object of the present invention is to provide a novel measuring method and a diagnostic kit for detecting diseases accompanied by abnormal production of calpastatin using the amount of calpastatin in serum, plasma, or urine as an indicator.
本発明を概説すれば、本発明の第１の発明はカルパスタ
チン産生異常を伴う疾病の検出方法に関し、血清、血漿
又は尿中のカルパスタチン量を正常値と比較測定するこ
とを特徴とする。To summarize the present invention, the first aspect of the present invention relates to a method for detecting a disease accompanied by abnormal production of calpastatin, and is characterized by measuring the amount of calpastatin in serum, plasma, or urine in comparison with a normal value.
また、第２の発明はカルパスタチン産生異常を伴う疾病
の検出キットに関し、抗カルパスタチン抗体を含有する
ことを特徴とする。Moreover, the second invention relates to a detection kit for a disease accompanied by abnormal production of calpastatin, and is characterized by containing an anti-calpastatin antibody.
本発明者らは上記現状にかんがみて、鋭意研究を重ね、
血清、血漿又は尿中のカルパスタチン量を測定すること
により、カルパスタチン産生異常を伴う疾病が検出でき
ること、及び血清、血漿又は尿中のカルパスタチン■が
簡便に高感度で定量できるキットを作製し、本発明を完
成させた。In view of the above-mentioned current situation, the present inventors have conducted extensive research,
By measuring the amount of calpastatin in serum, plasma, or urine, diseases accompanied by abnormal production of calpastatin can be detected, and we have created a kit that can easily quantify calpastatin ■ in serum, plasma, or urine with high sensitivity. , completed the present invention.
以下、本発明の詳細な説明する。The present invention will be explained in detail below.
カルパスタチン産生異常を伴う疾病とは下記（１）、（
２）、（３）のごとき疾病である。Diseases associated with abnormal calpastatin production are as follows (1), (
These are diseases such as 2) and (3).
（１） カルパスタチン含量の比較的多い細胞、臓器
自体の破壊による疾患。(1) Diseases caused by destruction of cells and organs themselves that contain relatively high amounts of calpastatin.
（２）カルパスタチン含量の比較的多い細胞、臓器自体
の量的異常による疾患。(2) Diseases caused by quantitative abnormalities in cells or organs themselves that contain relatively high amounts of calpastatin.
（３）カルパスタチン含量の比較的多い細胞、臓器自体
の質的異常による疾患。(3) Diseases caused by qualitative abnormalities in cells or organs themselves that contain relatively high amounts of calpastatin.
上記（１）、（２）、（３）を具体的に説明すると、（
１）の例としては心筋梗塞、脳梗塞等、（２）の例とし
ては癌等、（３）の例としては動脈硬化症、血栓症等が
挙げられる。To specifically explain (1), (2), and (3) above, (
Examples of 1) include myocardial infarction and cerebral infarction; examples of (2) include cancer; and examples of (3) include arteriosclerosis and thrombosis.
本発明者らは脳血栓症、脳梗塞、心筋梗塞、癌由来の血
清、血漿又は尿中には、健常人よりも多量のカルパスタ
チンが存在することを確認し、血清、血漿又は尿中カル
パスタチンがカルパスタチン産生異常を伴う疾病の指標
として使用できることを見出した。The present inventors confirmed that greater amounts of calpastatin are present in serum, plasma, or urine derived from cerebral thrombosis, cerebral infarction, myocardial infarction, or cancer than in healthy individuals, and found that calpastatin in serum, plasma, or urine We found that this can be used as an indicator of diseases associated with abnormal calpastatin production.
本発明において、血清、血漿又は尿中のカルパスタチン
量を測定する方法としては特に限定はなく、例えば、活
性測定方法及び免疫学的方法が挙げられるが、感度・簡
便さにおいて免疫学的方法が好ましい。免疫学的方法に
おいては抗体を用いる方法があるが、この場合、１種又
は２種以上の抗体を用いた免疫学的測定法が使用できる
。抗体としてはモノクローナル抗体及びポリクローナル
抗体が用いられるが、その特異性、親和性の点からモノ
クローナル抗体を使用することが望ましい。一方、免疫
学的測定方法には酵素免疫測定法、ラジオイムノアッセ
イ法、ラテックス凝集法、免疫比濁法、蛍光・発光免疫
測定法、ウェスタンブロッティング法等があり、感度、
簡便さ、非放射能という点から酵素免疫測定法が望まし
い。In the present invention, the method for measuring the amount of calpastatin in serum, plasma, or urine is not particularly limited, and examples thereof include activity measurement methods and immunological methods, but immunological methods are preferred in terms of sensitivity and simplicity. preferable. Immunological methods include methods using antibodies, and in this case, immunoassay methods using one or more types of antibodies can be used. Monoclonal antibodies and polyclonal antibodies can be used as antibodies, and monoclonal antibodies are preferably used in terms of their specificity and affinity. On the other hand, immunoassay methods include enzyme immunoassay, radioimmunoassay, latex agglutination method, immunoturbidimetry, fluorescence/luminescence immunoassay, and Western blotting.
Enzyme immunoassay is desirable because it is simple and non-radioactive.
カルパスタチン量の測定に用いる抗カルパスタチン抗体
をキットとしておくことで、試料中のカルパスタチン量
を簡便に測定することができる。キットに用いる試薬は
溶液状でも良いし、凍結乾燥品でも良い。By preparing the anti-calpastatin antibody used for measuring the amount of calpastatin as a kit, the amount of calpastatin in the sample can be easily measured. The reagents used in the kit may be in solution form or may be in lyophilized form.
以下に本発明を実施例をもって説明するが、本発明が以
下の実施例の範囲のみに限定されるものではない。The present invention will be explained below with reference to examples, but the present invention is not limited to the scope of the following examples.
実施例１
２種のモノクローナル抗体を用いたサンドイッチＥＩＡ
法による臓器型カルパスタチン量の測定
（１）モノクローナル抗体結合ビーズの作製）１ｙｂｒ
ｉｄｏｍａ ＣＳ Ｌ ４−７ （微工研菌寄第１０９
１４号（ＰＢＲＭ Ｐ−１０９１４） ］が産生ずるモ
ノクローナル抗体Ｃ３Ｌ４−７を特願平１−２１１２５
８号明細書記載の方法に準じ調製し、その１ ｍｇを含
有する０、１Ｍリン酸バッファー （ｐＨ８，０）５０
−にポリスチレンボール（種水化学社製、粒径６．３５
ｍｍ） １００個を加え、５℃で１６時間反応させ抗
体をビーズに固定化させた。ビーズを生理食塩水で洗浄
後、１％ウシ血清アルブミン（ＢＳ＾）を含むリン酸緩
衝生理食塩水に浸し、５℃で一晩放置し、抗カルパスタ
チンモノクローナル抗体結合ビーズを得た。Example 1 Sandwich EIA using two types of monoclonal antibodies
Measurement of organotypic calpastatin amount by method (1) Preparation of monoclonal antibody-bound beads) 1ybr
idoma CS L 4-7
No. 14 (PBRM P-10914)] monoclonal antibody C3L4-7 produced by Patent Application No. 1-21125
0, 1M phosphate buffer (pH 8,0) containing 1 mg prepared according to the method described in Specification No. 8, 50
-Polystyrene balls (manufactured by Tanezu Kagaku Co., Ltd., particle size 6.35)
mm) were added and reacted at 5°C for 16 hours to immobilize the antibody on the beads. After washing the beads with physiological saline, they were immersed in phosphate buffered saline containing 1% bovine serum albumin (BS^) and left overnight at 5°C to obtain anti-calpastatin monoclonal antibody-conjugated beads.
（２）酵素標識モノクローナル抗体の作製Ｈｙｂｒｉｄ
ｏｍａ ＣＳ Ｌ １−３ （微工研菌寄第１０９１
３号（ＦＢＲＭ Ｐ−１０９１３） ］が産生ずるモノ
クローナル抗体Ｃ３ＬＩ−３を特願平１−２１１２５８
号明細書記載の方法に準じ調製し、該抗体にペルオキシ
ダーゼ（ベーリンガーーマンノ１イム社製）をナカネ（
Ｎａｋａｎｅ）らの方法〔ジャーナル 才ブ ヒストケ
ミストリー アンド シトケ ミ ス ト リ − （
Ｊ、）ｌｉｓｔｏｃｈｅｍ、 Ｃｙｔｏｃｈｅｍ、）
第２２巻、第１０８４頁（１９８７）〕１によって
結合させ、標識抗体を得た。すなわち１０ｍｇのペルオ
キシダーゼを２−の精製水に溶かし、０．１Ｍ過ヨウ素
酸カリウムを０．２ ｍｌ！加える。室温で２０分間反
応させた後１ ｍＭ酢酸バッファー（ｐＨ９，０）に対
し４℃で一晩透析する。一方、Ｃ３Ｌ１−３抗体２ ｍ
ｇを１．５−のリン酸緩衝生理食塩水（ｐＨ７，４）に
溶かし、１０ｍＭ炭酸バッファ （ｐＨ９，５）に対
し一晩４℃で透析しておき、これを上記の過ヨウ素酸処
理したペルオキシダーゼと混合し、室温で２時間反応さ
せた後、水素化ホウ素ナトリウム（４ｍｇ／ｍｌりを０
．１１ｎｌ添加し、４℃で２時間反応後、リン酸緩衝生
理食塩水（ｐ）１７．４）で平衡化したウルトロゲルＡ
ｃ＾２２（ＬＫＢ社製）を用いゲルろ過により分画し、
ペルオキシダーゼ活性と抗体活性の一致する両分を集め
、メルチオレートナトリウムを終濃度０．０１％となる
ように添加し、４℃で保存した。(2) Production of enzyme-labeled monoclonal antibody Hybrid
oma CS L 1-3
No. 3 (FBRM P-10913)] monoclonal antibody C3LI-3 produced by Patent Application No. 1-211258.
The antibody was prepared according to the method described in the specification, and peroxidase (manufactured by Boehringer Mannoim) was added to the antibody (
[Journal of Histochemistry and Cytochemistry - (
J,) Listochem, Cytochem,)
Vol. 22, p. 1084 (1987)]1 to obtain a labeled antibody. That is, dissolve 10 mg of peroxidase in 2- purified water, and add 0.2 ml of 0.1M potassium periodate! Add. After reacting at room temperature for 20 minutes, the mixture was dialyzed against 1 mM acetate buffer (pH 9,0) at 4°C overnight. On the other hand, C3L1-3 antibody 2 m
g was dissolved in 1.5-phosphate buffered saline (pH 7.4) and dialyzed against 10 mM carbonate buffer (pH 9.5) overnight at 4°C, which was then treated with periodic acid as described above. After mixing with peroxidase and reacting at room temperature for 2 hours, add sodium borohydride (4 mg/ml to 0.0
．． After adding 11nl of Ultrogel A and reacting at 4°C for 2 hours, it was equilibrated with phosphate buffered saline (p17.4).
Fractionated by gel filtration using c^22 (manufactured by LKB),
Both portions with matching peroxidase activity and antibody activity were collected, sodium merthiolate was added to a final concentration of 0.01%, and the mixture was stored at 4°C.
（３）カルパスタチンの測定
ＥＩＡ法は以下のようにして行った。試料３００μｌを
チューブに入れ、固相化抗体ビーズをチューブの中に１
個ずつ入れ３７℃で２０分間第１インキユベーシヨンを
行う。次に、ビーズを３−の生理食塩水で３回洗い、標
識抗体液（３００倍希釈）３００μｌをビーズの入った
チューブに入れ、３７℃で２０分間第２インキユベーシ
ヨンを行う。(3) Measurement of calpastatin The EIA method was performed as follows. Put 300 μl of the sample into a tube, and add 1 immobilized antibody bead into the tube.
The first incubation was performed at 37° C. for 20 minutes. Next, the beads are washed three times with physiological saline, 300 μl of labeled antibody solution (300-fold dilution) is placed in the tube containing the beads, and a second incubation is performed at 37° C. for 20 minutes.
次にビーズを３１ｎｌの生理食塩水で３回洗いビーズを
別のチューブに移し、これに発色試薬（０−）ユニレン
ジアミンｌ ｍｇ ／ −１■、０２の０．０１％を含
む０．１Ｍクエン酸ノくツファ−ｐＨ５，０）を加え、
室温で１５分間反応させ、ＩＮのＨ，ＳＯ，を１ｍｌ加
え、反応を停止させた。波長４９２ｎｍの吸光度を測定
しｊこ。The beads were then washed three times with 31 nl of saline and the beads were transferred to another tube containing 0.1% of the coloring reagent (0-) unilene diamine l mg/-1■, 0.1M. Add citric acid (pH 5,0),
The reaction was allowed to proceed at room temperature for 15 minutes, and 1 ml of IN H,SO was added to stop the reaction. Measure the absorbance at a wavelength of 492 nm.
臓器型カルパスタチン濃度の増加に伴って発色の吸光度
は増加し、本測定方法により臓器型カルパスタチンの測
定が可能であることが明らかとなった。The absorbance of the color development increased as the organotypic calpastatin concentration increased, and it became clear that organotypic calpastatin could be measured by this measurement method.
実施例２
２種のモノクローナル抗体を用いたサンドイッチＥＩＡ
法による臓器型及び赤血球型カルパスタチン全体量の測
定
（１）モノクローナル抗体結合ビーズの作製）１ｙｂｒ
ｉｄｏｍａ ＣＳ Ｌ ５−１２ （微工研菌寄第１
０９１５号（ＦＢＲＭ Ｐ−１０９１５） ）が産生ず
るモノクローナル抗体Ｃ３Ｌ５−１２を特願平１−２１
１２５８号記載の方法に準じ調製し、該抗体を用い実施
例１−（１）と同様にモノクローナル抗体結合ビーズを
作製した。Example 2 Sandwich EIA using two types of monoclonal antibodies
Measurement of total amount of organ type and erythroid type calpastatin by method (1) Preparation of monoclonal antibody-conjugated beads) 1ybr
idoma CS L 5-12 (Microtechnical Laboratory 1st
No. 0915 (FBRM P-10915)) has filed a patent application for the monoclonal antibody C3L5-12 produced by
The antibody was prepared according to the method described in No. 1258, and monoclonal antibody-bound beads were produced in the same manner as in Example 1-(1) using the antibody.
（２）酵素標識モノクローナル抗体の作製上記記実施例
１−（２）と同様に作製した。(2) Preparation of enzyme-labeled monoclonal antibody It was prepared in the same manner as in Example 1-(2) above.
（３）臓器型及び赤血球型カルパスタチン全量の測定
実施例２−（１）で作製した抗体結合ビーズと、実施例
２−（２）で作製した酵素標識抗体を用い、実施例１−
（３）と同様に測定を行った。臓器型及び赤血球型カル
パスタチン量の増加に伴って発色の吸光度は増加し、本
測定法によりカルパスタチン全量の測定が可能であるこ
とが明らかとなった。(3) Measurement of organ type and erythroid type calpastatin total amount Using the antibody-bound beads prepared in Example 2-(1) and the enzyme-labeled antibody prepared in Example 2-(2), Example 1-
Measurement was performed in the same manner as in (3). The absorbance of the color development increased with the increase in the amount of calpastatin in organ type and erythroid type, and it became clear that the total amount of calpastatin could be measured by this measurement method.
実施例３
臓器型カルパスタチン測定ＥＩＡキットの製造と各種患
者血清中臓器型カルパスタチン量の測定
（１）ＥＩＡキットの製造
ＥＩＡキット （１回分）の組成を以下に示す。Example 3 Measurement of organ-type calpastatin Production of an EIA kit and measurement of the amount of organ-type calpastatin in the serum of various patients (1) Production of an EIA kit The composition of the EIA kit (1 batch) is shown below.
１、 抗体固相化ビーズ １個（前出モ
ノクローナル抗体Ｃ３Ｌ４−７が固相化されたビーズ）
２、 ペルオキシダーゼ標識抗体 ０．３−（前出
モノクローナル抗体Ｃ３ＬＩ−３をペルオキシダーゼに
より標識した抗体）
３、 発色試薬 ０．３−（０−
フェニレンジアミンＨＣＩの１０ｍｇ／−１過酸化水素
０．０１％を含むクエン酸バッファーｐＨ５，０）
４、 反応停止液（ＩＮ硫酸） １． Ｏｉｆ
５、標準品（ｇｉ器梨型カルパスタチン３０．９０．２
７０．８１ Ｑ ｐｍｏｌ含む１％ ＢＳＡ／ＴＢＳ
）
（２））患者血漿中臓器型カルパスタチンの測定上記Ｅ
ＩΔキットを用い健常人２３例、脳梗塞患者７例、心筋
梗塞患者７例、脳血栓患者５例、胃癌患者３例の血清を
用いて、実施例１に基づいて臓器型カルパスタチン量を
測定した。1. Antibody-immobilized beads 1 (beads on which the monoclonal antibody C3L4-7 mentioned above is immobilized) 2. Peroxidase-labeled antibody 0.3- (antibody labeled with the monoclonal antibody C3LI-3 mentioned above with peroxidase) 3 , coloring reagent 0.3-(0-
Citric acid buffer pH 5,0 containing 10 mg/-1 of phenylenediamine HCI and 0.01% hydrogen peroxide) 4. Reaction stop solution (IN sulfuric acid) 1. Oif
5. Standard product (gi device pear-shaped calpastatin 30.90.2
70.81 Q 1% BSA/TBS including pmol
) (2)) Measurement of organ-type calpastatin in patient plasma E above
The amount of organotypic calpastatin was measured based on Example 1 using the IΔ kit using serum from 23 healthy subjects, 7 patients with cerebral infarction, 7 patients with myocardial infarction, 5 patients with cerebral thrombosis, and 3 patients with gastric cancer. .
標準品を用いて検量線を求めた結果第１図に示すような
検量線が得られた。第１図のグラフにおいて、横軸はカ
ルパスタチン濃度（ｐｍｏｌ／β）を縦軸は吸光度（Ａ
４ｓ２ｎｍ）を示す。試料を測定して得られた吸光度か
らこの検量線を用いて臓器型カルパスタチン量を求めた
。As a result of determining a calibration curve using a standard product, a calibration curve as shown in FIG. 1 was obtained. In the graph in Figure 1, the horizontal axis is calpastatin concentration (pmol/β), and the vertical axis is absorbance (A
4s2nm). The amount of organ-type calpastatin was determined from the absorbance obtained by measuring the sample using this calibration curve.
血清中臓器型カルパスタチンの測定結果を第２図に示す
。すなわち、第２図は本発明により測定した血清中カル
パスタチン量を症例別にプロットしたグラフである。The measurement results of organotypic calpastatin in serum are shown in FIG. That is, FIG. 2 is a graph in which the amount of calpastatin in serum measured according to the present invention is plotted for each case.
第２図に示すように、健常人と脳梗塞患者、心筋梗塞患
者、脳血栓患者、胃癌患者の血清中の臓器型カルパスタ
チン量の分布に違いが見られた。すなわち上記疾患患者
において血清中臓器型カルパスタチン量が健常人よりも
多いということが見出された。As shown in FIG. 2, differences were observed in the distribution of organotypic calpastatin levels in serum between healthy subjects and patients with cerebral infarction, myocardial infarction, cerebral thrombosis, and gastric cancer. That is, it was found that the amount of organotypic calpastatin in the serum of patients with the above disease was higher than that of healthy individuals.
実施例４
臓器型及び赤血球型カルパスタチン全量測定ＥＩＡキッ
トの製造と各種患者血清中カルパスタチン全量の測定
（１）ＥＩＡキットの製造
ＥＩＡキット（１回分）の組成を以下に示す。Example 4 Production of EIA kit for measurement of total amount of calpastatin in organ type and red blood cell type and measurement of total amount of calpastatin in serum of various patients (1) Production of EIA kit The composition of the EIA kit (one batch) is shown below.
１、 抗体固相化ビーズ １個（前出モ
ノクローナル抗体Ｃ３Ｌ５−１２が固相化されたビーズ
）
２、 ペルオキシダーゼ標識抗体 ０．３ｍｊ’（
前出モノクローナル抗体Ｃ３ＬＩ−３をペルオキシダー
ゼにより標識した抗体）
３、 発色試薬 ０．３艷（０−
フェニレンジアミンＨＣＩの１０■／−１過酸化水素０
．０１％を含むクエン酸バッファーｐＨ５，０）
４、 反応停止液（ＩＮ硫酸＞ ｉ、 ｏ −
５、標準品（赤血球型カルパスタチンを３０．９０．２
７０．８１０ ｐｍｏｌ含む１％Ｏ３＾／ＴＢＳ ）
（２））患者血清中カルパスタチン全量の測定上記ＥＩ
Ａキットを用いて実施例３−（２）と同一の試料を用い
て、実施例２に基づいてカルパスタチン全量を測定した
。その結果第２図とほぼ一致した測定結果が得られた。1. 1 antibody-immobilized bead (bead on which the monoclonal antibody C3L5-12 mentioned above is immobilized) 2. Peroxidase-labeled antibody 0.3 mj' (
3. Coloring reagent 0.3 (0-
10■/-1 hydrogen peroxide of phenylenediamine HCI 0
．． 4. Reaction stop solution (IN sulfuric acid > i, o -
5. Standard product (red blood cell type calpastatin 30.90.2
(1% O3^/TBS containing 70.810 pmol) (2)) Measurement of total amount of calpastatin in patient serum EI above
The total amount of calpastatin was measured based on Example 2 using the A kit and using the same sample as in Example 3-(2). As a result, measurement results almost consistent with those shown in FIG. 2 were obtained.
実施例５
心筋梗塞発症後の血清中カルパスタチン全量の経時変化
実施例４のキットを用いて心筋梗塞患者３例の発症後の
血清中カルパスタチン全量の経時変化を調べた。その結
果を第３図に示す。すなわち第３図は発作後日数（日、
横軸）とカルパスタチン濃度（ｐｍｏｌ／ｉ、縦軸）と
の関係を示すグラフである。第３図に示すごとく発作後
１日後には血清中カルパスタチン全量は著しく増加して
おり、かつ２〜１３日間高値を持続していた。この結果
、従来、心筋梗塞の生化学的診断法として用いられてき
たクレアチニンキナーゼは心筋壊死は数週間にわたって
持続しているにもかかわらず、梗塞後３〜４日で正常化
してしまい、診断法として不十分であったことを考える
と、発作後数日が経過して入院した症例でも、心筋梗塞
と診断できる新規な検出方法及びキットが提供されたこ
とを示すものである。Example 5 Change over time in total amount of calpastatin in serum after onset of myocardial infarction Using the kit of Example 4, changes over time in total amount of calpastatin in serum after onset of myocardial infarction were investigated in three patients with myocardial infarction. The results are shown in FIG. In other words, Figure 3 shows the number of days after the attack (days,
It is a graph showing the relationship between calpastatin concentration (pmol/i, vertical axis) and calpastatin concentration (pmol/i, vertical axis). As shown in Figure 3, the total amount of calpastatin in the serum increased significantly one day after the attack, and remained high for 2 to 13 days. As a result, creatinine kinase, which has traditionally been used as a biochemical diagnostic method for myocardial infarction, normalizes within 3 to 4 days after myocardial infarction, even though myocardial necrosis persists for several weeks. Considering that this was insufficient, this shows that a new detection method and kit have been provided that can diagnose myocardial infarction even in patients hospitalized several days after the attack.
〔発明の効果〕
以上詳細に述べたように、本発明により、血清、血漿又
は尿中のカルパスタチン量が心筋梗塞症、脳梗塞症、脳
血栓症、癌等に代表される様なカルパスタチン産生異常
を伴う疾病のマーカーとなることが見出され、上記疾病
の新たな検出方法及び定量用キットが開発された。[Effects of the Invention] As described in detail above, the present invention can reduce the production of calpastatin in cases where the amount of calpastatin in serum, plasma, or urine is typified by myocardial infarction, cerebral infarction, cerebral thrombosis, cancer, etc. It was discovered that it serves as a marker for diseases accompanied by abnormalities, and new detection methods and quantitative kits for the above-mentioned diseases were developed.
第１図はカルパスタチン量測定で使用する検量線を示す
グラフ、第２図は血清中のカルパスタチンの測定結果を
示すグラフ、第３図は心筋梗塞発症後の血清中カルパス
タチン全量の経時変化を示すグラフである。Figure 1 is a graph showing the calibration curve used to measure the amount of calpastatin, Figure 2 is a graph showing the measurement results of calpastatin in serum, and Figure 3 is the change over time in the total amount of calpastatin in serum after the onset of myocardial infarction. This is a graph showing.
Claims (1)
比較測定することを特徴とするカルパスタチン産生異常
を伴う疾病の検出方法。 ２、カルパスタチン産生異常を伴う疾病の検出用キット
であって、抗カルパスタチン抗体を含有することを特徴
とするカルパスタチン産生異常を伴う疾病の検出用キッ
ト。[Scope of Claims] 1. A method for detecting a disease accompanied by abnormal production of calpastatin, which comprises measuring the amount of calpastatin in serum, plasma, or urine in comparison with a normal value. 2. A kit for detecting a disease accompanied by abnormal calpastatin production, the kit comprising an anti-calpastatin antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP12700390A JP2866151B2 (en) | 1990-05-18 | 1990-05-18 | Method and kit for detecting calpastatin abnormality |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP12700390A JP2866151B2 (en) | 1990-05-18 | 1990-05-18 | Method and kit for detecting calpastatin abnormality |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0424554A true JPH0424554A (en) | 1992-01-28 |
| JP2866151B2 JP2866151B2 (en) | 1999-03-08 |
Family
ID=14949276
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP12700390A Expired - Fee Related JP2866151B2 (en) | 1990-05-18 | 1990-05-18 | Method and kit for detecting calpastatin abnormality |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2866151B2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996036708A1 (en) * | 1995-05-19 | 1996-11-21 | Progen Biotechnik Gmbh | Autoantigen which can be used to detect a tendency to thrombosis |
| WO2001072320A1 (en) * | 2000-03-28 | 2001-10-04 | Autogen Research Pty Ltd | A method of treatment of metabolic disorders and agents useful for same |
-
1990
- 1990-05-18 JP JP12700390A patent/JP2866151B2/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996036708A1 (en) * | 1995-05-19 | 1996-11-21 | Progen Biotechnik Gmbh | Autoantigen which can be used to detect a tendency to thrombosis |
| WO2001072320A1 (en) * | 2000-03-28 | 2001-10-04 | Autogen Research Pty Ltd | A method of treatment of metabolic disorders and agents useful for same |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2866151B2 (en) | 1999-03-08 |
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