Grant-in-Aid for Developmental Scientific Research.
|Allocation Type||Single-year Grants|
|Research Institution||Kyoto University|
UEDA Kunihiro(1990-1991) Kyoto University, Fac. of Med., Assoc. Prof., 医学部, 助教授 (00027070)
上田 国寛(1990-1991) 京都大学, 医学部, 助教授
村地 孝(1989) 京都大学, 医学部, 教授 (10089104)
YOSHIMURA Manabu Kyoto Prefectural Medical University, Prof., 教授 (40094453)
ENDO Jiro Shimane Medical University, Prof., 教授 (20026892)
TOTANI Masayuki Nat. Inst. of Health and Nutrition, Sect. Chief, 栄養研究所, 部長 (70163988)
TABATA Masayoshi Kyoto University, Col. of Med. Tech., Asoc. Prof., 医療技術短期大学部, 助教授 (70115880)
KANNAGI Reiji Aichi Cancer Center, Res. Inst., Sect. Chief, 部長 (80161389)
上田 國寛 京都大学, 医学部, 助教授 (00027070)
|Project Period (FY)
1989 – 1991
Completed(Fiscal Year 1991)
|Budget Amount *help
¥14,700,000 (Direct Cost : ¥14,700,000)
Fiscal Year 1991 : ¥2,900,000 (Direct Cost : ¥2,900,000)
Fiscal Year 1990 : ¥3,500,000 (Direct Cost : ¥3,500,000)
Fiscal Year 1989 : ¥8,300,000 (Direct Cost : ¥8,300,000)
|Keywords||Calpain / Protease inhibitor / Calpastatin / Enzyme immunoassay / Monoclonal antibody / Domain structure / Immunohistochemistry / beta-Thalassemia / 高血圧症 / アルツハイマ-病 / ELISA / 繰り返しドメイン / リン酸化 / リンパ球 / HTLVーI / モノクロナル抗体 / プロテオリシス|
This study aimed at the development of an enzyme immunoassay (ELISA) system for calpastatin, i. e., an endogenous inhibitor of a Ca^<2+>-dependent cysteine protease (calpain), and the evaluation of its clinical value. Our main findings are sununarized as follows :
1. Preparation of anti-calpastatin antibodies -- By using calpastatins purified from porcine heart and human erythrocytes as immunogens, we obtained polyclonal as well as monoclonal antibodies.
2. Characterization of antibodies -- We identified the epitopes of these antibodies at various sites of domain 1 and 3 of calpastatin.
3. Immunohistochemistry of calpastatin -- By immunohistochernical techniques, we showed the localization of calpastatin in various tissues at a rm'croscopic, level.
4. Development of ELISA of calpastatin -- A sandwich-type ELISA system was developed employing two monocloiial antibodies with discrete epitopes.
5. Characterization of ELISA --- Our new ELISA enabled us to measure calpastatin as little as<100 pmol/L and also to differentiate between erythrocyte-type and organ-type calpastatins.
6. Clinical application of ELISA -- We quantitated calpastatin in blood, body fluid and tissue specimens obtained from patients of various diseases, such as beta-thalassemia, hypertension, and arthritis. Disease-specific changes in the calpain level suggested potential usefulness of this new ELISA for clinical-diagnosis.