1991 Fiscal Year Final Research Report Summary
Development and practical application of enzyme immunoassay of calpastatin
| Project/Area Number |
01870114
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| Research Category |
Grant-in-Aid for Developmental Scientific Research
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| Allocation Type | Single-year Grants |
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| Research Field |
Laboratory medicine
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| Research Institution | Kyoto University |
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Principal Investigator |
UEDA Kunihiro Kyoto University, Fac. of Med., Assoc. Prof., 医学部, 助教授 (00027070)
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| Co-Investigator(Kenkyū-buntansha) |
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)
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| Project Period (FY) |
1989 – 1991
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| Keywords | Calpain / Protease inhibitor / Calpastatin / Enzyme immunoassay / Monoclonal antibody / Domain structure / Immunohistochemistry / beta-Thalassemia |
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| Research Abstract |
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.
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