| Project/Area Number |
07557353
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
内分泌・代謝学
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
KOSUGI Shinji Kyoto University School of Medicine, Assistant, 医学研究科, 助手 (50252432)
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| Co-Investigator(Kenkyū-buntansha) |
SASAJIMA Masaaki Ciba-Corning Diagnostics, Investigator, 開発部開発課, 研究員
SUGAWA Hiodeo Kyoto University School of Medicine, Lecturer, 医学研究科, 講師 (70162857)
AKAMIZU Takashi Kyoto University School of Medicine, Assistant, 医学研究科, 助手 (20231813)
森 徹 京都大学, 医学研究科, 教授 (40026894)
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| Project Period (FY) |
1995 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1997: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1996: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Keywords | recombinant receptor / Chemiluminescence / CHO cells / tagged receptor / thyrotropin receptor / receptor autoantibody / monoclonal antibody / hydrophobic chromatography / 固相化 / TSH受容体 / 組替え受容体 / 抗受容体抗体 / 甲状腺 / CHO-K1細胞 / Hela S-3細胞 |
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| Research Abstract |
We investigated to introduce a specific, easy and highly sensitive method for TBII (Thyrotropin Binding Inhibitory Immunoglobulin) assay using chemiluminescence and recombinant thyrotropin receptor (TSHR) protein instead of radioisotpe and porcine thyroid membrane. As a recombinant TSHR protein, we examined expression of fulllength and extracellular domain of the TSHR using bacteria, baculovirus and mammalian cells. Because bacteria and baculovirus systems produced a lot of TSHR protein but glycan component and 3-dimensional structure were different from native receptor, it turned out to be difficult to use them in a practical assay. TSHR protein produce by mammalian cells (such as CHO cells) transfected with TSHR cDNA was considered to be functionally and structurally native. The extracellular domain of the TSHR can ben obtained as a secreted from. It is quite advantageous for mass production and handling of the receptor protein molecule. Further, it has become possible to highly purify the receptor protein by inserting histidine-tag and/or FLAG-tag in the amino terminal portion of the receptor.
By hydrophobic chromatography, the specificity of labelled TSH receptor increased but binding affinity of the receptor was slightly decreased ; further improvement and investigation is necessary.
Several kinds of monoclonal antibodies with positive TBII activities were obtained by transforming B cells from patients with autoimmune thyroid disease. These can be used to standardize the assay.
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