1997 Fiscal Year Final Research Report Summary
Development of an assay for thyrotropin receptor antibody using recombinant receptor and chemiluminescence.
| 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)
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| Project Period (FY) |
1995 – 1997
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| Keywords | recombinant receptor / Chemiluminescence / CHO cells / tagged receptor / thyrotropin receptor / receptor autoantibody / monoclonal antibody / hydrophobic chromatography |
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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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Research Products
(43 results)
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[Publications] Yoshida A, Sasaki N, Mori A, Taniguchi S, Mitani Y, Ueta Y, Hattori K, Sato R, Hisatome I, Mori T, Shigemasa C, Kosugi S.: "Different electrophysiological character of I^-,ClO_4^- and SCN^- in the transport by Na^+/I^- symporter." Biochem Biophys Res Commun. 231. 731-734 (1997)
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[Publications] Yoshida A,Sasaki N,Mori A,Taniguchi S,Mitani Y,Ueta Y,Hattori K,Sato R,Hisatome I,Mori T,Shigemasa C,Kosugi S.: "Different electrophysiological character of I^-, CIO_4^- and SCN^- in the transport by Na^+/I^- symporter" Biochem Biophys Res Commun. 231. 731-734 (1997)
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[Publications] Kohn L.D., Ban T., Okajima F., Shimura H., Shimura Y., Hidaka A., Guiliani C., Napolitano G., Kosugi S., Ikuyama S., Akamizu T., Tahara K., Saji M.: Cloning and regulation of glycoprotein hormone receptor genes. In : Molecular Endoclinology : Basic Concepts and Clinical Correlations. (ed.Weintraub B.D.). Raven Press, New York, 133-153 (1995)
Description
「研究成果報告書概要(欧文)」より
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