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Development of urtrasensitive noncompetitive enzyme immunoassay for hypothalamic hormone and it's clinical application

Research Project

Project/Area Number 04671485
Research Category

Grant-in-Aid for General Scientific Research (C)

Allocation TypeSingle-year Grants
Research Field 内分泌・代謝学
Research InstitutionThe University of Tokushima

Principal Investigator

AZUMA Hiroyuki  The University of Tokushima, School of Medicine Instructor, 医学部附属病院, 助手 (10241275)

Co-Investigator(Kenkyū-buntansha) NISHIDA Toshinobu  The University of Tokushima, School of Dentistry Instructor, 医学部附属病院, 助手 (50156080)
SHUNTANI Yasumi  The University of Tokushima, School of Medicine Instructor, 医学部附属病院, 助手 (10235773)
YOKOGOSAHI Yutaka  The University of Tokushima, School of Medicine Instructor, 医学部, 助手 (00253203)
Project Period (FY) 1992 – 1993
Project Status Completed (Fiscal Year 1993)
Budget Amount *help
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1993: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1992: ¥1,600,000 (Direct Cost: ¥1,600,000)
KeywordsGrowth hormone-releasing hormone / Somatostain / Enzyme immunoassay / Urtrasensitive / Streptavidin / Biotin / Sandwich assay / Growth hormone-releasing hormoe
Research Abstract

Sensitive noncompetitive enzyme immunoassay (EIA) for hypothalamic hormone (growth hormone-releasing hormone, GHRH ; somatostation, SRIF) was developed using sandwich method.
Specific antibody was raised in rabbits by immunizing them with an antigen-carrier protein complex. We first tried to obtain two kinds od antibody, which recognize the C-terminal or the N-terminal portion of the peptides, to establish sandwich EIA.However, only antibodies that C-terminal portion but not the N-terminal portion of GHRH(1-44), and the middle portion of SRIF(1-14) were obtained. Thus these antisera were purified by affinity chromatography, and their IgG fractions were used to EIA.
EIA was conducted by sandwich method utilizing high affinity of avidin (streptavidin) to biotin and antigen-antibody reaction. The peptides were biotinylated using biotinamidocaproate N-hydroxysccinimide ester and reacted with avidin, which had been immobilized to a plate, and the purified specific antibody described above. Th … More is complex formed was further reacted with goat anti-rabbit IgG-peroxidase conjugate and o-phenylenediamine (OPD), and the color development at 420 nm was measured. By means of this assay method, the peptides could be quantified in a range of 5.0-1,000 pg/tube (GHRH) and 0.61-100 pg/tube (SRIF). The assay sensitivity was almost the same as that by the conventional competitive radioimmunoassay. In normal subjects, the Plasma GHRH and SRIF levels were 27 (〕SY.+-.〔) 9.7 pg/ml and 15 (〕SY.+-.〔) 6.4 pg/ml(mean (〕SY.+-.〔) SD), respectively.
The problem to be solved was a relatively strong color development due to nonspecific reaction irrespective of the good dose-response curves. However, the color development of OPD due to non-specigic reaction was decreased to a negligible level by adding normal goat serum (10%) to the assay system, and by the use of antiserum with low non-specific reaction. In the future, Fluorometry at 320 nm, 405 nm, instead of colorimetry using OPD will be considered to increase the assay sensitivity. To reduce non-specific reaction, the method to trap biotinylated peptide to specific anti-peptide IgG-coated polystirene balls is now under investigation. Less

Report

(3 results)
  • 1993 Annual Research Report   Final Research Report Summary
  • 1992 Annual Research Report
  • Research Products

    (24 results)

All Other

All Publications (24 results)

  • [Publications] Tsuchihashi T.,Saito S.: "Production of immunoreactive corticotropin-releasing hormone in various neuroendocrine tumors" Jpn J Clin Oncol. 22. 232-237 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Takahashi H.,Bando H.,Saito S.: "Mechanism of impaired growth hormone secretion in patients with Cushing's disease" Acta Endocrinol. 127. 13-17 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] 高橋秀夫、新谷保実、土橋孝之、斎藤史郎: "ラット下垂体におけるソマトスタチン濃度の変動-脳内微小透析法による検討" 日内分泌会誌(抄録). 68. 320 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Takahashi H.,Bando H.,Saito S.: "Study on regulatory mechanism of somatostatin release in the rat using brain microdialysis technique" Ninth International Congress of Endocrinology(Abstr). 301 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Takahashi H.,Bando H.,Saito S.: "Measurement of somatostatin release in the rat brain by microdialysis" J Neurosci Method. 9(in press). (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] 高橋秀夫、斎藤史郎: "脳内微小透析法による神経ペプチドの解析" 日内分泌会誌. 70(印刷中). (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] 板東浩、高橋秀夫、新谷保実、斎藤史郎: "研究年報 Annual Research Reports 16" 財団法人 成長科学協会, 401-410 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Tsuchihashi T, Yamaguchi K, Abe K, Yanaihara N, Saito S: "Production of immunoreactive corticotropin-releasing hormone in various neuroendocrine tumors" Jpn J Clin Oncol. 22. 232-237 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Takahashi H, Bando H, Zhang CY, Yamasaki R, Saito S: "Mechanism of impaired growth hormone secretion in patients with Cushing's disease." Acta Endocrinol. 127. 13-17 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Bando H, Zhang CY, Yamasaki R, Saito S, Oshima T, Matsumoto K: "Differences in pathological findings and growth hormone response in patients with growth hormone-producung pituitary adenoma" Endocrinol Japan. 39. 355-363 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Takahashi H, Shintani Y, Bando H, Saito S: "Study on regulatory mechanism of somatostatin release in the rat using brain microdialysis technique" Ninth International Congress of Endocrinology(Abstract). 435 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Takahashi H, Shintani Y, Bando H, Saito S: "Measurement of somatostatin release in the rat brain by microdialysis" J Neurosci Method. 9(in press). (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Tsuchihashi T.,Saito S.: "Production of immunoreareactive corticotropin-releasing hormone in various neuroendocrine tumors" Jpn J Clin Oncol. 22. 232-237 (1992)

    • Related Report
      1993 Annual Research Report
  • [Publications] Takahashi H.,Bando H.,Saito S.: "Mechanism of impaired growth hormone secretion in patients with Cushing's disease" Acta Endocrinol. 127. 13-17 (1992)

    • Related Report
      1993 Annual Research Report
  • [Publications] 岡内泰弘、高橋秀夫、新谷保実、板東浩、斎藤史郎: "ラット視床下部におけるThyrotropin-Releasing Hormone(TRH)の分泌動態-脳内微小透析法による検討" 日内分泌会誌(抄録). 69. 727 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Takahashi H.,Bando H.,Saito S.: "Study on regulatory mechanism of somatostatin release in the rat using brain microdialysis technique" Ninth International Congress of Endocrinology(Abstr). 301 (1992)

    • Related Report
      1993 Annual Research Report
  • [Publications] Takahashi H.,Bando H.,Saito S.: "Measurement of somatostatin release in the rat brain by microdialysis" J Neurosci Method. 9 (in press). (1994)

    • Related Report
      1993 Annual Research Report
  • [Publications] 高橋秀夫、斎藤史郎: "脳内微小透析法による神経ペプチドの解析" 日内分泌会誌. 70 (印刷中). (1994)

    • Related Report
      1993 Annual Research Report
  • [Publications] 板東浩、高橋秀夫、新谷保実、斎藤史郎: "研究年報 Annual Research Reports 16" 財団法人 成長科学協会, 401-410 (1992)

    • Related Report
      1993 Annual Research Report
  • [Publications] Takayuki Tsuchihashi: "Production of immunoreactive corticotropin-releasing hormone in various neuroendocrine tumors" Japanese Journal of Clinical Oncology. 22. 232-237 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] 高橋 秀夫: "ラット下垂体におけるソマトスタチン濃度の変動 -脳内微小透析法による検討" 日本内分泌学会雑誌(抄録). 68. 320 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] 岡内 泰弘: "脳内微小透析法によるThyrotropin-Releasing Hormoneの測定" 日本内分秘学会雑誌(抄録). 68. 435 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] 高橋 秀夫: "ラット下垂体におけるソマトスタチン濃度の日内変動 -脳内微小透析法による検討" 日本内分泌学会雑誌(抄録). 68. 808 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Hideo Takahashi: "Study on regulatory mechanism of somatostatin release in the rat using brain microdialysis technique" International Congress of Endocrinology(abst). 301 (1992)

    • Related Report
      1992 Annual Research Report

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Published: 1992-04-01   Modified: 2016-04-21  

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