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1993 Fiscal Year Final Research Report Summary

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
KeywordsGrowth hormone-releasing hormone / Somatostain / Enzyme immunoassay / Urtrasensitive / Streptavidin / Biotin / Sandwich assay
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

  • Research Products

    (12 results)

All Other

All Publications (12 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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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

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Published: 1995-03-27  

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