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

下丘脑激素超灵敏非竞争性酶联免疫分析方法的研制及其临床应用

• 批准号: 04671485
• 负责人: AZUMA Hiroyuki
• 金额: $ 1.34万
• 依托单位: The University of Tokushima
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04671485/
• 关键词: Growth hormone-releasing hormone; Somatostain; Enzyme immunoassay; Urtrasensitive; Streptavidin; Biotin; Sandwich assay; Growth hormone-releasing hormoe

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

本文采用双抗体夹心法建立了下丘脑激素（生长激素释放激素（GHRH）和生长抑素（SRIF））的非竞争性酶免疫分析（EIA）方法。我们首先尝试获得两种能识别C端和N端的抗体，建立夹心酶免疫分析，但只获得了识别GHRH（1-44）C端而不识别N端的抗体和SRIF（1-14）中间部分的抗体。用亲和层析法纯化抗血清，并将其IgG组分用于EIA。EIA采用夹心法，利用亲和素（链霉亲和素）对生物素的高亲和力和抗原抗体反应进行。用生物素氨基己酸N-羟基琥珀酰亚胺酯将肽生物素化，并与固定在平板上的抗生物素蛋白和上述纯化的特异性抗体反应。日 ...更多信息 将形成的复合物进一步与山羊抗兔IgG-过氧化物酶结合物和邻苯二胺（OPD）反应，并在420 nm处测量显色。通过该测定方法，肽可以在5.0- 1，000 pg/管（GHRH）和0.61-100 pg/管（SRIF）的范围内定量。该方法的灵敏度与传统的竞争性放射免疫分析法相当。在正常受试者中，血浆GHRH和SRIF水平为27（）SY。±-。（）9.7pg/ml，15（）SY.（）6.4pg/ml（平均值）SY. ±.待解决的问题是由于非特异性反应而导致的相对强的显色，而与良好的剂量-响应曲线无关。但在测定体系中加入10%正常山羊血清和使用非特异性反应低的抗血清，可使OPD因非特异性反应而显色降至可忽略的水平。将来，将考虑在320 nm、405 nm处进行荧光测定，而不是使用OPD的比色法，以提高测定灵敏度。为了减少非特异性反应，目前正在研究将生物素化肽捕获到特异性抗肽IgG包被的聚苯乙烯球上的方法。少

项目成果

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Bando H、Zhang CY、Yamasaki R、Saito S、Oshima T、Matsumoto K：“生长激素产生的垂体腺瘤患者的病理结果和生长激素反应的差异”Endocrinol Japan。

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)

Yasuhiro Okauchi：“通过脑内微透析测量促甲状腺激素释放激素”日本秘密研究学会杂志（摘要）68. 435（1992）。

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)

Hideo Takahashi、Yasumi Shintani、Takayuki Tsuchihashi、Shiro Saito：“大鼠垂体中生长抑素浓度的变化 - 使用脑内微透析的研究”日本内分泌学会杂志（摘要）68. 320（1992）。

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)

Hideo Takahashi：“大鼠垂体中生长抑素浓度的昼夜变化 - 使用脑内微透析方法的研究”日本内分泌学会杂志（摘要）68. 808（1992）。

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)

Takahashi H.，Bando H.，Saito S.：“利用脑微透析技术研究大鼠生长抑素释放的调节机制”第九届国际内分泌学大会（Abstr）。