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Development of the ultra sensitive sandwich assay of human inhibin and it's clinical applocations

人抑制素超灵敏夹心法的研制及其临床应用

基本信息

批准号：
08557091
负责人：
IBUKI Yoshito
金额：
$ 8.45万
依托单位：
Gunma University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08557091/
关键词：
Inhibin Sandwich Assay Clinical Applications アッセイ法高感度化

项目摘要

Inhibin shows molecular heterogeneity and there exist not only biologically active dimers but also immunologically active and biologically inactive monomers in human body fluid. It is also widely recognized that several proteins including follistatin and alpha 2 macroglobulin binding to inhibin and structurally related activin interfere with the assay of inhibin.In order to establish a highly sensitive and specific sandwich assay of biologically active inhibin, we investigated the character of human inhibin, produced a variety of antibodies against inhibin and studied several methods of labeling the antibodies.First we established a large scale purification system for inhibin from human follicular fluid. It was then possible to obtain the required amount of inhibin for a standard in the assay kit. Recombinant human inhibin B was expressed, purified and characterized and we confirmed that this recombinant inhibin is useful as an inhibin B assay standard (Hasegawa et al.J Reprod Develop … More 42 : 23-25,1996. Inhibin and Activin pp139-154,1996. Hasegawa et al. Sexual Differentiation and Maturation pp139-154,1996).Next, we investigated labeling methods using many kinds of polyclonal and monoclonal antibodies. Using an anti alpha subunit polyclonal antibody coated on a bead and anti beta_A subunit monoclonal antibody labeled by ^<125>I,we established an inhibin A radioimmunometric assay (Inhibin A IRMA) (Hasegawa et al.Inhibin, Activin, and Follistatin, pp104-117,1997). Changes in inhibin A concentrations in circulating blood during normal human pregnancy and abnormal pregnancy such as pregnancy induced hypertension and intrauterine growth retardation were clarified using this inhibin A IRMA (Mizunuma et al. Inhibin, Activin, and Follistatin pp151-161,1997). Because this inhibin A IRMA is not sensitive enough to measure the inhibin concentrations in circulating blood in non-pregnant women, further investigation has been done in order to improve its sensitivity. The polyclonal antibody was purified by an inhibin coupled column and an activin coupled column and thus an anti alpha subunit antibody and an anti beta_A subunit antibody were obtained. Inhibin A immunofluorometric assay (Inhibin A IFMA) was established using microtiter plates coated with the anti beta_A subunit antibody and the anti alpha subunit antibody labeled with Europium. This inhibin A IFMA is sensitive enough to measure inhibin A concentrations in the circulating blood of non-pregnant women. The study of the change of inhibin A concentrations in various physiological and pathological conditions is continuing. Less

抑制素表现出分子异质性，人体液中不仅存在具有生物活性的二聚体，还存在具有免疫活性和生物非活性的单体。人们还广泛认识到，包括卵泡抑素和α2巨球蛋白在内的多种蛋白质与抑制素和结构相关的激活素结合会干扰抑制素的测定。为了建立生物活性抑制素的高灵敏度和特异性夹心测定法，我们研究了人抑制素的特性，生产了多种针对抑制素的抗体，并研究了几种标记抑制素的方法。首先，我们建立了从人卵泡液中提取抑制素的大规模纯化系统。然后可以获得检测试剂盒中标准品所需量的抑制素。重组人抑制素 B 被表达、纯化和表征，我们证实该重组抑制素可用作抑制素 B 测定标准（Hasekawa 等人，J Reprod Develop … More 42：23-25，1996。Inhibin 和 Activin pp139-154，1996。Hasekawa 等人，性分化和成熟 pp139-154,1996）。接下来，我们研究了使用多种多克隆和单克隆抗体的标记方法。使用包被在珠子上的抗α亚基多克隆抗体和用125 I标记的抗β_A亚基单克隆抗体，我们建立了抑制素A放射免疫测定法(抑制素A IRMA)(Hasekawa等人，Inhibin、Activin和Follistatin，第104-117页，1997)。使用该抑制素 A IRMA 阐明了正常人类妊娠和异常妊娠（例如妊娠高血压和子宫内生长迟缓）期间循环血液中抑制素 A 浓度的变化（Mizunuma 等人，Inhibin、Activin 和 Follistatin，第 151-161 页，1997）。由于这种抑制素 A IRMA 的灵敏度不足以测量非孕妇循环血液中的抑制素浓度，因此进行了进一步的研究以提高其灵敏度。将多克隆抗体通过抑制素偶联柱和激活素偶联柱纯化，得到抗α亚基抗体和抗β_A亚基抗体。使用涂有抗β_A亚基抗体和铕标记的抗α亚基抗体的微量滴定板建立抑制素免疫荧光测定（抑制素A IFMA）。这种抑制素 A IFMA 足够灵敏，可以测量非孕妇循环血液中的抑制素 A 浓度。关于各种生理和病理条件下抑制素A浓度变化的研究仍在继续。较少的