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

抑制素具有分子异质性，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无免疫活性的单体。人们也普遍认识到，与抑制素和结构相关的激活素结合的卵泡抑素和α 2巨球蛋白等几种蛋白质会干扰抑制素的测定。为了建立一种高灵敏度、高特异性的生物活性抑制素夹心检测方法，我们研究了人抑制素的特性，制备了多种抑制素抗体，并研究了几种抗体的标记方法。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。然后可以在测定试剂盒中获得所需的抑制素量。重组人抑制素B的表达，纯化和鉴定，我们证实了重组抑制素B是有用的抑制素B测定标准（Hasegawa et . j .生殖与发展杂志42:23-25,1996）。抑制素与激活素[j]，1996。Hasegawa等人。性别分化和成熟pp139-154,1996)。接下来，我们研究了多种多克隆和单克隆抗体的标记方法。利用包被在球头上的抗α亚基多克隆抗体和标记为^<125>I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（inhibin a IRMA） （Hasegawa et al.Inhibin, Activin, and Follistatin, pp104-117,1997）。使用该抑制素A IRMA （Mizunuma et al.）阐明了正常妊娠和异常妊娠（如妊高症和宫内生长迟缓）期间循环血液中抑制素A浓度的变化。抑制素，激活素和卵泡抑素，pp151-161,1997)。由于该抑制素A IRMA对非孕妇循环血液中抑制素浓度的测量不够敏感，为了提高其敏感性，我们进行了进一步的研究。该多克隆抗体经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。采用包被抗β A亚基抗体和标记Europium的抗α亚基抗体的微滴板，建立了抑制素A免疫荧光测定法（IFMA）。这种抑制素A IFMA足够敏感，可以测量非孕妇循环血液中的抑制素A浓度。抑制素A浓度在各种生理病理条件下的变化研究仍在继续。少

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