A SENSITIVE IMMUNORADIOMETRIC ASSAY FOR HUMAN ADRENOCORTICOTROPIC HORMONE
A SENSITIVE IMMUNORADIOMETRIC ASSAY FOR HUMAN ADRENOCORTICOTROPIC HORMONE
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人肾上腺皮质激素的灵敏免疫放射测定法
DOI：
10.2220/biomedres.10.491
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发表时间：
1989
影响因子：
1.2
通讯作者：
N. Yanaihara
中科院分区：
文献类型：
--
作者：
A. Ito;M. Ohbayashi;M. Hane;H. Takeda;Fumiko Iimuro;N. Yonezawa;M. Okada;H. Ohno;K. Iguchi;T. Mochizuki;N. Yanaihara
以合成的人促肾上腺皮质激素（ACTH）[1 - 39]为免疫原，制备了抗ACTH单克隆抗体和抗ACTH多克隆抗体，建立了一种简便、灵敏的人ACTH免疫放射分析法（IRMA）。将对人ACTH的18-39序列具有特异性的单克隆抗体固定在用于固相的聚苯乙烯珠上，并对I-24序列具有特异性的多克隆抗血清进行放射性标记。该方法依赖于由1251标记的多克隆抗体、人ACTH和固相单克隆抗体组成的免疫复合物的形成。固相上的放射性与标本中ACTH的含量成正比。该测定系统的最低检测限约为5 pg/ml。用该测定系统测量的正常受试者血浆中的免疫反应性ACTH水平的范围为10.4pg/ml（平均值± 1 SD）至51.7pg/ml（平均值± 1 SD）。批内和批间变异系数的范围分别为3.16.70/0和12.6-14.1”/0。对正常受试者血浆的HPLC分析揭示了单一免疫反应性形式，其以与合成人ACTH相同的保留时间洗脱（1-39）。该测定法对于测量生物体液和组织中的人ACTH将是非常有用的。免疫放射测定（IRMA）已成功用于测量几种循环多肽激素，包括人促肾上腺皮质激素（ACTH）（1，4，8）。与传统的放射免疫分析相比，这种方法的理论优势包括增强的灵敏度和特异性。本报告描述了一个敏感和可靠的IRMA测量血浆ACTH在未提取的标本的发展。该测定系统依赖于1251标记的ACTH氨基端特异性兔多克隆抗体、人ACTH和ACTH羧基端特异性固相单克隆抗体的免疫复合物的形成。使用该测定系统，测量正常受试者的血浆ACTH水平。材料和方法
A simple and sensitive immunoradiometric assay (IRMA) for intact human adrenocorticotropic hormone (ACTH) was developed using a monoclonal and a polyclonal antiserum both of which were raised against synthetic human ACTH(]—39) as an immunogen. A monoclonal antibody, specific for the 18-39 sequence of human ACTH, was immobilized on polystyrene beads for the solid phase, and a polyclonal antiserum specific for the I-24 sequence was radiolabelled. This method relies on the formation ofan immune complex consisting ofa 1251-labelled polyclonal antibody, human ACTH, and the solid phase monoclonal antibody. Radioactivity on the solid phaseis directly proportional to the amount of ACTH present in the specimen. The minimal detection limit of this assay system was approximately 5 pg/ml. Immunoreactive ACTH levels in plasma of normal subjects, as measured with this assay system, ranged from 10.4 pg/ml (mean+l SD) to 51.7 pg/ml (mean+ l SD). The ranges for the intraand interassay coefficients of variance were 3.16.70/0 and 12.6-14.1"/0, respectively. HPLC analysis of plasma of normal subjects revealed a single immunoreactive form which eluted with the same retention time as that of synthetic human ACTH(1-39). This assay will be extremely useful for the measurement of human ACTH in biologic fluids and tissues. The immunoradiometric assay (IRMA) has been successfully used for the measurement of several circulating polypeptide hormones, including human adrenocorticotropic hormone (ACTH) (l, 4, 8). The theoretical advantages of this approach include both enhanced sensitivity and specificity compared to the conventional radioimmunoassay. The present report describes the development of a sensitive and reliable IRMA for the measurement of plasma ACTH in unextracted specimens. This assay system depends upon the formation of an immune complex of 1251labelled rabbit polyclonal antibody specific for the amino-terminal ofACTH, human ACTH, and a solid phase monoclonal antibody specific for the carboxy-terminal ofACTH. Using this assay system, plasma ACTH levels in normal subjects were measured. MATERIALS AND METHODS