A functional quantitative receptor assay using flluoscein labeled hormones with a carbohydrate chain for the evaluation of the hormonal replacement therapy

使用带有碳水化合物链的荧光素标记激素进行功能定量受体测定，以评估激素替代疗法

基本信息

批准号：
21590621
负责人：
ONO Takaaki
金额：
$ 3万
依托单位：
Hamamatsu University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2009
资助国家：
日本
起止时间：
2009 至 2011
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-21590621/
关键词：
ホルモン TSH ビオチン化フローサイトメトリー糖鎖受容体

项目摘要

We have studied a non-isotopic ligand binding assay with the flow cytometry using biotinylated cytokines and fluorescent dye labeled streptavidin. Also, we have enabled to analyze a very small amount of receptors quantitatively according to the cell surface antigen by multicolor flow cytometry even in the heterogeneous cell population. In this study, we examined the possibility of applying the non-isotopic ligand binding assay with the flow cytometry into the hormone receptor assay. We examined the possibility of analysis of the thyrotropin(TSH) receptor expression by using recombinant human TSH(rhTSH) and FRTL-5 cell line which expresses TSH receptors. We biotinylated the rhTSH according to the previous studies in cytokines. D value, which is one of the components of Kormogonov-Smirnov statistics in flow cytometry, is useful for quantitative analysis of hormone receptor. If the assay system has enough sensitivity and specificity, D value is significantly high(p<0. 01). In clinical usage including the analyses using human serum, maximum D value should be needed more than 0. 50. We improved the efficacy of biotinylation to rhTSH and incubation methods. In this study, we tried to label the fluorescent dye to the rhTSH directly. Maximum D value improved to 0. 12, which meant specific binding. However, it should be considered about the more effective intensification of biotinylation or fluorescent labeling of the rhTSH.

我们使用生物素化细胞因子和荧光染料标记的链霉亲和素，通过流式细胞术研究了非同位素配体结合测定。此外，即使在异质细胞群中，我们也能够通过多色流式细胞术根据细胞表面抗原定量分析极少量的受体。在本研究中，我们研究了将流式细胞术非同位素配体结合测定应用于激素受体测定的可能性。我们检验了使用重组人TSH(rhTSH)和表达TSH受体的FRTL-5细胞系分析促甲状腺素(TSH)受体表达的可能性。我们根据之前的细胞因子研究对rhTSH进行了生物素化。 D值是流式细胞术中Kormogonov-Smirnov统计的组成部分之一，可用于激素受体的定量分析。如果检测系统具有足够的灵敏度和特异性，则 D 值显着较高（p<0. 01）。在临床使用中，包括使用人血清进行分析，最大D值应大于0。50。我们改进了rhTSH生物素化的功效和孵育方法。在这项研究中，我们尝试将荧光染料直接标记到rhTSH上。最大D值提高到0. 12，这意味着特异性结合。然而，应该考虑更有效地强化rhTSH 的生物素化或荧光标记。