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

我们研究了一种非同位素配体结合分析与流式细胞仪使用生物素标记的细胞因子和荧光染料标记的链霉亲和素。另外，即使在异质细胞群中，也能够根据细胞表面抗原通过流式细胞术定量分析非常少量的受体。本研究探讨了将非同位素配体结合分析技术与流式细胞术结合应用于激素受体分析的可能性。本研究应用重组人促甲状腺激素（rhTSH）和表达促甲状腺激素受体的FRTL-5细胞系，探讨了分析促甲状腺激素受体表达的可能性。根据细胞因子的研究，我们将rhTSH生物素化。D值是流式细胞术中Kormogonov-Smirnov统计量的组成部分之一，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值显著较高（p<0. 05）。01）。在临床应用中，包括使用人血清的分析，最大D值应大于0。50.我们改进了生物素化rhTSH的效果和孵育方法。本研究尝试将荧光染料直接标记到rhTSH上。最大D值提高到0。12，这意味着特定的约束力。然而，应该考虑更有效地加强rhTSH的生物素化或荧光标记。

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.