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

我们用生物素化细胞因子和荧光染料标记的链霉亲和素，用流式细胞术研究了非同位素配体结合分析。此外，我们还能够根据细胞表面抗原通过多色流式细胞术对极少量的受体进行定量分析，即使在异质细胞群体中也是如此。在这项研究中，我们探讨了将非同位素配体结合分析与流式细胞术应用于激素受体分析的可能性。我们研究了利用重组人促甲状腺激素(RhTSH)和表达TSH受体的FRTL-5细胞株分析促甲状腺激素(TSH)受体表达的可能性。根据前人对细胞因子的研究，我们对重组人促甲状腺激素进行了生物素化。D值是流式细胞术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度，则D值显著较高(p&lt；0。01)。在临床应用中，包括用人血清进行分析，最大D值应大于0。50美元。我们改进了生物素化重组人TSH的效果和孵育方法。在本研究中，我们尝试将荧光染料直接标记到重组人促甲状腺激素上。最大D值改进为0。12，这意味着特定的约束。然而，应该考虑更有效的强化生物素化或荧光标记的重组人促甲状腺激素。

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.