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

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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.