流式细胞术检测IgD与IgD受体结合实验方法的建立

Novel fluorescence based ligand-receptor binding assay : study on IgD receptor

目的建立非放射性荧光标记的配体受体结合实验,检测Ig D与其受体结合的作用特性。方法以配体受体结合理论为基础,利用荧光标记抗体技术,建立非放射性配体受体结合实验方法。利用流式细胞术检测人CD4+T细胞和人滑膜细胞上Ig D与Ig D受体结合的亲和力。结果人CD4+T细胞上Ig D与Ig D受体结合的解离常数(KD)和受体最大结合容量(Bmax)分别是8.10×10-10mol/L和6 605相对值/104细胞,人滑膜细胞上Ig D受体与Ig D结合的KD和Bmax值分别是7.02×10-10mol/L和5 977相对值/104细胞。结论该方法避免了传统受体与配体结合分析中放射性标记的问题,操作简便、稳定性好、灵敏度及选择性均较高,可为分析配体受体结合提供新的方法。

Objective To establish a fluorescence method for the detection of receptor binding assay and to detect the affinity of IgD ligand receptor binding. Methods Based on the experimental results of the ligand receptor bind- ing assay, fluorescence based receptor binding assay was established using fluorescent labeled antibody. The bind- ing affinity of IgD receptors on human CD4 ＋ T cells and human fibroblast-like synoviocytes （FLS） was detected by flow cytometry. Results The dissociation constant （ KD） and the maximum binding capacity （ B ） of IgD receptors on human CD4 ＋ T cells were 0. 810 nmol/L and 6 605 arbitrary unit/104 cells, respectively. The KD and Bmzx value of IgD receptors on human FLS was 0. 702 nmol/L and 5 977 arbitrary unit/104 cells respectively. Conclusion This method avoids the defects of the radioactive label in the binding of the receptor and the ligand, and offers simple operation, good stability, high sensitivity, which can provide a new way for the ligand and receptor binding.