【摘要】 目的建立一种灵敏的定量检测人外周血单个核细胞(PBMC)细胞浆和细胞核内糖皮质激素受体α同型(GRα)的生物素-亲和素-酶联免疫吸附试验(ABS-ELISA)方法。方法分别提取人PBMC细胞浆和细胞核总蛋白,用GRα纯品或总蛋白浓度相等的细胞提取液包被酶标板,加入针对GRα的特异性兔抗人多克隆抗体(一抗)和山羊抗兔IgG(二抗),应用生物素-亲和素系统(ABS)进行放大,采用多因素棋盘滴定方法确定最佳实验条件,用GRα纯品建立ELISA标准曲线,定量检测GRα的含量。结果该研究确定应用0.1 mol/L碳酸盐缓冲液(pH 9.6)包被GRα纯品或细胞提取液,一抗浓度500 ng/100μL,二抗浓度400 ng/100μL,使用ABS放大系统,以TMB作为辣根过氧化物酶(HRP)的底物,于450 nm处检测吸光度值。该ABS-ELISA方法灵敏度为1μg/L,工作浓度范围1~100μg/L,批内变异系数(CV)为5.0%~6.9%,批间CV为7.1%~9.6%,不同操作者批内和批间的CV分别为4.7%和6.2%,均在实验允许范围内,可重复性良好。另外,该法无明显交叉反应,有一定特异性。结论该研究建立了一种特异性高、可重复性好、敏感、快速、简便的定量测定PBMC细胞浆和细胞核内GRα的新方法,对进一步探讨自身免疫病的激素疗效与患者GR的关系等方面具重要价值。更多还原

【Abstract】 Objective To establish a highly sensitive,repeatable quantitative avidin biotin system enzyme-linked immunosorbent assay(ABS-ELISA) to detect human glucocorticoid receptor α(hGRα) in peripheral blood mononuclear cells(PBMC).Methods PBMC from health control was incubated with dexamethasone(DEX) or PBS.PBMC total protein was extracted from cytoplasm and nucleus respectively by NE-PER extraction kits.The hGRα(1 ng/mL to 100 ng/mL) was coated with carbonate buffer. The working concentration of polyclonal antibody(rabbit anti-human) against hGRα was 5 μg/mL,and that of the goat anti-rabbit IgG was 4 μg/mL.To increase the sensitivity,biotin and streptavidin amplifying system was used.Results Of this ABS-ELISA,the sensitivity in detecting hRGα was 1 ng/mL,and the CVs was less than 10%.Conclusions A highly sensitive and repeatable ABS-ELISA method is established for detecting hGRα in the extraction of PBMC.更多还原