用于蛋白酪氨酸激酶抑制剂高通量筛选的均相时间分辨荧光免疫方法的建立

Establishment of homogeneous time-resolved fluorescence immunoassay for high throughput screening of protein tyrosine kinase inhibitors

目的建立一种均相时间分辨荧光免疫分析方法用于蛋白酪氨酸激酶抑制剂的体外高通量筛选。方法根据铕联穴状化合物(EuK)和异藻蓝蛋白(XL-665)两个荧光化合物在激发后的能量共振转移所发出的特异性荧光,采用多功能酶标仪在波长为612nm和670nm处测定荧光信号的变化;以血管内皮生长因子2(VEGFR-2)为蛋白酪氨酸激酶,检测蛋白酪氨酸激酶抑制剂Sunitinib的抑制活性。结果建立了一种用于蛋白酪氨酸激酶抑制剂体外高通量筛选的均相时间分辨荧光免疫分析方法。在反应体系中,蛋白酪氨酸激酶VEGFR-2、三磷酸腺苷(ATP)和多肽底物浓度分别为5ng/μl、100μmol/L和1μmol/L。在上述条件下,检测到Sunitinib对VEGFR-2酪氨酸激酶抑制活性的IC50为86.7nmol/L,与文献报道的结果近似。结论本实验所建立的均相时间分辨荧光免疫分析方法操作简便,重复性好,可用于蛋白酪氨酸激酶抑制剂的体外高通量筛选。

Objective To establish an in vitro homogeneous time-resolved fluorescence immunoassay method for high throughput screening of protein tyrosine kinase (PTK) inhibitors. Methods Specific fluorescence signals at 670 and 612 nm were measured by multifunctional microplate reader when the fluorescence was emitted through a resonance energy transfer between fluorescent materials (EuK and XL-665). The inhibitory activity of Sunitinib, a standard PTK inhibitor, on vascular endothelia growth factor receptor 2 (VEGFR-...