CAJ | 학술논문

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612 nm和670 nm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/μ1、100 μmol/L和1 μmol/L.在上述条件下,检测到Sunitinib 对 VEGFR-2酪氨酸激酶抑制活性的IC_(50)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.
목적 건립일충균상시간분변형광면역분석방법용우단백락안산격매억제제적체외고통량사선.방법 근거유련혈상화합물(EuK)화이조람단백(XL-665)량개형광화합물재격발후적능량공진전이소발출적특이성형광,채용다공능매표의재파장위612 nm화670 nm처측정형광신호적변화;이혈관내피생장인자2(VEGFR-2)위단백락안산격매,검측단백락안산격매억제제Sunitinib적억제활성.결과 건립료일충용우단백락안산격매억제제체외고통량사선적균상시간분변형광면역분석방법.재반응체계중,단백락안산격매VEGFR-2、삼린산선감(ATP)화다태저물농도분별위5ng/μ1、100 μmol/L화1 μmol/L.재상술조건하,검측도Sunitinib 대 VEGFR-2락안산격매억제활성적IC_(50)위86.7nmol/L,여문헌보도적결과근사.결론 본실험소건립적균상시간분변형광면역분석방법조작간편,중복성호,가용우단백락안산격매억제제적체외고통량사선.
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-2) kinase activity was investigated. Results A homogeneous time-resolved fluorescence immunoassay was established for high throughput screening of PTK inhibitor. In this system, the concentrations of VEGFR-2, adenosine triphosphate (ATP) and poly-peptide substrate were 5 ng/μ1, 100 μmol/L and 1 μmol/L,respectively. Sunitinib inhibited VEGFR-2 kinase activity with an IC_(50) value of 86.7 nmol/L, which was close to the values tested using other methods. Conclusion The homogeneous time-resolved fluorescence immunoassay we established can be easily used for high throughput screening of PTK inhibitors.