基于ELISA、TRFIA及AlphaScreen技术的血管内皮生长因子受体3拮抗剂筛选模型的构建及比较

Construction and comparison of the screening assay methods for vascular endothelial growth factor receptor-3 tyrosine kinase based on ELISA,TRFIA and AlphaScreen assays

血管内皮生长因子受体3(VEGFR-3)为淋巴管特异标记物,与肿瘤淋巴管生成和转移具有明显的相关性,VEGFR-3胞内域的酪氨酸激酶(TK)直接参与了胞内信号转导途径,因此VEGFR-3 TK抑制剂可能成为抑制肿瘤淋巴道转移的有效药物。采用大肠杆菌成功表达人源VEGFR-3酪氨酸激酶(VEGFR-3 TK),并以其为靶点,分别构建了基于ELISA,TR-FIA及AlphaScreen技术的高通量药物筛选模型,并从反应条件、方法学参数及相关性等方面对这三种模型进行了评估及比较。AlphaScreen采用均相反应系统,反应速度最快。TRFIA和AlphaScreen由于采用了稀土离子作为标记物,其灵敏度和检测范围大大高于ELISA,且自动化程度高。样品检测结果表明,三种方法的相关性较好,其中TRFIA和AlphaS-creen技术适用于大规模药物筛选,具有很好的应用前景。

Vascular endothelial growth factor receptor 3（VEGFR-3） is expressed on lymphatic endothelium and may be involved in tumor angiogenesis and growth,thus it is an attractive target for researches seeking to prevent tumor growth and metasta-sis.In the present study,the tyrosine kinase of VEGFR-3 was purified inEscherichia coliexpression system and the enzyme-linked immunoassay（ELISA）,time-resolved fluoroimmunoassay（TRFIA） and light initiated chemiluminescence assay（Al-phaScreen） were established as the screening assay for inhibitor of VEGFR-3 TK.The assay conditions,assay parameters and correlation of these 3 methods were compared.The results showed AlphaScreen was least time consuming,TRFIA and Al-phaScreen had the best sensitivity and excellent dynamic range and seemed to be automatic.The correlation of these 3 methods was highly significant,in which TRFIA and AlphaScreen assay formats should be suiable for the drug screening in large scale and they may have good outlook of application for the identification of VEGFR-3 TK inhibitors.