DNA-linked inhibitor antibody assay (DIANA) as a new method for screening influenza neuraminidase inhibitors
作者:Milan Kožíšek、Václav Navrátil、Kateřina Rojíková、Jana Pokorná、Carlos Berenguer Albiñana、Petr Pachl、Jitka Zemanová、Aleš Machara、Pavel Šácha、Jason Hudlický、Ivana Císařová、Pavlína Řezáčová、Jan Konvalinka
DOI:10.1042/bcj20180764
日期:2018.12.12
effective against resistant influenza strains. A facile, sensitive, and inexpensive screening method would help achieve this goal. Recently, we described a multiwell plate-based DNA-linked inhibitor antibody assay (DIANA). This highly sensitive method can quantify femtomolar concentrations of enzymes. DIANA also has been applied to high-throughput enzyme inhibitor screening, allowing the evaluation of inhibition
流感神经氨酸酶负责从感染的细胞表面逸出新的病毒颗粒。几种神经氨酸酶抑制剂在临床上用于治疗患者或因紧急情况而储存。然而,对批准的抑制剂的病毒抗性的不断发展强调了需要开发对抗性流感病毒株有效的新型抗病毒剂。简便,敏感且便宜的筛选方法将有助于实现此目标。最近,我们描述了基于多孔板的DNA连接的抑制剂抗体测定(DIANA)。这种高度灵敏的方法可以定量酶的飞摩尔浓度。DIANA也已应用于高通量酶抑制剂的筛选,可从单一抑制剂浓度评估抑制常数。在这里,我们报告设计,连接到报告DNA寡核苷酸的tamiphosphor衍生物的合成和结构表征,以开发用于筛选潜在流感神经氨酸酶抑制剂的DIANA型测定。神经氨酸酶首先被固定的抗体捕获,然后测试化合物与寡聚连接的检测探针竞争与酶的结合,然后通过qPCR进行定量。我们通过与标准荧光分析法进行比较验证了该新方法,并证明了其对敏感的神经氨酸酶检测以及潜在的新型神经氨酸酶