Macromolecular binding and kinetic analysis with optically sectioned planar format assays
Macromolecular binding and kinetic analysis with optically sectioned planar format assays
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DOI：
10.1039/c2an35134j
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发表时间：
2012-01-01
期刊：
影响因子：
4.2
通讯作者：
Hanley, Quentin S.
中科院分区：
文献类型：
--
作者：
Ghafari, Homanaz;Parambath, Mithun;Hanley, Quentin S.
由于溶液荧光的背景干扰，通常不使用荧光强度方法进行大分子相互作用和配体与表面上受体的竞争性结合的实时分析。具有共聚焦检测的无分离光学切片平面格式测定（OSPFA）消除了这个问题。我们报告OSPFAs的指示剂位移和动力学评估的结合。商业雄激素受体结合结构域指示剂置换测定法适用于OSPFA，对于睾酮产生6.5 nM的IC 50，Z' = 0.77。这些测量的IC 50和Z'值在适合于药物筛选应用的范围内，其中Z' > 0.5表明良好至优异的筛选性。应用OSPFA研究抗体与平面表面上的夹心免疫测定的结合速率。Langmuir拟合提供了2 x 10（3）M-1 s（-1）至6 x 10（4）M-1 s（-1）范围内的正向速率常数和1 x 10（-4）s（-1）至4 x 10（-3）s（-1）范围内的反向常数，这涵盖了表征探针-靶标相互作用的有用范围。这项工作证明了OSPFAs在免洗涤条件下使用荧光研究动力学和结合相互作用的适用性。OSPFA是其他无分离方法的替代方法，例如声学、表面等离子体共振、椭圆偏振、荧光偏振和其他相关方法。OSPFA应进行小分子和大分子的实际动态结合研究，包括研究中的反应在表面上没有明显质量变化的情况。
Real-time analysis of macromolecular interactions and competitive binding of ligands to receptors on surfaces are not typically performed using fluorescence intensity methods due to background interference from solution fluorescence. Separation-free optically sectioned planar format assays (OSPFAs) with confocal detection remove this problem. We report OSPFAs for indicator displacement and kinetic assessment of binding. A commercial androgen receptor binding domain indicator displacement assay adapted as an OSPFA yielded an IC50 of 6.5 nM for testosterone with Z' = 0.77. These measured IC50 and Z' values are in ranges suitable for drug screening applications with Z' > 0.5 indicating good to excellent screenability. An OSPFA was applied to study the rate of antibody binding to a sandwich immunoassay on a planar surface. Langmuir fits provided forward rate constants in the range 2 x 10(3) M-1 s(-1) to 6 x 10(4) M-1 s(-1) and reverse constants 1 x 10(-4) s(-1) to 4 x 10(-3) s(-1) which cover a useful range for characterising probe-target interactions. This work demonstrates the suitability of OSPFAs for investigating kinetics and binding interactions using fluorescence under wash-free conditions. OSPFAs are an alternative to other separation-free methods such as acoustic, surface plasmon resonance, ellipsometry, fluorescence polarisation and other related methods. OSPFAs should make practical dynamic binding studies for small and large molecules including cases where the reaction under investigation results in no appreciable mass change on a surface.