Development of Kinetic Ligand-binding Assays Using a Fiber Optic Sensor.
Development of Kinetic Ligand-binding Assays Using a Fiber Optic Sensor.
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使用光纤传感器开发动力学配体结合测定。
DOI：
10.1093/clinchem/45.9.1683
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发表时间：
1999
影响因子：
9.3
通讯作者：
Wittliff
中科院分区：
文献类型：
--
作者：
Smith;Lemon;Erb;Erb;Downward;Ulrich;Wittliff
配体结合分析在生化研究和临床测定中普遍存在。在大多数常见的测定方法中，结合进行直到已经获得平衡条件，这导致相对长的孵育时间。在信号检测之前将反应混合物中的结合试剂与游离试剂分离的要求排除了对结合事件的直接观察。为了减少测定的持续时间并促进动力学分析，基于渐逝场技术的方法最近已用于测定开发中。这些包括光纤荧光传感器（1）（2）（3）和表面等离子体共振（4）。具体地，倏逝场技术基于以下观察：当光以接近全内反射的临界角的角度行进通过波导时，在波导的表面上产生倏逝场。这个场福尔斯随着离表面的距离呈指数下降，并且对波导表面和表面所在的介质的折射率非常敏感。在表面等离子体共振中，这种对折射率的敏感性用于测量结合到波导表面的物质的量。在渐逝场荧光测定法中，场刺激荧光团，其通过特异性结合相互作用附着于表面（2）。本文描述了使用渐逝光纤荧光传感器表征IgG和单价Fab（均对雌酮-1-葡糖苷酸（E1g）具有特异性）以及人雌激素受体α（hER-α）与携带E1g或特异性雌激素反应元件（ERE）的纤维的配体结合，并证明了表观缔合和解离结合常数的测定。
将从Polymicro Technologies获得的熔融二氧化硅光纤切割成11.5-cm长，并使用Fluorinert ™（3M）从光纤的7.0-cm部分去除包层。为了评估IgG、Fab和hER-α的结合动力学，纤维被…
Ligand-binding assays are ubiquitous in biochemical research and clinical determinations. In most common assay methods, binding proceeds until an equilibrium condition has been obtained, which leads to relatively long incubation times. The requirement for separating bound from free reagents in the reaction mixture before signal detection precludes direct observation of binding events. To reduce the duration of an assay and to facilitate kinetic analysis, methods based on evanescent field technology have recently been used in assay development. These include fiber optic fluorometric sensors (1)(2)(3) and surface plasmon resonance (4). Specifically, evanescent field technology is based on the observation that when light travels through a waveguide at angles approaching the critical angle for total internal reflection, an evanescent field is produced on the surface of the waveguide. This field falls off exponentially with distance from the surface and is exquisitely sensitive to the refractive indices of the waveguide surface and the medium in which the surface resides. In surface plasmon resonance, this sensitivity to refractive index is used to measure the amount of substance that binds to the waveguide surface. In evanescent field fluorometry, the field stimulates fluorophores, which become attached to the surface through specific binding interactions (2). We describe here the use of evanescent fiber optic fluorometric sensors to characterize ligand binding of IgG and monovalent Fab, both specific for estrone-1-glucuronide (E1g), and of the human estrogen receptor α (hER-α) to fibers bearing E1g or the specific estrogen response element (ERE) and demonstrate the determination of apparent association and dissociation binding constants.
Fused silica optical fibers obtained from Polymicro Technologies were cleaved in 11.5-cm lengths, and the cladding was removed from a 7.0-cm portion of the fiber using Fluorinert™ (3 M). To assess the binding kinetics of IgG, Fab, and hER-α, fibers were sensitized by …
雌激素结构对核结合和受体复合物诱导孕激素受体的影响。
影响因子：
2.9
作者：
VanderKuur,JA;Wiese,T;Brooks,SC
通讯作者：
Brooks,SC