Kinetic determinations of molecular interactions using Biacore -: minimum data requirements for efficient experimental design
Kinetic determinations of molecular interactions using Biacore -: minimum data requirements for efficient experimental design
复制标题
DOI：
10.1002/jmr.745
复制
发表时间：
2005-07-01
影响因子：
2.7
通讯作者：
Andersson, K
中科院分区：
文献类型：
--
作者：
Önell, A;Andersson, K
可靠的动力学估计，可以从显着较少的数据比今天常用的，特别是在表征1：1的相互作用，涉及低分子量化合物和蛋白质。我们设计了一种合理且具有成本效益的策略，使用Biacore的基于表面等离子体共振的生物传感器来确定动力学常数，并表明可以大大减少精确动力学测定所需的测量次数，从而提高样品通量并节省样品材料。对一系列可能的1：1相互作用物的模拟和测量数据进行了研究，以找到用于动力学分析的数据集的最低要求。结果表明，在1：1相互作用模型中，在10（4）< k（a）< 10（7）M ~（-1）s ~（-1））（缔合）和10（-4）< k（d）< 10（-1）s ~（-1）（解离）范围内的动力学常数很容易确定。由于Biacore数据的信息密集性，仅需要两种样品浓度即可可靠地确定动力学。由10 μ M和1 nM之间的10倍稀释液组成的标准样品浓度系列一致地提供了至少两个浓度，该浓度具有关于该区域相互作用的足够信息。在这个区域之外，常数的测定变得越来越不可靠。如果速率常数被证明在指定区域之外或数据与1：1-MTL模型拟合不佳，则需要更多的实验。提供了一个具有成本效益的测定，以提供可靠的动力学测量设计的一般建议。版权所有（c）2005年约翰威利父子有限公司。
Reliable kinetic estimates can be obtained from significantly less data than is commonly used today, particularly in the characterization of 1:1 interactions involving low molecular weight compounds and proteins. We have designed a rational and cost-effective strategy to determine kinetic constants using Biacore's surface plasmon resonance-based biosensors and show that the number of measurements necessary for accurate kinetic determinations can be greatly reduced, increasing sample throughput and saving sample material. Simulated and measured data for a range of possible 1:1 interactants were studied to find the minimum requirements of a data set for kinetic analysis. The results showed that kinetic constants in the region 10(4) < k(a) < 10(7) M-1 s(-1) (association) and 10(-4) < k(d) < 10(-1) s(-1) (dissociation) could easily be determined in a 1:1 interaction model. Owing to the information-dense nature of Biacore data, only two sample concentrations were necessary to reliably determine the kinetics. A standard sample concentration series consisting of 10-fold dilutions between similar to 10 mu M and similar to 1nM consistently provided at least two concentrations with sufficient information about the interaction in this region. Determinations of the constants became increasingly unreliable outside this region. If the rate constants prove to be outside the specified region or the data fits poorly to the 1:1-MTL model, more experiments are required. General recommendations for the design of a cost-effective assay to deliver reliable kinetic measurements are provided. Copyright (c) 2005 John Wiley & Sons, Ltd.