A quantitative assessment of heterogeneity for surface-immobilized proteins
A quantitative assessment of heterogeneity for surface-immobilized proteins
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DOI：
10.1021/ac000523p
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发表时间：
2001-02-01
影响因子：
7.4
通讯作者：
Leckband, DE
中科院分区：
文献类型：
--
作者：
Vijayendran, RA;Leckband, DE
许多生物技术应用使用固定在固体支持物上的蛋白质受体。尽管在溶液中，这些受体对其互补配体表现出同质的结合亲和力和缔合/解离动力学，但它们在固定后通常表现出异质的结合特征。在这项研究中，使用基于荧光的光纤生物传感器来量化与可溶性分析物（荧光标记的三硝基苯）与表面固定的单克隆抗 TNT 抗体结合相关的异质性。通过五种不同的固定策略将抗体固定在二氧化硅光纤探针上。我们使用 Sips 等温线来评估和比较这些不同固定方案的抗体结合亲和力和动力学速率参数的异质性。此外，我们使用二室传输动力学模型对动力学数据进行了全局分析，以分析分析物-抗体动力学的异质性。这些分析提供了一种定量工具，可用于评估不同抗体制剂的相对同质性。我们的结果表明，更均匀的蛋白质制剂表现出更均匀的亲和力和动力学常数。
Many biotechnological applications use protein receptors immobilized on solid supports. Although, in solution, these receptors display homogeneous binding affinities and association/dissociation kinetics for their complementary ligand, they often display heterogeneous binding characteristics after immobilization. In this study, a fluorescence-based fiber-optic biosensor was used to quantify the heterogeneity associated with the binding of a soluble analyte, fluorescently labeled trinitrobenzene, to surface-immmoblized monoclonal anti-TNT antibodies. The antibodies were immobilized on silica fiber-optic probes via five different immobilization strategies. We used the Sips isotherm to assesses and compare the heterogeneity in the antibody binding affinity and kinetic rate parameters for these different immobilization schemes. In addition, we globally analyzed kinetic data with a two-compartment transport-kinetic model to analyze the heterogeneity in the analyte-antibody kinetics. These analyses provide a quantitative tool by which to evaluate the relative homogeneity of different antibody preparations. Our results demonstrate that the more homogeneous protein preparations exhibit more uniform affinities and kinetic constants.