Poulsen Nicklas N, Andersen Nina Z, Østergaard Jesper, Zhuang Guisheng, Petersen Nickolaj J, Jensen Henrik
Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark.
Analyst. 2015 Jul 7;140(13):4365-9. doi: 10.1039/c5an00697j.
Rapid and sensitive quantification of protein based biomarkers and drugs is a substantial challenge in diagnostics and biopharmaceutical drug development. Current technologies, such as ELISA, are characterized by being slow (hours), requiring relatively large amounts of sample and being subject to cumbersome and expensive assay development. In this work a new approach for quantification based on changes in diffusivity is presented. The apparent diffusivity of an indicator molecule interacting with the protein of interest is determined by Taylor Dispersion Analysis (TDA) in a hydrodynamic flow system. In the presence of the analyte the apparent diffusivity of the indicator changes due to complexation. This change in diffusivity is used to quantify the analyte. This approach, termed Flow Induced Dispersion Analysis (FIDA), is characterized by being fast (minutes), selective (quantification is possible in a blood plasma matrix), fully automated, and being subject to a simple assay development. FIDA is demonstrated for quantification of the protein Human Serum Albumin (HSA) in human plasma as well as for quantification of an antibody against HSA. The sensitivity of the FIDA assay depends on the indicator-analyte dissociation constant which in favourable cases is in the sub-nanomolar to picomolar range for antibody-antigen interactions.
对基于蛋白质的生物标志物和药物进行快速、灵敏的定量分析,是诊断学和生物制药药物研发中的一项重大挑战。当前的技术,如酶联免疫吸附测定(ELISA),具有检测速度慢(数小时)、需要相对大量的样本且检测方法开发繁琐且昂贵等特点。在这项工作中,提出了一种基于扩散系数变化的定量分析新方法。在流体动力学流动系统中,通过泰勒分散分析(TDA)来测定与目标蛋白质相互作用的指示分子的表观扩散系数。在存在分析物的情况下,指示分子的表观扩散系数会因络合作用而发生变化。这种扩散系数的变化被用于定量分析物。这种方法被称为流动诱导分散分析(FIDA),其特点是速度快(数分钟)、具有选择性(可在血浆基质中进行定量分析)、全自动且检测方法开发简单。FIDA已被用于定量分析人血浆中的蛋白质人血清白蛋白(HSA)以及抗HSA抗体。FIDA检测的灵敏度取决于指示物 - 分析物解离常数,在有利情况下,对于抗体 - 抗原相互作用,该常数处于亚纳摩尔至皮摩尔范围内。