Microfluidic Measurement of Antibody-Antigen Binding Kinetics from Low-Abundance Samples and Single Cells
Microfluidic Measurement of Antibody-Antigen Binding Kinetics from Low-Abundance Samples and Single Cells
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DOI：
10.1021/ac101956e
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发表时间：
2010-10-15
影响因子：
7.4
通讯作者：
Hansen, Carl L.
中科院分区：
文献类型：
--
作者：
Singhal, Anupam;Haynes, Charles A.;Hansen, Carl L.
我们提出了一种简单的微流控荧光珠法，可以用标准倒置荧光显微镜准确地测量抗体-抗原结合动力学。我们测量了抗体-抗原相互作用的结合和解离速率常数，跨越平衡结合亲和力(30 PM-100 NM)近4个数量级。提出了两种不同版本的检测方法，可以通过使用荧光标记的抗原直接测量解离率，或者使用未标记的抗原间接测量解离率。我们还使用光谱分离和空间定位相结合的方法，展示了多种抗体-抗原相互作用的同时、多路结合测量。与表面等离子共振(SPR)光谱和替代测量技术相比，微流控装置中固定的8×10(4)抗体分子(类似于132个齐托莫尔)和少于2×10(6)个抗体(类似于3个attomoles)的完整抗体-抗原结合动力学被测量，检测下限和样品消耗减少了4个数量级。我们发现，微流控微珠试验，当与小体积隔室相结合时，能够直接测量单个杂交瘤细胞分泌的抗体的抗原结合动力学。我们预计，该方法将作为一种常规的分析工具，用于研究分子相互作用以及筛选从免疫动物中分离的初级抗体分泌浆细胞。
We present a simple microfluidic fluorescence bead assay for accurately measuring antibody-antigen binding kinetics with a standard inverted fluorescent microscope. We measured association and dissociation rate constants from antibody-antigen interactions spanning nearly 4 orders of magnitude in equilibrium binding affinity (30 pM-100 nM). Two versions of this assay are presented, which allow for dissociation rate measurements either directly, by use of fluorescently labeled antigen, or indirectly, by use of unlabeled antigen. We also demonstrate simultaneous, multiplexed binding measurements of multiple antibody-antigen interactions using a combination of spectral separation and spatial localization. Complete antibody-antigen binding kinetics were measured for as little as 8 x 10(4) antibody molecules (similar to 132 zeptomoles) immobilized on a single bead and less than 2 x 10(6) antibodies (similar to 3 attomoles) loaded into the microfluidic device, a reduction in detection limit and sample consumption of 4 orders of magnitude when compared to surface plasmon resonance (SPR) spectroscopy and alternative measurement techniques. We show that the microfluidic bead assay, when combined with small volume compartmentalization, enables direct measurement of antigen binding kinetics of antibodies secreted from single hybridoma cells. We anticipate that this assay will be useful as a routine analytical tool for studying molecular interactions as well as for screening primary antibody-secreting plasma cells isolated from immunized animals.