期刊
ANALYTICAL CHEMISTRY
卷 82, 期 20, 页码 8671-8679出版社
AMER CHEMICAL SOC
DOI: 10.1021/ac101956e
关键词
-
资金
- Natural Sciences and Engineering Research Council of Canada (NSERC)
- Canadian Institutes for Health Research (CIHR)
- NSERC
- Michael Smith Foundation for Health Research (MSFHR)
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.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据