Protein Quantification in Complex Mixtures by Solid Phase Single-Molecule Counting
Protein Quantification in Complex Mixtures by Solid Phase Single-Molecule Counting
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DOI：
10.1021/ac901068x
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发表时间：
2009-09-01
影响因子：
7.4
通讯作者：
Mitra, Robi D.
中科院分区：
文献类型：
--
作者：
Tessler, Lee A.;Reifenberger, Jeffrey G.;Mitra, Robi D.
在这里，我们提出了一种程序，通过计数结合的抗体来量化附着在表面上的单个蛋白质分子。我们系统地研究了阻碍表面固定蛋白质的健壮的单分子检测的许多参数。我们发现，化学吸附的牛血清白蛋白表面有助于用荧光抗体有效地检测单个目标分子，并且我们证明这些抗体结合的时间长度足以对数十亿个单独的蛋白质分子进行成像。这种表面表现出低水平的非特异性蛋白吸附，因此结合的抗体可以直接计数，而不需要使用双色符合检测。我们通过计算结合的抗体分子来准确地量化蛋白质丰度，并在现实世界的血清样本中稳健地执行这项工作。我们定量的抗体分子的数量与固定的蛋白质分子的数量线性相关(R-2=0.98)，我们的精确度(1-50%CV)有助于可靠地检测丰度的微小变化(7%)。因此，我们的程序允许以高灵敏度检测单个表面固定的蛋白质分子，并通过计数结合的抗体分子来准确地定量。有希望的是，我们可以用抗体多次探测流动细胞，这表明在未来应该有可能进行多重的单分子免疫分析。
Here we present a procedure for quantifying single protein molecules affixed to a surface by counting bound antibodies. We systematically investigate many of the parameters that have prevented the robust single-molecule detection of surface-immobilized proteins. We find that a chemically adsorbed bovine serum albumin surface facilitates the efficient detection of single target molecules with fluorescent antibodies, and we show that these antibodies bind for lengths of time sufficient for imaging billions of individual protein molecules. This surface displays a low level of nonspecific protein adsorption so that bound antibodies can be directly counted without employing two-color coincidence detection. We accurately quantify protein abundance by counting bound antibody molecules and perform this robustly in real-world serum samples. The number of antibody molecules we quantify relates linearly to the number of immobilized protein molecules (R-2 = 0.98), and our precision (1-50% CV) facilitates the reliable detection of small changes in abundance (7%). Thus, our procedure allows for single, surface-immobilized protein molecules to be detected with high sensitivity and accurately quantified by counting bound antibody molecules. Promisingly, we can probe flow cells multiple times with antibodies, suggesting that in the future it should be possible to perform multiplexed single-molecule immunoassays.