Tessler Lee A, Reifenberger Jeffrey G, Mitra Robi D
Center for Genome Sciences, Department of Genetics, Washington University in St. Louis School of Medicine, 4444 Forest Park Avenue, St. Louis, Missouri 63108, USA.
Anal Chem. 2009 Sep 1;81(17):7141-8. doi: 10.1021/ac901068x.
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 (1-5% 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.
在此，我们介绍一种通过计数结合的抗体来定量固定在表面的单个蛋白质分子的方法。我们系统地研究了许多阻碍对表面固定蛋白质进行稳健单分子检测的参数。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高效检测单个靶分子，并且我们表明这些抗体结合的时间长度足以对数十亿个单个蛋白质分子进行成像。该表面显示出低水平的非特异性蛋白质吸附，因此结合的抗体可以直接计数，而无需采用双色符合检测。我们通过计数结合的抗体分子准确地定量蛋白质丰度，并在实际血清样本中稳健地进行此操作。我们定量的抗体分子数量与固定的蛋白质分子数量呈线性关系（R² = 0.98），并且我们的精密度（1 - 5% CV）有助于可靠地检测丰度的微小变化（7%）。因此，我们的方法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进行准确的定量。有希望的是，我们可以用抗体多次探测流动池，这表明未来应该有可能进行多重单分子免疫分析。
