Center for Biotechnology and Interdisciplinary Studies, Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York, USA.
Biophys J. 2011 Oct 5;101(7):1749-57. doi: 10.1016/j.bpj.2011.08.036.
Monoclonal antibodies are typically monomeric and nonviscous at low concentrations, yet they display highly variable associative and viscous behavior at elevated concentrations. Although measurements of antibody self-association are critical for understanding this complex behavior, traditional biophysical methods are not capable of characterizing such concentration-dependent self-association in a high-throughput manner. Here we describe a nanoparticle-based method, termed self-interaction nanoparticle spectroscopy, that is capable of rapidly measuring concentration-dependent self-interactions for three human monoclonal antibodies with unique solution behaviors. We demonstrate that gold nanoparticles conjugated with antibodies at low protein concentrations (<40 μg/mL) display self-association behavior (as measured by the interparticle distance-dependent plasmon wavelength) that is well correlated with static light-scattering measurements obtained at three orders of magnitude higher antibody concentrations. Using this methodology, we find that the antibodies display a complex pH-dependent self-association behavior that is strongly influenced by the solution ionic strength. Importantly, we find that a polyclonal human antibody is nonassociative for all solution conditions evaluated in this work, suggesting that antibody self-association is more specific than previously realized. We expect that our findings will guide rational manipulation of antibody phase behavior, and enable studies that elucidate sequence and structural determinants of antibody self-association.
单克隆抗体在低浓度下通常为单体且无粘性，但在高浓度下表现出高度可变的缔合和粘性行为。尽管抗体自身聚集的测量对于理解这种复杂行为至关重要，但传统的生物物理方法无法以高通量的方式对这种浓度依赖性的自身聚集进行表征。在这里，我们描述了一种基于纳米颗粒的方法，称为自相互作用纳米颗粒光谱法，该方法能够快速测量三种具有独特溶液行为的人源单克隆抗体的浓度依赖性自身相互作用。我们证明，在低蛋白浓度（<40μg/mL）下与抗体偶联的金纳米颗粒显示出自身聚集行为（如通过颗粒间距离依赖的等离子体波长测量），与在三个数量级更高的抗体浓度下获得的静态光散射测量结果高度相关。使用这种方法，我们发现抗体显示出复杂的 pH 依赖性自身聚集行为，强烈受到溶液离子强度的影响。重要的是，我们发现多克隆人抗体在本工作中评估的所有溶液条件下均不具有聚集性，这表明抗体自身聚集比以前认识到的更为特异。我们期望我们的发现将指导抗体相行为的合理操作，并能够阐明抗体自身聚集的序列和结构决定因素。
