Nano-Microscopy of Therapeutic Antibody Aggregates in Solution
Nano-Microscopy of Therapeutic Antibody Aggregates in Solution
复制标题
溶液中治疗性抗体聚集体的纳米显微镜观察
DOI：
10.1007/978-1-0716-1450-1_13
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发表时间：
2022
影响因子：
--
通讯作者：
Honda Shinya
中科院分区：
文献类型：
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作者：
Senga Yukako;Ogura Toshihiko;Imamura Hiroshi;Honda Shinya
扫描电子辅助介电显微镜（SE-ADM）是一种用于观察水溶液中生物样品精细结构的新型显微镜技术。SE-ADM的一个主要优点是不需要样品预处理，包括脱水、干燥和染色，而传统的扫描电子显微镜（SEM）中不可缺少的预处理会导致样品变形。此外，样品在SE-ADM中没有直接用电子束照射，进一步避免了损伤。SE-ADM的分辨率高于光学显微镜，光学显微镜通常用于观察溶液中的生物样品，可以观察样品的详细结构。考虑到这些优点，我们应用SE-ADM观察了水溶液中不同大小和形状的治疗性免疫球蛋白G （IgG）的聚集。在本章中，我们概述了使用SE-ADM观察单克隆抗体聚集的逐步过程，以及随后使用SE-ADM图像数据分析颗粒分布和计算分形维数的过程。所提出的颗粒分析方法在尺寸测量方面具有很高的可靠性，可以确定样品的直径，准确度为±20%，精密度为±10%，定量下限≤50 nm。此外，通过计算图像的分形维数，可以对聚集体的形状进行分类，并确定聚集体的机制。
Scanning electron-assisted dielectric microscopy (SE-ADM) is a new microscope technology developed to observe the fine structure of biological samples in aqueous solution. One main advantage of SE-ADM is that it does not require sample pretreatment, including dehydration, drying, and staining, which is indispensable in conventional scanning electron microscopy (SEM) and can cause sample deformation. In addition, the sample is not directly irradiated with an electron beam in SE-ADM, further avoiding damage. The resolution of SE-ADM is higher than that of an optical microscope, which is typically used for observing biological samples in a solution, allowing for the observation of the detailed structure of samples. Considering these advantages, we applied SE-ADM to observe aggregates of therapeutic immunoglobulin G (IgG) of various sizes and shapes in an aqueous solution. In this chapter, we outline the step-by-step procedure for observing aggregates of monoclonal antibodies using SE-ADM and the subsequent analysis of the particle distribution and calculation of the fractal dimension using SE-ADM image data. The proposed method for particle analysis is highly reliable with respect to size measurement and can determine the diameter of a sample with an accuracy of ±20%, a precision of ±10%, and a lower limit of quantification of ≤50 nm. Further, by calculating the fractal dimension of the image, it is possible to classify the shape of the aggregates and determine the mechanism of aggregation.