Detection and characterization of protein aggregates by fluorescence microscopy
Detection and characterization of protein aggregates by fluorescence microscopy
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DOI：
10.1016/j.ijpharm.2006.08.024
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发表时间：
2007-02-01
影响因子：
5.8
通讯作者：
Arvinte, Tudor
中科院分区：
文献类型：
--
作者：
Demeule, Barthelemy;Gurny, Robert;Arvinte, Tudor
蛋白质药物的聚集会影响药物的稳定性和生物活性。蛋白质聚集体的检测在蛋白质表征过程中以及在药物制剂的优化过程中是需要的。本文介绍了一种技术，它包括分析蛋白质聚集体的荧光显微镜染色后，与疏水探针尼罗红。不需要对样品进行稀释、过滤或其他修饰。在高浓度蛋白质样品（193 mg/ml）中可以评估聚集。荧光显微镜观察允许检测和表征的蛋白质聚集体不容易检测到的光谱技术。尼罗红被证明是非常敏感的免疫球蛋白聚集体的检测和分析。比较了尼罗红、刚果红和硫黄素T染色。尼罗红和硫磺素T荧光共定位。测定免疫球蛋白聚集体的直径，并将聚集体的数量与900个光散射测量值相关联。人降钙素聚集体的研究揭示了人降钙素聚集机制的新方面。因此，尼罗红染色不仅可以检测非常低水平的蛋白质聚集体，而且有助于更好地理解蛋白质聚集的复杂机制。(c)2006 Elsevier B. V.保留所有权利。
Aggregation may compromise the stability as well as the biological activity of protein drugs. Detection of protein aggregates is needed ill the process of protein characterization and during optimization of pharmaceutical formulations. This paper describes a technique, which consists of analysing protein aggregates by fluorescence microscopy after staining with the hydrophobic probe Nile Red. Dilution, filtration or other modifications of the sample are not needed. Assessment of aggregation was possible in highly concentrated protein samples (193 mg/ml). Fluorescence microscopy observations allowed the detection and characterization of protein aggregates not easily detected by spectroscopic techniques. Nile Red was shown to be very sensitive for the detection and analysis of immunoglobulin aggregates. Nile Red, Congo red and Thioflavine T stainings were compared. Nile Red and Thioflavine T fluorescence were colocalized. The diameter of immunoglobulin aggregates was determined, and the number of aggregates was correlated with 900 light scattering measurements. Studies of human calcitonin aggregates brought to light new aspects of the human calcitonin aggregation mechanisms. Thus, Nile Red staining not only allows detection of very low levels of protein aggregates, but also contributes to a better understanding of the complex mechanisms governing protein aggregation. (c) 2006 Elsevier B.V. All rights reserved.