Vaccine Analytical Development, Merck Research Laboratories, West Point, PA, USA.
Electrophoresis. 2012 Sep;33(17):2790-7. doi: 10.1002/elps.201200095.
Many CE-based technologies such as imaged capillary IEF, CE-SDS, CZE, and MEKC are well established for analyzing proteins, viruses, or other biomolecules such as polysaccharides. For example, imaged capillary isoelectric focusing (charge-based protein separation) and CE-SDS (size-based protein separation) are standard replacement methods in biopharmaceutical industries for tedious and labor intensive IEF and SDS-PAGE methods, respectively. Another important analytical tool for protein characterization is a Western blot, where after size-based separation in SDS-PAGE the proteins are transferred to a membrane and blotted with specific monoclonal or polyclonal antibodies. Western blotting analysis is applied in many areas such as biomarker research, therapeutic target identification, and vaccine development. Currently, the procedure is very manual, laborious, and time consuming. Here, we evaluate a new technology called Simple Western™ (or Simon™) for performing automated Western analysis. This new technology is based on CE-SDS where the separated proteins are attached to the wall of capillary by a proprietary photo activated chemical crosslink. Subsequent blotting is done automatically by incubating and washing the capillary with primary and secondary antibodies conjugated with horseradish peroxidase and detected with chemiluminescence. Typically, Western blots are not quantitative, hence we also evaluated the quantitative aspect of this new technology. We demonstrate that Simon™ can quantitate specific components in one of our vaccine candidates and it provides good reproducibility and intermediate precision with CV <10%.
许多基于毛细管电泳（CE）的技术，如成像毛细管等电聚焦（IEF）、CE-SDS、CZE 和 MEKC，已经被广泛用于分析蛋白质、病毒或其他生物分子，如多糖。例如，成像毛细管等电聚焦（基于电荷的蛋白质分离）和 CE-SDS（基于大小的蛋白质分离）分别是生物制药行业中繁琐而费力的 IEF 和 SDS-PAGE 方法的标准替代方法。用于蛋白质特征分析的另一个重要分析工具是 Western blot，其中在 SDS-PAGE 基于大小的分离之后，蛋白质被转移到膜上，并与特异性单克隆或多克隆抗体进行印迹。Western blot 分析在许多领域得到了应用，如生物标志物研究、治疗靶标识别和疫苗开发。目前，该程序非常手动、费力且耗时。在这里，我们评估了一种称为 Simple WesternTM（或 SimonTM）的新技术，用于执行自动化 Western 分析。这项新技术基于 CE-SDS，其中分离的蛋白质通过专有的光激活化学交联附着在毛细管壁上。随后通过孵育和用辣根过氧化物酶偶联的一抗和二抗洗涤毛细管自动进行印迹，并通过化学发光进行检测。通常，Western blot 不是定量的，因此我们还评估了这项新技术的定量方面。我们证明，SimonTM 可以定量我们候选疫苗之一的特定成分，并且它提供了良好的重现性和中间精度，CV<10%。
