Sage Jessica M, Hall LaSharice, McVey April, Barohn Richard J, Statland Jeffrey M, Jawdat Omar, Dimachkie Mazen M, Agbas Abdulbaki
Department of Basic Sciences, Kansas City University of Medicine and Biosciences.
Rice University School of Medicine.
J Vis Exp. 2020 Feb 10(156). doi: 10.3791/60638.
Capillary electrophoresis immunoassay (CEI), also known as capillary western technology, is becoming a method of choice for screening disease relevant proteins and drugs in clinical trials. Reproducibility, sensitivity, small sample volume requirement, multiplexing antibodies for multiple protein labeling in the same sample, automated high-throughput ability to analyze up to 24 individual samples, and short time requirement make CEI advantageous over the classical western blot immunoassay. There are some limitations of this method, such as the inability to utilize a gradient gel (4%-20%) matrix, high background with unrefined biological samples, and commercial unavailability of individual reagents. This paper describes an efficient method for running CEI in a multiple assay setting, optimizing protein concentration and primary antibody titration in one assay plate, and providing user-friendly templates for sample preparation. Also described are methods for measuring pan TDP-43 and phosphorylated TDP-43 derivative in platelet lysate cytosol as part of the initiative in blood-based biomarker development for neurodegenerative diseases.
毛细管电泳免疫分析（CEI），也称为毛细管蛋白质免疫印迹技术，正成为在临床试验中筛选疾病相关蛋白质和药物的首选方法。可重复性、灵敏度、对样本量要求小、能在同一样本中使用多种抗体对多种蛋白质进行标记、具备自动高通量分析多达24个独立样本的能力以及所需时间短，这些使得CEI比传统的蛋白质免疫印迹免疫分析更具优势。该方法存在一些局限性，例如无法使用梯度凝胶（4%-20%）基质、未提纯的生物样本背景较高以及个别试剂无法商业化获取。本文描述了一种在多重分析设置中进行CEI的有效方法，在一个分析板中优化蛋白质浓度和一抗滴定，并提供用户友好的样本制备模板。还介绍了作为神经退行性疾病血液生物标志物开发计划一部分，在血小板裂解液胞质溶胶中测量泛TDP-43和磷酸化TDP-43衍生物的方法。
