Microparticle immunocapture assay for quantitation of protein multimer amount and size.
Microparticle immunocapture assay for quantitation of protein multimer amount and size.
复制标题
DOI：
10.1016/j.crmeth.2022.100214
复制
发表时间：
2022-05-23
期刊：
影响因子：
--
通讯作者：
中科院分区：
文献类型：
--
作者：
细胞压力和毒性通常与蛋白质多聚体或聚集体的形成有关。许多退行性疾病，包括阿尔茨海默氏病、帕金森氏病和亨廷顿病、Pron传播性疾病、肌萎缩侧索硬化症、心脏淀粉样变性和糖尿病，都以聚集的蛋白质沉积为特征。目前的方法在评估多聚体大小和多聚体定量以及结合一个或多个辅助特征(包括靶标特异性、操作简单性和处理速度)方面受到限制。在这里，我们报告了一种微粒子免疫捕获分析的发展，它结合了单抗固有的优势：蛋白质相互作用和高度定量的流式细胞术分析。使用成熟的试剂来构建我们的平台，并使用易于聚集的淀粉样β1-42肽(Aβ42)和α-突触核蛋白来演示原理证明，我们的结果表明，该方法是一种高度适应性的方法，可以在适用于实验室和临床样本的技术简化的工作流程中同时测量多聚体的大小和数量。一种简单、直接和快速的鉴定蛋白质聚集体的方法同时评估蛋白质聚集体的大小和数量，以适应存在合适的抗体试剂的任何蛋白质，证明了将蛋白质聚集体应用于脑脊液样本，也称为蛋白质多聚体或功能失调或无序的蛋白质沉积，已知与神经退行性疾病和其他疾病的病理性相关。在体外工作和临床样本中准确测量蛋白质聚集体是当务之急。然而，目前可用的方法在提供蛋白质聚集大小的测量方面是有限的。我们提出了一种基于微球的免疫捕获试验，它同时提供了蛋白质聚集量和大小的测量。该分析方法简单、特异、定量和快速，在一天的实验时间内就能提供结果。蛋白质聚集体的形成与几种人类疾病有关，是药物开发中的一个复杂因素。古特内克赫特等人。提出了一种微粒子、抗体介导的捕获平台，允许在简化的工作流程中对蛋白质聚集量和大小进行特定检测和定量，同时忽略单体。
Cellular stress and toxicity are often associated with the formation of protein multimers, or aggregates. Numerous degenerative disorders, including Alzheimer’s, Parkinson’s, and Huntington’s disease, prion-propagated disease, amyotrophic lateral sclerosis, cardiac amyloidosis, and diabetes, are characterized by aggregated protein deposits. Current methods are limited in the ability to assess multimer size along with multimer quantitation and to incorporate one or more ancillary traits, including target specificity, operative simplicity, and process speed. Here, we report development of a microparticle immunocapture assay that combines the advantages inherent to a monoclonal antibody:protein interaction with highly quantitative flow cytometry analysis. Using established reagents to build our platform, and aggregation-prone amyloid beta 1-42 peptide (Aβ42) and alpha-synuclein to demonstrate proof of principle, our results indicate that this assay is a highly adaptable method to measure multimer size and quantity at the same time in a technically streamlined workflow applicable to laboratory and clinical samples. A simple, straightforward, and quick assay to characterize protein aggregates Simultaneous assessment of protein aggregate size and amount Adaptable to any protein for which suitable antibody reagents exist Demonstrates application to CSF samples Protein aggregates, also known as protein multimers or dysfunctional or disordered protein deposits, are known to be associated pathogenically with neurodegenerative and other diseases. Accurate measurement of protein aggregates during in vitro work and with clinical samples is imperative. However, currently available methods are limited in providing a measure of protein aggregate size. We present a microparticle bead-based immunocapture assay that provides, at the same time, measurement of protein aggregate amount and size. The assay is simple, specific, quantitative, and quick, providing results within a single experimental day. Protein aggregate formation is associated with several human pathologies and is a complicating factor in the development of pharmaceutical agents. Gutknecht et al. present a microparticle, antibody-mediated capture platform that permits the specific detection and quantitation of protein aggregate amount and size, while ignoring monomers, in a streamlined workflow.