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均质96孔板PEA免疫分析方法具有高灵敏度、高特异性和出色的可扩展性。

Homogenous 96-plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability.

作者信息

Assarsson Erika, Lundberg Martin, Holmquist Göran, Björkesten Johan, Thorsen Stine Bucht, Ekman Daniel, Eriksson Anna, Rennel Dickens Emma, Ohlsson Sandra, Edfeldt Gabriella, Andersson Ann-Catrin, Lindstedt Patrik, Stenvang Jan, Gullberg Mats, Fredriksson Simon

机构信息

Olink Bioscience, Uppsala, Sweden.

Section for Molecular Disease Biology and Sino-Danish Breast Cancer Research Centre, Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

出版信息

PLoS One. 2014 Apr 22;9(4):e95192. doi: 10.1371/journal.pone.0095192. eCollection 2014.

Abstract

Medical research is developing an ever greater need for comprehensive high-quality data generation to realize the promises of personalized health care based on molecular biomarkers. The nucleic acid proximity-based methods proximity ligation and proximity extension assays have, with their dual reporters, shown potential to relieve the shortcomings of antibodies and their inherent cross-reactivity in multiplex protein quantification applications. The aim of the present study was to develop a robust 96-plex immunoassay based on the proximity extension assay (PEA) for improved high throughput detection of protein biomarkers. This was enabled by: (1) a modified design leading to a reduced number of pipetting steps compared to the existing PEA protocol, as well as improved intra-assay precision; (2) a new enzymatic system that uses a hyper-thermostabile enzyme, Pwo, for uniting the two probes allowing for room temperature addition of all reagents and improved the sensitivity; (3) introduction of an inter-plate control and a new normalization procedure leading to improved inter-assay precision (reproducibility). The multiplex proximity extension assay was found to perform well in complex samples, such as serum and plasma, and also in xenografted mice and resuspended dried blood spots, consuming only 1 µL sample per test. All-in-all, the development of the current multiplex technique is a step toward robust high throughput protein marker discovery and research.

摘要

医学研究对全面高质量数据生成的需求日益增长,以实现基于分子生物标志物的个性化医疗的前景。基于核酸邻近性的方法——邻近连接和邻近延伸分析,凭借其双报告分子,在多重蛋白质定量应用中显示出缓解抗体缺点及其固有交叉反应性的潜力。本研究的目的是基于邻近延伸分析(PEA)开发一种强大的96重免疫分析方法,以改进蛋白质生物标志物的高通量检测。这通过以下方式实现:(1)一种改进的设计,与现有的PEA方案相比,减少了移液步骤的数量,并提高了分析内精度;(2)一种新的酶系统,使用超嗜热酶Pwo将两个探针结合在一起,允许在室温下添加所有试剂并提高了灵敏度;(3)引入板间对照和新的标准化程序,提高了分析间精度(重现性)。发现多重邻近延伸分析在复杂样品(如血清和血浆)以及异种移植小鼠和重悬的干血斑中表现良好,每次测试仅消耗1微升样品。总而言之,当前多重技术的发展是朝着强大的高通量蛋白质标志物发现和研究迈出的一步。

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