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Multiplex bead binding assays using off-the-shelf components and common flow cytometers
Journal of Immunological Methods ( IF 2.2 ) Pub Date : 2020-12-25 , DOI: 10.1016/j.jim.2020.112952
Takamitsu Hattori 1 , Akiko Koide 2 , Tatyana Panchenko 3 , Larizbeth A Romero 4 , Kai Wen Teng 3 , Alexis D Corrado 3 , Shohei Koide 1
Affiliation  

The ability to quantify protein-ligand interactions in an accurate and high-throughput manner is important in diverse areas of biology and medicine. Multiplex bead binding assays (MBBAs) are powerful methods that allows for simultaneous analysis of many protein-ligand interactions. Although there are a number of well-established MBBA platforms, there are few platforms suitable for research and development that offer rapid experimentation at low costs and without the need for specialized reagents or instruments dedicated for MBBA. Here, we describe a MBBA method that uses low-cost reagents and standard cytometers. The key innovation is the use of the essentially irreversible biotin-streptavidin interaction. We prepared a biotin-conjugated fluorescent dye and used it to produce streptavidin-coated magnetic beads that are labeled at distinct levels of fluorescence. We show the utility of our method in characterization of phage-displayed antibodies against multiple antigens of SARS-CoV-2, which substantially improves the throughput and dramatically reduces antigen consumption compared with conventional phage ELISA methods. This approach will make MBBAs more broadly accessible.



中文翻译:

使用现成组件和普通流式细胞仪进行多重珠结合测定

以准确和高通量的方式量化蛋白质-配体相互作用的能力在生物学和医学的各个领域都很重要。多重珠结合测定 (MBBA) 是一种强大的方法,可以同时分析许多蛋白质-配体相互作用。尽管有许多成熟的 MBBA 平台,但很少有适合研究和开发的平台能够以低成本提供快速实验,并且不需要专门用于 MBBA 的专门试剂或仪器。在这里,我们描述了一种使用低成本试剂和标准细胞仪的 MBBA 方法。关键创新是使用基本上不可逆的生物素-链霉亲和素相互作用。我们制备了一种生物素偶联的荧光染料,并用它来生产链霉亲和素包被的磁珠,这些磁珠被标记为不同的荧光水平。我们展示了我们的方法在表征针对 SARS-CoV-2 多种抗原的噬菌体展示抗体方面的效用,与传统的噬菌体 ELISA 方法相比,这大大提高了通量并显着减少了抗原消耗。这种方法将使 MBBA 更广泛地普及。

更新日期:2020-12-25
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