R&D Department, Promega Corporation, Madison, WI, USA.
Abbvie, North Chicago, IL, USA.
Methods Mol Biol. 2023;2612:195-224. doi: 10.1007/978-1-0716-2903-1_15.
Traditional immunoassays to detect secreted or intracellular proteins can be tedious, require multiple washing steps, and are not easily adaptable to a high-throughput screening (HTS) format. To overcome these limitations, we developed Lumit, a novel immunoassay approach that combines bioluminescent enzyme subunit complementation technology and immunodetection. This bioluminescent immunoassay does not require washes or liquid transfers and takes less than 2 h to complete in a homogeneous "Add and Read" format. In this chapter, we describe step-by-step protocols to create Lumit immunoassays for the detection of (1) secreted cytokines from cells, (2) phosphorylation levels of a specific signaling pathway node protein, and (3) a biochemical protein-protein interaction between a viral surface protein and its human receptor.
传统的免疫分析方法可用于检测分泌型或细胞内蛋白，但操作繁琐，需要多次洗涤步骤，并且不容易适应高通量筛选 (HTS) 格式。为了克服这些限制，我们开发了 Lumit，这是一种新颖的免疫分析方法，结合了生物发光酶亚基互补技术和免疫检测。这种生物发光免疫分析不需要洗涤或液体转移，并且在均相“添加和读取”格式下不到 2 小时即可完成。在本章中，我们描述了创建 Lumit 免疫分析的分步协议，用于检测 (1) 细胞分泌的细胞因子，(2) 特定信号通路节点蛋白的磷酸化水平，以及 (3) 病毒表面蛋白与其人类受体之间的生化蛋白-蛋白相互作用。
