Toward a Point-of-Need Bioluminescence-Based Immunoassay Utilizing a Complete Shelf-Stable Reagent
Toward a Point-of-Need Bioluminescence-Based Immunoassay Utilizing a Complete Shelf-Stable Reagent
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DOI：
10.1021/acs.analchem.0c05074
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发表时间：
2021-03-17
影响因子：
7.4
通讯作者：
Dart, Melanie L.
中科院分区：
文献类型：
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作者：
Hall, Mary P.;Kincaid, Virginia A.;Dart, Melanie L.
酶联免疫吸附测定（ELISA）广泛用于临床诊断以及基础研究中的生物分子的检测和定量。虽然被广泛使用，但ELISA固有的复杂性排除了其用于直接的需求点测试的实用性，其中速度和简单性是必不可少的。考虑到这一点，我们开发了一种基于生物发光的免疫测定形式，为检测临床样品中的生物分子提供了一种灵敏而简单的方法。我们利用由两个小肽（11聚体，13聚体）和一个17 kDa多肽组成的三元分裂NanoLuc荧光素酶互补报告基因与发光底物结合，以创建一个完整的，货架稳定的添加和读取测定检测试剂。定向进化用于优化报告子组分序列以赋予化学和热稳定性以及溶解性，而制剂优化用于稳定可在水性缓冲液或样品基质中重构的一体化试剂。这些努力的结果是一个强大的，第一代基于生物发光的均相免疫测定报告平台，其中所有测定组件都可以配置成一个稳定的冻干饼，支持在复杂的人类样品中进行均相，快速和灵敏的一步生物分子定量。该技术代表了一种有前途的替代免疫测定形式，具有使关键诊断分子检测测试更接近需求点的巨大潜力。
Enzyme-linked immunosorbent assays (ELISAs) are used extensively for the detection and quantification of biomolecules in clinical diagnostics as well as in basic research. Although broadly used, the inherent complexities of ELISAs preclude their utility for straightforward point-of-need testing, where speed and simplicity are essential. With this in mind, we developed a bioluminescence-based immunoassay format that provides a sensitive and simple method for detecting biomolecules in clinical samples. We utilized a ternary, split-NanoLuc luciferase complementation reporter consisting of two small peptides (11mer, 13mer) and a 17 kDa polypeptide combined with a luminogenic substrate to create a complete, shelf-stable add-and-read assay detection reagent. Directed evolution was used to optimize reporter constituent sequences to impart chemical and thermal stability, as well as solubility, while formulation optimization was applied to stabilize an all-in-one reagent that can be reconstituted in aqueous buffers or sample matrices. The result of these efforts is a robust, first-generation bioluminescence-based homogenous immunoassay reporter platform where all assay components can be configured into a stable lyophilized cake, supporting homogeneous, rapid, and sensitive one-step biomolecule quantification in complex human samples. This technology represents a promising alternative immunoassay format with significant potential to bring critical diagnostic molecular detection testing closer to the point-of-need.