Toward a Point-of-Need Bioluminescence-Based Immunoassay Utilizing a Complete Shelf-Stable Reagent
| Autor: | Stuart K Forsyth, Emily Jost, Monika G. Wood, Valerie T Ressler, Dan Lazar, Kris Zimmermann, Connor Fitzgerald, Thomas A. Kirkland, Mary P. Hall, Thomas Machleidt, Thomas P. Smith, Kincaid Virginia, Keith V. Wood, Robin Hurst, Lance P. Encell, Melanie Dart |
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| Rok vydání: | 2021 |
| Předmět: |
Immunoassay
chemistry.chemical_classification Chromatography medicine.diagnostic_test Chemistry Biomolecule 010401 analytical chemistry Substrate (chemistry) Enzyme-Linked Immunosorbent Assay Immunologic Tests 010402 general chemistry Directed evolution 01 natural sciences 0104 chemical sciences Analytical Chemistry Homogeneous Reagent medicine Humans Bioluminescence Indicators and Reagents Luciferase Luciferases |
| Zdroj: | Analytical Chemistry. 93:5177-5184 |
| ISSN: | 1520-6882 0003-2700 |
| DOI: | 10.1021/acs.analchem.0c05074 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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