1. Sequential Protein Capture in Multiplex Single Molecule Arrays: A Strategy for Eliminating Assay Cross-Reactivity.
- Author
-
Gilboa T, Maley AM, Ogata AF, Wu C, and Walt DR
- Subjects
- COVID-19 blood, COVID-19 virology, Calibration, Cytokines blood, Humans, Reproducibility of Results, SARS-CoV-2 physiology, Biological Assay, Cross Reactions immunology, Proteins analysis
- Abstract
Measurements of multiple biomolecules within the same biological sample are important for many clinical applications to enable accurate disease diagnosis or classification. These disease-related biomarkers often exist at very low levels in biological fluids, necessitating ultrasensitive measurement methods. Single-molecule arrays (Simoa), a bead-based digital enzyme-linked immunosorbent assay, is the current state of the art for ultrasensitive protein detection and can detect sub-femtomolar protein concentrations, but its ability to achieve high-order multiplexing without cross-reactivity remains a challenge. Here, a sequential protein capture approach for multiplex Simoa assays is implemented to eliminate cross-reactivity between binding reagents by sequentially capturing each protein analyte and then incubating each capture bead with only its corresponding detection antibody. This strategy not only reduces cross-reactivity to background levels and significantly improves measurement accuracies, but also enables higher-order multiplexing. As a proof of concept, the sequential multiplex Simoa assay is used to measure five different cytokines in plasma samples from Coronavirus Disease 2019 (COVID-19) patients. The ultrasensitive sequential multiplex Simoa assays will enable the simultaneous measurements of multiple low-abundance analytes in a time- and cost-effective manner and will prove especially critical in many cases where sample volumes are limited., (© 2020 Wiley-VCH GmbH.)
- Published
- 2021
- Full Text
- View/download PDF