Cell-Free Dot Blot: an Ultra-Low-Cost and Practical Immunoassay Platform for Detection of Anti-SARS-CoV-2 Antibodies in Human and Animal Sera

• DOI: 10.1128/spectrum.02457-22
• Publication Date: 2023-01-31T14:06:42Z
• Authors: Masoud Norouzi; Thang Truong; Katariina Jaenes; Bryce M. Warner; Robert Vendramelli; Kevin Tierney; Darwyn Kobasa; Nikesh Tailor; Pamela Plant; Claudia dos Santos; Shawn Babiuk; Aruna Ambagala; Keith Pardee
• Keywords: Dot blot; 2019-20 coronavirus outbreak

Since its emergence in late 2019, the coronavirus disease 2019 (COVID-19) pandemic has caused severe disruption to key aspects of human life globally and highlighted need for timely, adaptive, accessible response strategies. Here, we introduce cell-free dot blot (CFDB) method, a practical ultra-low-cost immune diagnostic platform capable rapid mass immunity screening current future pandemics. Similar mechanism widely used enzyme-linked immunosorbent assays (ELISAs), our method is novel advantageous that (i) it uses linear DNA produce target viral antigen fused SpyTag peptide expression system without traditional cloning purification, (ii) SpyCatcher2-Apex2, an Escherichia coli-produced peroxidase conjugate as universal secondary detection reagent, obviating commercial or sophisticated enzyme conjugates, (iii) sera are spotted directly on nitrocellulose membrane, enabling simple "dipping" downstream incubation washing steps, opposed individual processing wells multiwell plate. To demonstrate utility performed CFDB detect anti-severe acute respiratory syndrome 2 nucleocapsid protein antibodies precharacterized (23 negative 36 positive COVID-19) hamster (16 COVID-19), including independent testing at collaborating laboratory, show assay performance comparable conventional ELISAs. At similar capacity 96-well plate ELISA kits, one costs only ~$3 USD. We believe can become valuable tool adaptive sero-surveillance animal populations.