Notably, the molecular detection reagents used in CFDB can be produced rapidly in-house, using established protocols and basic laboratory infrastructure, minimizing reliance on strained commercial reagents. Based on the principles of cell-free synthetic biology and alternative dot blotting procedures, our CFDB immunoassay is designed to provide for timely, practical, and low-cost responses to existing and emerging public health threats, such as the COVID-19 pandemic, at a similar throughput and comparable performance as conventional ELISAs. To address this need, we describe the development and functional validation of a novel immunoassay technique termed the cell-free dot blot (CFDB) method. IMPORTANCE The recent COVID-19 pandemic has highlighted the need for diagnostic platforms that are rapidly adaptable, affordable, and accessible globally, especially for low-resource settings. We believe that CFDB can become a valuable pandemic response tool for adaptive and accessible sero-surveillance in human and animal populations. At a similar capacity to 96-well plate ELISA kits, one CFDB assay costs only ~$3 USD. To demonstrate the utility of our method, we performed CFDB to detect anti-severe acute respiratory syndrome coronavirus 2 nucleocapsid protein antibodies in precharacterized human sera (23 negative and 36 positive for COVID-19) and hamster sera (16 negative and 36 positive for COVID-19), including independent testing at a collaborating laboratory, and we show assay performance comparable to that of conventional ELISAs. Similar in mechanism to the widely used enzyme-linked immunosorbent assays (ELISAs), our method is novel and advantageous in that (i) it uses linear DNA to produce the target viral antigen fused to a SpyTag peptide in a cell-free expression system without the need for traditional cloning and antigen purification, (ii) it uses Sp圜atcher2-Apex2, an Escherichia coli-produced peroxidase conjugate as a universal secondary detection reagent, obviating the need for commercial or sophisticated enzyme conjugates, and (iii) sera are spotted directly on a nitrocellulose membrane, enabling a simple “dipping” mechanism for downstream incubation and washing steps, as opposed to individual processing of wells in a multiwell plate. Here, we introduce the cell-free dot blot (CFDB) method, a practical and ultra-low-cost immune diagnostic platform capable of rapid response and mass immunity screening for the current and future pandemics. 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 the need for timely, adaptive, and accessible pandemic response strategies.
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