12/26/2023 0 Comments Dot blot 96 wells![]() ![]() 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 Spåatcher2-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. Pardee K National Centre for Foreign Animal Diseases, Canadian Food Inspection Agency, Winnipeg, Manitoba, Canada.Ambagala A National Centre for Foreign Animal Diseases, Canadian Food Inspection Agency, Winnipeg, Manitoba, Canada.Babiuk S Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada.Michael's Hospital, Unity Health Toronto, Toronto, Ontario, Canada. Dos Santos C Keenan Research Centre for Biomedical Science, St.Plant P Keenan Research Centre for Biomedical Science, St.Tailor N National Centre for Foreign Animal Diseases, Canadian Food Inspection Agency, Winnipeg, Manitoba, Canada.Kobasa D Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada.Tierney K Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada.Vendramelli R Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada.Warner BM Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada.Jaenes K Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada.Truong T Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada.Norouzi M Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada. ![]()
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