Single-Chain Variable Fragment-Based Dot Blot, Single, and Multiple Assays for Rapid SARS-CoV-2 Diagnostics

Antigenic detection is reliably utilized in rapid diagnostic tests and provides a significant time advantage during pandemics and epidemics. Therefore, the rapid detection of viral infections is of great importance and will remain crucial in the future. The SARS-CoV-2 outbreak, which resulted in severe losses, is the most recent example of this necessity. Among rapid diagnostic tests, lateral flow assays (LFAs) are the most practical and do not require specialized equipment, typically being developed using antibody pairs.

This study aimed to recombinantly produce a single-chain variable fragment (scFv) specific to the SARS-CoV-2 spike receptor-binding domain (sRBD) and to employ it in the development of LFAs utilizing both antibody and aptamer pairs and an aptamer cocktail.

Gold nanoparticles were employed as labeling agents, while both the scFv and full length forms of CR3022, along with aptamers specific to the S and N proteins, were utilized in a sandwich assay format.

scFv was produced at a higher concentration and biologically active. It demonstrated effective viral detection in single LFA, dot blot assay (DBA), and multiplex LFA. While single LFA successfully detected only the synthetic target, DBA and multiplex LFA selectively identified the virus in nasopharyngeal and oropharyngeal swab samples.

Findings highlight the differences and effectiveness of using scFv in combination with other capture agents and different assay principles for the development of cost-effective and rapid diagnostic tests.

scFvs exhibit variable binding in sandwich assays depending on the combinations employed. When used in combination with an aptamer cocktail, scFvs demonstrate enhanced target binding, which is shown for the first time in this study. The use of multiple testing strategies enables a more effective viral diagnosis.