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

Area of Science:

• Biotechnology
• Immunology
• Molecular Diagnostics

Background:

• Rapid diagnostic tests are crucial for managing pandemics, offering significant time advantages.
• Lateral flow assays (LFAs) are practical, equipment-free diagnostic tools, often using antibody pairs.
• The SARS-CoV-2 pandemic highlighted the need for efficient viral detection methods.

Purpose of the Study:

• To recombinantly produce a single-chain variable fragment (scFv) targeting the SARS-CoV-2 spike receptor-binding domain (sRBD).
• To develop LFAs using scFv in combination with antibody and aptamer pairs, including an aptamer cocktail.
• To evaluate the diagnostic performance of these novel assay formats.

Main Methods:

• Recombinant production of SARS-CoV-2 specific scFv.
• Development of sandwich assays using gold nanoparticles as labels.
• Utilized scFv, CR3022 antibody, and aptamers against S and N proteins in LFA, Dot Blot Assay (DBA), and multiplex formats.

Main Results:

• Biologically active scFv was produced at high concentrations.
• LFAs, DBAs, and multiplex assays demonstrated effective viral detection.
• DBA and multiplex LFA successfully identified SARS-CoV-2 in patient swab samples, outperforming single LFAs on synthetic targets.

Conclusions:

• scFv, when combined with aptamers, enhances target binding in sandwich assays, a novel finding.
• Different assay configurations and capture agent combinations influence scFv effectiveness.
• Multiple testing strategies and novel combinations offer improved viral diagnosis and cost-effective rapid tests.