A Nanobody-Based Lateral Flow Assay for Point-of-Care Diagnostics.

Lateral flow assays (LFAs) are among the most successful technologies for point-of-care and at-home testing, but further advances are needed to reduce costs and accelerate development. Alpaca-derived nanobodies (Nbs), single-domain antibody fragments, are promising immunoassay reagents across diverse applications. Their small size and ease of recombinant production make them particularly well suited for diagnostics. Here, we present a paper-based LFA targeting the SARS-CoV-2 nucleocapsid (N) protein that exclusively uses Nbs for direct antigen detection. We also demonstrate in-house synthesis of Nb-coated gold nanoparticles, enabling instrument-free visual readout and detection of N protein down to 40 ng/mL. This design avoids components that require mammalian cell culture and can be produced entirely from in-house reagents, simplifying manufacturing and lowering component costs. Because the assay is read visually without an external reader, it is well suited for deployment in resource-limited settings. Together, these results highlight the speed and practicality of developing Nb-based LFAs and suggest a broadly applicable strategy for detecting other clinically important disease biomarkers.