Nanobody-Based Lateral Flow Assay for Rapid Zika Virus Detection

Zika virus infections remain severely underdiagnosed due to their initial mild clinical symptoms. However, recent outbreaks have revealed neurological complications in adults and severe deformities in newborns, emphasizing the critical need for accurate diagnosis. Lateral flow assays (LFAs) provide a rapid, cost-effective, and user-friendly method for antigen testing at point-of-care, bedside, or in home settings. LFAs utilizing nanobodies have multiple benefits over traditional antibody-based techniques, as nanobodies are much smaller, more stable, and simpler to manufacture. We introduce a nanobody-based LFA for the rapid identification of Zika virus antigens. Starting from two previously reported nanobodies recognizing the Zika nonstructural protein 1 (NS1), we evaluate periplasmic and cytosolic nanobody expression and test different purification tags and immobilization strategies. We quantify nanobody binding kinetics and validate their mutually noncompetitive binding. Avidity effects boost the capture of the tetrameric target protein by 3 orders of magnitude and point to a general strategy for higher sensitivity LFA sensing. The nanobody LFA detects Zika NS1 with a limit of detection ranging from 25 ng/mL in buffer to 1 ng/mL in urine. This nanobody-LFA has the potential to facilitate on-site and self-diagnosis, improve our understanding of Zika infection prevalence, and support public health initiatives in regions affected by Zika virus outbreaks.