A rapid detection of Nipah virus nucleocapsid antigen in inactivated viral cultures

Nipah virus (NiV) is a highly lethal zoonotic henipavirus that causes severe respiratory disease and encephalitis and remains a priority pathogen for outbreak preparedness. Timely diagnosis is essential for containment, yet nucleic acid-based testing is often impractical in outbreak-prone, resource-limited settings. Here, we report the development of a rapid antigen test for NiV based on a cellulose nanobead (CNB)-enhanced lateral flow immunoassay (LFIA) targeting the viral nucleocapsid protein (NP). Anti-NiV NP antibodies were isolated from human synthetic phage-display libraries (scFv and Fab) by iterative bio-panning, reformatted as full-length IgGs, and screened using ELISA and immunofluorescence assays. Four lead antibodies were advanced to a sandwich-pairing matrix, which identified an optimized capture-detection configuration (mAb #5 as capture; mAb #2 as detection). Bio-layer interferometry confirmed nanomolar binding affinities for the selected antibodies (K_D = 39.50 nM for mAb #5; K_D = 26.36 nM for mAb #2). The finalized CNB-LFIA produced results within 15 min and achieved an analytical limit of detection of 1 ng/mL for recombinant NiV NP. The assay observed no cross-reactivity with SARS-CoV-2, SARS-CoV, MERS-CoV, or influenza A, whereas weak cross-reactivity for measles virus NP was observed only at a high antigen concentration (2 µg/mL). Using γ-irradiated cultured NiV quantified by RT-ddPCR, the LFIA detected viral load down to 4.1 × 10⁷ copies/mL (4.1 × 10⁶ copies/reaction) by a portable reader and 8.3 × 10⁷ copies/mL (8.3 × 10⁶ copies/reaction) by the naked eye. Collectively, this CNB-LFIA provides rapid, equipment-free NiV detection, supporting its potential utility for point-of-care diagnosis in resource-limited settings.