A rapid lateral flow immunoassay for infectious spleen and kidney necrosis virus (ISKNV)

A rapid, instrument-free lateral flow immunoassay (LFIA) device was developed and validated for screening of the ISKNV antigen. Based on a sandwich immunoassay format, two monoclonal antibodies targeting distinct viral epitopes were employed for capture and detection. The detection antibody was conjugated to gold nanoparticles (AuNPs), while the capture antibody was immobilized on a nitrocellulose membrane at the test spot. In the presence of ISKNV antigen, a distinct red signal was generated via formation of an antibody-antigen-antibody sandwich complex. Operating under an optimized 0.010 M borate buffer system (pH 8.5), containing 1% w/v sucrose, 0.5% w/v BSA, 0.1% v/v Tween-20, and 0.05% w/v NaN₃, the developed LFIA strip yielded a visual limit of detection (LOD) of 0.25 μ g mL^-1 within a 15 min assay. Incorporating a rapid post-assay gold enhancement step successfully drove a 5-fold sensitivity enhancement, lowering the refined visual LOD of 0.050 μ g mL^-1. This enhancement step required an additional 10 min, resulting in a total detection time of 25 min. The LFIA platform exhibited acceptable precision, with intra-day and inter-day %RSD values remaining below 11.50% and 12.17%, respectively. Accelerated aging at 55 °C indicated over 80% relative signal intensity, corresponding to an estimated equivalent storage period of 32 days at 25 °C. When applied to analyze real sea bass mucus samples, the assay results strongly aligned with standard PCR confirmation, demonstrating freedom from matrix effects and false-positive interference. Overall, this portable and cost-effective platform demonstrates strong potential as a rapid and user-friendly point-of-care tool for on-site ISKNV screening and aquaculture disease monitoring.