Abstract
Human infections with the H10 subtype avian influenza virus (AIV) have been reported in recent years, raising concern about potential human-to-human transmission. Effective field detection methods are essential for monitoring and controlling the spread of this virus. The objective of this study was to develop a rapid, highly sensitive, and specific detection system for the H10 subtype AIV. Two monoclonal antibodies targeting the hemagglutinin (HA) protein of H10 AIV were generated and characterized. One antibody was conjugated to europium-chelate fluorescent nanospheres and used as a tracer, while the other was immobilized on the test line to capture antigen. A time-resolved fluorescence immunochromatographic strip was then constructed and evaluated for analytical performance. The assay achieved a detection limit of 0.015 hemagglutination units for virus-containing allantoic fluid and 1.1 ng/mL for purified HA protein, which represents a substantial improvement in sensitivity compared with conventional immunochromatographic assays. The test correctly identified all H10 subtype strains examined, including H10N2, H10N3, H10N5, H10N7, and H10N8, and showed no cross-reactivity with nine other subtype AIVs or seven non-influenza avian pathogens. Reproducibility was high, with relative standard deviations below 10% across repeated assays. Receiver operating characteristic analysis yielded areas under the curve of 0.9896 and 0.9722 for virus- and protein-based evaluations, respectively, confirming excellent diagnostic accuracy. Field evaluation with 174 avian samples, including cloacal swabs, throat swabs, and fecal samples, demonstrated 100% concordance with real-time polymerase chain reaction. The entire test procedure was completed within 15 minutes, requiring minimal equipment and technical expertise. In conclusion, the developed time-resolved fluorescence immunochromatographic strip provides a rapid, sensitive, and highly specific tool for detecting the H10 subtype AIV. Its superior performance and ease of use make it well suited for routine surveillance and early warning in poultry farms and live bird markets, thereby supporting disease control and protecting public health.