Development of an Immunoassay for Highly Pathogenic Avian Influenza (H5N1) across Diverse Sample Matrices

Avian influenza of the highly pathogenic subtype H5N1 has emerged as a global health concern, becoming endemic in wild birds and increasingly transmitting to poultry, livestock, and humans. This study aimed to develop a robust immunoassay for the rapid detection of the H5N1 highly pathogenic avian influenza virus across various sample matrices, including sera, milk, eggs, and bird samples. The assay targets the hemagglutinin (HA) protein, chosen for its abundance and accessibility on the virus surface. Utilizing gold nanospheres conjugated with α-HA IgG antibodies, the assay generated distinct colorimetric signals for both negative and positive samples. The test initially demonstrated an effective colorimetric response with a limit of detection (LOD) of 0.16 nM in human serum and was further optimized for running in whole milk, exhibiting an LOD of 1.72 nM. The assay exhibited versatility across different serum types and dairy products, although high-viscosity samples like heavy cream presented challenges. Furthermore, the immunoassay successfully detected HA of H5N1 in complex sample matrices such as oral, cloacal, and fecal samples from birds. This rapid and sensitive immunoassay represents a significant advance in HPAI surveillance tools, improving prospects for real-time detection to control outbreaks.