Development and application of an enzyme-linked immunosorbent assay (ELISA) using a soluble recombinant nucleoprotein for the detection of antibodies to avian influenza virus

Avian influenza (AI) causes significant impact on industrial poultry farming, besides infecting a variety of vertebrates. The detection of antibodies against viral antigens by serological methods is important for the epidemiology, control and prevention of AI because their high simplicity and speed for assaying a large number of samples. Obtaining antigenic preparations used for detection of anti-avian influenza virus (AIV) antibodies usually requires complex and expensive procedures and Escherichia coli system expression may be an alternative. The nucleoprotein (NP) of AIV is an ideal antigen candidate because it is highly conserved across AIV strains, resulting in high cross-reactivity and immunogenicity for avian hosts. The NP gene segment was cloned and expressed from AIV isolate H4N6 in E. coli fused to a small ubiquitin-like modifier (SUMO) polypeptide and a poly-histidine tag, obtaining a soluble recombinant NP (rNP) containing the most important epitopes. After purification, the rNP was used as an antigen to develop an indirect rNP-enzyme-linked immunosorbent assay (ELISA) to effectively detect anti-AIV antibodies in chicken serum samples. This rNP-ELISA had high sensitivity (95%), specificity (97%), accuracy (96.7%) and agreement (k=0.88) in a comparative analysis with a commercial ELISA kit. The results suggest that rNP-ELISA offers a viable alternative to improve immunodiagnosis of AIV infection in chickens.