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

Abstract

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.

Monoclonal Antibody Preparation Against Nucleoprotein of Avian Influenza Virus Subtype H9N2

“…Avian influenza is a significant viral disease worldwide. H9N2 viruses have relatively fewer pathogenesis types, whereas over the past decade, there have been many reports of damage or loss in these strains (3,14,33). NP is the most important influenza virus internal protein, which can help detect influenza viruses A, B, and C. Previously research have shown that NP can be a proper target for the antiviral drugs development against influenza virus different types by stimulating immune responses (34,35).…”

Purification of Nucleoprotein of H9N2 Avian Influenza Virus Strain by Electroelution

“…It has been reported that patients benefit from antiviral treatment, especially in the first 48 hours of illness (16). Currently, serological diagnostic techniques such as agar gel propagation (AGP), hemagglutination inhibition, and enzyme-linked immunosorbent assay (ELISA) are considered as important tools for the antibodies detection against viral antigens (17). These techniques are common and relatively inexpensive, and generate results more rapidly (18,19).…”