Production and Characterization of Monoclonal Antibodies Against Nucleoprotein of Avian Influenza Virus

Abstract

The present study was carried out with an aim to develop anti-nucleoprotein (anti-NP) monoclonal antibodies (MAbs) for use in immunodiagnostic testing for detection of avian influenza virus (AIV) antigen or antibodies. The NP gene of AIV, cloned in pET vector, was expressed in Escherichia coli BL 21 strain to produce a 6x-His tagged recombinant NP (rNP) antigen of ∼61 kDa molecular weight as soluble fraction. The rNP antigen was detected in soluble fraction of bacterial cell lysate with anti-His HRPO conjugate and reacted with the reference AIV antibody positive serum in immunoblotting. The rNP was used to immunize BALB/c mice to produce hybridoma secreting anti-NP MAbs. Out of 11 anti-NP MAbs produced, 8D2-H5, 8D2-H9, and 6D11-A7 were of IgM isotype and 5D10-C9 and 5D10-F11 were of IgG2b type, while 3F3-D2, 7D2-C9, 7D2-G7, and 7D2-G8 were of IgG1 isotype. The MAbs 3F3-D2 and 7D2-G8 showed high intensity positive reaction with rNP and a low intensity reaction with H5N1 virus in Western blot analysis. The anti-NP MAbs produced in the present work may be valuable in developing a competitive ELISA or immunochromatographic strip test-based assays for the rapid diagnosis of avian influenza.