Differentiation between Vaccinated and Infected Poultry in Group by Avian Influenza Virus on the Basis of Antibody against NS1 by Indirect ELISA

Abstract

The DNA encoding NS1 protein of H9N2 avian influenza virus (AIV) was prepared by RT-PCR and inserted into pET-32a(+) to construct the expression plasmid pET-NSl. The recombinant plasmid was transformed into <i xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">E.coli</i> BL21 and expressed by IPTG induction at an optimized temperature. The recombinant NS1 protein was analyzed by SDS-PAGE and Western blot. The purified NS1 protein was plated as coating antigen to develop indirect ELISA for the detection of antibody against NS1. AIV negative sera and sera collected from H9N2 AIV infected birds and vaccinated birds were tested to determine the cut-off value between vaccinated poultry and virus infected poultry, and the minimum samples statistically. The results showed that a 46 KDa recombinant NS1 protein was expressed and the optimal coating concentration was 40 mug/mL, the optimal serum dilution was 1:200, the optimal HRP-IgG dilution was 1:10000 and the optimal reaction time for antigen and antibody was lh. The cut-off value was determined 0.5. To determine the antibody level against NS1 in a flock of poultry, at least 30 sera samples should be tested. The indirect-ELISA can be applied to differential diagnosis of vaccinated or infected poultry by AIV H9N2 in group.