Quantitative ELISA sandwich for a new vaccine against avian influenza virus H5N1.

Analytical techniques are essential in the process of standardizing and validating vaccines. In this study we described a methodology to establish an ELISA sandwich for the quantification of a new vaccine against avian influenza virus H5N1 based on the main antigenic determinant of the virus, the extracellular domain of the glycoprotein hemagglutinin (HA), fused to the extracellular domain of the chicken CD154 glycoprotein (HACD). The chimerical proteins HA and HACD were produced in SiHa cells and the experiments were performed by using three monoclonal antibodies (MAb-HA1, MAb-HA2 and MAb-HA3), alone or conjugated to horseradish peroxidase (HRP-HA1, HRP-HA2 and HRP-HA3). The hemagglutination inhibition assay was carried out with a negative and a positive H5N2 reference serum, together with the antigen H5N1 A/Mallard/Italy/3401/05, all purchased from the "Istituto Zooprofilattico delle Venezie", Italy. After demonstrating the similar recognition pattern between the HA and the HACD proteins, the MAb-HA2 at a concentration of 2,5 μg/mL was selected as the capture antibody and the HRP-HA3 at a dilution of 1/20000 was selected as the detection antibody due to their optimal values of optical density at these conditions. The best dynamic range of the standard curve using the protein HACD was achieved at concentrations from 100 to 1,56 ng/mL. There were no significant differences when five batches of HACD were quantified by the ELISA sandwich and the bicinchoninic acid method linked to densitometry. In conclusion, the final parameters for the quantification of the chimeric protein HACD using an ELISA sandwich were described, which could contribute to develop a large-scale process for the final vaccine production.