Fig. 6: HA-specific IgM, IgY, and IgA antibody levels in the serum of chickens immunised with 35 μg dose of rH9HA, rH9HA-CD83 scFv, and inactivated H9N2 virus vaccines.

The HA-specific isotypes of the antibodies were determined in 200-fold diluted sera collected at day 6, 14, 21, and 28 post primary vaccination by ELISA. The plates were coated with 1 μg of recombinant H9HA protein overnight at 4 °C. For detection, the plates were incubated with respective sera for 1 h at room temperature. This was followed by further incubation for 1 h with 1:3000 diluted anti-chicken IgM, IgY, and IgA antibodies. The colorimetric detection was carried out by adding tetramethylbenzidine (TMB) substrate and absorbance at 450 nm was recorded. The amount of HA-specific IgM, IgY, and IgA antibodies were expressed as a sample to reference ratio (relation of absorbance of a tested serum sample to the absorbance of the reference serum). Data are presented as mean ± SD and analysed by one-way ANOVA followed by Tukey’s multiple comparison test. ****p < 0.0001 ***p < 0.001 **p < 0.01 *p < 0.05. ppv post primary vaccination.