Fig. 7: Virus neutralising antibody titre measured by virus micro-neutralisation (MNT) test.

Virus MNT was carried out on day 28 post primary vaccination serum samples (35 μg dose group). The serum was first inactivated at 56 °C for 30 min and two-fold serum dilutions were made in phosphate-buffered saline. Then, the inactivated serum was incubated with 150 TCID₅₀ of H9N2 virus (UDL 01/08) for 1 h at 37 °C. The virus-serum mixture was then added onto the MDCK cells and further incubated for 1 h at 37 °C. After 1 h of incubation, the virus-serum mixture was removed from the cells and fresh media containing 2 μg/ml trypsin was added. The cells were stained with 0.1% crystal violet solution after 3 days. The virus neutralisation titre was expressed as the reciprocal of the highest serum dilution at which virus infection is blocked and the cells survive. Data are presented as mean ± SD and analysed by one-way ANOVA followed by Tukey’s multiple comparison test. **p < 0.01.