*2.5. Hemagglutination Inhibition Assay*

The hemagglutination inhibition assay (HAI) was used to quantify the antibodies against viral influenza A particles in serum from individual mice, as described previously [27]. An HI titre ≥ 40 was defined as a protective amount of serum antibodies [28]. Briefly, serum from individual mice were treated with receptor-destroying enzyme (RDE) overnight at 37 ◦C to remove the non-specific serum HAI inhibitors [29]. RDE was inactivated by incubation at 56 ◦C for 30 min., followed by the addition of 350 μL NaCl 0.9%. The HAI assay was initiated by adding 25 μL PBS to each well of a microtitre plate, followed by the addition of 50 μL of RDE treated serum. Serum was diluted in two-fold serial dilutions. 25 μL of influenza A/H1N1/SI or A/H1N1/CA09pdm containing four haemagglutinating units (HU) was added to each well. The plate was shaken, covered, and incubated at 20–25 ◦C for 15 min. Subsequently, 50 μL guinea pig erythrocytes were added, mixed, and followed by incubation for one hour at 4 ◦C. Thereafter, the plate was evaluated for hemagglutination and the degree of hemagglutination inhibition (HAI).
