ELISA was conducted to measure HA-specific antibody (IgG) responses in immunized rabbits and mice. The 96-well flat-bottom plates were coated with 100 ml of ConA (50 mg/ml) for 1 hour at room temperature, and washed 5 times with PBS containing 0.1% Triton X-100. Subsequently, the plates were incubated overnight at 4uC with 100 ml of transiently expressed HA antigen at 1 mg/ ml. After being washed 5 times as above, the plates were then blocked with 200 ml/well of blocking buffer (5% non-fat dry milk, 4% Whey, 0.5% Tween-20 in PBS at pH7.2) for 1 hour. After five washes, 100 ml of serially diluted rabbit or mouse serum was added in duplicate wells and incubated for 1 hour. After another set of washes, the plates were incubated for 1 hour at 37uC with 100 ml of biotinylated anti-rabbit or anti-mouse IgG (Vector Laboratories, Burlingame, CA) diluted at 1:1000 in Whey dilution buffer (4% Whey, 0.5% Tween-20 in PBS). Then, 100 ml of horseradish peroxidase-conjugated streptavidin (Vector Laboratories) diluted at 1:2000 in Whey buffer was added to each well and incubated for 1 hour. After the final washing, the plates were developed with 3,39,5,59 Tetramethybenzidine (TMB) solution at 100 ml per well (Sigma, St. Louis, MO) for 3.5 minutes. The reactions were stopped by adding 25 ml of 2 M H 2 SO 4 , and the plates were read at OD 450 nm. The end titration titer was determined as the highest serum dilution that has an OD reading above twice of that from the negative control serum.