Fig. 2: Analysis of the oligomeric form and activity of recombinant H9HA ectodomain containing foldon.

a BS3 cross-linking experiment for determining the native structure of H9HA ectodomain with foldon. Lane 1: rH9HA without BS3 Lane 2: rH9HA with 10 mM BS3 Lane 3: rH9HA-CD83 scFv without BS3 Lane 4: rH9HA-CD83 scFv with 10 mM BS3. About 15 µg of the recombinant protein was mixed with BS3 to a 10 mM final concentration and incubated for 1 h at room temperature. The cross-linking reaction was stopped by the addition of 1 M Tris-HCl pH 8.0 to a final concentration of 50 mM and incubated for 15 min at room temperature. After cross-linking, proteins were separated on 8% SDS-PAGE under reducing conditions, blotted, and analysed by western blot using anti-H9HA monoclonal antibody. M: monomer (70 kDa* 100 kDa^) D: dimer (140 kDa* 200 kDa^) T: Trimer (210 kDa* 300 kDa^) *rH9HA ^rH9HA-CD83 scFv. All blots were derived from the same experiment and were processed in parallel. b Haemagglutination assay to test the activity of recombinant H9HA with foldon to agglutinate chicken red blood cells. 1. rH9HA 2. rH9HA–CD83 scFv 3. Negative control (PBS). For the haemagglutination assay, a two-fold serial dilution of 35 μg of the recombinant HA proteins was carried out in 96-well plates. About 50 μl of 1% chicken RBCs was added. The plates were incubated at 4 °C for 1 h and the highest dilution of the protein causing the agglutination of the RBCs was noted.