*2.10. Surface Plasmon Resonance (SPR) Analysis*

Interactions between the influenza HA and the compounds were analyzed using the Berthold bScreen LB 991 (LabXMedia group, Midland, Canada) at 4 ◦C. Recombinant influenza HA protein (Abcam, Cambridge, England) from virus strain A/Vietnam/1203/2004 (H5N1) was immobilized on a sensor chip (Photo-cross-linker SensorCHIP™) using an amine coupling kit (GE Healthcare, Buckinghamshire, UK). Subsequently, compounds were injected as analytes at various concentrations, and we used PBST (10 mM phosphate buffer with 0.1% Tween 20, pH 5.0) as running buffer. For binding studies, analytes were applied at corresponding concentrations in running buffer at a flow rate of 30 μL/min with a contact time of 600 s and a dissociation time of 360 s. Chip platforms were regenerated with regeneration buffer (Glycine-HCl, pH = 2.0) after each test cycle. The processing and analyses of association and dissociation rate constants (Ka/Kon and Kd/Koff respectively) and the equilibrium dissociation constant (KD, kd/ka) were performed using the data analysis software of the bScreen LB 991 unlabelled microarray system according to a single-site binding model (1:1 Langmuir binding) with mass transfer limitations for binding kinetics determination.
