Cell-based HA binding assay MDCK cells were grown in 96-well plate until complete confluence, then fixed with 4% paraformaldehyde (PFA, Sigma), washed three times in PBS and blocked for at least 2 hrs in 5% BSA. The indicated amount of purified soluble HA proteins, measured by the Bradford assay, were added to the wells in duplicates or triplicates and incubated overnight at 4uC. Cells were washed three times in PBS and then incubated with anti-FLAG antibody (Origene, Rockville, MD, USA; 1:1000 dilution for 2 hrs at room temperature) to detect sHA proteins bound to cell surface. After washing for three more times in PBS to remove unbound sHA proteins, cells were incubated with goat-anti-mouse secondary antibody conjugated with HRP (ZymedH, Invitrogen) at a 1:5000 dilution for 1 hr at room temperature. Unbound secondary antibody was removed by washing three times in PBS, and ABTS substrate (Invitrogen) was added to the plate according to the manufacturer's instructions. Forty minutes after the addition of substrate, absorbance at 415 nm (OD415) was measured using a Sunrise TM plate reader (Tecan, Mä nnedorf, Switzerland).