The oligomerization status of the sH5 3 proteins was determined by analyzing the elution profile using a Superdex200GL 10-300 column (GE Healthcare). Sialic acid-binding activity of sH5 3 was assessed using a fetuin solid phase assay. 1 mg/ml fetuin per well was used to coat 96-well Nunc MaxiSorp plates. When indicated in the figure legend, fetuin was treated with Vibrio Cholera derived neuraminidase (VCNA; Roche) followed by three washing steps. sH5 3 was pre-complexed with horseradish peroxidase (HRP)linked anti-Strep-tag antibody (2:1 molar ratio) for 30 min at 0uC prior to incubation of limiting dilutions on the fetuin-coated plates (60 min, room temperature [RT]). HA-binding was subsequently detected using tetramethylbenzidine substrate (BioFX) in an ELISA reader (EL-808 [BioTEK]), reading the OD at 450 nm.