The binding activity of PN-SIA49 was assayed using full-length wild type and mutants HAs. Human epithelial kidney HEK293T cells (ATCC CRL-1573) were transfected in 6 wells plate (Corning, Corning, NY, USA) (16106 cells/well) with 4 mg of pcDNA 3.1D/V5-His-TOPO vector containing the HA nucleotide sequences described above. After centrifugation and fixation with 4% paraformaldehyde for 15 minutes at RT, the transfected cells were incubated for 30 minutes at room temperature with PN-SIA49 or conformational controls for H1N1 (RB62) at 10 mg/ml. Additionally, the isotype control, e137 (10 mg/ml) was introduced as well as untransfected cells and a mouse anti-influenza A HA (H1 subtype) monoclonal antibody (GeneTex Inc., Irvine, CA, USA) directed against a linear epitope to evaluate the transfection efficiency and the expression level for each HA. The cells were then washed with PBS and incubated for 30 minutes at room temperature with FITC-conjugated anti-human (Sigma Aldrich) or anti-mouse (Argene, Shirley, NY, USA) antibody. Afterwards, the cells were washed with PBS and analyzed by FACS. The FACS data were analyzed using the software Weasel w 2.5 (Waler+Eliza Hall, Institute of Medical Research, Parkville Victoria, Australia). The binding of PN-SIA49 to the different HA-mutants was then expressed as a binding percentage compared to wild-type. The data showing the PN-SIA 49 binding decrease between H1N1 wild type HA and H1N1 HA mutants, were obtained normalizing each PN-SIA 49 binding value to corresponding anti-H1 expression control values.