Fluorescence inhibition assay. Each viral isolate was titrated by the limiting dilution method and the viral titer calculated by the Reed-Muench formula. Neutralizing assays were carried out in 96 wells plate using MDCK cells (46104cells/ well). Serial dilutions, 30 mg/ml-0.03 mg/ml, of IgG PN-SIA28 were preincubated for 1 hour at 37uC with 100 TCID50 of each H1N1 or H3N2 virus. Following incubation, 100 ml of the mix antibody-virus were added to the cells and incubated for another hour at 37uC in 5% CO2. At the end of this incubation, cells were washed with PBS and 100 ml of MEM TPCK-Trypsin (2 mg/ml) were added in each well. Cells were incubated for 7 hours at 37uC in 5% CO2 and then washed with PBS, fixed and permeabilized with ice-cold ethanol. Cells were incubated with anti-influenza A mouse antibody (Argene, Shirley, NY, USA) for 30 minutes at 37uC in a humid chamber. The cells were then washed and incubated for 30 minutes at 37uC in a dark humid chamber with a FITC-conjugated secondary antibody (Argene, Shirley, NY, USA). Nuclei staining was obtained with Hoechst 33342 (Sigma Aldrich). An infection control without antibody was included, as well as a negative control with an anti-HCV/E2 antibody (e137). Each neutralization assay was performed in triplicate and repeated in two different sessions.