The following human H1N1 and H3N2 reference strains were acquired from the American Type and Culture Collection , 100 mg/ml of streptomycin (Gibco Invitrogen, Carlsbad, CA, USA) and 2 mg/ml TPCK-trypsin (Roche Applied Science). The A/swine/Parma/1/97 isolate was analogously grown on Newborn Swine Kidney (NSK) cells, kindly provided by the Zooprophylactic Institute of Brescia, Italy. At 80% confluence, cells in MEM supplemented with 2 mg/ml serum-free TPCK-trypsin, were infected with each strain at a MOI of 0.001. After 1 hour of infection, cells were washed with phosphate buffered saline (PBS); MEM supplemented with 2 mg/ml TPCK-trypsin was then added and cells were incubated at 37uC in 5% CO2 atmosphere. Cells were observed daily to monitor the cytopathic effect and, usually after 72-96 hours, the supernatant was collected, centrifuged at 1000 rcf for 10 minutes to eliminate cells debris and filtered with 0.22 mm filters (Millipore, Billerica, MA, USA). The supernatant was then aliquoted and stored at 280uC as cell-free virus. well). Serial dilutions, 10 mg/ml-0.03 mg/ml, of IgG PN-SIA49 were preincubated for 1 hour at 37uC with 100 median tissue culture infective doses (TCID50) of 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 with PBS 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 the anti-HCV/E2 antibody e137. Each neutralization assay was performed in triplicate and repeated in two different sessions.