solution containing the antibody was dialyzed against PBS and then concentrated using Amicon Ultra-15 Centrifugal Filter Devices (Millipore, Billerica, MA, USA). Antibody concentration was determined by SDS-PAGE gel and by spectrophotometric measurement at 280 nm. An anti-influenza A antibody directed against the HA (H1N1 subtype), named RB62, and an anti-HCV E2 glycoprotein antibody, named e137, produced and purified with an identical procedure were used as controls in all experiments. Viruses and cells The following reference strains were tested in the BLS3 laboratory of the Vita-Salute San Raffaele University: (H1N1) A/Puerto Rico/8/1934 (ATCC n. VR-1469); (H1N1) A/Wilson Smith/1933 (ATCC n. VR-1520); (H1N1) A/Malaya/302/1954 (ATCC n. VR-98); (H3N2) A/Hong Kong/8/1968 (ATCC n. VR-544); (H3N2) A/Aichi/2/1968 (ATCC n. VR-547); (H3N2) A/Victoria/3/1975 (ATCC n. VR-822); (H3N2) A/Port Chal- mers/1/1973 (ATCC n. VR-810). The swine origin influenza virus (S-OIV) (H1N1v) A/Milan/UHSR1/2009 was previously isolated in our laboratory [19,20]. A swine strain ((H1N1) A/ swine/Parma/1/1997) was kindly provided by the Zooprophy- lactic Institute of Brescia, Italy. The following H2N2, H3N2, H5N1, H7N2 and H9N2 viruses were tested at the Centers for Disease Control and Prevention of Atlanta, Georgia, USA: (H2N2) A/Ann-Arbor/6/1960; (H3N2) A/Philippines/01/1982; (H3N2) A/England/648/1989; (H3N2) A/Fukui/20/2004; (H3N2) A/Washington/01/2007; (H3N2) A/NewHampshire/ 01/2009; (H5N1) A/Vietnam/1203/2004 clade 1; (H5N1) A/ duck/Vietnam/NCVD98/2007 clade 2.3.4; (H7N2) A/New York/107/2003; (H9N2) LAIV A/chicken/Hong Kong/G9/ 1997. All viruses, excluding the A/swine/Parma/1/1997, were cultured on MDCK (Madin-Darby Canine Kidney) (ATCC CCL-34) cells propagated in Modified Eagle Medium (MEM) (Gibco Invitrogen, Carlsbad, CA, USA) supplemented with 10% inactivated fetal bovine serum (Gibco Invitrogen, Carlsbad, CA, USA), 50 mg/ml of penicillin (Gibco Invitrogen, Carlsbad, CA, USA), 100 mg/ml of streptomycin (Gibco Invitrogen, Carlsbad, CA, USA). The A/swine/Parma/1/97 isolate was analogously grown on NSK (Newborn Swine Kidney) 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 (Roche Applied Science), were infected with each strain at a MOI of 0.001. After 1 hour of infection, cells were washed with PBS (Phosphate buffered saline); MEM supplemented with 2 mg/ ml 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 stock. Virus neutralization assays 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 Cross-Neutralizing mAb to Influenza Viruses 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. The neutralization activity for each antibody concentration was expressed as the percentage reduction of fluorescent nuclei compared with the nuclei count in the infection control. Nuclei counting was performed by using the GE Healthcare’s IN Cell Analyzer 1000, an automated epifluorescence based microscope system. The neutralization curves were then fit by non-linear regression with the GraphPad Prism software, allowing IC50 calculation. Colorimetric assay. Each viral isolate was titrated to establish working dilution that produces 15–30 foci forming units per well in 96 tissue culture plates. Neutralizing assays were carried out in 96 wells plate using MDCK/SIAT-1 cells. Serial dilutions, 30 mg/ml-0.37 mg/ml, of IgG PN-SIA28 were preincubated for 1 hour at 37uC with the subset of viruses. Following this incubation, 100 ml of the antibody-virus mix was added to the cells and incubated for another hour at 37uC in 5% CO2. At the end of this incubation, the cells were washed twice in PBS and 100 ml of virus growth media containing 2 mg/ml of TPCK treated trypsin was added. Cells were incubated for 12–16 hours at 37uC in 5% CO2 and then washed with PBS, fixed and permeabilized with ice cold methanol/acetic acid (95:5) for 30 min at 220uC. Cells were incubated with anti-NP antibodies (Millipore, Billerica, MA, USA) for 30 minutes at 37u. The cells were then washed and incubated for 30 additional minutes at 37uC with a mouse HRP-conjugated secondary antibody. True Blue chromogenic substrate (KPL) was used to count the number of foci. Plaque reduction assay. Each viral isolate was titrated by the limiting dilution method and the viral titre calculated by the Reed-Muench formula. The viral titre was also calculated as plaque forming units (PFU) on six-well flat-bottomed plates (Corning, Corning, NY, USA). Neutralizing assays were carried out in 6 wells plates using MDCK cells (56105 cells/well). Two dilutions, 1–0.1 mg/ml, of IgG PN-SIA28 were preincubated for 1 hour at 37uC with 100 TCID50 each of H1N1 or H3N2 virus. Following this incubation, 1 ml each of virus-antibody mix was added on MDCK monolayer and the plate was incubated 1 hour at 37uC in 5% CO2. After this incubation, the medium was removed and the monolayer washed twice with PBS. Two ml of MEM-agarose 0.8% supplemented with penicillin (50 mg/ml) (Gibco Invitrogen, Carlsbad, CA, USA), streptomycin (100 mg/ml) (Gibco Invitrogen, Carlsbad, CA, USA), L-glutamine (2 mM) (Gibco Invitrogen, Carlsbad, CA, USA) and trypsin (2 mg/ml) (Roche Applied Sciences) were gently added to each well and the plates were incubated 48 hours at 37uC in 5% CO2. After this incubation the agarose medium was removed from each well and 1 ml of 70% methanol-crystal violet 1% (w/v) was added to each well at room temperature. Finally, the wells were washed with tap water and dried. An infection control without antibody was added as well as a negative control with anti-HCV/E2 e137 mAb. The neutralization was determined counting the PFU reduction in presence of antibodies in comparison with the infection control. PLoS ONE | www.plosone.org 8 December 2011 | Volume 6 | Issue 12 | e28001