J. Okada and others contribution of Abs that recognize site C should be considered (Koelle et al., 2006; Smith et al., 2004). METHODS Viruses. Twelve influenza A vaccine strains of type H3N2 (A/Aichi/2/ 68, A/Fukuoka/1/70, A/Tokyo/6/73, A/Yamanashi/2/77, A/Niigata/ 102/81, A/Fukuoka/C29/85, A/Guizhou/54/89, A/Kita-kyushu/159/93, A/Sydney/5/97, A/Panama/2007/99, A/Wyoming/3/2003 and A/New York/55/2004) were used in this study. Preparation of mAbs. The human mAbs with neutralizing activity used in this study were described previously (Okada et al., 2010). The Fab-PP (P denotes a single Fc-binding domain of protein A) form of Abs (Ito & Kurosawa, 1993) was purified with IgG Sepharose (GE Healthcare) and used in FCM analysis as well as in the HI assay. Alexa 488-anti-human IgG and Alexa 488-anti-mouse IgG used as the second Abs in FCM were purchased from Invitrogen. IgG was prepared by using a high expression vector (Bebbington et al., 1992) and purified with Protein A Sepharose (GE Healthcare). Purified IgGs were used in the HI assay. Construction of plasmid DNAs for cell-surface expression of HA and chimaeric HAs. Details are described in Supplementary Fig. S1 and the Supplementary Methods (available in JGV Online). Cell culture and transfection. 293T cells were maintained in Dulbecco’s modified Eagle’s medium (Wako) containing 10 % FBS and penicillin/streptomycin (Gibco). For FCM analysis, 293T cells in a 150 mm dish were transfected with 30 mg plasmid DNA (HA-chimaera expression vector) and 75 ml Lipofectamine LTX (Invitrogen) and were assayed 24 h after transfection. Cell-surface HA expression was verified by using anti-HA Ab F49 (Ueda et al., 1998). Haemadsorption assay. Haemadsorption activity of HA expressed on the cell surface was checked as described previously Nakajima et al. (2005) with some modifications. In brief, 0.3 ml of 2 % guinea-pig red blood cells was mixed with 0.3 ml of HA-expressed 293T cell suspension (56105) in a microtube, and the mixture was rotated slowly at 4 uC. After 1 h, the microtube was allowed to stand for 10– 15 min, so that 293T cells were precipitated at the bottom of the microtube while almost all of the free blood cells remained suspended. The suspended blood cells were removed, and the cell pellet was suspended in 1 ml of PBS in 0.05 % NaN3. The microtube was allowed to stand for 10–15 min and the suspended blood cells were removed again. The resulting cell pellet was suspended in 40 ml of PBS in 0.05 % NaN3, and the complexes of the HA-expressed 293T cells and the blood cells were observed under an optical microscope. FCM analysis. Cells transfected with HA expression vector (56105 cells per well) were incubated with 2.5 % goat serum in 2.5 % BSA for 30 min, then incubated with 5 mg Fab-PP ml21 or a 1 : 200 dilution of mouse mAb F49 for 1 h. Cells were washed with 2.5 % BSA, followed by incubation with Alexa 488-conjugated anti-human IgG or Alexa 488-conjugated anti-mouse IgG for 1 h. Then, cells were washed with 2.5 % BSA twice and resuspended in PBS at 56105 cells ml21 for analysis on a FACScan flow cytometer (Cytomics FC 500; Beckman Coulter). HR1-007, a haemorrhagic factor of Habu venom that recognizes the Fab region of the Ab, was used as a negative control for Fab-PP, and a 1 : 200 dilution of mouse serum was used as a negative control for F49. All reactions were performed on ice. HI assay. The HI test was performed as described previously (Okuno et al., 1990). Briefly, serial dilutions of 100 mg purified Fab-PP ml21 or 100 mg purified IgG ml21 in PBS were prepared. Serial dilutions of 334 Fab-PP were preincubated with 4 HA units of virus per well. Guinea- pig red blood cells were added to a final concentration of 0.5 %, and the plate was incubated at room temperature for 30–60 min. ACKNOWLEDGEMENTS This work was supported in part by a grant-in-aid for the 21st Century Center of Excellence (COE) program of Fujita Health University from the Ministry of Education, Culture, Sports, Science and Technology and a grant from Research on Pharmaceutical and Medical Safety from the Ministry of Health, Labour and Welfare of Japan. REFERENCES Bebbington, C. R., Renner, G., Thomson, S., King, D., Abrams, D. & Yarranton, G. T. (1992). High-level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker. Biotechnology (N Y) 10, 169–175. Ekiert, D. C., Bhabha, G., Elsliger, M. A., Friesen, R. H. E., Jongeneelen, M., Throsby, M., Goudsmit, J. & Wilson, I. A. (2009). Antibody recognition of a highly conserved influenza virus epitope. Science 324, 246–251. Gerhard, W., Yewdell, J., Frankel, M. E. & Webster, R. (1981). Antigenic structure of influenza virus haemagglutinin defined by hybridoma antibodies. 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