the number of collected B cells was estimated to be around 109, they probably represented 0.1–1 % of his total B-cell population. By using phage-display technology (Winter et al., 1994), we constructed a large Ab library composed of 361010 clones. The library was screened against inacti- vated virus particles of 12 different vaccine strains isolated between 1968 and 2004. The clones that bound to the antigens (Ags) were isolated, and mAbs that specifically bound to H3-strain viruses were selected. Finally, their binding activity to the 12 strains and neutralizing activity were studied. The 1143 clones were analysed. The sequence analysis revealed that they were composed of 153 unique mAbs with different VH sequences. Of the 153 clones, 113 showed both binding activity and virus-neutralizing activity, while the remaining 40 clones showed binding activity but did not show neutralizing activity. The majority of clones with neutralizing activity were anti-HA Abs that could be divided into three major groups showing distinct strain specificity: 1968–1973, 1977–1993 and 1997– 2003. In the present study, we analysed the location of the epitopes recognized by these clones. RESULTS A new method for identifying the location of epitopes recognized by mAbs with neutralizing activity Although epitopes could be identified by analyses of escape mutants that can grow in the presence of neutralizing mAb (Gerhard et al., 1981), this method is time-consuming and labour-intensive, especially since we wanted to identify the epitopes recognized by many mAbs. Therefore, we developed a simpler method for epitope mapping that is based on the analysis of chimaeras. After HA was artificially expressed on the cell surface, the binding activity of respective mAbs to HA was examined by flow cytometry (FCM). Since the range of specificity of the majority of clones was 1968–1973, 1977–1993 or 1997–2003 (Okada et al., 2010), HAs of the following five strains were selected: A/Aichi/2/68, A/Yamanashi/2/77, A/Fukuoka/C29/85, A/ Sydney/5/97 and A/Wyoming/3/2003. To identify the epitope, several chimaeric HA variants for each strain were constructed. We assumed that the epitopes would be localized in one of the seven sites (A, B1, B2, C1, C2, D or E), and we constructed chimaeric variants in which one of the seven sites was replaced by some other strain-derived sequence. Fig. 1(a) summarizes the amino acid sequences of the original five HAs and their chimaeric variants, and Fig. 1(b) shows the three-dimensional (3D) location of amino acid residues changed in the chimaeric HA of the Sydney/97 strain. In the case of the Aichi/68 strain, the original HA and nine kinds of chimaeric HA were constructed. To determine if the 3D structures of HAs artificially expressed on the cell surface were properly formed, the haemadsorption activity of the transfected cells was examined. As shown in Fig. 2, several reticulocytes http://vir.sgmjournals.org Epitope mapping of neutralizing Abs against H3N2 virus were attached to cells expressing the original HA and nine chimaeric HAs of the Aichi/68 strain. Essentially the same results were obtained for the other four strain-derived HA- transfected cells, with the exception of S97/50C1. These results indicate that the receptor-binding sites of HAs expressed on the cell surface were properly formed. To further examine whether these constructs could function as Ags, we performed pilot experiments with three representative clones: F033-161, F032-072 and F008- 007. Fig. 3 shows the FCM profiles. Clone F033-161, which binds and neutralizes the 1968–1973 strains, was tested for its ability to bind the HAs of the Aichi/68 strains (Fig. 3a). Clone F49, which binds to the epitope commonly present on HAs of all the H3 strains, was used as a positive control (Ueda et al., 1998). Clone F49 bound to the original HA- transfected cells and the nine chimaeric HA-transfected cells, suggesting that all of the HAs were expressed on the cell surface. F033-161 did not bind to either A68/188B2- or A68/219D-encoding DNA-transfected cells, but it did bind to all of the other HA-encoding DNA-transfected cells. These results suggest that F033-161 could not bind to HAs encoded by A68/188B2 and A68/219D, although they were present on the cell surface. We interpreted these results as evidence showing that the epitope recognized by clone F033-161 is located in site B2/D. Clone F032-072 was also tested (Fig. 3b). Since it binds and neutralizes the 1977– 1993 strains, HAs of Yamanashi/77 and Fukuoka/85 were used as binding targets. The results indicate that F032-072 does not bind to Y77/50C1, Y77/276C2, F85/50C1 or F85/ 276C2; however, it does bind to all of the other HAs. We concluded that clone F032-072 bound to site C1/C2. Finally, clone F008-007 was examined (Fig. 3c). Since it binds and neutralizes the 1997–2003 strains, HAs of Sydney/97 and Wyoming/2003 were used as binding targets. F49 did not react with S97/50C1; therefore, we determined that this chimaeric HA molecule was not properly expressed on the cell surface. F008-007 did not bind to S97/155B1, S97/189B2 or S97/50C1. Since F008- 007 also did not bind to W03/156B1 or W03/189B2 but did bind to all of the other HAs of the Sydney/97 and Wyoming/2003 strains, we concluded that site B1/B2 may contain the epitope recognized by this clone. Based on the FCM results from the chimaeric HAs together with the examination of their haemadsorption activity, we concluded that the HA constructs of the five H3 strains, with the exception of S97/50C1, could function as Ags expressed on the cell surface and that the epitopes of these Ags could be recognized by respective mAbs. Thus, we termed this method EMAC (epitope mapping through analysis of chimaeras). Location of epitopes recognized by 98 mAbs that neutralize H3N2 strains We used the EMAC method to examine epitopes recognized by mAbs with neutralizing activity. Ninety- eight clones that had been classified into 27 groups (Okada 327