1 M Na2CO3/NaHCO3, pH 9.2, 50 μL/well) at 4 °C overnight. Plates were blocked with PBST and 3% (w/v) non-fat dry milk for

2 h at room temperature. Plates were incubated with 3-fold dilutions to endpoint titre of pooled serum samples (100 μL per well in PBST with 1% non-fat dry milk (starting concentration: 1:50 dilution) for 2 h at room temperature. Following three washes with 100 μL PBST, plates were incubated with horseradish-peroxidase-conjugated anti-mouse IgG (Santa Cruz Biotechnology, Dallas, TX) at a dilution of 1:3000 in PBST and non-fat dry milk (1%, v/v) for 1 h at room temperature. Unbound antibody was removed by three washes with 100 μL PBST and plates were developed using SigmaFAST OPD substrate (Sigma, St. Louis, MO) (100 μL/well) and stopped with 3 M HCl (50 μL/well). The colorimetric change was measured as the optical density (OD 490 nm) on a Synergy 4 (BioTek, Winooski, http://www.selleckchem.com/products/BIBW2992.html VT) microplate reader. The endpoint titre was defined as the reciprocal of the highest dilution that yields an OD-value above the mean plus three standard deviations of blank wells. The hemagglutination inhibition (HI) assay was used to assess functional antibodies to the HA able to inhibit agglutination of turkey red blood cells (tRBCs). Serum samples were treated with 4 volumes of a receptor-destroying enzyme of Vibrio cholera filtrate

(Sigma, St. Louis, MO) for 18 h at 37 °C. After addition of 3 volumes of 2.5% Selleckchem Buparlisib (v/v) sodium citrate, the serum samples were incubated at 56 °C for 30 min and diluted with PBS unless to yield a 1:10 dilution of the original serum sample. Serum samples were 2-fold serially diluted in PBS (25 μL sample volume) in Nunc® 96-well polystyrene V-bottom microwell plates (Thermo Fisher Scientific, Waltham, MA) and then incubated with recombinant reassortant virus (PR8:AH1, PR8:SH1, PR8:malNL00, PR8:malAlb01 or PR8:chickJal12) at 4 HAU/25 μL in PBS for 30 min at room

temperature. Then, 50 μL 0.5% tRBCs (Lampire Biological Laboratory, Pipersville, PA) were added and the mixture was incubated for 45 min at 4 °C. Sera from all groups were assayed individually for the challenge strain PR8:SH1 for HI activity. Divergent H7 strains were assayed with pooled sera. The HI titre was calculated from the reciprocal of the highest dilution that completely inhibited hemagglutination of red blood cells and the geometric mean titre (GMT) of two independent assays was reported as the final titre. Two negative HI readings were assigned <10, single negative results were scored a value of 5 for the calculation of geometric means. In preparedness for a potential H7N9 pandemic, it is highly desirable, not only for vaccine manufacturers, but also for health care providers, to develop an influenza vaccine that at low vaccine dose, most preferably with a single administration, stimulates good immune responses.