HAI and ELISA assays. Whole virus, recombinant HA, or vaccine-specific ELISA was performed on starting concentrations of 10 µg/ml of virus or recombinant HA and on 1:20 dilution of the vaccine, as previously described (Wrammert et al., 2008) . In brief, microtiter plates were coated with live virus strains totaling 8 HAU of total virus per well or with 1 µg/ml of recombinant HA protein. To standardize the various ELISA assays, common highaffinity antibodies with similar affinities and binding characteristics against each virus strain were included on each plate, and the plate developed when the absorbance of these controls reached 3.0 ± 0.1 OD units. Goat anti-human IgG (goat anti-human I-peroxidase-conjugate; Jackson ImmunoResearch Laboratories) was used to detect binding of the recombinant antibodies, followed by development with horseradish peroxidase substrate (Bio-Rad laboratories). Absorbencies were measured at OD415 on a microplate reader (Invitrogen). Affinity estimates were calculated by nonlinear regression analysis of curves from eight dilutions of antibody (10 to 0.125 ug/ml) using GraphPad Prism. The HAI titers were determined as previously described (Wrammert et al., 2008) . In brief, the samples were then serially diluted with PBS in 96-well v-bottom plates and 8 HAU (as determined by incubation with 0.5% turkey RBCs in the absence of serum) of live, egg-grown virus was added to the well. After 30 min at room temperature, 50 µl of 0.5% turkey RBCs prepared, and purified as previously described (Wrammert et al., 2008) . The hemagglutination inhibition activity was determined using turkey red blood cells (Lampire Biological Laboratories) as previously described (Wrammert et al., 2008) ELISPOT assay. Direct ELISPOT to enumerate the number of either total IgG-secreting, pandemic H1N1 influenza-specific, or vaccine-specific plasmablasts present in the PBMC samples were essentially done as previously described (Crotty et al., 2003) . In brief, 96-well ELISPOT filter plates (Millipore) were coated overnight with either the optimized amounts of purified pandemic H1N1 virions, recombinant HA from the pandemic H1N1 (as above), the 08/09 influenza vaccine at a dilution of 1/20 in PBS, or goat anti-human Ig (Invitrogen). Plates were washed and blocked by incubation with RPMI containing 10% FCS at 37°C for 2 h. Purified and extensively washed PBMCs or sorted plasmablasts were added to the plates in dilution series and incubated for 6 h. Plates were washed with PBS, followed by PBS containing 0.05% Tween, and then incubated with a biotinylated anti-huIgG () antibody (Invitrogen) and incubated for 1.5 h at room temperature. After washing, the plates were incubated with an avidin-D-HRP conjugate (Vector Laboratories) and, finally, developed using AEC substrate (3 amino-9 ethylcarbazole; Sigma-Aldrich). Developed plates were scanned and analyzed using an automated ELISPOT counter (Cellular Technologies, Ltd.).