Influenza Antibody Binding and ASC Quantification

Microneutralization (MN), HI assay were previously described78 (link). Sera and mucosal (BAL and nasal washes) Abs binding to influenza viruses were analyzed by ELISA. Briefly, Nunc 96 well plates (Maxi-sorb) were coated with 100 HAU WIV or 2 μg/ml His-tagged rHA proteins, then blocked with 4% BSA in PBS-Tween for 1 hr. Two or ten-fold dilutions of samples are added to the plates for 2 hr at room temperature or overnight at 4 °C. Plates were then developed by biotin-anti-mouse IgG/IgA followed by streptavidin (SA)-HRP (Southern Biotech). The signals were developed using 1 x TMB (ebioscience) and measured at 450 nm using a plate reader (Biotek). Ab binding strength was measured by biolayer interferometry on an Octet Red instrument (Fortebio, Inc.) using H1N1 recombinant HAs as previously described79 (link). The frequency of ASCs in spleen was measured by ELISpot assay as previously described80 (link). Briefly, ELISpot plates (Millipore) were coated with 100 HAU WIV overnight and blocked with cRPMI-1640 media. Dilutions of cells were added to plates and incubated overnight at 37 °C. Plate-bound Abs were probed by biotin-anti-mouse IgG, SA-alkaline phosphatase (Southern Biotech), then Vector Blue Substrate Kit (Vector Lab). Spots were counted using an ImmunoSpot^® ELISPOT reader (Cellular Technology Ltd.).