The levels of HA-reactive IgG were measured in plasma and
in vitro stimulated B cell culture supernatants using the mPlex-Flu assay, as previously described
43 (link). The assay panels included whole HA or the head segments of influenza group 1, group 2, B strain and chimeric HA, as listed in
Table 1 . Briefly, 25
µL of plasma dilution (1:5000) or undiluted culture supernatants were incubated with 25
µL of a panel of beads coupled with HAs at room temperature for two hours on a rotary shaker (500 rpm) in the dark. Then 150
µL of phycoerythrin (PE) conjugated goat anti-human IgG (Southern Biotech, Birmingham, Al) was added and incubated at room temperature for 2 hours on a rotary shaker (500 rpm) in the dark. After wells were washed twice with PBS (pH 7.2) containing 0.1% BSA (MP Biomedical, LLC, France) and 0.1% Brij-35 (Thermo Scientific, Waltham, MA), IgG levels were analyzed on Magpix Multiplex Reader (Luminex, Austin, TX). All samples were measured in duplicate.
in vitro stimulated B cell culture supernatants using the mPlex-Flu assay, as previously described
43 (link). The assay panels included whole HA or the head segments of influenza group 1, group 2, B strain and chimeric HA, as listed in
µL of plasma dilution (1:5000) or undiluted culture supernatants were incubated with 25
µL of a panel of beads coupled with HAs at room temperature for two hours on a rotary shaker (500 rpm) in the dark. Then 150
µL of phycoerythrin (PE) conjugated goat anti-human IgG (Southern Biotech, Birmingham, Al) was added and incubated at room temperature for 2 hours on a rotary shaker (500 rpm) in the dark. After wells were washed twice with PBS (pH 7.2) containing 0.1% BSA (MP Biomedical, LLC, France) and 0.1% Brij-35 (Thermo Scientific, Waltham, MA), IgG levels were analyzed on Magpix Multiplex Reader (Luminex, Austin, TX). All samples were measured in duplicate.
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