Mshan4b50

H1N1‐specific IgG⁺ PBMCs were detected using the ELISPOT kit (CT780‐PR5; U‐CyTech Biosciences) according to the manufacturer's protocol. Briefly, 96‐well plates (MSHAN4B50; Millipore) were coated overnight at 4°C with either 4 μg/ml influenza A H1N1 (A/Guangdong‐Maonan/SWL1536/2019) hemagglutinin protein (40717‐V08H; Sino Biological) or with a coating antibody (50 μl/well). The assay was performed in duplicate. After washing with PBS, the plates were blocked by incubation with blocking buffer (200 μl/well) at RT for 1 h. After removing the blocking solution, approximately 0.5 × 10⁵ PBMCs isolated from heparinized whole blood using Histopaque (10771; Sigma–Aldrich) according to the manufacturer's instructions were added (100 μl/well) and incubated overnight at 37°C and 5% CO₂. After washing with PBS and wash buffer, diluted biotinylated detection antibody was added (100 μl/well) and incubated at RT for 2 h. The plates were again washed (wash buffer), and diluted streptavidin‐HRP conjugate was added (100 μl/well) and incubated at RT for 2 h. After washing with wash buffer, the AEC substrate solution was added (100 μl/well) and incubated at RT for 30 min. After washing and air‐drying, the plates were analyzed using an ELISPOT reader (AID, ELR08 IFL). The ELISPOT assay image for H1N1‐specific IgG⁺ PBMCs is shown in Figure S2.

Total immunoglobulin M (IgM) was measured by ELISA with a plate coated with 0.5 μg/mL anti-IgM and then detected with horseradish peroxidase–conjugated anti-goat IgM Abs (Southern Biotech). ELISpot assay plates (Millipore MSHAN4B50) were coated with capture goat anti-IgM antibody (Southern Biotech 1021-01) at 0.5 μg/mL and blocked with complete growth media (RPMI, 10% FCS, β-mercaptoethanol, L-glutamine, HEPES, NEAA, L-sodium pyruvate solution, and penicillin–streptomycin). About 1 × 10⁴ stimulated FO B cells were plated on the top wells and serially triple-diluted and incubated overnight. Biotinylated goat anti-IgM AP (Southern Biotech 1020-04) capture antibody was used at 0.1 μg/mL. Spots were developed using 5-bromo-4-chloro-3-indolyl-phospate (BCIP)/nitro blue tetrazolium (NBT) liquid substrate.

Total immunoglobulin M (IgM) was measured by ELISA with a plate coated with 0.5 μg/mL anti-IgM and then detected with horseradish peroxidase–conjugated anti-goat IgM Abs (Southern Biotech). ELISpot assay plates (Millipore MSHAN4B50) were coated with capture goat anti-IgM antibody (Southern Biotech 1021-01) at 0.5 μg/mL and blocked with complete growth media (RPMI, 10% FCS, β-mercaptoethanol, L-glutamine, HEPES, NEAA, L-sodium pyruvate solution, and penicillin–streptomycin). About 1 × 10⁴ stimulated FO B cells were plated on the top wells and serially triple-diluted and incubated overnight. Biotinylated goat anti-IgM AP (Southern Biotech 1020-04) capture antibody was used at 0.1 μg/mL. Spots were developed using 5-bromo-4-chloro-3-indolyl-phospate (BCIP)/nitro blue tetrazolium (NBT) liquid substrate.