Np2 bsa

For hapten NP-specific enzyme-linked immunosorbent assay (ELISA), NP2-BSA (bovine serum albumin) or NP14-BSA (10 μg/ml; LGC Biosearch Technologies) was coated in PBS. NP2-BSA was used to detect high-affinity Abs, and NP14-BSA was used to detect high- and low-affinity Abs. After blocking with 3% FCS–RPMI 1640, diluted serum samples were added into the plates. Anti-IgG Abs (1:3000 dilution; Rockland), anti-IgG1 Abs (1:500 dilution; A10551; Invitrogen), anti-IgG2b Abs (1:500 dilution; SL257799, Invitrogen), and anti-IgG2c Abs (1:1000 dilution; PA1-29288, Invitrogen) were used for the detection of each isotype. For the virus HA and nuclear protein–specific ELISA, recombinant H3N2 influenza A virus HA protein or recombinant H3N2 influenza A virus nuclear protein (2 μg/ml) was coated on the MaxiSorp ELISA plate (Thermo Fisher Scientific) in KPL coating solution (Seracare/LGC Clinical Diagnostics, Inc.). After blocking with 0.5% milk powder in 0.1% Tween 20 containing PBS, diluted serum samples were added into the plates. Concentrations of each isotypes were analyzed by area under the curve (AUC).

Fluorophore-labeled anti-B220 (RA3-6B2) and anti-FAS (Jo2) antibodies were from BD; anti-CD138 (281-2), anti-CD21/35 (CR2/CR1), and anti-IgD (11-26c2a) antibodies were from BioLegend; anti-GL7 (GL7), anti-CD38 (90), and anti-rabbit IgG antibodies were from Invitrogen; rabbit anti-cCasp3 and the matching isotype control antibodies were from Cell Signaling; and anti-GFP antibody was from Abcam. The fixable viability dye, zombie yellow, was from BioLegend. NP₁₈-OVA, NP₃₀-Ficoll, NP₂-BSA, NP-PE, and 4-hydroxy-3-iodo-5-nitrophenylacetyl (NIP)-BSA-biotin were from Biosearch Technologies. NP-APC conjugates were made by allowing NP-Osu (Biosearch Technologies) and APC (BioLegend) to react for 4 h at room temperature in a buffer containing 0.1 M NaHCO₃ and 0.15 M NaCl₂ (pH 8), at a molar ratio of 20:1. Dextran (200 kD, 31398) and Dextran-FITC (2,000 kD, FD2000S) were from Sigma-Aldrich.

Maxisorb Plates (Nunc) were coated with 0.05 μg/well CTH522 overnight at 4°C. Individual mouse sera were analyzed in duplicate. After blocking, serum was added in PBS with 2% BSA, starting with a 30-fold dilution for antigen-specific IgM, IgG or IgG subclasses. HRP-conjugated secondary antibody [rabbit anti-mouse IgG (Zymed), Goat anti-mouse IgG1 (Southern Biotech) or IgG2c (Thermofischer)] was diluted in PBS with 1% BSA. For detection of IgM, serum was added in PBS with 5% skimmed milk and detection was done using biotin conjugated anti-mouse IgM (Southern Biotech) for 1 h followed by streptavidin-HRP (BD Biosciences). After 1 h of incubation, antigen-specific antibodies were detected using TMB substrate as described by the manufacturer (Kem-En-Tec Diagnostics). The absorbance values were plotted as a function of the reciprocal dilution of serum samples. Antibody titers were determined as the highest serum dilution corresponding to a cut-off of ≥0.2 OD450. To measure anti-NP antibody responses, ELISA plates were coated with 0.1 μg/well of BSA coupled with different ratios of NP (NP2-BSA and NP13-BSA) (Biosearch Technologies).