Anti igg or igm biotin

Ninety-six-well plates were coated with 20 μg ml⁻¹ NP₂₂CGG or OVA, 512 HA units per mL PR8 virus, or 10 μg ml⁻¹ LCMV virus and blocked with complete RPMI (10% fetal bovine serum, 1% penicillin/streptomycin, 1% HEPES and 50 μM 2-mercaptoethanol). Bone marrow and splenic cells isolated from immunized mice were serially diluted in complete RPMI and incubated 16 h at 37 °C. The plates were then treated with either anti-IgG- or -IgM-biotin (Southern Biotechnology) followed by incubation with streptavidin-alkaline phosphatase (Sigma). Plates were then developed with 5-bromo-4-chloro-3-indolylphosphate (Sigma) until spots developed and spots counted with CTL Immunospot software.

96 well plates were coated with 100 HA of A/California/07/2009;H1N1, A/Michigan/45/2015;H1N1,A/Victoria/361/2011;H3N2, A/Singapore/INFIMH‐160019/2016;H3N2, B/Massachusetts/2/2012, B/Phuket/3073/2013, A/PR/8/34, 10 µg mL⁻¹ Rubeola Measles antigen (Meridian Life Science), or 10 µg mL⁻¹ varicella zoster virus antigen (Meridian Life Science10⁶ pfu mL⁻¹ HAd, 5 µg mL⁻¹ PspA, or 5 µg mL⁻¹ IgG or IgM (Southern Biotech). All plates were allowed to coat overnight at 4 °C and the next day blocked with complete media. Cultured human PBMCs or lung cells isolated from immunized mice were serially diluted in complete media and incubated overnight at 37 °C. The plates were then treated with either anti‐IgG‐ or ‐IgM‐biotin (Southern Biotech), followed by incubation with streptavidin‐alkaline phosphatase (Southern Biotech). Plates were then developed with the Vector Blue Alkaline Phosphatase Substrate Kit (Vector Laboratories) until spots developed and spots counted and analyzed with CTL immunospot software.