Np50 bsa

To measure anti-NP antibody titers, Maxisorp immunoplates (Thermo Fisher Scientific) were coated with NP₅₀-BSA (Biosearch Technologies), followed by blocking with 0.5% BSA in PBS containing 0.05% Tween-20 (PBST). Serially diluted sera were added to the wells, followed by incubation for 5–6 h at room temperature. After washing with PBST, HRP-conjugated goat anti–mouse IgM or IgG1 (Southern Biotech) was added to the wells, followed by overnight incubation at 4°C. The HRP activity was detected with tetramethylbenzidine substrate (KPL), and absorbance at 450 nm was measured using an iMark microplate reader (Bio-Rad Laboratories) after quenching the reaction with 1 N HCl. To measure the amount of noradrenaline, LNs were homogenized in 0.01 N HCl in the presence of 1 mM EDTA and 4 mM sodium metabisulfite. The pH of the collected supernatants was adjusted to ∼7 by adding 1 N NaOH. The concentration of noradrenaline in the supernatants was measured with a kit (Labor Diagnostika Nord). The amount of noradrenaline was normalized to the weight of the LNs.