ELISA plates (Evergreen Scientific, CA) were coated with 0.1 μg of protein per well in coating buffer [0.05 M sodium carbonate–sodium bicarbonate (pH 9.6)] overnight at 4°C. After washing three times with PBS-T buffer, the plates were blocked with PBS–3% BSA buffer for 1 hour at 37°C. The concentrations of antigen-specific IgG/G1/G2a in sera were monitored using a 5-fold dilution series beginning with an initial 100-fold dilution in PBS–1% BSA. The diluted serum samples were added to each well, and the plates were incubated at 37°C for 1 hour and washed five times with PBS-T buffer. The secondary goat anti-mouse IgG-HRP antibody (HRP-conjugated goat anti-mouse IgG1 or IgG2a secondary antibodies were used for IgG1/IgG2a subtypes; Invitrogen) was then added to each well at a 1:5000 dilution and incubated for 1 hour at 37°C, followed by washing five times with PBS-T buffer. Next, the TMB (3,3′,5,5′-tetramethylbenzidine) Microwell Peroxidase Substrate System (KPL) was applied in the dark for color development. After 10 min, the enzymatic reaction was quenched by adding TMB BlueSTOP (KPL) solution, and plates were read within 30 min at 650 nm using an ELISA reader (VersaMax; Molecular Devices). Endpoint titers were presented as the sample dilution resulting in an OD650 (optical density at 650 nm) equal to twice the mean background (negative serum) of the assay. For CXCL13 quantification in sera, the Mouse CXCL13 ELISA Kit (Boster Biological Technology, CA) was used by following the manufacturer’s instructions.

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