Igg2a

We used a previously described method (Legorreta-Herrera et al., 2004 (link)). Briefly, 1 µg of Pb ANKA antigen diluted in 100 µl of carbonate buffer was added to each well of the ELISA microplate (Corning, NY, USA) and incubated for 2 h at 37°C. The plate was washed with 0.5% Tween 20 in PBS and blocked with 3% skimmed milk in PBS for 2 h at 37°C. The plate was washed and incubated with 100 µl of (1:20) diluted test plasma for 1 h at 37°C; the plate was washed and precalibrated dilutions of Ab goat anti-mouse specific for IgM, IgG, IgG1, IgG2a, IgG2b, and IgG3 (Zymed, San Francisco California, USA) were added for 1 h at 37°C; the plates were washed and incubated with streptavidin-peroxidase (Sigma-Aldrich) after washing; and the plates were incubated with ortho-phenylenediamine at 0.4 mg/ml in citrate buffer with 0.03% hydrogen peroxide and incubated for 20 min in the dark. The absorbance was measured at 492 nm using a Stat-Fax 2100 microplate reader (Awareness Technology Inc. USA).

For cell culture and virus preparation, Dulbecco's Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were purchased from Life Technologies (Grand Island, NY, USA) and Lonza (Basel, Switzerland), respectively. To measure cytokine concentrations, cytometric bead array (CBA) mouse inflammation/Th1/Th2/Th17 cytokine kit, mouse IL-5 and IL-13 flex sets were purchased from BD Biosciences (San Diego, CA, USA). All reagents for flow cytometry including Golgi Plug, Cytofix/Cytoperm solution, anti-mouse CD16/32 (Mouse BD Fc Block^TM), CD4-APC-Cy7, CD44-FITC, IFN-γ-APC, IL-17-PE, IL-5-APC, CD11c-FITC, CD45-PerCP, Siglec-F-PE, and Ly6G-PE-Cy7 were purchased from BD Biosciences. For development of ELISA, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG was purchased from Southern Biotechnology (Birmingham, AL, USA). HRP-conjugated goat anti-mouse IgG1 and IgG2a were purchased from Zymed Laboratories (San Francisco, CA, USA).