Bvd6 24g2

Levels of IL-2, IFN-γ, IL-4, IL-6, and IL-17 in culture supernatants were quantified using a sandwich ELISA. The following pairs of capture and biotinylated detection rat anti-mouse mAbs were used: for IFN-γ, anti-IFN-γ (P4-6A2, Biolegend) and biotin-conjugated anti-IFN-γ (XMG1.2, Biolegend) Abs; for IL-2, anti-IL-2 (JES6-1A12, BD Biolegend) and biotin-conjugated anti-IL-2 (JES6-5H4, BD Biolegend) Abs; for IL-4, anti-IL-4 (11B11, Biolegend) and biotin-conjugated anti-IL-4 (BVD6-24G2, Biolegend) Abs; for IL-6, anti-IL-6 (MP5-20F3, BD Biosciences) and biotin-conjugated anti-IL-6 (MP5-32C11, BD Biosciences); for IL-17, anti-IL-17 Ab (TC11-18H10, BD biosciences) and biotin-conjugated anti-IL-17Ab (TC11-8H4, BD Biosciences). Capture Abs (2 μg/ml) were coated onto 96-well plates. After blocking with 0.5% BSA in PBS containing 0.05% Tween 20, the diluted samples and recombinant protein standards were incubated for 1 h at room temperature. Plates were then incubated with biotin-conjugated detection Abs (1 μg/ml) for 1 h at 37°C and reacted with streptavidin-conjugated horseradish peroxidase, followed by o-phenylenediamine. The reaction was terminated by addition of 0.5 M H₂SO₄. The absorbance at 490 nm was then measured, and a graph was created by analyzing three samples.

Mouse IFN-γ (R&D Systems) and IL-12p40 (BD Biosciences) ELISA kits were used according to the manufacturer’s protocols to determine cytokine concentrations in mouse serum. Serum concentrations of IL-4 were determined using a sandwich ELISA. Maxisorp plates (Nunc) were coated with anti-mouse IL-4 capture antibody (2 µg/mL; 11B11; BioLegend) in 50 mM sodium bicarbonate buffer (pH 9.4) overnight. Plates were washed with 0.05% Tween 20 (Sigma-Aldrich) in PBS, blocked with 4% bovine serum albumin (BSA; Roche) in PBS for 2 h, and then samples and standards (mouse rIL-4; Peprotech), serially diluted two-fold from 2,000 pg/mL, were added for 1 h. Anti-mouse IL-4 detection antibody (0.5 µg/mL; BVD6-24G2, BioLegend) diluted in 4% BSA in PBS was added for 1 h, followed by HRP-conjugated streptavidin (2 µg/mL; Sigma-Aldrich) for 30 min. The plates were developed with TMB substrate (BD Biosciences), and the reaction stopped with 1 M H₂SO₄ (Sigma-Aldrich).