Goat anti mouse ig hrp

To determine antibody levels in supernatants of single GC B cell cultures or in the serum of immunized mice, ELISAs were performed as described before.^{20 (link)} 96-well high-binding half-area microplates (Greiner or Corning) were coated overnight at 4°C with antigen or capture antibody in PBS (25 μl per well). In between each step, plates were washed with PBS + 0.05% Tween20. After overnight incubation, plates were blocked with 2.5% bovine serum albumin (BSA, Sigma) in PBS for 2 hours at room temperature. For single GC B cell cultures experiments, 25 μl of undiluted supernatant was added to wells coated with 1 μg/ml goat anti-mouse Ig (Southern Biotech) or HA_PR8 and for serum samples 3-fold serial dilutions of serum samples starting at 1/100 were added to wells coated with NP₄-BSA (10 μg/ml, Biosearch) or HA_PR8 (1 μg/ml). After washing, detection occurred with goat anti-mouse Ig-HRP (Southern Biotech) or IgG-HRP (Jackson Immunoresearch) for single cell cultures and serum ELISAs respectively, followed by development with TMB (slow kinetic form, Sigma). The reaction was stopped with 1N hydrochloric acid and the absorbance was measured at 450 nm on a Fisher Scientific accuSkan FC plate reader.