Biotinylated rat anti mouse igg1

The levels of L. major-specific IgG1 and IgG2b antibodies were determined by ELISA in the sera of infected BALB/c mice as previously described [17 (link)]. Sera were obtained 40 days post infection at the end of the experiment. Biotinylated goat anti-mouse IgG2 (Southern Biotech) and biotinylated rat anti-mouse IgG1 (BD Pharmingen) were used. Plates were read at an optical density of 490 nm. A titration curve was performed for all samples.

T. muris specific IgG1 and IgG2a antibody responses were investigated in the serum of day 19 infected mice. ELISA plates were coated with 5 μg/ml T. muris ES antigen diluted in carbonate bicarbonate buffer (15 mM Na₂CO₃, 35 mM NaHCO₃ adjusted to pH 9.6) and incubated overnight at 4 °C. Plates were washed with PBS Tween (PBS containing 0.05% Tween20, Sigma-Aldrich) and blocked with 3% BSA in PBS Tween. Serum was diluted 1:20–1:2560 in PBS Tween, added to the plate at 50 μl/well, and incubated for 1 h at 37 °C. Plates were washed and incubated with 50 μl/well of biotinylated rat anti-mouse IgG1 (BD Biosciences, Oxford, UK) or IgG2a (BD Biosciences) for 1 h at room temperature. After washing, plates were incubated with 75 μl/well streptavidin β peroxidase (Roche Diagnostics GmbH, Germany) for 1 h at room temperature. Plates were then washed and developed with 100 μl/well of 3,3′, 5,5′ tetramethylbenzidine (Ultra TMB ELISA substrate, Thermo Fisher Scientific). The color development was stopped by adding 100 μl/well of 2 N sulphuric acid (R&Dsystems, Abingdon, UK). Absorbance was read using a Dynex MRX11 plate reader (Dynex Technologies, West Sussex, UK) at 405 nm with a reference of 490 nm.