Detection of autoreactive IgM and IgG by ELISA

Serum samples from naïve and infected animals at 30 dpi were used to examine the presence of mouse brain lysate-specific IgM and IgG using a colorimetric approach. For this purpose, polysorb ELISA plates (Biolegend) were coated overnight with 50 μg/ml either T. brucei Antat 1.1E whole cell lysate prepared in house, or mouse brain lysate (Novus Biologicals) in 1× coating buffer (Biolegend). After extensive washes with 1× ELISA washing buffer (Biolegend), total mouse IgM or IgG were detected in mouse serum (1:50 to 1:10,000 dilution in 1× PBS) or human CSF (1:400 in 1× PBS) by using Horseradish peroxidase-conjugated antibodies specific for mouse IgM (Thermo) or IgG (all isotypes; Sigma) using the recommended concentrations, and the resulting absorbance was detected at 450 nm using an ELISA Multiskan plate reader (Thermo).