Quantification of Antigen-Specific IgG by ELISA

To measure antigen-specific IgG, ELISAs were performed, as described previously (29 (link)). High-binding 96-well ELISA plates (Greiner Bio-one, Kremsmünster, Austria) were coated overnight at 4°C with 1 μg/ml recombinant protein (produced as described elsewhere (12 (link), 18 (link), 19 (link), 21 (link))), washed with PBS–Tween (phosphate-buffered saline–0.05% Tween) and incubated with blocking buffer (1% BSA in PBS, pH 6.5) for 2 h at room temperature. Mouse sera (collected at day 42 before tick challenge) were diluted in blocking buffer, added to the wells and incubated for 1 h at room temperature. Plates were washed and incubated for 1 h with horseradish peroxidase (HRP)-linked anti-mouse IgG (Cell Signaling, Beverly, MA, USA) diluted 1:1,000 in blocking buffer. The plates were washed again and developed using TMB substrate [50 µl TMB chromogene in 5 ml TMB substrate buffer (8,2 gr NaAc and 21 gr citric acid monohydrate dissolved in 1 liter H₂O + 10 µl 3% H₂O₂, pH 5)] and optical density was measured in a Biotek (Winooski, VT, USA) ELISA plate reader at 450–655 nm.