The total IgG concentration in mouse serum was determined using the IgG (Total) Mouse Uncoated ELISA Kit (Invitrogen, Cat no. 88-50400) according to the manufacturer’s instructions. Serum samples were diluted 1:10,000 before addition to ELISA plates. The optical density was measured at 450 nm with a Multiscan FC Plate Reader (Thermo Scientific).
To assess Tp2-specific IgG production, Nunc MaxiSorp™ Flat bottom 96-well ELISA plates were coated with Tp2 monomer (10 µg mL-1, 100 µL per well) at 4°C overnight. After blocking (1xPBS + 0.1% Tween™20 + 1% BSA) for 2h at RT, mouse serum samples (diluted 1:30) were added to each well and incubated with cross-absorbed anti-mouse IgG HRP polyclonal antibody (diluted 1:10,000) (STAR117P, Bio-Rad) for 2h at RT on a Multiscan FC Plate Reader (Thermo Scientific) at medium shaking speed. After four washes in 1xPBS with 0.05% Tween™20, plates were developed with TMB substrate solution (Invitrogen) for 20 min at RT. The enzymatic reaction was stopped by adding 2N H2SO4 (R&D Systems) to each well and the plates read at 450 nm using a Multiscan FC Plate Reader (Thermo Scientific). ELISA results were presented as normalised OD450 with the mean OD450 values of blank wells subtracted from the sample well values.
To assess Tp2-specific IgG production, Nunc MaxiSorp™ Flat bottom 96-well ELISA plates were coated with Tp2 monomer (10 µg mL-1, 100 µL per well) at 4°C overnight. After blocking (1xPBS + 0.1% Tween™20 + 1% BSA) for 2h at RT, mouse serum samples (diluted 1:30) were added to each well and incubated with cross-absorbed anti-mouse IgG HRP polyclonal antibody (diluted 1:10,000) (STAR117P, Bio-Rad) for 2h at RT on a Multiscan FC Plate Reader (Thermo Scientific) at medium shaking speed. After four washes in 1xPBS with 0.05% Tween™20, plates were developed with TMB substrate solution (Invitrogen) for 20 min at RT. The enzymatic reaction was stopped by adding 2N H2SO4 (R&D Systems) to each well and the plates read at 450 nm using a Multiscan FC Plate Reader (Thermo Scientific). ELISA results were presented as normalised OD450 with the mean OD450 values of blank wells subtracted from the sample well values.