Antibody Titer Measurement with ELISA

The multiepitope peptide rOmp22 was first diluted to an optimal concentration (10 μg/mL) to coat a 96-well plate. The resulting solution was then added to each well (100 μL per well) and incubated for 12–18 h at 4°C. After washing five times with PBS plus 0.05% Tween 20 (PBST), 200 μL of 2% bovine serum albumin (BSA) was added to each well and incubated for 2 h at 37°C to block the unoccupied sites. After washing, serial dilutions of the pooled serum in each group ranging from 1:200 to 1:51,200 were added to the wells in triplicate and incubated at 37°C for 2 h. The plates were washed 5 times as described above. Then, 100 μL per well of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (diluted 1:10,000) was added and the plates were incubated for 1 h. The plates were then washed 5 times and incubated with 100 μL per well of 3,3ʹ,5,5ʹ-tetramethylbenzidine solution (TMB) as the substrate until the desired absorbance was reached. The reaction was stopped by the addition of 100 μL of 2 M H₂SO₄. The optical density of the samples was measured at 450 nm using an ELISA plate reader. The endpoint titer was defined as the highest dilution for which the OD at 450 nm was at least 0.1 above that of the background well.