1.Mapping of the monoclonal antibody epitopes was performed using linear peptides that were 15 amino acids in length and that overlapped by 11 residues spanning the full length of the peptide.
2.The ELISA plates were coated with 100 μl of peptides (10 μg/ml). Following overnight incubation, plates were blocked with PBS + 0.05% Tween-20 and 1% BSA for one h.
3.Plates were incubated with 100 μl of monoclonal antibodies or controls at varying concentrations (0.00006–5.0 μg/ml).
4.After one h, plates were incubated with 100 μl/well of the secondary antibody, at a 1:20,000 dilution in PBST-1% BSA. All the incubation steps were done at room temperature.
5.Plates were washed six times with PBS-Tween between each step.
6.After a final wash, samples were incubated for about 15 min with the TMB Plus Reagent according to the manufacturer's instructions. The optical density was read at 450 nm after addition of stopping solution (100 μl/well).