Plates were coated with 100 mL/well of purified REV gp90 antigen diluted in carbonate-bicarbonate buffer (pH 9.6) for incubation overnight at 4uC. Following 4 washes with 200 mL/ well of PBS/0.05% Tween-20, the plates were blocked with 200 mL/well of blocking buffer (PBS containing 5% skim milk) for 1 h at 37uC. The supernatant of hybridoma culture (100 mL/well) was added in duplicate and the plates were incubated for 1 h at 37uC. After washing three times with PBS, 100 mL of horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin G (IgG, 1:5,000 dilution,Sigma, St Louis, MO, USA) was added to each well and incubated for 1 h at 37uC. Plates were washed three times with PBS and incubated with 100 mL/well of o-phenylenediamine dihydrochloride (OPD, Sigma, St Louis, MO, USA) containing 0.3% H 2 O 2 for 5 minutes at room temperature in the dark. The reaction was stopped with 50 mL/well of 2 M H 2 SO 4 and the absorbance measured at 492 nm.