O phenylenediamine dihydrochloride substrate tablets

For virus capture ELISA assay, Microlon 600 96-well plates (VWR, Radnor, PA) were coated overnight at 4 °C with a mixture of 100 ng of 4G2/2H2 Abs in 0.1 ml coating buffer (Sigma, 08058). Unbound Abs were washed off with PBS containing 0.05% Tween 20, and blocked with 3% BSA (Fisher). Virus was diluted in blocking buffer to optimal concentrations, and 0.1 ml was added to each well and incubated 1 h at 37 °C. Unbound viruses were removed by washing, and serum samples diluted in blocking buffer were added and incubated for another hour. Unbound serum was removed by washing and then incubated for 1 h at 37 °C with goat-anti-monkey secondary Ab conjugated with HRP (Fitzgerald, MA, USA). Unbound secondary Ab was washed off, and signals were developed with o-phenylenediamine dihydrochloride substrate tablets (Sigma, 34006). OD was read at 492 nm.