ELISA assay was performed with antigens at 0.5µg/ml and antibody at 1µg/ml. ELISA plates were coated on bench overnight in 0.1M Carbonate pH 9.6, 100µl/well, washed once in wash buffer 300µl/well (TBS + 0.1% Tween 20) and blocked for 2 hours in 1% BSA in D-PBS (300µl/well). Antibodies, diluted to working strength in diluent (DPBS + 1% BSA + 0.05% Tween 20 + 0.2% Proclin 950), were added at 100µl/well and incubated for 2 hours (shaken at ambient temperature), followed by 3 x 300µl/well washes. Goat anti Mouse IgG-HRP (Biorad103005) diluted 1 in 2500 in diluent, was added at 100µl/well and incubated with shaking for 1 hour at ambient temperature. Plate was then washed 6 x 300µl/well. TMB (KPL Sureblue 5120-0077) was added at 100µl/well. Reaction for screening assay was stopped by addition of 1M HCl (100µl/well) and absorbance read at 450nm.
Immunofluorescence was carried out in Vero cells. Cells were seeded on coverslips and infected with DENV (serotypes 1, 2, 3 and 4) for 48h at MOI 1. Control coverslips consisted of uninfected Vero cells. After fixation with 4% PFA, samples were stained with Mouse anti-Dengue virus NS1 antibody (AbDENVNS1-DA034 or MAB12136). Antibody was diluted 1:500 and Triton X-100 was used as detergent. Imaging was performed using a Leica SP5 confocal microscope. Immunofluorescence was observed in DENV serotype 1 and DENV serotype 3 infected cells, using AbDENVNS1-DA034 or MAB12136. DENV serotype 1 infected cells (DV1), stained with AbDENVNS1-DA034 are shown above (Virology Research Services Ltd).