Antibody-Mediated Neutralization of Dengue Virus

U937 cells expressing DC-SIGN were seeded overnight on 96-well plates [10] (link). A six point dilution series of protein G–purified mAbs diluted in RPMI 1640 medium without FCS were incubated with a constant amount of DENV-1 05K2916 (EU081234), DENV-2 TSV01, DENV-3 VN32/96, or DENV-4 2641Y08 at an MOI of 1 for 30 min at 37 °C. Plasma or mAb–virus mixtures were then transferred onto the U937-DC-SIGN cells and incubated for 2 h at 37 °C before adding RPMI 1640, 10% FCS. After incubation overnight, cells were stained intracellularly with Abs against NS1 and E protein and analyzed on a FACSVerse cytometer (Becton Dickinson). Data were analyzed using FlowJo software (TreeStar). The proportions of infected cells were plotted against the dilution factor, and the EC50 was calculated with Prism5 (GraphPad Software), applying a three-parameter non-linear curve fit.