Maxisorp plates (Nalgene-Nunc, Grand Island, NY, USA) were coated with lysate from RVFV-infected Vero-E6 cells or with lysate from uninfected Vero-E6 cells (negative control) diluted 1:2000 in phosphate-buffered saline (PBS)36 (link) and allowed to adsorb overnight at 4 °C. Plates were blocked in 5% fetal bovine serum (FBS) and 5% skim milk in PBS 1 h at 37 °C. Patient serum samples were assayed in duplicate and were serially diluted in blocking buffer and then incubated on blocked plates for 1 h at 37 °C. After three washes in PBS with 0.1% Tween 20 (PBST), plates were incubated for 1 h at 37 °C with anti-human IgG HRP (Jackson ImmunoResearch Inc, West Grove, PA, USA) diluted 1:5000 in blocking solution with 0.1% Tween 20. Following 3 PBST washes, the plates were incubated in TMB substrate (KPL) for 10 min; reactions were stopped with the addition of 1% HCl, and plates were read at 450 nm. Data were analyzed using Excel (Microsoft Corp, Redmond, WA, USA) and Prism (GraphPad Software Inc, La Jolla, CA, USA) software. Raw OD values from the negative control Vero-E6 plate were subtracted from those of the RVFV lysate plate. The endpoint titer was defined as the dilution of the serum that gave a value at least three standard deviations above the average value obtained from the negative control serum.
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