RVFV Antibody Detection Assay

Maxisorp plates (Nalgene-Nunc, Rochester, NY) were coated with RVFV lysate prepared from infected Vero-E6 cells as previously described [24 (link)], diluted 1:2000 in PBS, and allowed to adsorb overnight at 4°C. Plates were blocked in 5% milk in PBS with 0.1% Tween-20 (PBST) with 5% FBS for 1 hour at 37°C. Patient samples and controls were serially diluted in blocking buffer and incubated on plates for 2 hours at 37°C. Plates were washed three times in PBST then incubated for 1 hour at 37°C with anti-human IgG HRP or anti-human IgM HRP (Jackson ImmunoResearch Inc, West Grove, PA) diluted 1:5000 in blocking solution. Plates were washed three times in PBST before incubation in TMB substrate (KPL, Milford, MA) for 10 minutes. Reactions were stopped with TMB stop solution, and plates were read at 450nm. Data were analyzed using Excel (Microsoft Corp, Redmond, WA) and Prism (GraphPad Software Inc, La Jolla, CA).