RVFV Antibody Detection ELISA

MaxiSorp plates (Thermo Scientific) were coated with lysate, diluted 1:1,000 in PBS, from RVFV-infected Vero-E6 cells or with lysate from uninfected Vero-E6 cells to act as a negative control (McElroy et al., 2009 (link)). Plates were left at 4°C overnight, and then blocked in blocking buffer (5% non-fat milk in PBS-0.1% Tween 20) at 37°C for 1 h. Terminal mouse serum samples were serially diluted in blocking buffer, and then incubated on blocked plates at 37°C for 2 h. All serum samples were assayed in duplicate alongside normal mouse serum as a negative control. After incubation with sera, plates were washed three times with PBS-0.1% Tween 20 (PBST), and then incubated for 1 h at 37°C in anti-mouse IgG-HRP (Jackson ImmunoResearch) diluted 1:5,000 in blocking buffer. Following three PBST washes, the plates were incubated in tetramethylbenzidine (TMB) substrate, and then stopped with TMB stop solution. Plates were read at 450 nm and the raw data were analyzed in Excel by subtracting the negative control absolute values from those of the RVFV lysate plate. The endpoint ELISA titer for each mouse was defined as the highest dilution of serum that resulted in an OD value at least two standard deviations above the average obtained from all negative mouse serum control wells.