MAYVBeAr was heat inactivated at 56°C for 30 minutes and diluted in PBS to 9.4x104 PFU/100 μl was added to each well of high binding flat bottom 96 well plates (Corning). Plates were sealed and incubated at 4°C for 4 days to allow for virus coating. Plates were blotted dry and blocked for 1 h with 100 μl of 5% milk in PBS with 0.05% Tween-20 (ELISA buffer). Prior to use the plates were washed three times with 100 μl of ELISA buffer. Heat-inactivated sera from mice was diluted 1:50 in ELISA buffer and serially diluted 1:3 for a total of 6 dilutions. 100 μl of each dilution was added to wells and incubated for 1.5 h at room temperature. Plates were washed three times with PBS with 0.05% Tween-20 (ELISA wash buffer) and blotted dry. Secondary antibodies (goat anti-mouse IgG1 HRP conjugated–Rockland 610-103-040; goat anti-mouse IgG2b HRP conjugated–Rockland 610-103-042; goat anti-mouse IgG3 HRP conjugated–Rockland 610-103-043; goat anti-mouse IgG + IgM HRP conjugated–Rockland 610-103-115) were diluted 1:10,000 in ELISA buffer and 100 μl was added to wells and incubated for 1 h at RT. Plates were then washed three times with ELISA buffer, blotted dry and developed with OPD substrate. The reaction was stopped with 1 M HCl 10 minutes after exposure. Plates were read at 490 nm on a BioTek plate reader. Biological replicates were used to evaluate treatment groups.

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