To determine endpoint anti-NS1 antibody titers, Nunc MaxiSorp flat-bottom 96-well plates (Thermo Fisher Scientific) were coated overnight at 4°C with ZIKV NS1 protein (Sino Biological) at 1 μg/ml in PBS as described previously. The plates were washed four times with PBS +0.05% Tween 20 (PBST) and then blocked with StartingBlock Block Buffer (Thermo Fisher Scientific) for 5 min at room temperature. Serially diluted mouse serum samples were added to wells and incubated at 37°C for 1 hour, and the plates were washed four times. Bound antibodies were detected using HRP-conjugated goat anti-mouse IgG (GE Healthcare Life Sciences), and the optical density (OD) was read at 492 nm. To confirm that the IgG2a antibody isotype predominates the anti-NS1 humoral response, antibodies were captured as described above, and bound antibodies were detected using HRP-conjugated anti-mouse IgG2a (GE Healthcare Life Sciences). Endpoint titers were determined as the reciprocal of the highest serum sample dilution with an OD reading above the cutoff, set as 2SD above the mean OD of serum samples from prevaccinated or naïve mice.

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