High binding 96 well elisa plates

To measure antigen-specific IgG, ELISAs were performed, as described previously (29 (link)). High-binding 96-well ELISA plates (Greiner Bio-one, Kremsmünster, Austria) were coated overnight at 4°C with 1 μg/ml recombinant protein (produced as described elsewhere (12 (link), 18 (link), 19 (link), 21 (link))), washed with PBS–Tween (phosphate-buffered saline–0.05% Tween) and incubated with blocking buffer (1% BSA in PBS, pH 6.5) for 2 h at room temperature. Mouse sera (collected at day 42 before tick challenge) were diluted in blocking buffer, added to the wells and incubated for 1 h at room temperature. Plates were washed and incubated for 1 h with horseradish peroxidase (HRP)-linked anti-mouse IgG (Cell Signaling, Beverly, MA, USA) diluted 1:1,000 in blocking buffer. The plates were washed again and developed using TMB substrate [50 µl TMB chromogene in 5 ml TMB substrate buffer (8,2 gr NaAc and 21 gr citric acid monohydrate dissolved in 1 liter H₂O + 10 µl 3% H₂O₂, pH 5)] and optical density was measured in a Biotek (Winooski, VT, USA) ELISA plate reader at 450–655 nm.

High-binding 96-well ELISA plates (Greiner) were coated with 30ng 4G2 antibody (clone D1-4G2-4-15) in carbonate buffer (pH 9.6) overnight at 4°C. Plates were blocked in Tris-buffered saline containing 0.05% Tween-20 and 5% normal goat serum (cat.# G6767, Sigma-Aldrich, St. Louis, MO) for 1 hour at 37°C, followed by incubation with either ZIKV (PRVABC59, BEI) or DENV-2 (New Guinea C, BEI) for 1 hour at 37°C. Heat inactivated plasma was tested at an 1:12.5 starting dilution in 8 serial 4-fold dilutions in duplicate, incubating for 1 hour at 37°C. Horseradish peroxidase (HRP)-conjugated goat anti-monkey IgG antibody (Abcam, Cambridge, MA) was used at a 1:2,500 dilution, followed by the addition of SureBlue reserve TMB substrate (KPL, Gaithersburg, MD). Reactions were stopped by stop solution (KPL, Gaithersburg, MD). Optical densities (OD) were detected at 450 nm. The limit of detection was defined as an OD value of the 1:12.5 dilution greater than three times the background OD of ZIKV/DENV naive macaque plasma. The log₁₀ 50% effective dilutions (ED₅₀) were defined as the plasma dilutions at which there was a 50% decline in the maximum IgG virion binding based on the OD. Whole virion IgG binding responses were compared between 0 and 28 dpi time points.

ZIKV-specific binding IgG was detected using a whole virion ELISA previously described (48 (link)). Briefly, high-binding 96-well ELISA plates (Greiner) were coated with 40 ng/well of 4G2 antibody (clone D1-4G2-4-15) in carbonate buffer (pH 9.6 overnight at 4°C). Plates were blocked in Tris-buffered saline containing 0.05% Tween-20 and 5% normal goat serum for 1 hour at 37°C, followed by an incubation with ZIKV (PRVABC59 strain from BEI). Rhesus plasma was tested at 1:12.5 starting dilution in 8 serial 4-fold dilutions, incubating for 1 hour at 37°C. HRP-conjugated goat anti-human IgG monkey ads-HRP (Southern Biotech, 2049-05) was used at a 1:2500 dilution, followed by the addition of SureBlue reserve TMB substrate, followed by stop solution (KPL). Optical densities were detected at 450 nm (PerkinElmer, Victor). Half of maximal effective dilution (ED₅₀) values were calculated with the sigmoidal dose-response (variable slope) curve fit in Prism 7 (GraphPad), which uses a least squares fit. The positive control was plasma from a ZIKV-infected monkey at 6 weeks after infection, and the negative control was plasma from an uninfected monkey. Samples with an ED₅₀ below the limit of detection of 50 were plotted at the limit.