MN50 Microneutralization assay

A high-throughput ZIKV microneutralization (MN) assay was used for measuring ZIKV-specific neutralizing antibodies, as previous described [21,22]. Briefly, serum samples were serially diluted threefold in 96-well microplates, and 100 μl of ZIKV-PR (PRVABC59) containing 100 PFU was added to 100 μl of each serum dilution and incubated at 35°C for 2 hours. Supernatants were then transferred to microtiter plates containing confluent Vero cell monolayers (World Health Organization, NICSC-011038011038). After incubation for 4 days, cells were fixed with absolute ethanol/methanol for 1 hour at −20°C and washed three times with PBS. The pan-flavivirus monoclonal antibody 6B6-C1 conjugated to HRP (6B6C-1 was a gift from J. T. Roehrig, U.S. Centers for Disease Control and Prevention) was then added to each well, incubated at 35°C for 2 hours, and washed with PBS. Plates were washed, developed with TMB for 50 min at room temperature, and stopped with 1:25 phosphoric acid, and absorbance was read at 450 nm. For a valid assay, the average absorbance at 450 nm of three noninfected control wells had to be ≤ 0.5, and virus-only control wells had to be ≥ 0.9. Normalized absorbance values were calculated, and the MN50 titer was determined by a log midpoint linear regression model. The MN50 titer was calculated as the reciprocal of the serum dilution that neutralized ≥ 50% of ZIKV, and seropositivity was defined as a titer ≥ 10, with the maximum measurable titer of 7290. Log10 MN50 titers are reported.