Quantitative ZIKV and DENV-VLP Binding Assay

The Luminex assay was conducted by FlexMap 3D (Luminex, Austin, TX, USA), and the conjugation of VLP was previously reported (70 (link)). Briefly, 65 μg ZIKV and DENV-VLP was conjugated to 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride, ECD/N-hydroxy-sulfo-succinimide, NHS (Thermo Fisher, Waltham, MA, USA), and activated in 12.5 million MagPlex beads (Luminex, Austin, TX, USA) in 50 mM 2-(N-morpholino)ethanesulfonic acid buffer, pH 7.0 or 6.0, for 120 min at room temperature. After conjugation, excess active residues were blocked by sample buffer (1% bovine serum albumin [BSA] in d-PBS) overnight at 4°C. A total of 10,000 ZIKV- and DENV-VLP conjugated beads/mL and anti-ZIKV MAb was incubated at room temperature in sample buffer for 90 min and washed with phosphate-buffered saline plus 0.05% Tween 20 (PBST). After washing, the beads were incubated with 10 μg/mL phycoerythrin-labeled anti-rabbit IgG (Thermo Fisher, Waltham, MA, USA) for 60 min. The beads were washed and mixed with sheath fluid (Luminex, Austin, TX, USA). The plates were measured the fluorescence intensity by FlexMap 3D.