DENV-1 Neutralization and Binding Assays

Plaque reduction neutralization tests (PRNT) and pre- and post-attachment neutralization assays were performed with DENV-1 strain 16007 on Vero cells as previously described [13] (link), [64] (link). Binding of intact MAbs or Fabs (E103, E106, and a negative control WNV E16) to DENV-1 virions (strain 16007) was detected by capture ELISA [13] (link), [64] (link). Briefly, humanized DENV-1 E50 MAb (subcomplex DIII A-strand specific antibody) was coated at 2 µg/ml on MaxiSorp (Nunc) polystyrene 96-well microtiter plates in a sodium carbonate (pH 9.3) buffer. Plates were washed three times in wash buffer (PBS with 0.02% Tween 20) and blocked for one hour at 37°C with blocking buffer (DMEM with 10% FBS). DENV-1 virions (2.5×10⁵ PFU) diluted in DMEM with 10% heat-inactivated FBS were captured on plates for two hours at 37°C. Wells were washed thrice with blocking buffer and DENV-1 MAb or Fab was then added at 100 µg/ml and 4-fold serial dilutions to duplicate wells and incubated for two hours at 37°C. Plates were washed five times and then sequentially incubated with goat anti-mouse (whole molecule) IgG-HRP (Sigma, St Louis, MO) and tetramethylbenzidine substrate (Dako). The reaction was stopped with the addition of 2 N H₂SO₄ to the medium, and emission (450 nm) was read using an iMark microplate reader (Bio-Rad).