Quantifying Anti-NS Monoclonal Antibody Affinity

The affinity of anti-NS monoclonal antibodies (mAb)19 (link) for NS conformers with and without EMB was quantified by sandwich ELISA as described previously19 (link). Briefly, 96-well plates (Corning Inc., Costar 3590) were coated with antigen-purified rabbit polyclonal anti-NS antibody (2 μg/ml), washed (0.9% w/v NaCl, 0.05% v/v Tween20) and blocked with blocking buffer (PBS, 0.25% w/v bovine serum albumin, 0.05% v/v Tween20, 0.025% w/v Na azide). Gel filtered NS conformers and NS/EMB complexes were diluted in blocking buffer and incubated for 2 h. After washing, wells were incubated with each mAb (1A10, 10B8, 10G12 and 7C6) from 10 μg/ml with serial dilutions. Rabbit anti-mouse IgG-HRP labeled antibody was used for detection with TMB substrate solution, and HRP activity was measured in a GloMax plate reader (Promega) at 450 nm. The interplate and intraplate coefficient of variation were both less than 5%.