Competition ELISA with human anti-WNV antibodies. After purification and refolding, wild-type, K307N and N394K DIII were diluted (5 µg/ml) in 0.1 M Na carbonate buffer (pH 9.3) and adsorbed to 96-well plates overnight at 4 °C. After blocking with PBS, 2% BSA and 0.05% Tween 20 (PBS-BT), wells were preincubated for 1 h at 23 °C with PBS-BT containing no antibody, E16 IgG (50 µg/ml), E16 Fab (50 µg/ml) or E53 IgG (50 µg/ml). E53 serves as a negative control as it recognizes an epitope in domain I and II of WNV E protein. Subsequently, human plasma (1/40 dilution in PBS-BT, heat-inactivated) was directly added for an additional 1 h at 23 °C. We obtained the human samples with informed consent from seven different WNV-infected patients (gift of M. Busch and L. Tobler, Blood Systems Research Institute, San Francisco, CA). Because the samples were sequentially numbered and not linkable back to the original subjects, they satisfied the criteria for exemption from approval from the Human Studies Committee at Washington University. After six washes with PBS-BT, plates were serially incubated with biotin-conjugated goat anti-human IgG (1 µg/ml), streptavidin-horseradish peroxidase (2 µg/ml) and tetramethylbenzidine developing substrate (DAKO). We determined optical densities at 450 nm with an automatic ELISA plate reader (Tecan) and adjusted them after subtraction of the value obtained from nonimmune human plasma.