ELISA Quantification of HTNV Gn Antibodies

ELISAs were carried out as previously described (72 (link)). High-binding ELISA 96 half-well microplates (Corning) were coated with purified HTNV Gn (25 μl, 3 μg/ml in PBS) overnight at 4°C. Plates were washed five times with PBS containing 0.05% Tween 20 (PBS-T) and blocked with blocking buffer (5% nonfat milk in PBS-T) for 1 h at room temperature (RT). The blocking buffer was removed, and serially diluted Ab (starting at 20 μg/ml, 1:5 dilution in blocking buffer) or serum (starting at 1:50, 1:5 dilution in blocking buffer) was added for 2 h at RT. Plates were washed five times with PBS-T. Secondary Ab [goat anti-rabbit IgG F(ab′)₂, AP conjugate; Invitrogen; 1:1,000] was added for 1 h, and the plates were washed as described above. The p-nitrophenyl phosphate substrate (Sigma) was added to detect binding, and the optical densities (ODs) were measured at 405 nm.