High binding elisa 96 half well microplates

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.

High-binding ELISA 96 half-well microplates (Corning) were coated with purified NiV-F (25 μL, 3 μg/mL in PBS) overnight at 4 °C. Plates were washed 5 times with PBS containing 0.05% Tween20 (PBS-T) and blocked with blocking buffer (5% nonfat milk in PBS-T) for 1 h at room temperature. The blocking buffer was removed and serial diluted antibody (mAb66 purified from hybridoma cells and expressed from cloned mAb66) (starting at 50 μg/mL, 1:5 dilution in blocking buffer) was added for 2 h at room temperature. Plates were washed 5 times with PBS-T. Secondary antibody (goat anti-Rabbit IgG F(ab')₂, AP conjugate, Invitrogen, 1:1,000) was added for 1 h and plates were washed, as described above. The p-nitrophenyl phosphate substrate (Sigma) was added to detect binding and OD were measured at 405 nm (SI Appendix, Fig. S1).

High-binding ELISA 96-half-well microplates (Corning) were coated with purified JUNV GP1 (25 μL, 3 μg/mL in PBS) overnight at 4°C. Plates were washed five times with PBS-T (0.05% Tween 20) and blocked with blocking buffer (5% nonfat milk in PBS-T) for 1 h at room temperature. The blocking buffer was removed, and serially diluted JUN1 Fab (at 50 μg/mL, 1:5 dilution in blocking buffer) was added for 30 min at room temperature. JUN1 to JUN7 IgG was added at the 80% effective concentration (EC₈₀) and incubated for a further 1.5 h. Plates were washed five times with PBS-T. Secondary antibody (goat anti-mouse IgG Fc biotin conjugate; Thermo Fisher Scientific; 1:1,000) was added for 30 min, plates were washed as described above, and streptavidin-AP (1:10,000; Invitrogen) was added for 30 min. Following a final wash, p-nitrophenyl phosphate substrate (Sigma) was added to detect binding and the ODs were measured at 405 nm. The competition is reported for JUN1 Fab at 50 μg/mL and is reported relative to the percent competition measured for JUN1 IgG.