3 3 5 5 tetramethylbenzidine chromogenic substrate solution

His-capture ELISA was performed as previously described [34 ]. In brief, MaxiSorp plates (Thermo) were coated overnight at 4°C with 1.5 μg/ml of a mouse anti-His tag monoclonal antibody (mAb) (R&D Systems) in PBS, pH 7.5. The next day the plates were incubated at 4°C in blocking buffer (2% BSA in PBS, pH 7.5) for 2 h and the Env-derived soluble trimers was added to the plate at a concentration of 3 μg/ml in PBS and incubated at RT for 40 min. Serially diluted mAbs at a maximum concentration of 10 μg/ml or sera from vaccinated animals were added into wells, and following incubation and washing, the secondary antibodies of peroxidase-conjugated goat anti-human IgG or goat anti-rabbit IgG were added to all wells. Following incubation and washing, the signals were developed by addition of the 3,3’,5,5;-tetramethylbenzidine chromogenic substrate solution (Life Technologies) and detected at 450 nm. For direct-coat ELISA, trimers were added directly to the wells at 3 μg/ml and analyzed for antibody binding as described above.

His-capture ELISA was performed as previously described [7 (link)]. In brief, ELISA plates coated with 2 μg/ml of mouse anti-His mAb (R&D Systems) were first blocked in 5% nonfat milk and then used to capture the His₆-tagged trimers. Serially diluted mAbs or sera from vaccinated animals were added into wells, and following incubation and washing, the secondary antibodies of peroxidase-conjugated goat anti-human IgG or goat anti-guinea pig IgG were added to all wells. Following incubation and washing, the plates were developed with the 3,3’,5,5’-tetramethylbenzidine chromogenic substrate solution (Life Technologies). For direct-coat ELISA, trimers were added directly to the wells at 2 μg/ml and analyzed as above.