His-Tagged Trimer Binding ELISA

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.