Ni2 nitrilotriacetic acid affinity chromatography

To verify the specificity of MAbs, the RBDs of the MERS-CoV and SARS-CoV S proteins were prepared using a baculovirus expression system, as described previously (Fukuma et al., 2015 (link)). Briefly, the nucleotide sequences encoding amino acids 358–588 of the S protein of MERS-CoV and amino acids 318–510 of the S protein of SARS-CoV were tagged at the C-terminus with histidine-tags and cloned into the pAcYM1 transfer vector. Insect Sf9 cells were transfected with mixtures of the transfer vector and BD BaculoGold Linearized Baculovirus DNA (BD Biosciences, San Jose, CA) according to the manufacturer’s instructions, thus producing MERS-CoV RBD-expressing baculovirus (AcMERS-RBD-His) and SARS-CoV RBD-expressing baculovirus (AcSARS-RBD-His). Insect Tn5 cells were then infected with AcMERS-RBD-His or AcSARS-RBD-His, thus producing MERS-CoV RBD and SARS-CoV RBD, respectively. Three days post infection, the RBD proteins were purified from the culture supernatants by using by Ni²⁺-nitrilotriacetic acid affinity chromatography (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. These RBD proteins were used in conventional ELISAs. Briefly, 96-well ELISA plates (Thermo Fisher Scientific) were coated with 125 ng of the purified RBD proteins and an IgG-ELISA was performed as described previously (Iwata-Yoshikawa et al., 2016 (link)).