For competitive ELISA used in epitope mapping of mAbs, 2 μg/mL recombinant RBD-his (Sino Biological, Beijing, China) was added in 384-well plates and incubated at 4 °C overnight. 50 μg/mL mAbs per well were added. The plates were incubated at 37 °C for 1 h and then washed. Biotinylation of mAbs (the top 20 neutralizing Abs and 81A11, previously reported SARS-CoV CR302224 (link)) were performed using the EZ-link NHS-PEO Solid Phase Biotinylation Kit (Pierce) according to the manufacturer’s protocol and purified using MINI Dialysis Unit (ThermoFisher, 69576). 500 ng/mL biotinylated mAbs were added to each well, and the plates were incubated at 37 °C for 1 h. ALP-conjugated streptavidin (Mabtech, Sweden, 3310-10) was added at 1:1000, followed by an incubation of 30 mins at 37 °C. For the quantification of bound IgG, PNPP (Thermo Fisher) was added at 1 mg/mL and the absorbance at 405 nm was measured by the MultiSkan GO fluoro-microplate reader (Thermo Fisher).
Rbd his
Manufactured by Sino Biological
Sourced in China
RBD-his is a recombinant protein produced in a mammalian expression system. It contains the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein with a histidine (his) tag. The RBD-his protein can be used for research purposes.
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2 protocols using rbd his
1
Competitive ELISA for Epitope Mapping
2
Isolation of MERS-CoV RBD-specific mAbs
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Biopanning was utilized to isolate human mAbs specific to the MERS-CoV RBD from our previously established human combinatorial scFv antibody library. This process involved six rounds of selection using M-270 epoxy Dynabeads™ (Invitrogen, Waltham, MA, USA), which were covalently immobilized with 4 μg of recombinant His-tagged MERS-CoV RBD (RBD-His; Sino Biological, Beijing, China). The methodology for both the biopanning and subsequent procedures followed previously described protocols (Kim et al., 2022 (link)). After biopanning, 96 phage clones were randomly selected from the final output colonies. The reactivity of these clones to MERS-CoV RBD-His was evaluated using a phage enzyme-linked immunosorbent assay (ELISA), following established methods (Choi et al., 2023 (link)). Subsequent DNA sequencing of these clones led to the identification of four distinct scFv clones, each characterized by unique complementarity-determining region (CDR) sequences.
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