Mers cov rbd

Each scFv identified by phage ELISA was cloned into the Fc fusion vector and transiently expressed in Chinese hamster ovary (CHO) cells. Next, for the expression of full-length IgG, synthesized DNAs of heavy chain and light chain for each mAb were inserted into MarEx vectors (Celltrion) by enzymatic digestion with NheI (NEB)/PmeI (NEB) and HpaI (NEB)/ClaI (NEB), respectively. CR3022 antibody was reconstituted with variable sequences for light and heavy chain according to the published sequence information (US8,106,170B2). Thereafter, transient expression by co-transfection was performed in CHO cells. Each scFv-Fc and full-length IgG was purified with affinity chromatography on Protein A (GE Healthcare). For the production of SARS-CoV-2 RBD, DNA encoding the SARS-CoV-2 S protein RBD (YP_009724390.1: Arg319-Asn536) with a polyhistidine tag at the C-terminus was cloned into the MarEx vector and transiently expressed in CHO cells. SARS-CoV-2 RBD with polyhistidine tag was affinity purified using Ni-NTA Resin (Thermo Fisher). Recombinant proteins for RBD and its mutants (A435S, F342L, G476S, K458R, N354D, V367F, V483A, W436R), SARS-CoV S1, HCoV-HKU1 S1, and MERS-CoV RBD were commercial products (Sino Biological).

To determine the avidity of IgG antibodies, two sets of plates were prepared. Both were coated with 1 µg/ml MERS-CoV RBD, (a.a. 367–606) (Sino Biological, USA). After serum incubation, one set of plates was washed three times for 5 min with 50 µl/well 7 M Urea in PBS+0.05% Tween 20 whereas the other set was washed with the same amount of PBS+0.05% Tween. In-between the washing steps, all the plates were washed with PBS+0.01% Tween 20 with the ELISA washer. The rest of the procedure is identical as described above.