Fig 1: Evaluation of the involvement of hACE2, L-SIGN, and various glycan structures in the neutralization promoted by mAbs(A) Competition of mAbs binding to NTD by various glycans was determined by ELISA. Antibodies were pre-incubated on ice in the presence or absence of 2-O-methyl-α-Neu5Ac (Ac2Me; 8 mM), D-glucuronic acid (GlcA; 8 mM), or with sialoglycopeptides containing a mixture of N/O-glycans with a terminal Neu5Ac (GP; 0.06 mM). Inhibition is manifested by prevention of binding, resulting in a low level of yellow coloring. None of the antibodies were inhibited by GlcA or Ac2Me, whereas BLN4 and BLN12 were inhibited by the GP.(B−D) The ability of purified recombinant hACE2 and human L-SIGN receptors to bind S1 or NTD, in the presence of antibodies was tested by BLI. (B) Binding of hACE2 to S1 in the presence of neutralizing antibodies (representing each of the epitope groups, depicted by different colors, as indicated). Each of the biotinylated antibodies was immobilized on a streptavidin sensor, loaded with S1, washed, and incubated with recombinant hACE2 for 300 s. Time 0 represents the binding of the hACE2 to the antibody-S1 complex. MD29 and MD65 were used as positive and negative controls, respectively. (C) Binding of human L-SIGN to NTD and RBD. L-SIGN-Fc was immobilized on a protein A sensor and incubated with NTD or RBD. (D) Binding of human L-SIGN to NTD in the presence of neutralizing antibodies (representing each of the epitope groups). NTD-His was immobilized on Ni-NTA sensors and saturated with each of the antibodies separately; the NTD-mAb complex was then incubated with L-SIGN for 300 s. Time 0 represents the binding of the L-SIGN to the antibody-NTD complex.