Fig. 4. Biliverdin decreases binding to SARS-CoV-2 spike by a group of human monoclonal IgGs.

(A) Antibodies were titrated sixfold and assayed by direct ELISA for binding to recombinant S1 biliverdin-depleted by purification under acidic conditions (−biliverdin), same protein but supplemented with biliverdin (+biliverdin) or N121Q S1. Area under the curve (AUC) is shown for IgG that were sensitive to biliverdin and two unaffected control IgGs. AUC values are color-coded as per the key; fold change compared to WT protein are reported. (B) Biliverdin-sensitive IgGs were titrated 10-fold and incubated with 293T cells expressing full-length WT or N121Q SARS-CoV-2 spike with or without 10 μM biliverdin. Binding was detected using an anti-IgG antibody and reduction in binding in the presence of biliverdin is shown as % MFI reduction and color-coded as a heatmap of the quartile values. (C) Enzyme-linked immunosorbent assay (ELISA) titration curves for four neutralizing IgG including the biliverdin-insensitive control COVA1-18. (D) Relative MFI dose-dependent curves for four neutralizing IgG including the biliverdin-insensitive control COVA1-18. Relative MFI calculated by normalizing to the MFI of the biliverdin-insensitive COVA1-18 at the highest concentration against spike. (E) IgG indicated above each graph were titrated fivefold against SARS-CoV-2 spike pseudotype, in the presence and absence of 10 μM biliverdin, and a version of spike encoding the mutation N121Q. COVA1-18 was used as a biliverdin-insensitive control IgG. (F) Neutralization of SARS-CoV-2 (England 02/2020/407073) by IgGs was measured in the absence and presence of 10 μM biliverdin in Vero-E6 cells. P003_027 was used as a biliverdin-insensitive control IgG.