Goat anti human igg fc horseradish peroxidase hrp

The IgG ELISAs for spike protein and RBD were conducted as previously described (61 (link)). Briefly, ELISA plates were coated with recombinant Wuhan-Hu-1 (purified and prepared as described in (61 (link))) or Delta RBD (ACRO Biosystems SPD-C52Hh) and RBD (ACROBiosystems, SPD-C52Hp) antigens at 0.5 μg/mL. Five 3-fold serial dilutions of sera beginning at 1:100 or 1:500 were performed in PBS with 0.1% Tween with 1% nonfat dry milk. Secondary labeling was performed with goat anti-human IgG-Fc horseradish peroxidase (HRP) (1:3000, Bethyl Labs, A80–104P). Antibody binding was detected with TMB/E HRP substrate (Millipore Sigma, ES001) and 1 N HCl was used to stop the reaction. OD₄₅₀ was read on a Tecan infinite M1000Pro plate reader.

The IgG ELISAs for spike protein and RBD were conducted as previously described [71 (link)]. Briefly, ELISA plates were coated with recombinant B.1.351 spike (purified and prepared as described in [71 (link)]) and RBD (ACROBiosystems, SPD-C52Hp) antigens described in at 2 μg/mL. Five 3-fold serial dilutions of sera beginning at 1:500 were performed in PBS with 0.1% Tween with 1% Carnation nonfat dry milk. Dilution series of the synthetic sera comprised of the anti-RBD antibody REGN10987 [72 (link)], which binds to both Wuhan-1-like RBD and B.1.351 RBD, and pooled pre-pandemic human serum from 2017–2018 (Gemini Biosciences; nos. 100–110, lot H86W03J; pooled from 75 donors) were performed such that the anti-spike antibody was present at a highest concentration of 0.25 μg/mL. REGN10987 was recombinantly produced by Genscript. The REGN10987 is the same as that used in [73 (link)]. Pre-pandemic serum alone, without anti-RBD antibody depletion, was used as a negative control, averaged over 2 replicates. Secondary labeling was performed with goat anti-human IgG-Fc horseradish peroxidase (HRP) (1:3000, Bethyl Labs, A80-104P). Antibody binding was detected with TMB/E HRP substrate (Millipore Sigma, ES001) and 1 N HCl was used to stop the reaction. OD₄₅₀ was read on a Tecan infinite M1000Pro plate reader.

The IgG ELISAs for spike protein and RBD were conducted as previously described (71 (link)). Briefly, ELISA plates were coated with recombinant B.1.351 spike (purified and prepared as described in (71 (link))) and RBD (ACROBiosystems, SPD-C52Hp) antigens described in at 2 μg/mL. Five 3-fold serial dilutions of sera beginning at 1:500 were performed in PBS with 0.1% Tween with 1% Carnation nonfat dry milk. Dilution series of the synthetic sera comprised of the anti-RBD antibody REGN10987 (72 (link)), which binds to both Wuhan-1-like RBD and B.1.351 RBD, and pooled pre-pandemic human serum from 2017–2018 (Gemini Biosciences; nos. 100–110, lot H86W03J; pooled from 75 donors) were performed such that the anti-spike antibody was present at a highest concentration of 0.25 μg/mL. REGN10987 was recombinantly produced by Genscript. The REGN10987 is the same as that used in (73 (link)). Pre-pandemic serum alone, without anti-RBD antibody depletion, was used as a negative control, averaged over 2 replicates. Secondary labeling was performed with goat anti-human IgG-Fc horseradish peroxidase (HRP) (1:3000, Bethyl Labs, A80–104P). Antibody binding was detected with TMB/E HRP substrate (Millipore Sigma, ES001) and 1 N HCl was used to stop the reaction. OD₄₅₀ was read on a Tecan infinite M1000Pro plate reader.