Hrp conjugated goat anti human igg antibody

High binding 96-well ELISA plates (Costar) were coated with various MUC1 peptides by incubating 50 μl/well of peptide at 2 μg/ml in carbonate buffer at 37 °C for 2 hours. Free binding sites on the plate were blocked by addition of 300 μl/well of 5% fat free powdered milk diluted in 1X PBS with 0.1% tween 20 (MPBS-Tween) and incubation at 37 °C for 1 hour. Primary antibody or plasma was diluted in MPBS-Tween, 50 μl/well was added to the plates, which were then incubated at 37 °C for 2 hours. The wells were rinsed with PBS-Tween, then HRP-conjugated goat anti-human IgG antibody (SouthernBiotech) was added followed by addition of tetramethylbenzidine substrate and stop solution (Cell Signaling Technology). Signal level was measured by absorbance at 450 nm.

All SARS-CoV-2 proteins were purchased from Sino Biological (Beijing, China). About 200 ng/well of SARS-CoV-2 spike (#40589-V08B1), spike S1 subunit (#40591-V08H), spike NTD (#40591-V49H), spike S2 subunit (#40590-V08B), RBD (#40592-V08H), RBD T478K (#40592-V08H91), nucleocapsid (#40588-V08B), and envelope (#40609-V10E3) were coated on the 96-well ELISA plate overnight at 4 °C, respectively. Plates were washed by PBS supplemented with 0.05% Tween-20 (PBST) three times, followed by blocking with 5% BSA in PBST for 1 h at room temperature. Sera were diluted 20-fold for the first well and fourfold serial diluted for subsequent wells in 5% BSA in PBST and incubated at 4 °C overnight. Plates were washed three times by PBST and added with HRP-conjugated goat anti-human IgG antibody (#2040-05, SouthernBiotech, 1:3000) for 30 min at room temperature. Plates were washed five times with PBST. 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (#P0209, Beyotime) was added for 15 min and stopped by the stopping buffer (#C1058, Solarbio). OD450 was measured by Varioskan Lux Microplate Reader (Thermo Fisher).

The whole virion binding ELISA was performed as described previously with the addition of a second whole virion binding ELISA that included the use of a different secondary antibody: horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody (Southern Biotech, Birmingham, AL) to track the titer of human-derived Ig used to treat the animals [37 (link)]. Optical density (OD) was detected at 450 nm on a Victor X Multilabel plate reader (PerkinElmer, Waltham, MA). The limit of detection was defined as an OD value of the 1:12.5 dilution greater than three times the background OD of ZIKV naive macaque plasma. The log₁₀ 50% effective dilutions (ED50) were defined as the plasma dilutions at which there was a 50% decline in the maximum IgG virion binding based on the OD. Whole virion IgG binding responses were examined across all time points (0–62 dpi).