Anti hamster igg hrp

RBD-specific binding antibodies were assessed by ELISA as described in Tostanoski and colleagues [3 (link)]. Briefly, 96-well plates were coated with 1 μg ml−1 of SARS-CoV-2 RBD protein (Aaron Schmidt, Massachusetts Consortium on Pathogen Readiness) or 1 μg ml−1 of SARS-CoV-2 S protein (Sino Biological) in 1Å~Dulbeccoʼs phosphate-buffered saline (DPBS) and incubated at 4°C overnight. After incubation, plates were washed once with wash buffer (0.05% Tween-20 in 1Å~ DPBS) and blocked with 350 μl of casein block per well for 2–3 h at room temperature. After incubation, the block solution was discarded, and plates were blotted dry. Three-fold serial dilutions of heat-inactivated serum in casein block were added to wells, and plates were incubated for one h at room temperature. Plates were washed three times and then subsequently incubated for one h with 0.1 μg ml−1 of anti-hamster IgG HRP (SouthernBiotech) in casein block at room temperature in the dark. Plates were washed three times, and then 100 μl of SeraCare KPL TMB SureBlue Start solution was added to each well; plate development was halted by the addition of 100 μl of SeraCare KPL TMB Stop solution per well. The absorbance at 450 nm was recorded using a VersaMax or Omega microplate reader. ELISA endpoint titers were defined as the highest reciprocal serum dilution that yielded an absorbance two-fold above background.

SARS-CoV-2 Spike as well as HIV-1 Env specific binding antibodies were assessed by Enzyme-linked immunosorbent assays (ELISAs) as described previously [13 (link), 14 (link)]. 96-well plates were coated with 1 μg/ml of SARS-CoV-2 S protein (Sino Biological) or HIV-1 clade C Env 459C gp140 in 1× Dulbeccoʼs phosphate-buffered saline (DPBS) and incubated at 4 °C overnight. The next day, plates were washed once with wash buffer (0.05% Tween-20 in 1× DPBS) and blocked with 350 μl of casein block per well for 2–3 h at room temperature. Next, the block solution was discarded, and plates were blotted dry. Three-fold serial dilutions of serum in casein block were added to wells, and plates were incubated for 1 h at room temperature. Plates were washed three times and then subsequently incubated for 1 h with 0.1 μg ml−1 of anti-hamster IgG HRP (SouthernBiotech) in casein block at room temperature in the dark. Plates were washed three times, and then 100 μl of SeraCare KPL TMB SureBlue Start solution was added to each well; plate development was halted by the addition of 100 μl of SeraCare KPL TMB Stop solution per well. The absorbance at 450 nm was recorded using a VersaMax or Omega microplate reader. ELISA endpoint titers were defined as the highest reciprocal serum dilution that yielded an absorbance two-fold above background.

RBD-specific binding antibodies were assessed by ELISA essentially as described^{10 (link),11 (link)}. Briefly, 96-well plates were coated with 1 µg ml⁻¹ of SARS-CoV-2 RBD protein (Aaron Schmidt, Massachusetts Consortium on Pathogen Readiness) or 1 µg ml⁻¹ of SARS-CoV-2 S protein (Sino Biological) in 1× Dulbeccoʼs phosphate-buffered saline (DPBS) and incubated at 4 °C overnight. After incubation, plates were washed once with wash buffer (0.05% Tween-20 in 1× DPBS) and blocked with 350 µl of casein block per well for 2–3 h at room temperature. After incubation, the block solution was discarded and plates were blotted dry. Three-fold serial dilutions of heat-inactivated serum in casein block were added to wells, and plates were incubated for 1 h at room temperature. Plates were washed three times and then subsequently incubated for 1 h with 0.1 µg ml⁻¹ of anti-hamster IgG HRP (SouthernBiotech) in casein block at room temperature in the dark. Plates were washed three times, and then 100 µl of SeraCare KPL TMB SureBlue Start solution was added to each well; plate development was halted by the addition of 100 µl of SeraCare KPL TMB Stop solution per well. The absorbance at 450 nm was recorded using a VersaMax or Omega microplate reader. ELISA endpoint titers were defined as the highest reciprocal serum dilution that yielded an absorbance two-fold above background.