Mouse igg specific secondary antibody

ELISAs were performed as previously reported (Israelow et al., 2020 (link)). In short, Triton X-100 and RNase A were added to serum samples at final concentrations of 0.5% and 0.5 mg/ml, respectively, and incubated at room temperature for 3 h before use to reduce risk from any potential virus in serum. 96-Well MaxiSorp plates (#442404; Thermo Fisher Scientific) were coated with 50 µl/well of recombinant SARS-CoV-2 S1 protein (100 µg, #S1N-C52H3; ACROBiosystems) at a concentration of 2 µg/ml in PBS and incubated overnight at 4°C. The coating buffer was removed, and plates were incubated for 1 h at room temperature with 200 µl of blocking solution (PBS with 0.1% Tween-20 and 3% milk powder). Serum was diluted 1:50 in dilution solution (PBS with 0.1% Tween-20 and 1% milk powder), and 100 µl of diluted serum was added for 2 h at room temperature. Plates were washed three times with PBS-T (PBS with 0.1% Tween-20), and 50 µl of mouse IgG-specific secondary antibody (1:10,000, #405306; BioLegend) diluted in dilution solution added to each well. After 1 h of incubation at room temperature, plates were washed three times with PBS-T. Samples were developed with 100 µl of TMB Substrate Reagent Set (#555214; BD Biosciences), and the reaction was stopped after 15 min by the addition of 2 N sulfuric acid.

ELISAs were performed as previously reported.⁵⁶ (link) In short, Triton X-100 and RNase A were added to serum samples at final concentrations of 0.5% and 0.5mg/ml respectively and incubated at room temperature (RT) for 3 hours before use to reduce risk from any potential virus in serum. 96-well MaxiSorp plates (Thermo Scientific #442404) were coated with 50 μl/well of recombinant SARS Cov-2 S1 protein (ACROBiosystems #S1N-C52H3-100ug) at a concentration of 2 μg/ml in PBS and were incubated overnight at 4°C. The coating buffer was removed, and plates were incubated for 1h at RT with 200 μL of blocking solution (PBS with 0.1% Tween-20, 3% milk powder). Serum was diluted 1:50 in dilution solution (PBS with 0.1% Tween-20, 1% milk powder) and 100 μL of diluted serum was added for two hours at RT. Plates were washed three times with PBS-T (PBS with 0.1% Tween-20) and 50 μL of mouse IgG-specific secondary antibody (BioLegend #405306, 1:10,000) diluted in dilution solution added to each well. After 1h of incubation at RT, plates were washed three times with PBS-T. Samples were developed with 100 μL of TMB Substrate Reagent Set (BD Biosciences #555214) and the reaction was stopped after 15 min by the addition of 2 N sulfuric acid.

ELISAs were performed as previously reported(49 ). In short, Triton X-100 and RNase A were added to serum samples at final concentrations of 0.5% and 0.5mg/ml respectively and incubated at room temperature (RT) for 3 hours before use to reduce risk from any potential virus in serum. 96-well MaxiSorp plates (Thermo Scientific #442404) were coated with 50 μl/well of recombinant SARS Cov-2 S1 protein (ACROBiosystems #S1N-C52H3–100ug) at a concentration of 2 μg/ml in PBS and were incubated overnight at 4 °C. The coating buffer was removed, and plates were incubated for 1h at RT with 200μl of blocking solution (PBS with 0.1% Tween-20, 3% milk powder). Serum was diluted 1:50 in dilution solution (PBS with 0.1% Tween-20, 1% milk powder) and 100μl of diluted serum was added for two hours at RT. Plates were washed three times with PBS-T (PBS with 0.1% Tween-20) and 50μl of mouse IgG-specific secondary antibody (BioLegend #405306, 1:10,000) diluted in dilution solution added to each well. After 1h of incubation at RT, plates were washed three times with PBS-T. Samples were developed with 100μl of TMB Substrate Reagent Set (BD Biosciences #555214) and the reaction was stopped after 15 min by the addition of 2 N sulfuric acid.