Anti human igm peroxidase antibody

ELISAs were performed as previously described (Amanat et al., 2020 (link)). In short, Triton X-100 and RNase A were added to serum samples at final concentrations of 0.5% and 0.5mg/ml respectively and incubated at room temperature (RT) for 30 minutes 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 HRP anti-Human IgG Antibody (GenScript #A00166, 1:5,000) or anti-Human IgM-Peroxidase Antibody (Sigma-Aldrich #A6907, 1:5,000) diluted in dilution solution added to each well. After 1 h of incubation at RT, plates were washed three times with PBS-T. Plates 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. Plates were then read at a wavelength of 450 nm and 570nm.