Goat anti hamster igg h l hrp

Purified recombinant proteins (RBD, NTD, HR, N, RdRp) were coated onto 96-well high-binding polystyrene plates (Greiner Bio-one) at 2.5 μg/ml in 100 mM bicarbonate-carbonate coating buffer pH 9.6 overnight at 4 °C. The wells were then blocked with 200 μl of 5% skim milk for 1 h at 37 °C and then washed three times with 0.05% PBST. Heat-inactivated serum samples from mice and hamsters, intestinal lavage and lung homogenate samples were diluted in 2% skim milk and 100 μl of the respective serum dilutions were added to the wells. The sera were incubated in the plates for at least 1 h at 37 °C. The ELISA plates were again washed thrice in PBST, followed by the addition of 100 μl of 1:3000 HRP-conjugated goat anti-mouse IgG, IgG1, Ig2a or IgA antibody (Southern Biotech) in PBST. 1:10,000 dilution of goat anti-hamster IgG (H + L) HRP (Southern Biotech) was used for hamster sera. The plates were incubated at 37 °C for 1–2 h, washed thrice in 0.05% PBST and the assay was developed using OPD substrate for 10–20 min in the dark. The optical densities (OD) were read at 492 nm in microplate reader (Tecan) after stopping the reaction with 3 M H₂SO₄.