Alkaline phosphatase conjugated goat anti hamster igg

MaxiSorp plates (Nunc) were coated with 100 ng (2 µg/ml) whole spike protein diluted in PBS for overnight adsorption at 4 °C. Plates were washed in PBS/Tween (0.05% v/v) and wells blocked using casein (ThermoFisher Scientific) for at least 1 h at RT. Standard positive sera (pool of hamster serum from AZD1222-AZD2816 vaccinated animals with high endpoint titer against original spike protein), individual hamster serum, negative and internal control samples were added to plates and incubated for at least 2 h at RT. Following washing, bound antibodies were detected by addition of Alkaline Phosphatase-conjugated goat anti-hamster IgG (Sigma-Aldrich, SAB3700455) (1:1000 dilution) for 1 h at RT and addition of p-Nitrophenyl Phosphate, Disodium Salt substrate (Sigma-Aldrich) and optimal density reading at 405 nm. An arbitrary number of ELISA units (EU) were assigned to the reference pool and optical density values of each dilution were fitted to a 4-parameter logistic curve using SOFTmax PRO software. ELISA units were calculated for each sample using the optical density values of the sample and the parameters of the standard curve. All data was log-transformed for presentation and statistical analyses.

MaxiSorp plates (Nunc) were coated with 100ng (2μg/ml) whole spike protein diluted in PBS for overnight adsorption at 4°C. Plates were washed in PBS/Tween (0.05% v/v) and wells blocked using casein (ThermoFisher Scientific) for at least 1 hr at RT. Standard positive sera (pool of hamster serum from AZD1222-AZD2816 vaccinated animals with high endpoint titer against original spike protein), individual hamster serum, negative and internal control samples were added to plates and incubated for at least 2 hours at RT. Following washing, bound antibodies were detected by addition of Alkaline Phosphatase-conjugated goat anti-hamster IgG (Sigma-Aldrich, SAB3700455) (1:1000 dilution) for 1hr at RT and addition of p-Nitrophenyl Phosphate, Disodium Salt substrate (Sigma-Aldrich) and optimal density reading at 405nm. An arbitrary number of ELISA units (EU) were assigned to the reference pool and optical density values of each dilution were fitted to a 4-parameter logistic curve using SOFTmax PRO software. ELISA units were calculated for each sample using the optical density values of the sample and the parameters of the standard curve. All data was log-transformed for presentation and statistical analyses.