Goat anti human igg hrp antibody

The SARS-CoV-2 and RaTG13 RBD proteins were first diluted to 2 μg/mL with 0.05 M carbonate-bicarbonate buffer (pH 9.6) and then coated onto 96-well ELISA plates (Corning, USA) by overnight incubation at 4°C. Plates were blocked in 5% skim milk in PBST and incubated with serially diluted serum samples. Each serum sample was prepared in triplicate. Subsequently, plates were incubated with goat anti-human IgG-HRP antibody (Thermo Fisher, USA) and developed with 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate. The reactions were stopped with 2 M sulfuric acid. Absorbance at 450 nm was measured using a microplate reader (PerkinElmer, USA). The endpoint titer was defined as the highest reciprocal dilution of serum to give an absorbance greater than 2.1-fold that of the background values. Antibody titers below the limit of detection were determined to be half the limit of detection.

ELISA plates were coated overnight at 4°C with 200 ng per well of SARS-CoV-2 RBD or GX/P2V/2017 RBD protein in 0.05 M carbonate-bicarbonate buffer (pH 9.6) and blocked with 5% skimmed milk in PBS. The serum was serially diluted and added to each well. After incubation at 37°C for 1 h, the plates were washed three times with 0.05% Tween 20 in PBS and incubated with goat anti-human IgG-HRP antibody (Thermo Fisher Scientific) for 1 h. After intensive washing, 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was added to each well. The reaction was stopped with 2 M hydrochloric acid and the absorbance was measured at 450 nm using a microplate reader (PerkinElmer, USA). The endpoint titer was defined as the highest reciprocal dilution of serum to give an absorbance greater than 2.1-fold of the background values. The antibody titers below the limit of detection were determined as half of the limit of detection.