Rabbit anti human igg hrp antibody

ELISA microplates (Greiner bio-one) were coated with 1 nmole of synthetic peptides or 20 μg of the RBD protein (in medium) diluted in 50 μL PBS. Medium without RBD protein was used as a negative control. Following an overnight incubation at 4 °C, plates were washed 3 times with PBS containing 0.05% Tween 20 (PBST) and blocked with 100 μL PBST containing 5% FBS (5% FBS/PBST) for 1 h at room temperature (RT). Sera were diluted 1:100 in PBST containing 1% FBS (1% FBS/PBST) and added to the plates (100 μL/well) (in duplicate for monkey sera and one well for human sera) and incubated at RT for 2 h. After a 3-time wash, goat anti-monkey IgG HRP antibody (Abcam) diluted 1:10,000 in 1% FBS/PBST or rabbit anti-human IgG HRP antibody (Abcam) diluted 1:80,000 was added to the well (100 μL/well) and the plates were incubated at RT for 90 min. After washing, TMB substrate (BioLegend) was added (70 μL/well) to develop color. Following a 30-min incubation, the reaction was stopped by adding 30 μL 2 N sulfuric acid (H₂SO₄). Optical Density at the wavelength of 450 nm (OD450) was measured (MULTISKAN FC, Thermo scientific).

The quantity of RBD expressed in EBY200 surface was determined by quantitative ELISA, as described previously (23 (link)). In brief, 0.5 mg of dry power of S. cerevisiae pYD1-RBD/EBY200 cells was weighed in 1.5-ml microtubes and washed twice with PBS. Collected cell pellets were resuspended in 100 µl of a series of diluted Anti-Spike-RBD human IgG (Sanyou Bio, Shanghai, China) solutions [in PBS containing 1% (w/v) BSA] and incubated at room temperature for 1 h. Cell pellets were washed three times in PBS containing 0.05% Tween-20. After washing, cells were resuspended in 100 µl of PBS containing 0.1 µg of rabbit anti-human IgG-HRP antibody (Abcam, UK) and incubated at room temperature for 1 h. After washing in the same way, cells were developed with the addition of 100 µl of HRP substrate 3,3′,5,5′-tetramethylbenzidine (TMB) (YEASEN, Shanghai, China) at room temperature in darkness for 30 min and stopped reactions by addition of 100 μl of 2 mol/L H₂SO₄. Finally, absorbance of cell supernatant was measured at 450 nm using a microplate reader (Thermo Electron Corporation, USA).