Goat anti monkey 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).

Binding properties of sera to SARS-CoV-2 prototype-Beta chimeric RBD-dimer protein were determined by ELISA. 96-well plates (3590; Corning, USA) were coated over-night with 3 μg/ml of prototype-Beta chimeric RBD-dimer protein in 0.05 M carbonate-bicarbonate buffer, pH 9.6, and blocked in 5% skim milk in PBS. Serum samples from macaques were serially diluted and added to each well. The plates were incubated for 2 hours and then washed. The plates were incubated with goat anti-monkey IgG-HRP antibody (Abcam, ab112767), incubated for 1.5 hours and then washed. The plates subsequently developed with 3,3’,5,5’-tetramethylbenzidine (TMB) substrate. Reactions were stopped with 2 M hydrochloric acid, and the absorbance was measured at 450 nm using a microplate reader (PerkinElmer, USA). The endpoint titers were defined as the highest reciprocal dilution of serum to give an absorbance greater than 2.5-fold of the background values. Antibody titer below the limit of detection was determined as half the limit of detection.