Hrp conjugated anti human igg antibody

High binding 96-well plates (Corning) were coated with 100 μL of 2 μg/mL RBD-DP diluted in 0.1 M Sodium Carbonate/Bicarbonate buffer overnight in duplicates at 4 °C. The next day, the wells were blocked with 1% (w/v) BSA in PBS, 0.05% Tween (PBS-T). After four washes with PBS-T, 100 μL serially diluted ACE2-Fc (in-house, 9.75–20,000 ng/μl) was added to the wells. The plates were incubated at room RT for 2 h to allow ACE2-Fc to bind to RBD-DP. Plates were washed four times with PBS-T followed by the addition of 100 μL 1:2000 diluted HRP-conjugated anti-human IgG antibody (Life technologies) and incubated for 1 h at RT. After this binding step, the plates were washed four times with PBS-T and two times with ultra-pure water. Finally, 100 μL TMB substrate (Invitrogen) was added and incubated for 15 min in the dark. The reaction was terminated with 100 μL stop solution (Invitrogen) and the absorption was measured at 450 nm using a Synergy H1 microplate reader (BioTek). Binding signals were reported as the background subtracted signal divided by the background. Hill equation was used to fit RBD-DP:ACE2-Fc binding curve and apparent K_D was calculated based on this fit (GraphPad Software).

Alum-CpG control and different formulations of RBD-DP (Formulation 1, 2, 3) were blocked overnight with 5% (w/v) BSA. After three washes with PBS-T, serially diluted ACE2-Fc (in-house, 31.25–8000 ng/μl) was added and the samples were incubated for 2 h at RT. Then, the formulations and Alum-CpG were spun down at 13,000g for 5 min. Free ACE-2-Fc, which did not bind to the RBD on the Alum-CpG, remained in the supernatant. The ACE-2-Fc content in the supernatant was quantified by ELISA. For this, 96-well high binding plates (Corning) were coated overnight with 200 ng/well of RBD-DP protein. After blocking with 0.1% (w/v) BSA and four washes with PBS-T, 100 µL of each supernatant sample or ACE2-Fc standards (31.25–8000 ng/μl) were added to the wells in duplicates. Plates were washed four times with PBS-T and 100 μL 1:2000 diluted HRP-conjugated anti-human IgG antibody (Life technologies) was added and incubated at RT for 1 h. Plates were washed four times with PBS-T and two times with dH₂O before 100 µL TMB solution (Invitrogen) was added. The reaction was terminated with 100 μL stop solution (Invitrogen) and absorption readings were taken at 450 nm using a Synergy H1 microplate reader. The results were analyzed by considering ACE2-Fc standard signals as 100% binding.