New monoclonalantibodies targeting ACE2 receptor could treat the next waves of SARS-CoV-2 NatureMicrobiol
. Human ACE2 enzymatic activity was then measured using the ACE2 Inhibitor Screening Assay Kit following the manufacturer’s instructions. The intensity of the fluorescent product of the hACE2 reaction product was detected at 555 nm/585 nm with Clariostar Plus Microplate Reader . MLN-4760 served as a positive control ACE2 inhibitor.To evaluate the ability of anti-hACE2 mAbs to bind to cell surface ACE2 by flow cytometry, A549 cells were engineered to express ACE2.
To assess the impact of hACE2 variation on the interaction between hACE2 and human anti-hACE2 antibodies, human 293T cells were transfected with plasmids expressing 18 hACE2 variants, including S19P, K26R, K26E, T27A, E35K, E37K, K68E, M82I, P84T, E329G, D355N, P389H, P426A, D427Y, R559S, S692P, N720D, L731F and wild-type hACE2, respectively, along a GFP-expressing plasmid. Cells transfected with plasmids carrying GFP only served as the negative control.
Serially diluted mouse plasma was diluted in PBS buffer containing 2% bovine serum albumin and mixed with 30 ng of His-tagged hACE2-NanoLuc protein. After 1 h incubation at 4 °C, the mixture was incubated with 3 μl of Dynabeads Protein G magnetic beads . After 1 h rotation at 4 °C, the beads were washed three times and bound NanoLuc activity was measured using Nano-Glo Luciferase Assay System and a GloMax Navigator Microplate Luminometer .
All of the animal procedures and experiments were performed by following protocols approved by the Rockefeller University Institutional Animal Care and Use Committee.Six-week-old hACE2-knock-in female mice, in which human ACE2 cDNA replaces the endogenous mouse ACE2 sequences, were obtained from Jackson Labs -Ace2tm1Dwnt/J, strain 035000). The mice were housed at a temperature of 22 °C and a humidity of 30–70% under a 12 h–12 h light–dark cycle with ad libitum access to food and water.
Infection experiments in mice, and all procedures involved therein, were approved by the Rockefeller University Institutional Animal Care and Use Committee.HEK-293T/ACE2cl.22 cells were incubated with anti-hACE2 mAbs at concentrations of 50, 10 or 1 μg ml
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