Multi mode plate reader

The cells were seeded in 96 well plates, cultured for 24–72 h and incubated with 10 μl/well Cell Counting Kit-8 reagent (MedChemExpress HK-K0301) for 2 h at 37°C. The absorbance was measured at 450 nm using a multi-mode plate reader (Molecular Devices).

The inhibition potential of the mAbs was assessed using a competitive ELISA. Briefly, ACE-2 protein (Acro Biosystems, Newark, DE, USA) was coated onto ELISA plates (Greiner, Frickenhausen, Germany) at a 1 µg/mL concentration in PBS and was incubated (for 12–72 h) at 2–8 °C in a humid chamber. The plate was then blocked using a 2.0% solution of BSA (SD Fine Chem, Mumbai, India). Different concentrations of ZRC-3308-A7 (1500 to 2.059 ng/mL, three-fold serial dilutions) and ZRC3308-B10 (800 to 3.277 ng/mL, 2.5-fold serial dilutions) were incubated with 100 ng/mL of Biotinylated RBD (Acro Biosystems, Newark, DE, USA) on a plate shaker at room temperature for 60 min to allow the binding of the mAbs to RBD. This mixture was then loaded onto an ACE-2 coated plate to allow the unbound RBD to bind to the coated ACE2. Detection was accomplished using peroxidase conjugated streptavidin at 1/50 K dilution. TMB was used as a substrate (Sigma Aldrich, St. Louis, MO, USA). The reaction was terminated using 1 N sulfuric acid, and the absorbance was read at 450 nm in a multi-mode plate reader (Molecular devices, San Jose, CA, USA).

Physical characteristics, including body weight and BMI, were measured. Total and visceral fat mass and lean mass were assessed using dual X-ray absorptiometry (DXA; iDXA, General Electric Inc). Plasma concentrations of glucose and insulin were quantified in the fasting state as we previously described [17 (link)]. These levels were used to calculate the homeostasis model assessment for insulin resistance (HOMA-IR) (fasting insulin (µU/L) × fasting glucose (nmol/L)/22.5 [18 (link)]. Lipid profiles, including high-density lipoproteins (HDL), low-density lipoproteins (LDL), total cholesterol, and triglycerides, were measured via enzymatic assays using reagents from Roche Diagnostics (Indianapolis, IN, USA) as we previously described [17 (link)]. Hemoglobin A1c (HbA1c) was measured using a kit from Crystal Chem (Elk Grove Village, IL, USA), following the protocol recommended by the manufacturer. Nitric oxide (NO) levels were estimated via measuring its metabolites, nitrate and nitrite, in serum samples using Griess reaction (Cayman Chemicals, Ann Arbor, MI) as we previously published [19 (link)]. Briefly, nitrate was converted into nitrite utilizing nitrate reductase; then, the Griess reagents were added, converting nitrite into a dark purple azo compound. Optical density was measured at 540 nm using a multimode plate reader (Molecular Devices, San Jose, CA, USA).