Colorimetric microplate reader

Based on the protocol summary of Fe²⁺ assay, the samples were placed into 96-well plates. Each well was added with 5 µL assay buffer (Abcam, Cambridge, MA, USA) and incubated for 30 min at 37°C. Then, 100 µL Iron Probe (BioAssay Systems, CA, USA) was added to each well containing the Iron Standard and test samples and incubated for 1 h at 37°C protected from light. The absorbance at 593 nm was analyzed immediately with a colorimetric microplate reader (Bio-Rad Laboratories, Shanghai, China).

Biotinylated hACE2 was synthesized by incubating a 1:1 molar ratio of succinyl propionyl biotin (Dojin Chemical, Kumamoto, Japan, 346-06351) and hACE2 protein (SinoBiologicals, Wayne, NJ, USA. 10108H08H) on ice, overnight and at room temperature, for 1 h. A 50 μL aliquot containing 100 ng RBD protein was added to each well of a 96-well microtiter plate (NUNC, 44-2404-21), followed by overnight incubation at 4 °C. The plates were blocked by incubation with 3% BSA in PBS, followed by incubation with an appropriate dilution of biotinylated hACE2 on ice for 30 min. After washing with PBST, HRP-conjugated streptavidin was added to each well, and the plate was incubated for 30 min. The plate was washed four times with PBST, followed by the addition of the TMB substrate solution. The enzymatic reaction was stopped by the addition of 0.2 N H₂SO₄, and the absorbance of each well at 450 nm was measured with a colorimetric microplate reader (BioRad).