Pelargonidin at different concentrations (1–10-20–50 µM) was tested using the ACE2: SARS-CoV-2 Spike Inhibitor Screening Assay Kit (BPS Bioscience San Diego, CA, USA) according to the manufacturer's instructions. Briefly, we have thawed ACE2 protein on ice and diluted to 1 μg/ml in PBS. We have used 50 μL of ACE solution to coat a 96-well nickel-coated plate (1 h). The plate was washed 3 times and incubated for 10 min with a Blocking Buffer. Next, 10 μL of inhibitor solution containing the Pelargonidin was added to wells and incubated for 1 h at room temperature with slow shaking. For the “Positive Control” and “Blank,” 10 μL of inhibitor buffer (5% DMSO, Sigma-Aldrich, St. Louis, MO, USA, solution) were used. After the incubation (1 h), SARS-CoV-2 SPIKE (RBD)-Fc was thawed on ice and diluted to 0.25 ng/μL (≈5 nM) in Assay Buffer 1; 5 nM SPIKE protein was added to each well, except to the blank. The reaction was incubated for 1 h at room temperature, with slow shaking. After 3 washes and incubation with a Blocking Buffer (10 min), we have treated the plate with an Anti-mouse-Fc-HRP and the plate was incubated for 1 h at room temperature with slow shaking. Finally, HRP substrate was added to the plate to produce chemiluminescence, which was measured using FluoStar Omega microplate reader (BMG LABTECH Ortenberg, GE).

To confirm the validity of the assay used in this study, remnants of plasma samples used to test levels of anti-SARS CoV2 IgG in post COVID-19 patients were used. The original samples were collected at the blood bank of Azienda Ospedaliera of Perugia from post COVID-19 donors who participate to a program of plasma biobanking. The program’s protocol included the quantitative analysis of the anti-SARS-CoV-2 IgG antibodies directed against the subunits (S1) and (S2) of the virus SPIKE protein. An informed consent was obtained by each donor for the use of the plasma sample remnants and the protocol was approved by the Ethical Committee of the University of Perugia: authorization n. 61,843 (July 13,2020).

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.