Sigmafast opd reagent

Serum levels of SARS-CoV-2 RBD IgG were measured as previously described^{33 (link)}. Briefly, SARS-CoV-2 RBD was produced in Sf9 insect cells infected with recombinant baculoviruses (Invitrogen, CA, USA). Following purification, the protein was concentrated to 5 mg/mL by ultrafiltration. Ninety-six well microplates were coated with RBD at 1 μg/mL. Serum samples were diluted 1:500 in phosphate-buffered saline-Tween (PBS-T) containing 1% bovine serum albumin and run in triplicate (mean values are reported). The plates were incubated with 1:5000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Jackson Laboratories). After three washes with PBS-T, the binding was detected using SigmaFast OPD reagent (Sigma) according to the manufacturer’s recommendations. Color development was stopped with 3 M H₂SO₄ and read on a Multiskan FC (ThermoFisher Scientific) plate reader at 492 nm. Serial sera from individual patients were analyzed in the same run. The cut-off discriminating between positive and negative sera was set as the mean absorbance of control sera plus three times the standard deviation.

Wells were coated overnight at 4°C with 100 µg/ml GST-UbcH10 in PBS 1X, 1 mM TCEP, in the presence of an EDTA free protease inhibitor cocktail (Roche Diagnostics). Binding step was performed with different concentrations of biotinylated peptides L1, L2, ScrL2, U1, ScrU1, S2 (2.2, 11, 22, 44, 108 µM) in PBS 1X (with 1 mM TCEP for U1 and ScrU1). A blocking solution 1% BSA in PBS 1X, 0.05% Tween-20 was used. Washes were executed with PBS 1X, 0.05% Tween-20. To verify the interaction a 1∶10000 dilution of horseradish peroxidase-conjugated streptavidin (Sigma Aldrich) in 0.3% BSA, PBS 1X was incubated for 1 hour. The colorimetric reaction was carried out with SIGMA-FAST OPD reagent (Sigma Aldrich), according to the manufacturer's instructions. Finally, readings were performed at 490 nm on a Model680 MicroplateReader (Bio-Rad, Hercules, CA-USA), and data were recorded by Microplate Manager 5.2 program and elaborated by GraphPad Prism program. Negative control experiments with the fusion tag GST in coating were performed in the same conditions described above.

For ELISA assays, 5 μg/mL streptavidin in phosphate/citrate buffer pH 5.0 was incubated overnight at 37°C for coating. Firstly, wells were coated with 0.8 μM biotinylated Cul3^49-68 peptides in phosphate buffered saline (PBS) 1X for 1 h at room temperature. Binding step was performed with increasing concentrations (0.4, 0.75, 1.5, 3.8, 7.6 and 15.6 μM) of His-TrxA- KCTD11^BTB in PBS 1X. His-TrxA was used as negative control in the same concentrations. As blocking solution 3% gelatine, 0.05% Tween-20 in PBS 1X was used for 1 h at 37°C. Washes were executed with PBS 1X. To reveal the occurred interaction mouse anti-His monoclonal antibody was incubated in 1:1000 dilution at room temperature for 2 h; then, horseradish peroxidase-conjugated anti-mouse antibody (Pierce) was diluted 1:10000 in 1% gelatin, PBS 1X and incubated at 20°C for 1 h. The colorimetric reaction has been carried out with SIGMAFAST OPD reagent (Sigma Aldrich), according to the manufacturer’s instructions. Finally, a Model 680 Microplate Reader (Bio-Rad, Hercules, CA-USA) has been used for readings at 490 nm; data were processed by a Microplate Manager 5.2 program. The reported data are mean values of triplicate experiments.