Uv vis plate reader

Various doses, with a maximum concentration of 1.7 μM, of eGFP-Ep9, eGFP-FLAG, or full-length N protein (fl-N) were diluted in PBS (pH 8.0) and then immobilized on a 96-well Nunc MaxiSorp flat-bottom microtiter plate before incubation on a shaker (150 rpm) at 4°C for 12 to 18 h. After incubation, unattached proteins were removed through aspiration using a plate washer (BioTek) and wells were blocked with 100 μl ChonBlock blocking/sample dilution buffer (Chondrex, Inc.) for 30 min with shaking (150 rpm) at room temperature. The plate was then washed three times with wash buffer (0.05% [vol/vol] Tween 20 in PBS). Pooled plasma from five patients within each experimental group was diluted 100-fold in ChonBlock blocking/sample dilution buffer, and 100 μl was added to each well before incubating for 1 h with shaking (150 rpm) at room temperature. The plate was then washed three times with wash buffer. The detection antibody, IgG Fc goat anti-human–HRP (Invitrogen), was diluted 1:5000 in ChonBlock secondary antibody buffer, and 100 μl was added per well; the plate was incubated for 30 min at 150 rpm and room temperature. Following six washes with wash buffer, 1-Step Ultra TMB-ELISA substrate solution (100 μl per well; Thermo Scientific) was added. Absorbance of TMB substrate was measured twice at 652 nm by UV-Vis plate reader (BioTek) after 5 and 15 min of incubation.

The phage-displayed SARS-CoV-2 epitopes were used in phage ELISAs with patient plasma samples diluted 100-fold in coating buffer (50 mM Na₂CO₃, pH 9.6). After incubation in a 96-well Nunc MaxiSorp flat-bottom microtiter plate with shaking at 150 rpm at 4°C for 12 to 18 h, plasma was aspirated by a plate washer (BioTek). Next, the plate was treated with 100 μl per well of ChonBlock blocking/sample dilution buffer (Chondrex, Inc.) for 1 h with shaking at 150 rpm at room temperature and washed three times with wash buffer (0.05% [vol/vol] Tween 20 in PBS). The epitope displaying phage and controls were diluted to 1 nM in ChonBlock blocking/sample dilution buffer, and 100 μl was added to each well before incubating for 2 h with shaking (150 rpm) at room temperature. The plate was then washed three times with wash buffer. The primary antibody, anti-M13-horseradish peroxidase (HRP) (Creative Diagnostics), was diluted 1:5,000 in ChonBlock secondary antibody buffer, and 100 μl was added per well; the plate was incubated for 1 h at 150 rpm and room temperature. Following three washes with wash buffer, 1-Step Ultra 3,3′,5,5′-tetramethylbenzidine (TMB)-ELISA substrate solution (100 μl per well; Thermo Scientific) was added. Absorbance of TMB substrate was measured twice at 652 nm by a UV-visible (UV-Vis) plate reader (BioTek) after 5 and 15 min of incubation.