5 5 tetramethylbenzidine tmb substrate

Serum antibodies against SARS-CoV-2 were determined using an indirect ELISA. The 96-well ELISA microplates (Thermo Fisher Scientific Korea, Ltd.) were coated overnight at 4°C with 100 μL per well of 2 μg/mL of plant- and E. coli-expressed rNPs. The microplates were washed three times with washing buffer of PBS-T (0.05% Tween 20) and blocked with blocking buffer (PBS-T containing 5% of skim milk) for 2 h at 37°C. After four washes, the specimens were diluted 100-fold with blocking solution and incubated at 37°C for 2 h. The plates were then washed five times with washing buffer. Following this step, HRP-conjugated goat anti-human total Ab (Thermo Fisher Scientific; catalog no. 31418) was diluted in blocking solution (1:40,000) and added at a 100 μL volume per well and incubated at 37°C for 1 h. After an extensive washing step, 50 μL of 3,3′,5,5′-tetramethylbenzidine substrate (TMB; Sigma-Aldrich, USA) was added to each well at room temperature in the dark. After 30 min, the reaction was stopped with 25 μL of 1 M H₂SO₄, and the absorbance at 450 nm was measured in each well. The samples were tested in triplicate.

Serum antibodies against SARS-CoV-2 were determined using an indirect ELISA. The 96-well ELISA microplates (Thermo Fisher Scientific Korea, Ltd.) were coated overnight at 4°C with 100 μL per well of 2 μg/mL of plant- and E. coli-expressed rNPs. The microplates were washed three times with washing buffer of PBS-T (0.05% Tween 20) and blocked with blocking buffer (PBS-T containing 5% of skim milk) for 2 h at 37°C. After four washes, the specimens were diluted 100-fold with blocking solution and incubated at 37°C for 2 h. The plates were then washed five times with washing buffer. Following this step, HRP-conjugated goat anti-human total Ab (Thermo Fisher Scientific; catalog no. 31418) was diluted in blocking solution (1:40,000) and added at a 100 μL volume per well and incubated at 37°C for 1 h. After an extensive washing step, 50 μL of 3,3′,5,5′-tetramethylbenzidine substrate (TMB; Sigma-Aldrich, USA) was added to each well at room temperature in the dark. After 30 min, the reaction was stopped with 25 μL of 1 M H₂SO₄, and the absorbance at 450 nm was measured in each well. The samples were tested in triplicate.

The whole-cell enzyme-linked immunosorbent assay (ELISA) was carried out as described^{24 (link)}. For testing the DARPin surface display, 750 μL of L. lactis cell suspension in PBS with optical density A₆₀₀ = 1.0 was centrifuged (5,000 × g, 5 min, 4 °C) and washed twice with 500 μL PBS. Cells were then resuspended in 200 μL of fluorescein isothiocyanate (FITC)-conjugated human IgG (Jackson ImmunoResearch, West Grove, USA; diluted 1: 500 in PBS), and incubated 1 h at room temperature (RT) with gentle shaking. Cells were then washed twice with PBS and resuspended in 200 μL of peroxidase-conjugated mouse anti-human IgG (Jackson ImmunoResearch, West Grove, USA) solution diluted 1:2500 in PBS. After 1 h incubation at RT with gentle shaking, cells were washed, first with PBS and then with substrate buffer (150 mM Na₂HPO₄, 50 mM citric acid, pH 6.0). The cells were then resuspended in 1 mL of substrate buffer, and 100 μL of the appropriate dilutions (1:5 and 1:25) in substrate buffer were loaded on a microtiter plate. 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Sigma-Aldrich, Steinheim, Germany) was added and the reaction stopped after 15 min by adding 50 μL of 2 M sulphuric acid. Absorbances were read at 450 nm using an Infinite M1000 (Tecan, Salzburg, Austria).