3 3 5 5 tetramethylbenzidine substrate solution

MAXISORP NUNC-IMMUNO plates were coated with 50 μl/well anti-SARS-CoV-2 pAb at 1.5 µg/ml overnight at 4 °C. Plates were washed 5 × 5 min with Tris-buffered saline supplemented with 0.05% Tween-20 (TBS-T, pH 7.2), before blocking with 300 μl/well of 0.1 M NaH₂PO₄/Na₂HPO₄ (pH 7.2), 0.15 M NaCl, 0.05% (v/v) Tween-20 and 1% (w/v) bovine serum albumin (BSA) for 1 hr at room temp. The blocking buffer was removed and 50 μl/well sample added. Each plate also included a series of dilutions of recombinant SARS-CoV-2 Nc as an internal control. Antigen incubation was performed at RT for 1 h. The plates were then washed 5 × 5 min with TBS-T before addition of 1.25 μg/ml (50 μl/well) of biotinylated, affinity-purified rabbit α-SARS-CoV-2 IgG, diluted in 0.1 M NaH₂PO₄/Na₂HPO₄ buffer (pH 7.2), 0.15 M NaCl, 0.05% (v/v) Tween-20 and 10% (v/v) rabbit serum (Sigma). Plates were incubated for 1 h at room temp and washed again 5 × 5 min with TBS-T. Horseradish peroxidase (HRP) - Avidin conjugate (BioLegend) was diluted 1:2500 in blocking buffer and added at 50 μl/well. After 1 h incubation at RT, plates were washed 6 × 5 min with TBS-T before addition of 50 μl/well of 3,3′,5,5′-tetramethylbenzidine substrate solution (SeraCare), which was left to develop for 1 h at room temperature. Reactions were stopped with 50 μl/well of 1 M H₂SO₄ solution and a plate reader used to measure absorbance at 450 nm.