One step ultra tmb blotting solution

Serum was first diluted 100-fold (40-fold for optimized conditions) with serum-dilution buffer (1% bovine serum albumin, 1% casein, 0.5% sucrose, 0.2% polyvinylpyrrolidone, 0.5% Tween20 in 0.01 M phosphate-buffered saline, Ph 7.4). Thereafter, 100 ΜL of the diluted sample was added to each microarray well and incubated for 30 min (or 2 h) on a shaker (500 rpm, 37 °C); the well incubated with serum-dilution buffer only was set as an experimental control. Thereafter, the microarray was rinsed three times with washing buffer (0.01 M PBST) and incubated with 100 ΜL of horseradish peroxidase (HRP) conjugated goat anti-human IgG (ZSGB-BIO, Beijing, China) for another 30 min on a shaker (500 rpm, 37 °C). Human HRP-IgG was diluted 10,000-fold with peroxidase conjugate stabilizer/diluent (Thermo Scientific, Waltham, MA, USA) for experimental use. Finally, 100 ΜL of one-step Ultra TMB-Blotting Solution (Thermo Scientific) was used to detect the informative signal of IgGs against probes using a microarray imager (Suzhou Epitope, Suzhou, China). The data were processed using IBT software, which was also developed by Suzhou Epitope (Suzhou, China). The signal for each dot was calculated using the following equation: Signal dot = Signal readout − Signal background.

We performed ELISA to detect antibodies against SARS-CoV-2 as described previously (15 (link)). We incubated 96-well MaxiSorp microplates (ThermoFisher, https://www.thermofisher.com) with 2 μg/mL of the recombinant receptor-binding domain (RBD) of the spike protein, the whole length of the nucleoprotein, or phosphate-buffered saline (PBS) at 4°C overnight. We then incubated the microplates with 5% skim milk in PBS containing 0.05% tween-20. We incubated the antigen-coated microplates with the serum or plasma samples 40-fold diluted in 5% skim milk in PBS containing 0.05% tween-20, followed by the peroxidase-conjugated goat antihuman IgG, Fcγ fragment–specific antibody (Jackson ImmunoResearch Laboratories, https://www.jacksonimmuno.com). We added One-Step Ultra TMB-Blotting Solution (ThermoFisher) to each well and incubated for 3 min at room temperature. We stopped the reaction by adding 2 M H₂SO₄ and immediately measured the optical density at 450 nm (OD₄₅₀). We subtracted the OD₄₅₀ value of the PBS wells from the OD₄₅₀ value of the spike protein or nucleoprotein wells as background.