96-well plates (Corning, 3690) were coated with S, S1, NTD, S2 or RBD at 3μg/mL overnight at 4°C. The plates were washed (5 times with PBS/0.05% Tween-20, PBS-T), blocked with blocking buffer (5% skimmed milk in PBS-T) for 1 h at room temperature. Serial dilutions of serum, plasma, mAb or supernatant in blocking buffer were added and incubated for 2 hr at room temperature. Plates were washed (5 times with PBS-T) and secondary antibody was added and incubated for 1 hr at room temperature. IgM was detected using Goat-anti-human-IgM-HRP (horseradish peroxidase) (1:1,000) (Sigma: A6907) and IgG was detected using Goat-anti-human-Fc-AP (alkaline phosphatase) (1:1,000) (Jackson: 109-055-098). Plates were washed (5 times with PBS-T) and developed with either AP substrate (Sigma) and read at 405 nm (AP) or 1-step TMB (3,3′,5,5′-Tetramethylbenzidine) substrate (Thermo Scientific) and quenched with 0.5 M H2S04 before reading at 450 nm (HRP).
1 step tmb 3 3 5 5 tetramethylbenzidine substrate
Manufactured by Thermo Fisher Scientific
1-step TMB (3,3′,5,5′-Tetramethylbenzidine) substrate is a chromogenic substrate used in various immunoassays, such as enzyme-linked immunosorbent assays (ELISA), to detect and quantify the presence of specific analytes. It undergoes an enzymatic reaction that produces a colored product, allowing for colorimetric detection and measurement.
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2 protocols using 1 step tmb 3 3 5 5 tetramethylbenzidine substrate
1
SARS-CoV-2 Antibody Detection Assay
2
SARS-CoV-2 Protein-Specific Antibody Assay
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96-well plates (Corning, 3690) were coated with S, S1, NTD, S2 or RBD at 3 μg/mL overnight at 4°C. The plates were washed (5 times with PBS/0.05% Tween-20, PBS-T), blocked with blocking buffer (5% skimmed milk in PBS-T) for 1 h at room temperature. Serial dilutions of plasma, mAb or supernatant in blocking buffer were added and incubated for 2 hr at room temperature. Plates were washed (5 times with PBS-T) and secondary antibody was added and incubated for 1 hr at room temperature. IgM was detected using Goat-anti-human-IgM-HRP (horseradish peroxidase) (1:1,000) (Sigma: A6907) and IgG was detected using Goat-anti-human-Fc-AP (alkaline phosphatase) (1:1,000) (Jackson: 109-055-098). Plates were washed (5 times with PBS-T) and developed with either AP substrate (Sigma) and read at 405 nm (AP) or 1-step TMB (3,3′,5,5′-Tetramethylbenzidine) substrate (Thermo Scientific) and quenched with 0.5 M H2S04 before reading at 450 nm (HRP).
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