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20 protocols using tmb single component substrate solution

1

SARS-CoV-2 Antibody Binding Affinity by ELISA

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ELISA was applied to study the binding ability of antibodies with SARS-CoV-2 RBDs (Sino Biological) and S trimers (AcroBiosystems). Antigens were diluted with ELISA Coating Buffer (Solarbio) to 1.0 μg/mL and immobilized onto High Binding ELISA 96-Well Plate (BEAVER) with 100 μL per well overnight at 4 °C. Plates were washed four times with PBST (Solarbio) and blocked with 3% skim milk for 1 h at 37 °C. Then, serially diluted antibodies were added 100 μL per well and incubated at 37 °C for 1 h. After pipetting off the unbound antibodies, plates were washed four times with PBST and further incubated with 100 μL per well of goat anti-human IgG (Fc specific)-Peroxidase antibody (1:5000 dilution, Sigma) for 1 h at 37 °C. After a final four times washing with PBST, the binding of antibodies with SARS-CoV-2 antigens were visualized by adding 100 μL peroxidase substrate TMB Single-Component Substrate solution (Solarbio) and incubating for 15 min in dark. The reaction was terminated by adding 50 μL stop buffer (Solarbio) and the plates were immediately submitted to an ELISA microplate reader (TECAN Infinite M200 Pro) to measure the optical density (OD) at 450 nm. Data were analyzed with GraphPad Prism Version 9.0.0 and EC50 values were determined using a four-parameter nonlinear regression.
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2

Antibody Titer Detection in Large Yellow Croaker

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The antibody titer in serum of large yellow croaker collected above was detected by ELISA. Briefly, 96-well plates were coated with 100 µL of rDLD protein (1 µg/mL) in ELISA Coating Buffer (Solarbio, Beijing, China) per well and incubated at 4 • C overnight. After being washed with PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , 0.2% Tween) five times, the plates were blocked with 5% BSA (Yeasen Biotech, Shanghai, China) in PBS for 1.5 h at 37 • C. After PBST washing, the serum samples with serial dilutions were applied in blocking solution (5% skimmed milk powder) for 1 h at 37 • C. After PBS washing five times, the plates were incubated with mouse anti-large yellow croaker IgM monoclonal antibody for 1 h at 37 • C. After PBST washing, plates were incubated with HRP-conjugated sheep anti-mouse IgM antibody (Thermo Fisher; 1:4000) and then reacted with 100 µL TMB single-component substrate solution (Solarbio Science) for 10 min. Finally, the reaction was stopped by ELISA stop solution (Solarbio), and the absorbance of each well was measured in an INFINITE 200 PRO (Tecan Austria, Grödig, Austria) at 450 nm.
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3

SARS-CoV-2 S1 Protein Antibody Quantification

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Serum samples from various groups of mice were analyzed using standard indirect Enzyme-linked immunosorbent assay (ELISA) to assess the IgG and its isotype-specific antibody levels of S1 or N proteins. The purified S1 protein (WT, Beta, Delta, Lambda, and Omicron) was coated into ELISA plates by overnight incubation at 4 ℃ for subsequent binding assay. The next day, each well was blocked with 5% skim milk in PBST (PBS with 0.05% Tween-20) for 2 h at 37 ℃. Then, the serum was performed as a primary antibody and serially diluted with ELISA-blocking buffer was incubated for 1 h at 37 ℃. The S1-specific IgG, IgG1, and IgG2a antibodies were titrated by incubating with the horseradish peroxidase (HRP)‐conjugated Goat Anti-Mouse IgG (Promega; W4021), IgG1 or IgG2a (Jackson ImmunoResearch Laboratories; 115–035-205, 115–035-206) as detection antibodies for 1 h at 37 ℃. Each well was developed with TMB Single-Component Substrate solution (Solarbio; PR1200) in the dark for 10 min. 2 M H2SO4 was used to stop color development. Finally, the absorbance at 450 nm was performed immediately using a multi-functional enzyme standard instrument (Omega, Germany). Similar to the Method described above, indirect ELISA was employed to ascertain the endpoint titers of N-protein-specific IgG, IgG2a, and IgG1.
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4

Synthesis of Benzene Dicarboxylic Acid Compound

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2-amino benzene dicarboxylic acid (NH2–H2DBC) and direct turquoise blue (DTB5B) were purchased from Aladdin (Shanghai, China). Anhydrous ferric chloride (FeCl3), N, N-Dimethylformamide (DMF), hydrogen peroxide, and dimethyl sulfoxide (DMSO) were brought from Sinopharm (Beijing, China). TMB single-component substrate solution was obtained from Solarbio life science (Beijing, China). Deionized water and anhydrous ethanol were purchased from Beijing Chemical Industry Group Co., Ltd (Beijing, China). All chemicals were used without any purify.
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5

Quantitative Protein-Protein Interaction Assay

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DP1, GINS, or GAN (1 µg/100 µl of each protein) was fixed onto the surface of the wells shown in Figure 5 (left panel, red), and incubated at room temperature for 2 hr. Skim milk (5%, with PBS, 100 µl/well) was then applied, after which the wells were incubated at room temperature for 2 hr. The binding proteins (100 µl) shown in Figure 4 (left panel, black) were added into wells, after which samples were incubated and washed as described above. His‐HRP antibody (1:1,000 diluted) was then added into wells, the samples were incubated at room temperature for 2 hr, and then washed with phosphate buffered saline with tween 20 buffer five times to remove unbound proteins. TMB (3, 3′, 5, 5′‐tetramethylbenzidine) (TMB Single‐Component Substrate Solution from Solarbio) was added into wells (100 µl/each well) and incubated at 37°C for 10 min, the reaction was stopped by adding 50 µl 0.5 N HCl, and the absorbance at 460 nm was measured.
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6

Enzyme-Linked Immunosorbent Assay for Peptide Detection

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The peptides of tags and their mutants were diluted to 2.0 μg/ml and 50 μl each was added into 96 well plates for 1 hour to adsorb (n=3). After washing with phosphate buffer saline containing 0.05% Tween-20 (PBST) three times, the plate was blocked with a rapid-blocking buffer for 10 min. After washing with PBST three times, mouse anti-tags mAbs were added into each well (40 μl/well) and incubated at 37°C for 1 h. Next, after washing and blocking as described above, 100 μl of horseradish peroxidase (HRP)-labeled goat anti-mice IgG (1:2500) were added and incubated at 37°C for 1 h. The results were detected by the plate reader after color-developing with TMB single-component substrate solution (Solarbio, cat# PR1200, Beijing, China) and stopping. Peptide-free and mAb-free wells were performed as negative controls. The absorbance value at 450 nm (OD450) was measured for analysis.
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7

Quantifying Antigen-Specific IgG via ELISA

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Serum samples were examined for antigen-specific immunoglobulin G (IgG) by ELISA. ELISA was performed according to a previous study [18 (link)]. Briefly, 96-well plates were coated with SLS and fimbriae proteins (F4, F5, F6, F18 and F41), kept overnight at 4°C, washed with a washing solution containing 0.05% Tween 20 in PBS and then blocked with block buffer (PBS containing 0.1% Bovine Serum Albumin) at room temperature for 2 h. Following the washing step with a washing buffer (PBS containing 0.05% Tween 20), serially diluted serum samples were added to the plates. IgG was detected with a Goat Anti-Mouse IgG/HRP (Solarbio, China). ELISA plates were developed with TMB Single-Component Substrate solution (Solarbio) and stop solution (2 M H2SO4). After adding the stop solution, the plates were read at 450 nm optical density (OD450).
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8

ELISA for Detecting HSV-1 Antibodies in Rhesus Macaques

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ELISA plates (Corning, Shanghai, China) were coated with whole HSV-1 (F strain) as the antigen for serum IgG antibody detection. Briefly, Vero cells were infected with HSV-1 for 36 h, the cell suspension was harvested and sonicated for 30 s and 45 μg of cell lysate/well was coated on the ELISA plates at 4 °C overnight. The plates were washed 5 times with PBS/0.05% Tween 20 buffer and then blocked with PBS/5% BSA at 4 °C overnight. Each sample of the rhesus macaque serum was diluted at 1:500 in PBS/0.5% BSA, added to two wells, and then incubated at room temperature for 2 h. Next, the plates were washed as described above and incubated with HRP-conjugated mouse anti-rhesus IgG (Bioss, Shanghai, China) diluted at a ratio of 1:5000 in PBS /0.5% BSA at 37 °C for 1 h. TMB Single-Component Substrate solution (Solarbio, Beijing, China) was added to each well after 5 washes. The color reaction was stopped using ELISA Stop Solution (Solarbio, Beijing, China). Subsequently, the wells were read at an absorbance of 450 nm. Similarly, the ELISA plates coated with Vero cells were mock-infected as the control and the procedures were repeated as described above. The resulting values were determined by subtracting the values obtained for uninfected cell lysates from the values obtained with infected cell lysates [29 (link)].
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9

Sandwich ELISA for DAdV-3 Detection

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To develop sandwich ELISA for detection of DAdV-3, mAb 3D9 was purified and HRP-conjugated as previously described (Wang et al. 2018 (link)). In the sandwich ELISA, the purified mAb 3D9 diluted in 0.1 M carbonate buffer with the final concentration of 1.25 µg/mL was coated into a 96-well ELISA plate (100 µL/well) at 4 °C overnight. The following morning, the 96-well ELISA plate was blocked with 360 µL of 5% skim milk in PBST for 2 h at 37 °C. After the blocking and washes, the virus supernatants or samples diluted were added into the 96-well ELISA plate for 1 h at 37 °C and the 96-well ELISA plate was washed with PBST for three times. Then, the HRP-conjugated mAb 3D9, diluted by PBST with a final concentration of 0.625 µg/mL, was added into the 96-well ELISA plate for 30 min at 37 °C. After washed with PBST for three times, the 96-well ELISA plate was added with 100µL TMB single-component substrate solution (Solarbio) per well and reacted for 15 min at 37 °C. Finally, 50 µL of 2 M H2SO4 per well was used to stop the chromogenic reaction and an ELISA reader was used to measure the absorbance values at 450 nm.
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10

Epitope Correlation of SARS-CoV-2 Antibodies

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Competition enzyme-linked immunosorbent assay (ELISA) was performed to explore the epitope correlation of two antibodies. Briefly, the first antibody at the concentration of 2 μg/mL was coated on plates (BEAVER) and incubated at 4 °C overnight. Then, excess antibodies were washed away by PBS and blocked by 3% skim milk. SARS-CoV-2 S1 protein (Sino Biological) was biotinylated using an EZ-Link™ Sulfo-NHS-LC-LC-Biotin kit (ThermoFisher), followed by mixing with 50 μg/mL of the second competition antibodies or PBS blank control. After incubation at 37 °C for 1 h, plates were washed three times with PBS and the diluted Ultrasensitive Streptavidin-Peroxidase Polymer (Sigma) was added (1:2000) subsequently. Then the plates was incubated at 37 °C for 1 h again. TMB Single-Component Substrate Solution (Solarbio) was used to detect the S1 binding with the coated first antibodies. The absorbance at 450 nm was measured in an Infinite M200 PRO Multimode Microplate Reader (TECAN). Competitive percentage of two antibodies was calculated with reference to the PBS blank control.
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