Tmb single component substrate solution

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.

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.

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)].

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.