Tetramethylbenzidine substrate

SARS-CoV2 S1 protein (Sino Biological Inc., BJ) was coated on a high-binding 96-well plate (Thermo Scientific) at 4°C overnight. Plates were blocked with 5% non-fat milk in phosphate-buffered saline (PBS) for 1 h at room temperature, followed by incubation with 1:100 diluted plasmas in dilution buffer (PBS, 2% non-fat milk, and 0.05% Tween-20). A 1:4,000 dilution of horseradish peroxidase (HRP)-conjugated mouse anti-human IgG, IgM, and IgA antibodies (BaiaoTong Experiment Center, LY) was added and incubated for 1 h at room temperature. Wells were washed six times between each step with 0.05% Tween-20 in PBS (PBST). Finally, wells were developed using tetramethylbenzidine substrate (Beyotime Inc., WH) and were stopped by the addition of stop solution, followed by reading at 450 and 570 nm. The sample OD was calculated as OD450 subtracted by OD570.

To test the specific IgM levels against VP6 in the antisera of immunized fish, anti-IgM MAbs were prepared and kept in our laboratory. Serum antibody levels were determined by ELISA as described previously (23 (link)). Briefly, recombinant VP6 (rVP6) (24 (link)) (8 μg/mL in carbonate-bicarbonate buffer) was used as antigen to coat wells overnight at 4°C. After blocking with 5% skim milk at 37°C for 2 h, serum samples (1:50) were added and the plates were incubated for 2 h at 37°C and washed 3× with PBST and then incubated with rabbit anti-grass carp IgM (21 (link)) (1:1000) for 1 h at 37°C followed by incubation with HRP-conjugated goat anti-rabbit IgG (1:5000) for 1 h at 37°C. The wells were developed by reaction with tetramethylbenzidine substrate (Beyotime) for 10 min and the reaction was stopped by addition of 2 M H₂SO₄. OD values at 450 nm were taken as described above. Results of 10 samples were averaged to obtain the antibody titers of immunized fish at the different sampling times.

Recombinant PRA proteins were expressed and purified as previously described (Li et al., 2018 (link)). Indirect ELISAs were conducted to detect the interaction between recombinant SRCR1-4 and PRA proteins. Briefly, 96-well polystyrene microtiter plates (Corning, NY, United States) were coated with recombinant proteins at 400 ng/well in PBS buffer (pH 8.0) overnight at 4°C followed by blocking with 1% BSA in PBS-T buffer (PBS containing 0.5% Tween 20, Sigma-Aldrich) before use. 1, 0.1, 0.01, and 0.001 μg of the interaction partner were added to wells followed by incubation for 1 h at 37°C. After three washes with PBS-T buffer, the presence of an interaction was detected by the binding of anti-His or Mab2-5G2 antibodies followed by development with tetramethylbenzidine substrate (Beyotime) to form a visible product. The values of absorbance at 450 nm were evaluated using a Victor^TM X5 Multilabel Plate Reader (PerkinElmer, Waltham, MA, United States). Home-made recombinant swine hepatitis E virus ORF2 protein (239) or PBS were used as the negative or blank controls.