Tmb one substrate solution

Specific antibodies against A. suum were measured in the first experiment only. ELISA plates (Immunolon, Nunc, Thermo Fisher Scientific, Waltham, MA) were coated with 100 uL of 5 ug/mL of A. suum 3rd to 4th stage larvae (L3/L4) extract⁷³ (link) or 0.1 ug of lyophilized Bb12 in coating buffer (Sodium Carbonate buffer, pH 9.5). After an overnight incubation at 4°C, plates were washed six times (0.05% Tween 20 in 1X-PBS) and blocked with 0.05% Tween 20, 0.5% BSA in PBS for 30 minutes. Plates were washed and used for testing 100 uL of serum diluted at 1:3,000 and 1:6,000 (0.05% Tween 20 in PBS) or ileum fluid (contents obtained from the terminal 60 cm of pig intestine were centrifuged at 4C and 800 X g for 10 minutes to separate clear fluid from particulates). Test samples were run in duplicates and incubated for two hr at room temperature. After six washes, 100 uL of HRP-conjugated porcine anti-IgM, anti-IgA, anti-IgG1 or anti-IgG2 (Serotec, Raleigh, NC) were added at a 1:10,000 dilution and incubated for one hr. After a final wash, 100 uL of TMB-One substrate solution (Promega, Madison, WI) was added. Total anti-L3/L4 or anti-Bb12-specific antibody isotypes were determined after reading plates with an optical density (OD) at 450nm after 20 minutes. Reaction was stopped by addition of 1M HCl solution.

96-well plate (Corning, NY, USA) was coated with 2 µg/ml of plant-produced F-native protein, plant-produced SC-TM protein, and plant-produced SARS-CoV-2 Receptor binding domain protein [36 (link)] (as a negative control) in phosphate buffer (20 mM, pH 7.4), 25 µl per well, overnight at 4 °C. After washing three times with PBS-T, the plate was blocked with 200 µL of 5 % skim milk in PBS at 37 °C for 1 h. Then, the plate was washed three times with PBS-T and incubated with serially diluted plant-produced Motavizumab and incubated at 37 °C for another 2 h. After sample incubation, the solution was removed and washed three times with PBS-T. After washing, the plate was incubated with 1:2500 HRP-conjugated goat anti-human IgG kappa light chain (Southern Biotech, Birmingham, AL, USA), and incubation was continued at 37 °C for another hour. After secondary antibody incubation, the wells were emptied and washed. The TMB one substrate solution (Promega, Madison, WI, USA) was added and incubated for 10 min, the reaction was stopped by adding 1 M sulfuric acid and the absorbance was measured using a multimode reader (Perkin Elmer, MA, USA) at 450 nm.

96-well ELISA plates (Corning, NY, USA) were coated with 2 µg/ml of RSV-F his-tagged monomer protein (Sino biological, Beijing, China) in phosphate buffer (20 mM, pH 7.4), 25 µl per well, overnight at 4°C. After washing three times with PBS + 0.05% Tween 20 (PBS-T), the plate was blocked with 200 µL of 5% nonfat dried milk in PBS at 37°C for 1 h. Then, the plate was washed three times with PBS-T and incubated with a serially diluted plant-produced mAbs sample and incubated at 37°C for another 2 h. After sample incubation, the solution was removed and washed with PBS-T. After washing, the plate was incubated with 1:2,500 HRP-conjugated goat anti-human IgG (Southern Biotech, Birmingham, AL, USA), and incubation was continued at 37°C for another hour. After secondary antibody incubation, the wells were emptied and washed. A TMB one substrate solution (Promega, Madison, WI, USA) was added and incubated for 10 min, the reaction was stopped by adding 1 M sulfuric acid, and the absorbance was measured using a multimode reader (Perkin Elmer, MA, USA) at 450 nm. All experiments were performed in triplicate. The half maximal effective concentration (EC₅₀) value was calculated using nonlinear regression in GraphPad. Prism software v9.3.