Anti toxin b chicken igy

For antigen expression in monomicrobial culture, the transformed YS1646 strains were grown overnight in LB with 50 μg/ml of ampicillin at 37°C in 0% CO₂, centrifuged at 21,130 × g for 10 min, resuspended in PBS, and then mixed in with NuPAGE lithium dodecyl sulfate (LDS) sample buffer (Invitrogen) according to the manufacturer’s instructions. For antigen expression in RAW 264.7 macrophages, infection was allowed to proceed for either 1 h or 24 h. Samples were then collected, centrifuged, resuspended in PBS, and mixed with sample buffer as described above. All samples were heated for 10 min at 70°C and then cooled on ice. Proteins were separated on a 4 to 12% Bis-Tris protein gel (Invitrogen) and transferred to nitrocellulose membranes using a Trans-Blot Turbo RTA mini-nitrocellulose transfer kit (Bio-Rad, Hercules, CA). For detection of TcdA_5458–8130 and TcdB_5461–7080, the membranes were incubated first with anti-toxin A chicken IgY (1:5,000; Abnova, Taipei, Taiwan) and anti-toxin B chicken IgY (1:10,000; Abnova) antibodies, respectively, followed by goat anti-chicken IgY conjugated to horseradish peroxidase (1:10,000; Thermo Fisher Scientific). Immunoreactive bands were visualized using the SuperSignal West Pico Plus chemiluminescent substrate (Thermo Fisher Scientific) and autoradiography film (Denville Scientific, Holliston, MA).

For antigen expression in vitro, the transformed YS1646 strains were grown overnight in LB at 37°C in 0% CO₂. Overnight cultures were centrifuged at 21,130 × g for 10 min and resuspended in PBS. Six hundred seventy nanograms of each sample and 7 ng and 50 ng of positive controls were then mixed in with NuPAGE lithium dodecyl sulfate sample buffer (Invitrogen) according to the manufacturer’s instructions. All samples were heated for 10 min at 100°C and then cooled on ice. Proteins were separated on a 4% to 20% Bis-Tris protein gel (Invitrogen) and transferred to nitrocellulose membranes using a Trans-Blot Turbo RTA mini nitrocellulose transfer kit (Bio-Rad, Hercules, CA). For detection of TcdA_5458–8130 and TcdB_5461–7080, the membranes were incubated first with anti-toxin A chicken IgY (1:5,000; Abnova, Taipei, Taiwan) and anti-toxin B chicken IgY (1:10,000; Abnova) antibodies, respectively, followed by goat anti-chicken-IgY IgG conjugated to horseradish peroxidase (HRP; 1:10,000; Thermo Fisher Scientific). Immunoreactive bands were visualized using the SuperSignal West Pico Plus chemiluminescent substrate (Thermo Fisher Scientific) and autoradiography film (Denville Scientific, Holliston, MA).