Pvdf membranes

Vero cells were seeded and cultured in 24-well plates using serum-free medium (VP-SFM) (Gibco, Carlsbad, CA) containing 4 mM glutamine at 37°C with 5% CO₂, and the cells were grown to confluence. Varying amounts of TcdA rRBD were added to the wells, and PBS was used as the negative control. After a 30-min incubation, the wells were washed twice with cold PBS. Radioimmunoprecipitation assay buffer (RIPA buffer) (Millipore, Billerica, MA), a strong cell lysis buffer, was added to the cells. The total cell lysate was analyzed by SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes (Pall Corporation) pre-soaked in methanol. PVDF membranes were blocked with 5% (w/v) nonfat dry milk in PBS and sequentially exposed to 0.5 μg/mL TcdA-specific monoclonal antibodies (Clone PCG-4; GeneTex, Taiwan) and 50 ng/mL horseradish peroxidase (HRP) conjugated to a secondary antibody (GeneTex) to detect the amount of TcdA rRBD bound to Vero cells. Antibodies were diluted in PBS containing 1% nonfat dry milk (w/v). PVDF membranes were washed twice at every step with PBST (1× PBS containing 0.05% Tween 20) at room temperature. Finally, signals were detected using enhanced chemiluminescence (ECL) according to the manufacturer’s instructions (KPL, Gaithersburg, MD).