Initial tests to verify the reactivity in Western blotting of each MAb with the homologous partially purified strain were performed as previously described (21 (link)). Later, the cross-reactivity of one representative MAb (4A3) with all FMDV serotypes was confirmed as follows.
Virus lysates from IBRS-2 cells infected with different FMDV serotypes were denatured and reduced by heating at 95°C for 5 min in Red loading buffer and dithiothreitol (DTT) (NEB). The samples were resolved through the use of 12% Tris-glycine gels and transferred to nitrocellulose membranes (GE Healthcare) (0.45 μM) using a Mini-Protean Tetra cell (Bio-Rad). Membranes were placed in blocking buffer (20 mM Tris and 150 mM NaCl [pH 7.6] with 0.1% [vol/vol] Tween 20 [TBS-T] and 1% [wt/vol] bovine serum albumin [BSA] [Melford]) for 1 h at room temperature (RT) followed by incubation with hybridoma supernatants (MAbs) and anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (Dako) (1/5,000 in blocking buffer) in sequence for 1 h at RT. The incubations were separated by cycles of three washings with TBS-T. West Pico chemiluminescent substrate (Thermo Fisher Scientific, United Kingdom) was added to the membrane, and exposures of the membrane were collected and visualized using a G:Box Chemi XX6 system (Syngene).
Virus lysates from IBRS-2 cells infected with different FMDV serotypes were denatured and reduced by heating at 95°C for 5 min in Red loading buffer and dithiothreitol (DTT) (NEB). The samples were resolved through the use of 12% Tris-glycine gels and transferred to nitrocellulose membranes (GE Healthcare) (0.45 μM) using a Mini-Protean Tetra cell (Bio-Rad). Membranes were placed in blocking buffer (20 mM Tris and 150 mM NaCl [pH 7.6] with 0.1% [vol/vol] Tween 20 [TBS-T] and 1% [wt/vol] bovine serum albumin [BSA] [Melford]) for 1 h at room temperature (RT) followed by incubation with hybridoma supernatants (MAbs) and anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (Dako) (1/5,000 in blocking buffer) in sequence for 1 h at RT. The incubations were separated by cycles of three washings with TBS-T. West Pico chemiluminescent substrate (Thermo Fisher Scientific, United Kingdom) was added to the membrane, and exposures of the membrane were collected and visualized using a G:Box Chemi XX6 system (Syngene).