Fig. 4. Ab117 cross-specificity. (a) Sequence alignment of the representative FMDV serotypes [28,30,52]. Residues with red background are conserved. Residues at the Fab117–SAT2/ZIM/7/83 interface are boxed. Black triangles indicate appreciable contribution to the interface, whilst red triangles indicate a hydrogen bond interaction (contributions determined using PISA). Residues directly making contact with W138 (≤4.0 Å) are indicated using an asterisk. The secondary structure of the SAT2/ZIM/7/83 proteins is indicated above the alignment. Alignment was performed using CLUSTALW [60,61], and the figure was generated using ESPript [41]. (b) Heterologous sandwich ELISA shows pan-specific binding of Ab117 to thermally disrupted virus. Integrin α_vβ₆ was used as a universal capture for all intact viruses, whilst heated particles were trapped directly on the plates, with Ab117 applied for detection. SAT1/ZIM/22/89 inactivated virus was tested in an independent experiment. (c) Ab117 binding to SAT3/ZIM/4/81 [62] wild-type VLPs. Serum raised in animals infected with SAT3 was used as a positive control, whilst the lack of M3 binding to disrupted capsids showed the inability of this VHH to recognize SAT3-disrupted particles. Ab117 binds to untreated capsids, suggesting their instability. Heating at 56 °C increased the signal, consistent with increased disruption of particles, while heating at 65 °C exhibited a lower signal, which suggests the disruption of the conformational epitope recognized by Ab117 at higher temperatures. Cyan bars represent the signal using untreated capsids (4 °C), orange, and red bars represent the binding level using capsids heat treated for 15 min at 56 °C and 65 °C, respectively.