Figure 5
Immunoblot analysis of VEEV antigen detected by scFv phage or scFv-Fc fusions. VEEV VRS preparations were prepared at 56°C under non-reducing conditions and separated by 10% SDS-PAGE. After Western blot membranes were cut in stripes corresponding to 5–6 μg VEEV proteins. A. Immunostain was performed with 5 × 1010 (cfu) anti-VEEV scFv phage particles/mL, murine anti-VEEV mAbs 8/6 and 8747 (1:1000) and detected with mAb mouse anti-M13 HRP (1:4000), respectively goat anti-mouse IgG Fc specific HRP (1:10000). IIB6 is a non VEEV-specific control scFv phage. B. Additionally, VEEV VRS samples were prepared at 56°C under non-reducing or at 99°C under reducing conditions, respectively. Western blots were stained with 1 μg/mL scFv-Fc and murine anti-VEEV IgG (1:1000) and detected with goat anti-human (gamma chain specific) AP (1:5000), or goat-anti mouse (Fc specific) AP (1:10000), respectively. The marker bands were marked with a pencil.