Figure 2.

Specificity of double antibody sandwich (DAS) enzyme-linked immunosorbent assays (ELISAs) for inactivated foot-and-mouth disease virus (FMDV) antigen fractionated by sucrose density gradient (SDG). FMDV strains Asia 1 Shamir (A,D,G,J), SAT2 SAU/2/00 (B,E,H,K), and O₁ Manisa (C,F,I,L) were used. FMDV 146S particles purified by SDG were again layered on SDG without further treatment (A–C) or after prior acidification for conversion into 12S^A particles (D–F). Crude FMDV antigen was similarly fractionated on SDG without further treatment (G–I) or after prior acidification (J–L). Twenty fractions of each SDG were analyzed by ELISA using either a VHH that is specific for 146S particles (open diamonds) or that binds both 12S and 146S particles (closed squares). The different 146S-specific VHHs used were M332F (A,D,G,J), M379F (B,E,H,K), and M170F (C,F,I,L). The different 12S- and 146S-recognizing VHHs used were M98F (A,D,G,J), M311F (B,E,H,K), and M8F (C,F,I,L). In panel (I), the low amount of FMDV antigen detected in fractions 1–15 by M8 ELISA is visualized by plotting on a different scale (right axis; gray circles). FMDV antigen concentrations in fractions were calculated from titration series in ELISA against a standard of untreated FMDV antigen with known 146S content. Fraction 1 corresponds to top of gradient.