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. 2021 Jun 8;9(6):620. doi: 10.3390/vaccines9060620

Figure 4.

Figure 4

Specificity of novel VHHs for FMDV particles fractionated by SDG. SDG fractions were analyzed using various VHH based DAS-ELISAs. FMDV strains A22/IRQ/24/64 (a–c), A24/Cruzeiro/BRA/55 (d–f), A/TUR/14/98 (g–l), A/GDMM/CHA/2013 (m–o) and Asia1/Shamir/ISR/89 (p–r) were used. SDG were layered with a 1:1 mixture of FMDV particle preparations that were either untreated or heated for 1 h at 56 °C to ensure the presence of both 146S and 12S. The 20 fractions were analyzed by the measurement of absorbance at 254 nm to identify the 146S peak and by two ELISAs using the broadly reactive VHHs M3F and M8F to identify all peaks (a,d,g,m,p). The other panels contain the putative 146S-specific VHHs, with each panel containing clones from a single CDR3 group, except panel (j), which contains two VHHs from different CDR3 groups. Clones from the same CDR3 group have identical line colors except for the four VHHs of the same CDR3 group in panels (b,h,n) that were given two colors to allow discrimination of curves. Panel (r) represents the same data as panels (p,q) with a different Y-axis scale to enable visualization of lower peaks. Only for the M3F ELISAs, SDG fraction samples and standards were heated for 1 h at 56 °C prior to ELISA. The 12S peak appeared in fractions 1–5, while that of 75S appeared in fractions 7–10 and 146S in fractions 13–16.