Figure 3.

(A) Screening of VHH library. 94 individual phages-VHH clones were tested by TIA using TcTS611/2. Three clear groups are observed, NI: non-inhibitors, WI: weak inhibitors, SI: strong inhibitors. Each point represents phages prepared from a single colony. TIA values lower than 25% were considered as negative. (B) Purified VHHs inhibit recombinant TcTS activity. A fixed mass of 0.5 ng of purified TcTS611/2 was preincubated with increasing concentrations of each VHH and trans-sialidase activity was analyzed by TIA. The values represent the average of at least three independent determinations. (C) The selected VHHs recognize conformational epitopes. Recombinant proteins, all carrying a His-tag, were spotted onto nitrocellulose membrane as indicated in the panel 4: A) SI14, B) WI58, C) SI52, D) SI96, E) SI57, F) TcTS611/2, G) Denatured TcTS611/2 in 0.1% SDS, H) Non-related (non-anti-TcTS) VHH. Panel 1 and panel 2 were incubated with native TcTS and denatured TcTS in 0.1% SDS, respectively. Both panels were treated with rabbit anti-TcTS serum. Panel 3 was processed with a mouse anti-Histidine antibody as a control for protein immobilization. Filters were revealed with the corresponding HRP-conjugated-anti-serum for chemiluminescence generation.