Figure 1
Identification of anti-ETEC nanobodies from immunized llama and yeast display library. (A) Flowchart of llama immunization and panning. Two male llamas were subcutaneously immunized with N-terminal fragments of eight class 5 ETEC adhesins followed by PBMC isolation and generation of phage-displayed libraries of 108 in size. Libraries were panned on immobilized colonization factor antigens CS1 and CS2. Output from those pannings was used for a subsequent panning round on CfaE. Cross-reactive clones were sent for sequence determination and further characterization. (B) Flowchart of selection process of synthetic nanobodies. Binders to CfaE from the naïve yeast-displayed library were enriched over two rounds of selection by performing magnetic-activated cell sorting (MACS) on yeast that were stained with purified and labeled CfaE. High affinity binders were selected during fluorescence-activated cell sorting (FACS) and single cell clones were screened for binding with CS1 and CS2. Cross-reactive clones were sent for sequence determination and further characterization. (C) Flow analysis demonstrating example of binding of yeast displaying nanobody 2R23 to CfaE, CS1 and CS2 proteins. (D) Flow analysis demonstrating functional human monoclonal antibody 68-61 competing for binding to CfaE with yeast library derived nanobody 2R215 on yeast.
