Figure EV1. Anti‐BACE1 VHH production and selection.

1. Schematic summarizing the production procedure. A dromedary and a llama were immunized with recombinant human BACE1 ectodomain (amino acids 46–460). Blood lymphocytes from the immunized animals were collected for RNA extraction. cDNA was prepared and the variable fragments of heavy chain only IgGs were amplified by RT‐PCR and purified using agarose gel electrophoresis. cDNAs encoding VHH were cloned into the pHEN4 phagemid vector and phage libraries were prepared. Three rounds of consecutive phage panning were performed to enrich phage particles that bound recombinant BACE1 in an ELISA assay. Binding to BACE1 was confirmed in a phage ELISA. Finally, BACE1 binding VHHs were purified with initial testing of inhibitory activity using an in vitro APP cleavage assay (MBP‐C125sw enzymatic assay).
2. Protein sequences for anti‐BACE1 VHH identified as having inhibitory activity in the in vitro APP cleavage assay. Sequences are aligned, with framework and CDR regions indicated.