Optimization of blood sampling time for isolation of antigen binding single-domain antibodies from immunized llamas

Single-domain antibody fragments (VHHs) binding specific antigens are often isolated by llama immunization and phage display selection. We determined the optimal blood sampling time for generating phage display libraries with a high frequency of antigen-binding VHHs. Two llamas were immunized with 7 antigens. Blood samples collected with 2-3 day interval up to 11 days post immunization were used for deep sequencing and phage display selection of VHHs. The frequency of VHH clones was determined by the identification of unique VHH sequences in deep sequenced libraries. On average, the highest clone frequency was found in libraries generated 5 days post immunization. Polyclonal phage ELISA also suggested day 5 as the optimal blood sampling time.