Alanine scan of the HCDR3 region of MAN-1, MAS-1, and MAS-2. The amino acids of the HCDR3, which were substituted by alanine, are given. The binding properties of the HCDR3 mutants were determined by flow cytometry on activated Mac-1 receptor in a whole-blood assay. Binding of the scFv's in flow cytometry was detected by an Alexa Fluor 488–labeled anti–His-tag antibody. Results are shown as percentage of the mean fluorescence of the original clones binding to activated Mac-1. Representative results out of at least 5 experiments are shown.