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We describe a human cell display strategy to isolate high-affinity single-chain antibody fragments (scFvs) specific for CD22 for the treatment of B-cell malignancies. Our strategy uses flow cytometry and human embryonic kidney 293T (HEK-293T) cells that are widely used for transient protein expression. Flow cytometry enhances the screen’s sensitivity thereby allowing us to isolate high-affinity scFvs. Using human cell display, one could isolate and engineer scFvs, single domains, Fabs, or whole IgGs for increased affinity and other biological functions.