Affinity assessment of scFvs. A. Immunoblot analysis of binding of scFv to rhGPC3. The antigens including rhGPC3 and GST protein were spotted onto cellulose membrane (10 ng/each). After the blocking step the membrane were incubated with the scFvs antibody in room temperature for 1 h. The binding of scFv to antigen was detected by incubation with mouse anti-V5 mAb following by infrared dye IR680-labeled anti-mouse antibodies. PBS was used as negative control. B. Direct ELISA for affinity determination of scFv 3E11. Two different concentration of rhGPC3 protein (0.5 and 1.0 ug/ml) were coated and incubated with serial diluted scFv 3E11. For detection, mouse-sourced anti-V5 mAb following HRP-conjugated anti-mouse antibody and SureBlue substrate (measured at 450 nm) were used. C. EC50 values for 5 candidate scFvs determined by direct ELISA are shown. Assays were performed twice separately for each scFv.