Enrichment of hGPC3-reactive scFvs. A. Surface co-localization of hGPC3-reactive yeast cells after two rounds of MACS-sorting. Yeast display library were incubated with target antigens and MACS sorting were performed. hGPC3-reactive yeast are double-labeled with mouse anti-c-myc detected with anti-mouse Alexa Fluor 488 (green) secondary and biotinylated hGPC3-GST detected by streptavidin-phycoerythrin (red). B. Successful enrichment of hGPC3-reactive yeast cells by three rounds of FACS sorting. Representative FACS sorting using 29mer peptide hGPC3530-558 antigen was shown. Gradually decreasing concentrations of antigen were utilized in each round. Yeast cells without antigen incubation were used as control. In third FACS sorting, the PE-conjugated neutravidin was used in order to minimize enrichment of streptavidin-specific scFv also present in the library.