Validation of scFv specificity by ELISA. A. Preparation of soluble scFvs. scFv cDNA amplified from the enriched yeast population was co-transformed into YVH10 yeast using p416-BCCR vector. Yeast were induced to secrete scFvs with 2% galactose. Culture supernatant (5 ul) were loaded into SDS-PAGE gel for detection by anti-V5 mAb in Western Blot. Approximately 80% of yeast transformants produced soluble scFv. B. ELISA screening of 576 scFv for binding to hGPC3-GST. Maxsorb plates were coated with hGPC3-GST and GST protein. scFvs were incubated in plates then washed extensively. HRP-conjugated anti-V5 mAb was used for quantification of binding. Each scFv was tested in parallel for binding to hGPC3-GST and GST. C. scFv with highest hGPC3-GST/GST binding ratio were screened for binding to full-length hGPC3 protein expressed by mammalian cells by ELISA. G3-C1 is a VH only scFv isolated using GPC3530-558. The remainder of scFv were isolated using the truncated rhGPC3-GST fusion protein.