Validation of glypican-3-specific scFv isolated from paired display/secretory yeast display library.

BMC Biotechnology
Yonghai LiDavid E Kaplan

Abstract

Glypican-3 (GPC3) is a heparan-sulfate proteoglycan frequently expressed on the cell membrane of malignant hepatocytes in hepatocellular carcinoma. The capacity for screening potential antibodies in vitro using human hepatocellular lines is critical to ensure binding to this highly post-translationally modified glycophosphatidylinositiol-linked protein. We hypothesized that we could utilize a recently described paired display/secretory yeast library to isolate human-derived scFv against glypican-3 for potential diagnostic and/or therapeutic application. Using two different biotinylated antigen targets, a synthesized 29mer fragment GPC3(550-558) and a truncated GPC3(368-548) fused with glutathione S-transferase (GST) we enriched the yeast display library to greater than 30% target-specific yeast with both positive selection and depletion of streptavidin- and GST-specific clones. After cloning of scFv cDNA from the enriched sub-library, scFv specificity was validated by ELISA for binding to recombinant protein from prokaryotic and eukaryotic sources and ultimately naturally presented human protein on the cell membrane of human hepatocellular cell lines. Specificity was confirmed using non-expressing cell lines and shRNA knockdown...Continue Reading

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Citations

Aug 2, 2014·Immunopharmacology and Immunotoxicology·Safar FarajniaLeila Rahbarnia
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Apr 14, 2020·International Journal of Nanomedicine·Lijie XiaFuchun Zhang

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Methods Mentioned

BETA
xenograft
FACS
ELISA
dot
flow cytometry
transfection
PCR
Assay
phage display

Software Mentioned

PRISM
GraphPad
FlowJo
Ambion Silencer Select
IgBLAST

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