Screening of scFv-displaying phages recognizing distinct extracellular domains of EGF receptor by target-guided proximity labeling method

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• 作者：Chialun Chang^a ; ^b ; ¹ ; Atsushi Takayanagi^a ; ^b ; ¹ ; Tetsuhiko Yoshida^c ; Nobuyoshi Shimizu^a ; ^{smzgsp@dmb.med.keio.ac.jp}
• 关键词：scFv ; single-chain Fv antibody fragment ; VH ; heavy chain of an antibody Fv fragment ; VL ; light chain of an antibody Fv fragment ; sulfo-SBED ; Sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1 ; 3&prime ; -dithiopropionate ; CDR ; comp
• 刊名：Journal of Immunological Methods
• 出版年：2011
• 出版时间：30 September, 2011
• 年：2011
• 卷：372
• 期：1-2
• 页码：127-136
• 全文大小：1K

文摘

We recently constructed the scFv-displaying phage library with extremely high repertoire and have successfully utilized for screening scFv antibodies against various proteins, polysaccharides and glyco-lipids. Here, we developed a new screening strategy to isolate scFv antibodies against cell surface EGF receptor (EGFR). For this, we applied two slightly different methods of ¡°target-guided proximity labeling,?such as Proximity selection (ProxiMol) method and a new sulfo-SBED labeling method with the aide of monoclonal anti-human EGFR antibody B4G7 as a guide molecule. ProxiMol method relies on the Biotin-labeling of scFv-displaying phages that bound to the target in a vicinity of 100 ? from the guide molecule, whereas sulfo-SBED method transfers Biotin to scFv-displaying phages, which bound to the target in a distance of 20 ?. After two rounds of panning on the EGFR-overexpressing A431 cells starting from approx. 1 ¡Á 10¹² pfu, 47 each of Biotin-labeled scFv-displaying phages were recovered using Streptoavidin-coated magnetic beads, and among them total 11 scFv-phages were found to be definitely positive for binding to A431 cell surface by ELISA assay. Restriction mapping and sequencing analysis of these scFv-phage DNAs revealed that they encode 4 different scFv-nucleotide sequences in total. Immuno-fluorescent microscopy provided evidence that these 4 scFv antibodies bind specifically to EGFR on the A431 cells, showing slightly different staining patterns. Thus, ¡°target-guided proximity labeling?methods were powerful for isolating scFv-displaying phages that recognize distinct extracellular domains of the target receptor. This novel screening strategy could be applicable to many other cell surface antigens and receptors.