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

Chialun Chang, Atsushi Takayanagi, Tetsuhiko Yoshida, Nobuyoshi Shimizu

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

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 Å from the guide molecule, whereas sulfo-SBED method transfers Biotin to scFv-displaying phages, which bound to the target in a distance of 20 Å. After two rounds of panning on the EGFR-overexpressing A431 cells starting from approx. 1×1012pfu, 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.

Original languageEnglish
Pages (from-to)127-136
Number of pages10
JournalJournal of Immunological Methods
Volume372
Issue number1-2
DOIs
Publication statusPublished - 2011 Sep 30

Fingerprint

Epidermal Growth Factor Receptor
Bacteriophages
Single-Chain Antibodies
Biotin
Restriction Mapping
Antigen Receptors
Cell Surface Receptors
Surface Antigens
Polysaccharides
Microscopy
Enzyme-Linked Immunosorbent Assay
Staining and Labeling
Lipids
Antibodies
DNA
Proteins

Keywords

  • Antibody screening
  • Biotin
  • EGF receptor
  • Phage display

ASJC Scopus subject areas

  • Immunology
  • Immunology and Allergy

Cite this

Screening of scFv-displaying phages recognizing distinct extracellular domains of EGF receptor by target-guided proximity labeling method. / Chang, Chialun; Takayanagi, Atsushi; Yoshida, Tetsuhiko; Shimizu, Nobuyoshi.

In: Journal of Immunological Methods, Vol. 372, No. 1-2, 30.09.2011, p. 127-136.

Research output: Contribution to journalArticle

Chang, Chialun ; Takayanagi, Atsushi ; Yoshida, Tetsuhiko ; Shimizu, Nobuyoshi. / Screening of scFv-displaying phages recognizing distinct extracellular domains of EGF receptor by target-guided proximity labeling method. In: Journal of Immunological Methods. 2011 ; Vol. 372, No. 1-2. pp. 127-136.
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