title: Screening of scFv-displaying phages recognizing distinct extracellular domains of EGF receptor by target-guided proximity labeling method.
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 100Å 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 × 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.
authors.affiliation: Advanced Research Center for Genome Super Power, Keio University, 2 Okubo, Tsukuba, Ibaraki 300-2611, Japan.
authors.forename: Chialun
authors.initials: C
authors.lastname: Chang
authors.forename: Atsushi
authors.initials: A
authors.lastname: Takayanagi
authors.forename: Tetsuhiko
authors.initials: T
authors.lastname: Yoshida
authors.forename: Nobuyoshi
authors.initials: N
authors.lastname: Shimizu
mesh_terms: D000918:Antibody Specificity; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D000939:Epitopes / Q000276:immunology; D066246:ErbB Receptors / Q000276:immunology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology*; D019151:Peptide Library; D057127:Single-Chain Antibodies / Q000276:immunology*
chemical_list: D000939:Epitopes; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D057127:Single-Chain Antibodies; D066246:ErbB Receptors