title: Combining phage display and screening of cDNA expression libraries: a new approach for identifying the target antigen of an scFv preselected by phage display.
abstract: A potential method for identifying new tumor-specific antibody structures as well as tumor-associated antigens is by selecting scFv phage libraries on tumor cells. This phage display technique involves multiple rounds of phage binding to target cells, washing to remove non-specific phage and elution to retrieve specific binding phage. Although the binding properties of an isolated tumor-specific scFv can be evaluated by ELISA, FACS and immunohistochemistry, it still remains a challenge to define the corresponding antigen. Here, we provide evidence that the target antigen of a given scFv displayed on phages can be detected in an immobilized lambda phage cDNA expression library containing thousands of irrelevant clones. The library contained CD30-negative breast-cancer specific cDNA as well as human CD30 receptor cDNA. The interaction of anti-CD30 scFv phages and their target antigen after blotting onto nitrocellulose filters was documented under defined conditions. Screening of different ratios between CD30 receptor and breast cancer specific clones (1:1 and 1:200) revealed that the CD30 antigen could be detected by anti-CD30 scFv phages using at least 5x10(12) plaque forming units of filamentous phages per blot. These investigations demonstrate that it is possible to detect the target antigen of a preselected scFv displayed on filamentous phages in lambda phage cDNA expression libraries.
authors.affiliation: Department of Veterinary Science, Institute of Immunology and Molecular Biology, Luisenstr. 56, University of Berlin, 10117, Germany. stefan.barth@uni-koeln.de
authors.forename: S
authors.initials: S
authors.lastname: Barth
authors.forename: U
authors.initials: U
authors.lastname: Weidenmüller
authors.forename: M K
authors.initials: MK
authors.lastname: Tur
authors.forename: M F
authors.initials: MF
authors.lastname: Schmidt
authors.forename: A
authors.initials: A
authors.lastname: Engert
mesh_terms: D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology*; D010582:Bacteriophage lambda / Q000235:genetics; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D001943:Breast Neoplasms / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D004306:Dose-Response Relationship, Immunologic; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D015723:Gene Library; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D017730:Ki-1 Antigen / Q000235:genetics* / Q000276:immunology*; D016254:Mutagenesis, Insertional / Q000235:genetics; D019151:Peptide Library; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D007135:Immunoglobulin Variable Region; D017730:Ki-1 Antigen; D019151:Peptide Library