title: A novel approach for immunization, screening and characterization of selected scFv libraries using membrane fractions of tumor cells.
abstract: Isolation of cell-surface specific antibodies prerequisites the functional expressing of antigens on intact cells, which are maintained routinely by cell culturing. However, long-term culturing of tumor cells could alter their antigen expression patterns and stable fixation of whole cells is not guaranteed on plastic surfaces during stringent screening procedures. We prepared functional breast cancer cell-membrane fractions that express surface molecules in their native conformation. Specific binding phages were isolated from phage antibody libraries constructed from the spleen messenger RNA of mice immunized with breast cancer cell-membrane fractions. After negative selection on non-mammary carcinoma cells and four rounds of positive selection on breast carcinoma cell lines, phage antibodies were enriched that bound specifically to breast cancer cell lines as confirmed by phage enzyme linked immunosorbent assay (ELISA) using 96-well plates coated with breast cancer cell membranes. The isolated phage antibodies were highly specific for the breast cancer cell line 8701-BC but not on other carcinoma such as the Hodgkin-derived cell line L540Cy as demonstrated by ELISA and flow cytometry. This report describes a rapid and more versatile method for isolating antibody fragments compared to whole cell screening procedures. One single membrane preparation can be stored for at least 15 months at -80 degrees C and used to immunize mice or for screening of antibody libraries. The selection and screening strategy used should be generally applicable to identify novel cell-surface antigens and their corresponding antibodies.
authors.affiliation: Fraunhofer IME, Department of Pharmaceutical Product Development, D-52074 Aachen, Germany.
authors.forename: Mehmet Kemal
authors.initials: MK
authors.lastname: Tur
authors.forename: Achim
authors.initials: A
authors.lastname: Rothe
authors.forename: Michael
authors.initials: M
authors.lastname: Huhn
authors.forename: Ute
authors.initials: U
authors.lastname: Goerres
authors.forename: Alexander
authors.initials: A
authors.lastname: Klimka
authors.forename: Michael
authors.initials: M
authors.lastname: Stöcker
authors.forename: Andreas
authors.initials: A
authors.lastname: Engert
authors.forename: Rainerr
authors.initials: R
authors.lastname: Fischer
authors.forename: Ricarda
authors.initials: R
authors.lastname: Finner
authors.forename: Stefan
authors.initials: S
authors.lastname: Barth
mesh_terms: D000818:Animals; D000917:Antibody Formation / Q000276:immunology; D001943:Breast Neoplasms / Q000276:immunology; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000276:immunology*; D051379:Mice; D019151:Peptide Library; D014407:Tumor Cells, Cultured; D014611:Vaccination
chemical_list: D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D019151:Peptide Library