A novel approach for immunization, screening and characterization of selected scFv libraries using membrane fractions of tumor cells.
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.
Tur
Mehmet Kemal
MK
Fraunhofer IME, Department of Pharmaceutical Product Development, D-52074 Aachen, Germany.
Rothe
Achim
A
Huhn
Michael
M
Goerres
Ute
U
Klimka
Alexander
A
Stöcker
Michael
M
Engert
Andreas
A
Fischer
Rainerr
R
Finner
Ricarda
R
Barth
Stefan
S
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
D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D019151:Peptide Library