An optimized method for cell-based phage display panning.
BACKGROUND
Traditional methods of phage display panning, bind purified antigen to plates or other solid phases to which libraries are then applied, followed by vigorous washings in detergent-supplemented buffers to select for specific phage Fab. These methods are not directly applicable to antigens in their native environment on cell surfaces or in settings where the target antigen is unknown.


OBJECTIVE
To develop a model antigen system employing whole cells rather than purified protein immobilized on a substrate; to optimize methods for phage display panning using a cell-based system.


RESULTS
Specificity of binding of phage Fab to antigen on cells was demonstrated by output titer and by flow cytometry. Output titers showed a plateau and binding advantage, after four washes, corresponding to removal of most non-specifically bound phage. Enrichment advantage was independent of input phage number. Longer incubation times, to cell tolerance, improved specific binding. Temperature had modes impact as a variable in the panning and washing. An increase in output titers paralleled enrichment for specific phage Fab.


CONCLUSIONS
An optimized method applied to whole cells can productively enrich specific phage Fab in mixtures with large excesses of non-specific phage Fab over several rounds of panning.
Watters
J M
JM
Department of Medicine, Harvard Medical School, New England Deaconess Hospital, Boston MA 02215, USA.
Telleman
P
P
Junghans
R P
RP
D000818:Animals; D000954:Antigens, Surface / Q000032:analysis* / Q000276:immunology; D001435:Bacteriophages / Q000235:genetics* / Q000276:immunology; D016208:Databases, Factual; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D006825:Hybridomas; D007140:Immunoglobulin Fab Fragments / Q000276:immunology* / Q000302:isolation & purification; D007130:Immunoglobulin Idiotypes / Q000235:genetics / Q000276:immunology; D051379:Mice; D019151:Peptide Library; D015375:Receptors, Interleukin-2 / Q000276:immunology; D013696:Temperature
D000954:Antigens, Surface; D007140:Immunoglobulin Fab Fragments; D007130:Immunoglobulin Idiotypes; D019151:Peptide Library; D015375:Receptors, Interleukin-2; D015252:Deoxyribonucleases, Type II Site-Specific; C024403:GGCC-specific type II deoxyribonucleases