Pathfinder selection: in situ isolation of novel antibodies.
BACKGROUND
To devise a novel method for targeted recovery of binding molecules from phage libraries.


OBJECTIVE
To assess the potential of the novel technique to the selection of human antibodies to specific cell surface antigens in situ, including carcinoembryonic antigen (CEA), E- and P-selectins, and to the selection of novel antibodies which recognize immobilized purified antigen.


METHODS
Recovery of these antibodies from a naive human scFv library was effected using a 'pathfinder' molecule. Monoclonal and polyclonal antibodies, as well as natural ligands can serve as pathfinders when conjugated directly or indirectly to horseradish peroxidase (HRP). In the presence of biotin tyramine these molecules catalyze biotinylation of phage binding in close proximity to the target antigen, allowing specific recovery of 'tagged' phage from the total population using streptavidin. In this way, phage binding to the target itself, or in its immediate proximity, are selectively recovered.


RESULTS
This work demonstrates that an existing binding specificity can be used as a tool to select phage libraries in situ, obviating the need to purify or clone the target.


CONCLUSIONS
The speed and technical simplicity of this method should find a wide range of applications to phage display libraries, and could be applied to the discovery of new receptors and the elucidation of protein-protein interactions.
Osbourn
J K
JK
Cambridge Antibody Technology Limited, The Science Park, Melbourn, UK. jane.osbourn@camb-antibody.co.uk
Derbyshire
E J
EJ
Vaughan
T J
TJ
Field
A W
AW
Johnson
K S
KS
D000595:Amino Acid Sequence; D000906:Antibodies / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D000918:Antibody Specificity; D000954:Antigens, Surface / Q000276:immunology*; D002272:Carcinoembryonic Antigen / Q000276:immunology; D019040:E-Selectin / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D019007:P-Selectin / Q000276:immunology
D000906:Antibodies; D000954:Antigens, Surface; D002272:Carcinoembryonic Antigen; D019040:E-Selectin; D019007:P-Selectin