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Pathfinder selection: in situ isolation of novel antibodies
J K Osbourn1, E J Derbyshire, T J Vaughan
1Cambridge Antibody Technology Limited, The Science Park, Melbourn, UK. jane.osbourn@camb-antibody.co.uk
Background:
To devise a novel method for targeted recovery of binding molecules from phage libraries.
Objectives:
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.
Study Design:
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.
Conclusion:
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.