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Rescue and In Situ Selection and Evaluation (RISE): A Method for High-Throughput Panning of Phage Display Libraries

Department of Crop Protection, Faculty of Agricultural and Applied Biological Sciences, Ghent University, Ghent, Belgium, Bayer BioScience N.V., Ghent, Belgium

Christophe Ampe

Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium

Luc Tirry

Department of Crop Protection, Faculty of Agricultural and Applied Biological Sciences, Ghent University, Ghent, Belgium

Peter Denolf

Bayer BioScience N.V., Ghent, Belgium

Phage display has proven to be an invaluable instrument in the search for proteins and peptides with optimized or novel functions. The amplification and selection of phage libraries typically involve several operations and handling large bacterial cultures during each round. Purification of the assembled phage particles after rescue adds to the labor and time demand. The authors therefore devised a method, termed rescue and in situ selection and evaluation (RISE), which combines all steps from rescue to binding in a single microwell. To test this concept, wells were precoated with different antibodies, which allowed newly formed phage particles to be captured directly in situ during overnight rescue. Following 6 washing steps, the retained phages could be easily detected in an enzyme-linked immunosorbent assay (ELISA), thus eliminating the need for purification or concentration of the viral particles. As a consequence, RISE enables a rapid characterization of phage-displayed proteins. In addition, this method allowed for the selective enrichment of phages displaying a hemagglutinin (HA) epitope tag, spiked in a 10⁴-fold excess of wild-type background. Because the combination of phage rescue, selection, or evaluation in a single microwell is amenable to automation, RISE may boost the high-throughput screening of smaller sized phage display libraries.

Key Words: phage display • high-throughput screening • microwell rescue • Bacillus thuringiensis • hemagglutinin

Journal of Biomolecular Screening, Vol. 10, No. 2, 108-117 (2005)
DOI: 10.1177/1087057104271956


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