High-throughput screening and characterization of clones selected from phage display libraries

Cytometry A. 2007 Aug;71(8):625-31. doi: 10.1002/cyto.a.20417.

Abstract

Background: Clones from phage display libraries are generally selected by a number of rounds of panning and regrowth, followed by primary screening to identify hits and secondary characterization to identify clones with optimal affinity and specificity. Because functional screening for binding or other activity can be material-, time-, and labor-intensive, sequencing is often used to identify the emergence of a consensus sequence prior functional characterization. However, the consensus sequence is not always the optimal one because factors such as phage growth rates, nonspecific binding, and other selection pressures can bias the selection process.

Methods: To improve function-based phage display library screening and characterization, we developed a multiplexed approach employing optically-encoded microsphere arrays and flow cytometry.

Results: We show that capture of phage from crude culture supernatants enables the efficient screening of binding activity and the evaluation of binding avidity. The approach uses small volumes and a homogeneous no-wash format that minimizes reagent consumption and sample handling. The use of optically-encoded microspheres allows many phage to be screened simultaneously, greatly increasing throughput.

Conclusions: This approach is flexible, supporting primary and secondary screening for a range of functional assays, and scalable, potentially supporting the screening of thousands to hundreds of thousands of clones per hour.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Bacteriophages
  • Cholera Toxin / metabolism
  • Cloning, Molecular / methods*
  • Microspheres
  • Peptide Library*
  • Protein Subunits / metabolism
  • Reproducibility of Results

Substances

  • Peptide Library
  • Protein Subunits
  • Cholera Toxin