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J Vis Exp. 2015 Jan 17;(95):51492. doi: 10.3791/51492.

Scalable high throughput selection from phage-displayed synthetic antibody libraries.

Author information

  • 1The Recombinant Antibody Network; The Banting and Best Department of Medical Research, University of Toronto; shanemiersch@gmail.com.
  • 2The Recombinant Antibody Network; The Banting and Best Department of Medical Research, University of Toronto.
  • 3The Recombinant Antibody Network; Antibiome Center, University of California, San Francisco at Mission Bay.
  • 4The Recombinant Antibody Network; Department of Biochemistry and Molecular Biology, The University of Chicago.

Abstract

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

PMID:
25651360
PMCID:
PMC4354533
DOI:
10.3791/51492
[PubMed - indexed for MEDLINE]
Free PMC Article
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