Antibody Isolation from Human Synthetic Libraries of Single-Chain Antibodies and Analysis Using NGS

Adi Amir, David Taussig, Almog Bitton, Limor Nahary, Anna Vaisman-Mentesh, Itai Benhar*, Yariv Wine

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review


Antibody libraries came into existence 30 years ago when the accumulating sequence data of immunoglobulin genes and the advent of PCR technology made it possible to clone antibody gene repertoires. Phage display (most common) and additional display and screening technologies were applied to pan out desired binding specificities from antibody libraries. As other antibody discovery tools, phage display is not an off-the-shelf technology and not offered as a kit but rather requires experience and expertise for making it indeed very useful. Next-generation sequencing (NGS) coupled with bioinformatics is a powerful tool for analyzing large amount of DNA sequence output of the panning. Here, we demonstrate how NGS analysis of phage biopanning (phage-Seq) of complex antibody libraries can facilitate the antibody discovery process and provide insights regarding the biopanning process (see Fig. 1).

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Number of pages26
StatePublished - 2023

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


  • CDRs (complementarity determining regions)
  • FR (variable framework region)
  • HTS (high-throughput sequencing)
  • NGS (next-generation sequencing
  • Phage display
  • Phage-Seq
  • Single-chain antibodies
  • Synthetic library
  • VH (variable region of antibody heavy chain)
  • VL (variable region of antibody light chain)
  • scFv (single-chain variable fragment)


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