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Recombinant Antibody Selections by Combining Phage and Yeast Display

  • Fortunato Ferrara
  • Maria Felicia Soluri
  • Daniele SblatteroEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1904)

Abstract

In vitro display technologies have put together the generation of large antibody libraries with selection and screening procedures to identify lead candidates. Phage display antibody libraries allow selecting and identifying binders for a variety of antigens. Nonetheless, the procedure is limited by the possibility to quantitatively follow the enrichment during selection cycles and tune up the clones for specific binding proprieties (i.e., affinity). Yeast display allows the expression of thousands of copies of the antibody on each cell, simultaneously carrying the plasmid encoding that antibody, moreover the selection parameters can be accurately controlled by flow cytometry-based analysis and sorting.

The combination of phage and yeast display takes advantage of both platforms by starting with a vast number of antibodies in the phage display selections followed by the precise sorting of the clones specifically recognizing the target of interest.

In the present chapter, we illustrate protocols to generate and enrich - using fluorescence-activated cell sorting (FACS) - yeast display antibody libraries, using selection outputs obtained from phage antibody display libraries as starting material. The present methods can be easily applicable for the identification of monoclonal antibodies with desired binding properties.

Key words

Phage antibody display Yeast surface antibody display FACS Antigens Monoclonal antibody High-throughput scFv 

References

  1. 1.
    Clackson T et al (1991) Making antibody fragments using phage display libraries. Nature 352(6336):624–628CrossRefGoogle Scholar
  2. 2.
    Winter G et al (1994) Making antibodies by phage display technology. Annu Rev Immunol 12:433–455CrossRefGoogle Scholar
  3. 3.
    Feldhaus MJ et al (2003) Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library. Nat Biotechnol 21(2):163–170CrossRefGoogle Scholar
  4. 4.
    Boder ET, Wittrup KD (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol 15(6):553–557CrossRefGoogle Scholar
  5. 5.
    Sblattero D, Bradbury A (2000) Exploiting recombination in single bacteria to make large phage antibody libraries. Nat Biotechnol 18(1):75–80CrossRefGoogle Scholar
  6. 6.
    Bradbury AR et al (2011) Beyond natural antibodies: the power of in vitro display technologies. Nat Biotechnol 29(3):245–254CrossRefGoogle Scholar
  7. 7.
    Gera N, Hussain M, Rao BM (2013) Protein selection using yeast surface display. Methods 60(1):15–26CrossRefGoogle Scholar
  8. 8.
    Ferrara F et al (2015) Recombinant renewable polyclonal antibodies. MAbs 7(1):32–41CrossRefGoogle Scholar
  9. 9.
    D’Angelo S et al (2018) Selection of phage-displayed accessible recombinant targeted antibodies (SPARTA): methodology and applications. JCI Insight 3(9)Google Scholar
  10. 10.
    Ferrara F et al (2012) Using phage and yeast display to select hundreds of monoclonal antibodies: application to antigen 85, a tuberculosis biomarker. PLoS One 7(11):e49535CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Fortunato Ferrara
    • 1
  • Maria Felicia Soluri
    • 2
  • Daniele Sblattero
    • 3
    Email author
  1. 1.Specifica Inc.Santa FeUSA
  2. 2.Department of Health Sciences and IRCADUniversity of Piemonte Orientale (UPO)NovaraItaly
  3. 3.Department of Life SciencesUniversity of TriesteTriesteItaly

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