Phage Display and Subtractive Selection on Cells

  • Steffen U. EisenhardtEmail author
  • Karlheinz Peter
Part of the Springer Protocols Handbooks book series (SPH)

Phage display is a powerful tool to select antibodies for conformation-specific epitopes from antibody libraries. Based on the M13 pIII phage display technology we describe a cell suspension-based strategy, which allows panning against complex, multimeric, fully functional cell membrane epitopes without alteration of structure due to purification or immobilization. The method requires a subtractive panning strategy to avoid selection of phage that bind to the plethora of cell surface epitopes that are are not targeted. The panning can be carried out in fully physiological ex vivo conditions, so that the functional properties of the cells and the surface receptors can be preserved and thus phage can be specifically depleted or selected for neo-epitopes exposed after physiological alterations of the targeted molecules. This subtractive panning strategy is described in detail in this chapter and allows for the specific selection of single-chain antibodies directed against functionally regulated epitopes on cell surface molecules.


Phage Display Phage Library Antibody Library Resuspend Pellet Target Epitope 
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  1. Arap W, Kolonin MG, Trepel M, Lahdenranta J, Cardo-Vila M, Giordano RJ et al (2002) Steps toward mapping the human vasculature by phage display. Nat Med 8(2):121–127PubMedCrossRefGoogle Scholar
  2. Barbas CF 3 rd, Kang AS, Lerner RA, Benkovic SJ (1991) Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc Natl Acad Sci USA 88(18):7978–7982PubMedCrossRefGoogle Scholar
  3. Boder ET, Wittrup KD (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol 15(6):553–557PubMedCrossRefGoogle Scholar
  4. Clackson T, Hoogenboom HR, Griffiths AD, Winter G (1991) Making antibody fragments using phage display libraries. Nature 352(6336):624–628PubMedCrossRefGoogle Scholar
  5. de Wildt RM, Mundy CR, Gorick BD, Tomlinson IM (2000) Antibody arrays for high-throughput screening of antibody-antigen interactions. Nat Biotechnol 18(9):989–994PubMedCrossRefGoogle Scholar
  6. Dorsam H, Rohrbach P, Kurschner T, Kipriyanov S, Renner S, Braunagel M et al (1997) Antibodies to steroids from a small human naive IgM library. FEBS Lett 414(1):7–13PubMedCrossRefGoogle Scholar
  7. Eisenhardt SU, Schwarz M, Bassler N, Peter K (2007a) Subtractive single-chain antibody (scFv) phage-display: tailoring phage-display for high specificity against function-specific conformations of cell membrane molecules. Nat Protoc 2(12):3063–3073PubMedCrossRefGoogle Scholar
  8. Eisenhardt SU, Schwarz M, Schallner N, Soosairajah J, Bassler N, Huang D et al (2007b) Generation of activation-specific human anti-alphaMbeta2 single-chain antibodies as potential diagnostic tools and therapeutic agents. Blood 109(8):3521–3528PubMedCrossRefGoogle Scholar
  9. Fuchs P, Breitling F, Dübel S, Seehaus T, Little M (1991) Targeting recombinant antibodies to the surface of Escherichia coli: fusion to a peptidoglycan associated lipoprotein. Biotechnology (N Y) 9(12):1369–1372CrossRefGoogle Scholar
  10. Giordano RJ, Cardo-Vila M, Lahdenranta J, Pasqualini R, Arap W (2001) Biopanning and rapid analysis of selective interactive ligands. Nat Med 7(11):1249–1253PubMedCrossRefGoogle Scholar
  11. Griffiths AD (1993) Production of human antibodies using bacteriophage. Curr Opin Immunol 5(2):263–267PubMedCrossRefGoogle Scholar
  12. Hawlisch H, Muller M, Frank R, Bautsch W, Klos A, Kohl J (2001) Site-specific anti-C3a receptor single-chain antibodies selected by differential panning on cellulose sheets. Anal Biochem 293(1):142–145PubMedCrossRefGoogle Scholar
  13. Hoogenboom HR, de Bruine AP, Hufton SE, Hoet RM, Arends JW, Roovers RC (1998) Antibody phage display technology and its applications. Immunotechnology 4(1):1–20PubMedCrossRefGoogle Scholar
  14. Hust M, Maiss E, Jacobsen HJ, Reinard T (2002) The production of a genus-specific recombinant antibody (scFv) using a recombinant potyvirus protease. J Virol Methods 106(2):225–233PubMedCrossRefGoogle Scholar
  15. Hynes RO (1992) Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69(1):11–25PubMedCrossRefGoogle Scholar
  16. Kipriyanov SM, Moldenhauer G, Little M (1997) High level production of soluble single chain antibodies in small-scale Escherichia coli cultures. J Immunol Methods 200(1–2):69–77PubMedCrossRefGoogle Scholar
  17. Le Gall F, Kipriyanov SM, Moldenhauer G, Little M (1999) Di-, tri- and tetrameric single chain Fv antibody fragments against human CD19: effect of valency on cell binding. FEBS Lett 453(1–2):164–168PubMedCrossRefGoogle Scholar
  18. Levitan B (1998) Stochastic modeling and optimization of phage display. J Mol Biol 277(4):893–916PubMedCrossRefGoogle Scholar
  19. Little M, Welschof M, Braunagel M, Hermes I, Christ C, Keller A et al (1999) Generation of a large complex antibody library from multiple donors. J Immunol Methods 231(1–2):3–9PubMedCrossRefGoogle Scholar
  20. McCafferty J, Griffiths AD, Winter G, Chiswell DJ (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348(6301):552–554PubMedCrossRefGoogle Scholar
  21. Moghaddam A, Borgen T, Stacy J, Kausmally L, Simonsen B, Marvik OJ et al (2003) Identification of scFv antibody fragments that specifically recognise the heroin metabolite 6-monoacetylmorphine but not morphine. J Immunol Methods 280(1–2):139–155PubMedCrossRefGoogle Scholar
  22. Osbourn JK, Derbyshire EJ, Vaughan TJ, Field AW, Johnson KS (1998a) Pathfinder selection: in situ isolation of novel antibodies. Immunotechnology 3(4):293–302PubMedCrossRefGoogle Scholar
  23. Osbourn JK, Earnshaw JC, Johnson KS, Parmentier M, Timmermans V, McCafferty J (1998b) Directed selection of MIP-1 alpha neutralizing CCR5 antibodies from a phage display human antibody library. Nat Biotechnol 16(8):778–781PubMedCrossRefGoogle Scholar
  24. Parsons HL, Earnshaw JC, Wilton J, Johnson KS, Schueler PA, Mahoney W et al (1996) Directing phage selections towards specific epitopes. Protein Eng 9(11):1043–1049PubMedCrossRefGoogle Scholar
  25. Robert R, Jacobin-Valat MJ, Daret D, Miraux S, Nurden AT, Franconi JM et al (2006) Identification of human scFvs targeting atherosclerotic lesions: selection by single round in vivo phage display. J Biol Chem 281(52):40135–40143PubMedCrossRefGoogle Scholar
  26. Roberts RW, Szostak JW (1997) RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc Natl Acad Sci USA 94(23):12297–12302PubMedCrossRefGoogle Scholar
  27. Schwarz M, Rottgen P, Takada Y, Le Gall F, Knackmuss S, Bassler N et al (2004) Single-chain antibodies for the conformation-specific blockade of activated platelet integrin alphaIIbbeta3 designed by subtractive selection from naive human phage libraries. FASEB J 18(14):1704–1706PubMedGoogle Scholar
  28. Schwarz M, Meade G, Stoll P, Ylanne J, Bassler N, Chen YC et al (2006) Conformation-specific blockade of the integrin GPIIb/IIIa: a novel antiplatelet strategy that selectively targets activated platelets. Circ Res 99(1):25–33PubMedCrossRefGoogle Scholar
  29. Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228(4705):1315–1317PubMedCrossRefGoogle Scholar
  30. Takagi J, Springer TA (2002) Integrin activation and structural rearrangement. Immunol Rev 186:141–163PubMedCrossRefGoogle Scholar
  31. Zahnd C, Amstutz P, Pluckthun A (2007) Ribosome display: selecting and evolving proteins in vitro that specifically bind to a target. Nat Methods 4(3):269–279PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  1. 1.Department of Plastic and Hand SurgeryUniversity of Freiburg Medical CentreFreiburgGermany

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