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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
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):121–127
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):7978–7982
Boder ET, Wittrup KD (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol 15(6):553–557
Clackson T, Hoogenboom HR, Griffiths AD, Winter G (1991) Making antibody fragments using phage display libraries. Nature 352(6336):624–628
de Wildt RM, Mundy CR, Gorick BD, Tomlinson IM (2000) Antibody arrays for high-throughput screening of antibody-antigen interactions. Nat Biotechnol 18(9):989–994
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):7–13
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):3063–3073
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):3521–3528
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):1369–1372
Giordano RJ, Cardo-Vila M, Lahdenranta J, Pasqualini R, Arap W (2001) Biopanning and rapid analysis of selective interactive ligands. Nat Med 7(11):1249–1253
Griffiths AD (1993) Production of human antibodies using bacteriophage. Curr Opin Immunol 5(2):263–267
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):142–145
Hoogenboom HR, de Bruine AP, Hufton SE, Hoet RM, Arends JW, Roovers RC (1998) Antibody phage display technology and its applications. Immunotechnology 4(1):1–20
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):225–233
Hynes RO (1992) Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69(1):11–25
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):69–77
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):164–168
Levitan B (1998) Stochastic modeling and optimization of phage display. J Mol Biol 277(4):893–916
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):3–9
McCafferty J, Griffiths AD, Winter G, Chiswell DJ (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348(6301):552–554
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):139–155
Osbourn JK, Derbyshire EJ, Vaughan TJ, Field AW, Johnson KS (1998a) Pathfinder selection: in situ isolation of novel antibodies. Immunotechnology 3(4):293–302
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):778–781
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):1043–1049
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):40135–40143
Roberts RW, Szostak JW (1997) RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc Natl Acad Sci USA 94(23):12297–12302
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):1704–1706
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):25–33
Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228(4705):1315–1317
Takagi J, Springer TA (2002) Integrin activation and structural rearrangement. Immunol Rev 186:141–163
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):269–279
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer-Verlag
About this protocol
Cite this protocol
Eisenhardt, S.U., Peter, K. (2010). Phage Display and Subtractive Selection on Cells. In: Kontermann, R., Dübel, S. (eds) Antibody Engineering. Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-01144-3_12
Download citation
DOI: https://doi.org/10.1007/978-3-642-01144-3_12
Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-642-01143-6
Online ISBN: 978-3-642-01144-3
eBook Packages: Springer Protocols