Abstract
Antibody identification by phage display on protein or peptide targets is well established and many protocols are available. But there are many targets that cannot be expressed recombinantly or, like peptides, do not reflect correct folding of the protein. Most of these targets are cell surface receptors. Here, we describe a protocol for a panning strategy on cells to obtain specific binders to cell surface receptors. A depletion step is included to prevent enrichment of antibodies that bind to unwanted targets. Each step of the protocol is explained and variations of this protocol are given.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Köhler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495–497
Murphy AJ, Macdonald LE, Stevens S et al (2014) Mice with megabase humanization of their immunoglobulin genes generate antibodies as efficiently as normal mice. Proc Natl Acad Sci U S A 111:5153–5158. https://doi.org/10.1073/pnas.1324022111
Macdonald LE, Karow M, Stevens S et al (2014) Precise and in situ genetic humanization of 6 Mb of mouse immunoglobulin genes. Proc Natl Acad Sci U S A 111:5147–5152. https://doi.org/10.1073/pnas.1323896111
Brüggemann M, Osborn MJ, Ma B et al (2015) Human antibody production in transgenic animals. Arch Immunol Ther Exp 63:101–108. https://doi.org/10.1007/s00005-014-0322-x
Hanes J, Jermutus L, Weber-Bornhauser S et al (1998) Ribosome display efficiently selects and evolves high-affinity antibodies in vitro from immune libraries. Proc Natl Acad Sci 95:14130–14135. https://doi.org/10.1073/pnas.95.24.14130
Breitling F, Dübel S, Seehaus T et al (1991) A surface expression vector for antibody screening. Gene 104:147–153
McCafferty J, Griffiths AD, Winter G, Chiswell DJ (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348:552–554
Hoogenboom HR, Winter G (1992) By-passing immunisation. Human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro. J Mol Biol 227:381–388
Funahashi S-I, Suzuki Y, Nakano K et al (2017) Generation and characterization of monoclonal antibodies against human LGR6. J Biochem (Tokyo) 161(4):361–368. https://doi.org/10.1093/jb/mvw077
Khademi F, Mostafaie A, Parvaneh S et al (2017) Construction and characterization of monoclonal antibodies against the extracellular domain of B-lymphocyte antigen CD20 using DNA immunization method. Int Immunopharmacol 43:23–32. https://doi.org/10.1016/j.intimp.2016.11.035
Yoon H, Song JM, Ryu CJ et al (2012) An efficient strategy for cell-based antibody library selection using an integrated vector system. BMC Biotechnol 12:62. https://doi.org/10.1186/1472-6750-12-62
Cao J, Zhao P, Miao XH et al (2003) Phage display selection on whole cells yields a small peptide specific for HCV receptor human CD81. Cell Res 13:473–479. https://doi.org/10.1038/sj.cr.7290190
Shukla GS, Krag DN (2005) Phage display selection for cell-specific ligands: development of a screening procedure suitable for small tumor specimens. J Drug Target 13:7–18. https://doi.org/10.1080/10611860400020464
Eisenhardt SU, Schwarz M, Bassler N, Peter K (2007) Subtractive single-chain antibody (scFv) phage-display: tailoring phage-display for high specificity against function-specific conformations of cell membrane molecules. Nat Protoc 2:3063–3073. https://doi.org/10.1038/nprot.2007.455
Larsen SA, Meldgaard T, Fridriksdottir AJR et al (2016) Raising an antibody specific to breast cancer subpopulations using phage display on tissue sections. Cancer Genomics Proteomics 13:21–30
Jones ML, Alfaleh MA, Kumble S et al (2016) Targeting membrane proteins for antibody discovery using phage display. Sci Rep 6:26240. https://doi.org/10.1038/srep26240
Hust M, Meyer T et al (2011) A human scFv antibody generation pipeline for proteome research. J Biotechnol 152(4):159–170. https://doi.org/10.1016/j.jbiotec.2010.09.945
Kügler J, Wilke S et al (2015) Generation and analysis of the improved human HAL9/10 antibody phage display libraries. BMC Biotechnol 15:10. https://doi.org/10.1186/s12896-015-0125-0
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Fahr, W., Frenzel, A. (2018). Phage Display and Selections on Cells. In: Hust, M., Lim, T. (eds) Phage Display. Methods in Molecular Biology, vol 1701. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7447-4_17
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7447-4_17
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7446-7
Online ISBN: 978-1-4939-7447-4
eBook Packages: Springer Protocols