Skip to main content
SpringerLink
Log in

Isolation of Recombinant Antibodies That Recognize Native and Accessible Membrane Biomarkers

  • Conference paper
Nanotechnology to Aid Chemical and Biological Defense

Abstract

Monoclonal antibodies are indispensable reagents for diagnostics, but their isolation and production by means of conventional immunization and hybridoma technology is tedious, time-intensive, and expensive. Panning large pre-immune phage display libraries is a reliable and fast alternative; in addition, this approach presents the advantage that can be applied to toxic and non-immunogenic antigens. Furthermore, panning can be performed directly on whole cells to identify antibodies that will recognize their membrane-bound antigens in their native conformation and lipid environment. This opportunity is particularly meaningful when it is necessary to isolate antibodies that are able to bind accessible epitopes in vivo, as it is the case of biomarkers exposed at the cell membrane surface of pathogenic micro-organisms. Nevertheless, the effectiveness of biopanning can be undermined by different biases. In this presentation, the most common shortcomings of antibody phage display will be discussed, taking into account the most recently published reports as well as the experiments performed in our laboratory. Furthermore, some strategies useful to overcome these drawbacks will be illustrated and integrated with practical advice.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Köhler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495–497

    Article  Google Scholar 

  2. Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228:1315–1317

    Article  Google Scholar 

  3. McCafferty J, Griffiths AD, Winter G, Chiswell DJ (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348:552–554

    Article  Google Scholar 

  4. Ponsel D, Neugebauer J, Ladetzki-Bachs K, Tissot K (2011) High affinity, developability and functional size: the Holy Grail of combinatorial antibody library generation. Molecules 16:3675–3700

    Article  Google Scholar 

  5. Arbabi- Ghahroudi M, MacKenzie R, Tanha J (2009) Selection of non-aggregating VH binders from synthetic VH-phage display libraries. Methods Mol Biol 525:187–216

    Article  Google Scholar 

  6. Chen W, Zhu Z, Xiao X, Dimitrov DS (2009) Construction of a human antibody domain (VH) library. Methods Mol Biol 525:81–99

    Article  Google Scholar 

  7. Koide A, Tereshko V, Uysal S et al (2007) Exploring the capacity of minimalist protein interfaces: interface energetics and affinity maturation to picomolar KD of a single-domain antibody with flat paratope. J Mol Biol 373:941–953

    Article  Google Scholar 

  8. Liu JL, Anderson GP, Goldman ER (2007) Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library. BMC Biotechnol 7:78

    Article  Google Scholar 

  9. Mandrup OA, Friis NA, Lykkemark S, Just J, Kristensen P (2013) A novel heavy domain antibody library with functionally optimized complementarity determining regions. PLoS ONE 8:e76834

    Article  Google Scholar 

  10. Monegal A, Ami D, Martinelli C et al (2009) Immunological applications of single domain llama recombinant antibodies isolated from a naïve library. Protein Eng Des Sel 22:273–280

    Article  Google Scholar 

  11. Olichon A, de Marco A (2012) Preparation of a naïve library of Camelidae single domain antibodies: advantages and limitations of single-pot strategies. Methods Mol Biol 911:65–78

    Article  Google Scholar 

  12. Perelson AS, Oster GF (1979) Theoretical studies of clonal selection: minimal antibody repertoire size and reliability of self-non-self discrimination. J Theor Biol 81:645–670

    Article  Google Scholar 

  13. Griffiths AD, Williams SC, Hartley O et al (1994) Isolation of high-affinity human antibodies directly from large synthetic repertoires. EMBO J 13:3245–3260

    Google Scholar 

  14. Ewert S, Huber T, Honegger A, Plückthun A (2003) Biophysical properties of human antibody variable domains. J Mol Biol 325:531–553

    Article  Google Scholar 

  15. Christ D, Famm K, Winter G (2007) Repertoires of aggregation-resistant human antibody domains. Protein Eng Des Sel 20:413–416

    Article  Google Scholar 

  16. Lee CC, Perchiacca JM, Tessier PM (2013) Towards aggregation-resistant antibody by design. Trends Biotechnol 31:612–620

    Article  Google Scholar 

  17. Ma X, Barthelemy PA, Rouge L, Wiesmann C, Sidhu SS (2013) Design of synthetic autonomous VH domain libraries and structural analysis of a VH domain bound to vascular endothelial growth factor. J Mol Biol 425:2247–2259

    Article  Google Scholar 

  18. Saerens D, Pellis M, Loris R et al (2005) Identification of a universal VHH framework to graft non-canonical antigen-binding loops of camel single-domain antibodies. J Mol Biol 352:597–607

    Article  Google Scholar 

  19. Zabetakis D, Anderson GP, Bayya N, Goldman ER (2013) Contribution of the complementary determining regions to the thermal stability of a single-domain antibody. PLoS ONE 8:e77678

    Article  Google Scholar 

  20. Djender S, Schneider A, Beugnet A et al (2014) Bacterial cytoplasm as an effective cell compartment for producing functional VHH-based affinity reagents and Camelidae IgG-like recombinant antibodies. Microb Cell Fact 13:140

    Article  Google Scholar 

  21. Chan CEZ, Chan AHY, Lim APC, Hanson BJ (2011) Comparison of the efficiency of antibody selection from semi-synthetic scFv and non-immune Fab phage display libraries against protein targets for rapid development of diagnostic immunoassays. J Immunol Methods 373:79–88

    Article  Google Scholar 

  22. Tiede C, Tang AA, Deacon SE et al (2014) Adhiron: a stable and versatile peptide display scaffold for molecular recognition applications. Protein Eng Des Sel 27:145–155

    Article  Google Scholar 

  23. Even-Desrumeaux K, Nevoltris D, Lavaut MN et al (2014) Masked selection: a straight-forward and flexible approach for the selection of binders for specific epitopes and differentially expressed proteins by phage display. Mol Cell Proteomics 13:653–665

    Article  Google Scholar 

  24. Hoogenboom HR, Lutgerink JT, Pelsers MM et al (1999) Selection-dominant and nonaccessible epitopes on cell surface receptors revealed by cell-panning with a large phage antibody library. Eur J Biochem 260:774–784

    Article  Google Scholar 

  25. 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

    Article  Google Scholar 

  26. Belizaire AK, Tchistiakova L, St-Pierre Y, Alakhov V (2003) Identification of a murine ICAM-1-specific peptide by subtractive phage library selection on cells. Biochem Biophys Res Commun 309:625–630

    Article  Google Scholar 

  27. Geuijen CA, Bijl N, Smit RC et al (2005) A proteomic approach to tumour target identification using phage display, affinity purification and mass spectrometry. Eur J Cancer 41:178–187

    Article  Google Scholar 

  28. Kurosawa G, Akahori Y, Morita M et al (2008) Comprehensive screening for antigens overexpressed on carcinomas via isolation of human mAbs that may be therapeutic. Proc Natl Acad Sci U S A 105:7287–7292

    Article  Google Scholar 

  29. Poul MA, Becerril B, Nielsen UB, Morisson P, Marks JD (2000) Selection of tumor specific internalizing human antibodies from phage libraries. J Mol Biol 301:1149–1161

    Article  Google Scholar 

  30. Ridgway JBB, Ng E, Kern JA et al (1999) Identification of a human anti-CD55 single-chain Fv by subtractive panning of a phage library using tumor and non-tumor cell lines. Cancer Res 59:2718–2723

    Google Scholar 

  31. Kurosawa G, Sumitomo M, Ukai Y et al (2011) Selection and analysis of anti-cancer antibodies for cancer therapy obtained from antibody phage library. Cancer Sci 102:175–181

    Article  Google Scholar 

  32. Rockberg J, Löfblom J, Hjelm B, Uhlén M, Ståhl S (2008) Epitope mapping of antibodies using bacterial surface display. Nat Methods 5:1039–1045

    Article  Google Scholar 

  33. Hudson P, Hudson EP, Uhlen M, Rockberg J (2012) Multiplex epitope mapping using bacterial surface display reveals both linear and conformational epitopes. Sci Rep 2:706

    Article  Google Scholar 

  34. De Genst E, Silence K, Decanniere K et al (2006) Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies. Proc Natl Acad Sci U S A 103:4586–4591

    Article  Google Scholar 

  35. Hagay Y, Lahav J, Levanon A, Panet A (2003) Function-modulating human monoclonal antibodies against platelet-membrane receptors isolated from a phage-display library. J Thromb Haemost 1:1829–1836

    Article  Google Scholar 

  36. Abe Y, Yoshikawa T, Inoue M et al (2011) Fine tuning of receptor-selectivity for tumor necrosis factor-α using a phage display system with one-step competitive panning. Biomaterials 32:5498–5504

    Article  Google Scholar 

  37. Fransson J, Borrebaeck CA (2009) Selection and characterization of antibodies from phage display libraries against internalizing membrane antigens. Methods Mol Biol 480:113–127

    Article  Google Scholar 

  38. Jespers L, Schon O, Famm K, Winter G (2004) Aggregation-resistant domain antibodies selected on phage by heat denaturation. Nat Biotechnol 22:1161–1165

    Article  Google Scholar 

  39. Dolk E, van der Vaart M, Hulsik DL et al (2005) Isolation of llama antibody fragments for prevention of dandruff by phage display in shampoo. Appl Environ Microbiol 71:442–450

    Article  Google Scholar 

  40. Harmsen MM, van Solt CB, van Zijederveld-van Bemmel AM, Niewold TA, van Zijederveld FG (2006) Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy. Appl Microbiol Biotechnol 72:544–551

    Article  Google Scholar 

  41. Veggiani G, Ossolengo G, Aliprandi M, Cavallaro U, de Marco A (2011) Single-domain antibodies that compete with the natural ligand fibroblast growth factor block the internalization of the fibroblast growth factor receptor1. Biochem Biophys Res Commun 408:692–696

    Article  Google Scholar 

  42. de Marco A (2013) Methodologies for the isolation of alternative binders with improved clinical potentiality over conventional antibodies. Crit Rev Biotech 33:40–48

    Article  Google Scholar 

  43. Forsman A, Beirnaert E, Aasa-Chapman MMI et al (2008) Llama antibody fragments with cross-subtype human immuodeficency virus type 1 (HIV-1)-neutralizing properties and high affinity for HIV-1 gp120. J Virol 82:12069–12081

    Article  Google Scholar 

  44. Gruszka A, Martinelli C, Sparacio E, Pelicci PG, de Marco A (2012) The concurrent use of N- and C-terminal antibodies anti-nucleophosmin 1 in immunofluorescence experiments allows for precise assessment of its subcellular localisation in acute myeloid leukaemia patients. Leukemia 26:159–163

    Article  Google Scholar 

  45. Oliveira S, Schiffelers RM, van der Veeken J et al (2010) Downregulation of EGFR by a novel multivalent nanobody-liposome platform. J Control Release 145:165–175

    Article  Google Scholar 

  46. Roovers RC, Vosjan MJ, Laeremans T et al (2011) A bi-paratopic anti-EGFR nanobody efficiently inhibits solid tumor growth. Int J Cancer 129:2013–2024. doi:10.1002/ijc.26145

    Article  Google Scholar 

  47. Villa A, Lovato V, Bujak E et al (2011) A novel synthetic naïve human antibody library allows the isolation of antibodies against a new epitope of oncofetal fibronectin. MAbs 3:264–272

    Article  Google Scholar 

  48. Chasteen L, Ayriss J, Pavlik P, Bradbury ARM (2006) Eliminating helper phage from phage display. Nucleic Acids Res 34:e145

    Article  Google Scholar 

  49. Peters EA, Schatz PJ, Johnson SS, Dower WJ (1994) Membrane insertion defects caused by positive charges in the early mature region of protein pIII of filamentous phage fd can be corrected by prlA suppressor. J Bacteriol 176:4296–4305

    Google Scholar 

  50. Rodi DJ, Soares AS, Makowski L (2002) Quantitative assessment of peptide sequence diversity in M13 combinatorial peptide phage display libraries. J Mol Biol 322:1039–1052

    Article  Google Scholar 

  51. Brammer LA, Bolduc B, Kass JL et al (2008) A target-unrelated peptide in an M13 phage display library traced to an advantageous mutation in the gene II ribosome-binding site. Anal Biochem 373:88–98

    Article  Google Scholar 

  52. Matochko WL, Cory Li S, Tang SK, Derda R (2014) Prospective identification of parasitic sequences in phage display screens. Nucleic Acids Res 42:1784–1798

    Article  Google Scholar 

  53. Thomas WD, Golomb M, Smith GP (2010) Corruption of phage display libraries by target-unrelated clones: diagnosis and countermeasures. Anal Biochem 407:237–240

    Article  Google Scholar 

  54. Derda R, Tang SK, Li SC et al (2011) Diversity of phage-displayed libraries of peptides during panning and amplification. Molecules 16:1776–1803

    Article  Google Scholar 

  55. Matochko WL, Chu K, Jin B et al (2012) Deep sequencing analysis of phage libraries using Illumina platform. Methods 58:47–55

    Article  Google Scholar 

  56. ‘t Hoen PAC, Jirka SM, Ten Broeke BR (2012) Phage display screening without repetitious selection rounds. Anal Biochem 421:622–631

    Article  Google Scholar 

  57. Yu X, Barmina O, Burgoon M, Gilden D (2009) Identification of measles virus epitopes using an ultra-fast method of panning phage-displayed random peptide libraries. J Virol Methods 156:169–173

    Article  Google Scholar 

  58. Ravn U, Gueneau F, Baerlocher L et al (2010) By-passing in vitro screening – next generation sequencing technologies applied to antibody display and in silico candidate selection. Nucleic Acids Res 38, e193

    Article  Google Scholar 

  59. Ravn U, Didelot G, Venet S et al (2013) Deep sequencing of phage display libraries to support antibody discovery. Methods 60:99–110

    Article  Google Scholar 

  60. Zhang H, Torkamani A, Jones TM et al (2011) Phenotype-information-phenotype cycle for deconvolution of combinatorial antibody libraries selected against complex systems. Proc Natl Acad Sci U S A 108:13456–13461

    Article  Google Scholar 

  61. Kastelic D, Frković-Grazio S, Baty D et al (2009) A single-step procedure of recombinant library construction for the selection of efficiently produced llama VH binders directed against cancer markers. J Immunol Methods 350:54–62

    Article  Google Scholar 

  62. Zeng L, Skinner SO, Zong C, Sippy J, Feiss M, Golding I (2010) Decision making at a subcellular level determines the outcome of bacteriophage infection. Cell 141:682–691

    Article  Google Scholar 

  63. Ryvkin A, Ashkenazy H, Smelyanski L et al (2012) Deep panning: steps towards probing the IgOme. PLoS ONE 7:e41469

    Article  Google Scholar 

  64. Monegal A, Olichon A, Bery N et al (2012) Single heavy chain antibodies with VH hallmarks are positively selected during panning of llama (Lama glama) naïve libraries. Develop Comp Immunol 36:150–156

    Article  Google Scholar 

  65. Steiner D, Forrer P, Stumpp MT, Plückthun A (2006) Signal sequences directing cotranslational translocation expand the range of proteins amenable for phage display. Nat Biotechnol 24:823–831

    Article  Google Scholar 

  66. Boder ET, Wittrup KD (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol 15:553–557

    Article  Google Scholar 

  67. Ferrara F, Naranjo LA, Kumar S et al (2012) Using phage and yeast display to select hundreds of monoclonal antibodies: application to antigen 85, a tuberculosis biomarker. PLoS ONE 7:e49535

    Article  Google Scholar 

  68. Ferrara F, D’Angelo S, Gaiotto T et al (2015) Recombinant renewable polyclonal antibodies. MAbs 7:32–41

    Article  Google Scholar 

  69. Gai SA, Wittrup KD (2007) Yeast surface display for protein engineering and characterization. Curr Opin Struct Biol 17:467–473

    Article  Google Scholar 

  70. Beer M, Liu CQ (2012) Panning of a phage display library against a synthetic capsule for peptide ligands that bind to the native capsule of Bacillus anthracis. PLoS ONE 7:e45472

    Article  Google Scholar 

  71. Paoli GC, Chen CY, Brewster JD (2004) Single-chain Fv antibody with specificity for Listeria monocytogenes. J Immunol Methods 289:147–155

    Article  Google Scholar 

Download references

Acknowledgments

The author wishes to thank Aurelie Schneider for her assistance, Creative Core AHA-MOMENT grant from Slovene Ministry of Economic Development and Technology as well as the European Fund for Regional Development – Cross-Border Cooperation Programme Italy-Slovenia 2007–2013, (Project PROTEO, Code N. CB166) for having supported this work with research funds.

Author information

Authors and Affiliations

  1. Department of Biomedical Sciences and Engineering, University of Nova Gorica, Glavni Trg 9, SI-5261, Vipava, Slovenia

    Ario de Marco

Authors
  1. Ario de Marco
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ario de Marco .

Editor information

Editors and Affiliations

  1. Worcester Polytechnic Institute, Worcester, Massachusetts, USA

    Terri A. Camesano

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media Dordrecht

About this paper

Cite this paper

de Marco, A. (2015). Isolation of Recombinant Antibodies That Recognize Native and Accessible Membrane Biomarkers. In: Camesano, T. (eds) Nanotechnology to Aid Chemical and Biological Defense. NATO Science for Peace and Security Series A: Chemistry and Biology. Springer, Dordrecht. https://doi.org/10.1007/978-94-017-7218-1_4

Download citation

Publish with us

Policies and ethics

Search

Navigation