Evaluation of Recombinant Antibodies on Protein Microarrays Applying the Multiple Spotting Technique

  • Zoltán KonthurEmail author
  • Jeannine Wilde
Part of the Springer Protocols Handbooks book series (SPH)


The generation of recombinant antibodies by phage display in high-throughput demands fast downstream technologies and streamlined processes for the identification and initial characterisation of individual binders. Next to standard immunological methods such as enzyme-linked immunosorbent assays (ELISA) and Western-blot, protein microarrays offer a wide range of possibilities in the evaluation process of monoclonal binders. Here, we describe the application of a special protein microarray method – the multiple spotting technique (MIST) – for the simultaneous evaluation of hundreds of phage display derived soluble monoclonal antibody fragments on protein microarrays. The standard operating procedures provided include the expression of soluble antibody fragments in microtitre plates, the spotting protocols and data evaluation schemes. Additionally, we show the comparability of this protein microarray application to conventional ELISA on a recent target antigen in our semi-automated selection pipeline. Applying MIST allows to reduce time, material and waste, and extends automation beyond the selection process applying conventional microarray machinery.


Phage Display Antibody Fragment Recombinant Antibody Protein Array Microarray Slide 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This work was supported by the German Federal Ministry for Education and Research (BMBF) through the National Genome Research Network (NGFN-II) project “Antibody Factory” (Grant No. 01GR0427) and the Max Planck Society. ZK acknowledges additional support from EU-FP6 CA “Proteome Binders” (RICA 026008).


  1. Angenendt P, Glökler J, Konthur Z, Lehrach H, Cahill D (2003) 3D protein microarrays: performing multiplex immunoassays on a single chip. Anal Chem 75:4368–4372PubMedCrossRefGoogle Scholar
  2. Angenendt P, Wilde J, Kijanka G, Baars S, Cahill DJ, Kreutzberger J, Lehrach H, Konthur Z, Glökler J (2004) Seeing better through a MIST: evaluation of monoclonal recombinant antibody fragments on microarrays. Anal Chem 76:2916–2921PubMedCrossRefGoogle Scholar
  3. Ayriss J, Woods T, Bradbury A, Pavlik P (2007) High-throughput screening of single-chain antibodies using multiplexed flow cytometry. J Proteome Res 6:1072–1082PubMedCrossRefGoogle Scholar
  4. Buckler DR, Park A, Viswanathan M, Hoet RM, Ladner RC (2008) Screening isolates from antibody phage display libraries. Drug Discov Today 13:318–324PubMedCrossRefGoogle Scholar
  5. De Masi F, Chiarella P, Wilhelm H, Massimi M, Bullard B, Ansorge W, Sawyer A (2005) High throughput production of mouse monoclonal antibodies using antigen microarrays. Proteomics 5:4070–4081PubMedCrossRefGoogle Scholar
  6. de Wildt RM, Mundy CR, Gorick BD, Tomlinson IM (2000) Antibody arrays for high-throughput screening of antibody-antigen interactions. Nat Biotechnol 18:989–994PubMedCrossRefGoogle Scholar
  7. Hallborn J, Carlsson R (2002) Automated screening procedure for high-throughput generation of antibody fragments. Biotechniques Suppl, 30–37Google Scholar
  8. Hultschig C, Kreutzberger J, Seitz H, Konthur Z, Büssow K, Lehrach H (2006) Recent advances in protein microarrays. Curr Opin Chem Biol 10:4–10PubMedCrossRefGoogle Scholar
  9. Hust M, Steinwand M, Al-Halabi L, Helmsing S, Schirrmann T, Dübel S (2009) Improved microtiter plate production of single chain Fv fragments in Escherichia coli. N Biotechnol. 25:424–428Google Scholar
  10. Joos T, Bachmann J (2009) Protein microarrays: potentials and limitations. Front Biosci 14:4376–4385PubMedCrossRefGoogle Scholar
  11. Konthur Z (2007) Automation of selection and engineering. In: Dübel S (ed) Handbook of therapeutic antibodies. Wiley-VCH, Weinheim, pp 413–431Google Scholar
  12. Konthur Z, Walter G (2002) Automation of phage display for high-throughput antibody development. Targets 1:30–36CrossRefGoogle Scholar
  13. Konthur Z, Hust M, Dübel S (2005) Perspectives for systematic in vitro antibody generation. Gene 364:19–29PubMedCrossRefGoogle Scholar
  14. Krebs B, Rauchenberger R, Reiffert S, Rothe C, Tesar M, Thomassen E, Cao M, Dreier T, Fischer D, Höss A, Inge L, Knappik A, Marget M, Pack P, Meng XQ, Schier R, Söhlemann P, Winter J, Wölle J, Kretzschmar T (2001) High-throughput generation and engineering of recombinant human antibodies. J Immunol Methods 254:67–84PubMedCrossRefGoogle Scholar
  15. Schwenk JM, Lindberg J, Sundberg M, Uhlén M, Nilsson P (2007) Determination of binding specificities in highly multiplexed bead-based assays for antibody proteomics. Mol Cell Proteomics 6:125–132PubMedGoogle Scholar
  16. Taussig MJ, Stoevesandt O, Borrebaeck CA, Bradbury AR, Cahill D, Cambillau C, de Daruvar A, Dübel S, Eichler J, Frank R, Gibson TJ, Gloriam D, Gold L, Herberg FW, Hermjakob H, Hoheisel JD, Joos TO, Kallioniemi O, Koegl M, Konthur Z, Korn B, Kremmer E, Krobitsch S, Landegren U, van der Maarel S, McCafferty J, Muyldermans S, Nygren PA, Palcy S, Plückthun A, Polic B, Przybylski M, Saviranta P, Sawyer A, Sherman DJ, Skerra A, Templin M, Ueffing M, Uhlen M (2007) ProteomeBinders: planning a European resource of affinity reagents for analysis of the human proteome. Nat Methods 4:13–7PubMedCrossRefGoogle Scholar
  17. Turunen L, Takkinen K, Söderlund H, Pulli T (2009) Automated panning and screening procedure on micorplates for antibody generation from phage display libraries. J Biomol Screen 14:282–293PubMedCrossRefGoogle Scholar
  18. Walter G, Konthur Z, Lehrach H (2001) High-throughput screening of surface displayed gene products. Comb Chem High Throughput Screen 4:193–205PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2010

Authors and Affiliations

  1. 1.Department of Vertebrate GenomicsMax Planck Institute for Molecular GeneticsBerlinGermany

Personalised recommendations