Protein microarrays for validation of affinity binders MÅRTEN SUNDBERG KTH - Royal Institute of Technology School of Biotechnology Stockholm © Mårten Sundberg Stockholm, December 2011 KTH - Royal Institute of Technology School of Biotechnology Science for Life Laboratory SE-171 21 Solna Sweden Printed By: AJ E-print AB Oxtorgsgatan 9 111 57 Stockholm Sweden ISBN 978-91-7501-149-3 TRITA-BIO Report 2011:23 ISSN 1654-2312 Mårten Sundberg (2011): Protein microarrays for validation of affinity binders, School of Biotechnology, Royal Institute of Technology (KTH), Stockholm, Sweden. ISBN 978-91-7501-149-3 Abstract Is specificity an important issue regarding affinity reagents? What about the validation of affinity reagents today, is it good enough? This depends on the application and the producer of the reagent. Validation should be the most important marketing argument that can be found. Today there is a continuous growth of both the number of affinity reagents that are produced and the different types of affinity reagents that are developed. In proteomics they become more and more important in exploring the human proteome. Therefore, validated affinity reagents should be on top of every proteomic researcher’s list. How should this be accomplished? Better international agreements on how affinity reagents should be tested to be regarded as functional reagents are needed. One of the most important issues is the specificity of the affinity reagent. An international standard for which specific validation that is needed for different kinds of applications would be very useful. In this thesis, it is shown that the protein microarray platform that was established within the HPA project at KTH is a very good tool to determine the specificity of different affinity binders. In the first study, the production of mono-specific antibodies for tissue profiling in the Human Protein Atlas (HPA) project is presented. The section describing the use of protein microarrays for validation of the antibodies is relevant for this thesis. The implementation of protein microarrays in the HPA workflow was an important addition, because a deeper insight of the specificity of all the antibodies produced were now available. In a second study, bead based arrays were compared to planar protein microarrays used in the HPA project. In this study, 100 different bead identities were coupled with 100 different antigens and mixed together to generate an array. The correlation between the two types of assays was very high and the conclusion was that the methods can be used as backup to each other. A third study was a part of an international initiative to produce renewable affinity binders against proteins containing SH2 domain. Here, the HPA protein microarrays were modified to analyze different types of reagents produced at six laboratories around the world. Monoclonal antibodies, single chain fragment and fibronectin scaffolds were tested as well as mono-specific antibodies. It was shown to be possible to adapt protein microarrays used in the HPA project to validate other kinds of affinity reagents. Keywords: Microarray, protein, antibody, antigen, affinity, validation © Mårten Sundberg List of publications This thesis is based upon the following three papers. The papers are included in the Appendix. I. Nilsson, P., Paavilainen, L., Larsson, K., Odling, J., Sundberg, M., Andersson, A. C., Kampf, C., Persson, A., Al-Khalili Szigyarto, C., Ottosson, J., Bjorling, E., Hober, S., Wernerus, H., Wester, K., Ponten, F., and Uhlen, M. (2005) Towards a human proteome atlas: high-throughput generation of mono-specific antibodies for tissue profiling, Proteomics 5, 4327-4337. II. Schwenk, J. M., Lindberg, J., Sundberg, M., Uhlen, M., and Nilsson, P. (2007) Determination of binding specificities in highly multiplexed bead-based assays for antibody proteomics, Mol Cell Proteomics 6, 125-13. III. Sjoberg, R*., Sundberg, M*., Gundberg, A., Sivertsson, A., Schwenk, J, M., Uhlen, M., Nilsson, P (2011) Validation of various affinity reagents using protein microarrays. Manuscript submitted to New Biotechnology. All papers are reproduced with permission from the copyright holders *These authors contributed equally to this work. Related publications Colwill, K., Persson, H., Jarvik, N. E., Wyrzucki, A., Wojcik, J., Koide, A., Kossiakoff, A. A., Koide, S., Sidhu, S., Dyson, M. R., Pershad, K., Pavlovic, J. D., Karatt-Vellatt, A., Schofield, D. J., Kay, B. K., McCafferty, J., Mersmann, M., Meier, D., Mersmann, J., Helmsing, S., Hust, M., Dubel, S., Berkowicz, S., Freemantle, A., Spiegel, M., Sawyer, A., Layton, D., Nice, E., Dai, A., Rocks, O., Williton, K., Fellouse, F. A., Hersi, K., Pawson, T., Nilsson, P., Sundberg, M., Sjoberg, R., Sivertsson, A., Schwenk, J. M., Takanen, J. O., Hober, S., Uhlen, M., Dahlgren, L. G., Flores, A., Johansson, I., Weigelt, J., Crombet, L., Loppnau, P., Kozieradzki, I., Cossar, D., Arrowsmith, C. H., Edwards, A. M., and Graslund, S. (2011) A roadmap to generate renewable protein binders to the human proteome, Nat Methods 8, 551- 558. Rimini, R., Schwenk, J. M., Sundberg, M., Sjoberg, R., Klevebring, D., Gry, M., Uhlen, M., and Nilsson, P. (2009) Validation of serum protein profiles by a dual antibody array approach, J Proteomics 73, 252-266. Lundberg, E., Sundberg, M., Graslund, T., Uhlen, M., and Svahn, H. A. (2007) A novel method for reproducible fluorescent labeling of small amounts of antibodies on solid phase, J Immunol Methods 322, 40-49. Eriksson, C., Agaton, C., Kange, R., Sundberg, M., Nilsson, P., Ek, B., Uhlen, M., Gustafsson, M., and Hober, S. (2006) Microfluidic analysis of antibody specificity in a compact disk format, J Proteome Res 5, 1568-1574. Uhlen, M., Bjorling, E., Agaton, C., Szigyarto, C. A., Amini, B., Andersen, E., Andersson, A. C., Angelidou, P., Asplund, A., Asplund, C., Berglund, L., Bergstrom, K., Brumer, H., Cerjan, D., Ekstrom, M., Elobeid, A., Eriksson, C., Fagerberg, L., Falk, R., Fall, J., Forsberg, M., Bjorklund, M. G., Gumbel, K., Halimi, A., Hallin, I., Hamsten, C., Hansson, M., Hedhammar, M., Hercules, G., Kampf, C., Larsson, K., Lindskog, M., Lodewyckx, W., Lund, J., Lundeberg, J., Magnusson, K., Malm, E., Nilsson, P., Odling, J., Oksvold, P., Olsson, I., Oster, E., Ottosson, J., Paavilainen, L., Persson, A., Rimini, R., Rockberg, J., Runeson, M., Sivertsson, A., Skollermo, A., Steen, J., Stenvall, M., Sterky, F., Stromberg, S., Sundberg, M., Tegel, H., Tourle, S., Wahlund, E., Walden, A., Wan, J., Wernerus, H., Westberg, J., Wester, K., Wrethagen, U., Xu, L. L., Hober, S., and Ponten, F. (2005) A human protein atlas for normal and cancer tissues based on antibody proteomics, Mol Cell Proteomics 4, 1920-1932. Janzi, M., Odling, J., Pan-Hammarstrom, Q., Sundberg, M., Lundeberg, J., Uhlen, M., Hammarstrom, L., and Nilsson, P. (2005) Serum microarrays for large scale screening of protein levels, Mol Cell Proteomics 4, 1942-1947. Contents Introduction ................................................................................................................................. 1 Human Protein Atlas project ................................................................................................. 2 Affinity reagents used in protein microarrays ................................................................ 3 Antigens ..................................................................................................................................... 3 Antibodies ................................................................................................................................. 4 Polyclonal antibodies ........................................................................................................ 4 Monoclonal antibodies ...................................................................................................... 5 Antibody fragments .............................................................................................................. 6 Other kinds of affinity reagents ....................................................................................... 6 Protein microarrays - production and application ......................................................... 7 Different approaches of protein microarrays .............................................................. 7 Antigen microarray ............................................................................................................ 7 Antibody microarray ......................................................................................................... 8 Peptide microarray ............................................................................................................ 8 Reverse-phase microarrays ........................................................................................... 8 Production of protein microarrays ................................................................................... 9 Printing robots .................................................................................................................... 9 Surfaces and immobilization ....................................................................................... 10 Blocking of substrate ...................................................................................................... 11 Sample labeling and detection systems .................................................................. 12 Incubation, scanning and data evaluation ............................................................. 12