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Molecular & Cellular Proteomics 5:245-255, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

A Novel Subtractive Antibody Phage Display Method to Discover Disease Markers^*

Daniëlle Hof, Kalok Cheung, Hilde E. Roossien, Ger J. M. Pruijn and Jos M. H. Raats^,,¶

From the Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen, NL-6500 HB Nijmegen, The Netherlands and ModiQuest B. V., NL-6525 GA Nijmegen, The Netherlands

Today’s research demands fast identification of potential diagnostic and therapeutic targets. We describe a novel phage display strategy to identify disease-related proteins that are specifically expressed in a certain (diseased) tissue or cells. Phages displaying antibody fragments are selected on complex protein mixtures in a two-step manner combining subtractive selection in solution with further enrichment of specific phages on two-dimensional Western blots. Targets recognized by the resulting recombinant antibodies are immunoaffinity-purified and identified by mass spectrometry. We used antibody fragment libraries from autoimmune patients to discover apoptosis-specific and disease-related targets. One of the three identified targets is the U1-70K protein, a marker for systemic lupus erythematosus overlap disease. Interestingly the epitope on U1-70K recognized by the selected recombinant antibody was shown to be apoptosis-dependent, and such epitopes are believed to be involved in breaking tolerance to self-antigens. The other two proteins were identified as polypyrimidine tract-binding protein-associated splicing factor (PSF)/nuclear RNA- and DNA-binding protein of 54 kDa (p54^nrb) and heterogeneous ribonucleoprotein C.

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