Abstract
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 (p54nrb) and heterogeneous ribonucleoprotein C.
Highlights
Today’s research demands fast identification of potential diagnostic and therapeutic targets
Recombinant autoantibodies from patient libraries were selected on apoptotic cell extract, after subtraction on non-apoptotic cell extract, to identify disease-related targets that are present in the apoptotic material
Two of the three targets are well documented autoantigens with the U1-70K protein being an important marker for systemic lupus erythematosus (SLE) overlap disease. heterogeneous nuclear ribonucleoprotein C (hnRNP C) is autoantigenic as well, autoantibodies to this protein have been reported in a few patients only [40, 41]
Summary
One approach to discover “proteome-specific” proteins is to compare the proteome of a diseased cell or tissue with the proteome of the same cell or tissue in a normal (non-diseased) state This is commonly done by two-dimensional gel electrophoresis and mass spectrometry. A major advantage of subtractive phage display technology is the simultaneous generation of recombinant monoclonal antibodies recognizing potential disease markers. Such antigen-antibody pairs could be directly applicable in therapeutics or diagnostics. We present a novel subtractive antibody phage display method that enables the identification of proteome-specific, intracellular epitopes. After identification of proteome-specific phages, their targets are immunoaffinitypurified using these recombinant antibodies and identified by means of mass spectrometry.
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