A novel subtractive antibody phage display method to discover disease markers.
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.
Hof
Daniëlle
D
Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen, NL-6500 HB Nijmegen, The Netherlands.
Cheung
Kalok
K
Roossien
Hilde E
HE
Pruijn
Ger J M
GJ
Raats
Jos M H
JM
D000906:Antibodies / Q000235:genetics* / Q000276:immunology*; D000941:Antigens / Q000378:metabolism; D017209:Apoptosis; D001327:Autoimmune Diseases / Q000276:immunology* / Q000378:metabolism*; D015415:Biomarkers; D019921:Biotinylation; D002457:Cell Extracts; D003593:Cytoplasm / Q000378:metabolism; D000939:Epitopes / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D019151:Peptide Library; D011994:Recombinant Proteins / Q000378:metabolism; D012107:Research Design; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism
D000906:Antibodies; D000941:Antigens; D015415:Biomarkers; D002457:Cell Extracts; D000939:Epitopes; D019151:Peptide Library; D011994:Recombinant Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear