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Our research employs an unbiased selection of novel tumor antigens for the purpose of diagnosing cancer presymptomatically. Our goal is to use serum reactivity to proteins expressed in tumors as diagnostic or prognostic biomarkers. Serum reactivity toward a cellular protein occurs because of the presentation of a mutated form of the protein from the tumor cells or overexpression of the protein in the tumor cells. The antibody reaction to large numbers of these antigens/epitopes is detected in a highly parallel assay on robotically spotted protein microarrays. We call this approach of global profiling of tumor antigens “epitomics.” By assaying serum antibodies from patients and controls with two-color fluorescence detection on antigen microarrays, we can identify the presence of cancer in sera from subjects with cancer without false positives due to other benign syndromes classically confounding other diagnostic markers.
www.karmanos.org/∼tainskym
Recent advances in protein microarray methods have presented the field of proteomics with extraordinary opportunities for the unbiased selection of biomarkers. Protein arrays provide a useful platform for multiplexed assays of hundreds to thousands of proteins. To address the need for a comprehensive scan of the human repertoire of cancer antigens on microarrays, we developed a technology using phage display libraries printed on microarrays of phage clones expressing selected antigens, an approach adopted by other laboratories as well. Thus far, the amount of protein spotted on these arrays was limited by the titer of the bacteriophage. We devised an approach to rapidly prepare purified proteins from T7 phage clones using two PCR steps and a short incubation for in vitro transcription/translation followed by purification on a minicolumn for affinity chromatography. These purified proteins are suitable for high-throughput immunoscreening on protein microarrays.
Direct production and purification of T7 phage display cloned proteins selected and analyzed on microarrays, p. 220.
