- High density antibody arrays for early detection and diagnostic marker discovery
- Our project utilizes both broad proteomic and glycomic screens and follows up with targeted analyses of specific biomarker candidates discovered in our previous studies. The broad screens will utilize unique high-density antibody microarrays (up to 6000 different analytes) to discover novel biomarkers while current candidates come from previous array work in the Lampe laboratory and studies in the Hanash laboratory that utilize orthogonal 2-D protein fractionation system (IPAS), coupled with protein tagging and immuno-depletion of abundant proteins, to quantitatively profile and mass spectrometry to identify the plasma proteins. After a two-stage triage process, the best candidates will then be evaluated with more conventional ELISA and lectin based methods. Our approaches will specifically target proteomic and glycomic changes in proteins critical for the regulation of apoptosis, proliferation, angiogenesis, inflammation/ prostaglandins, insulin resistance, toll-like receptor (TLR), transforming growth factor (TGF)-β and STAT signaling pathway deregulation during CRC to discover biomarkers of early detection and diagnosis. To do this, we will interrogate unique prediagnostic plasma samples, tissue from primary CRC tumors, and plasma from CRC cases. We have the ability to array thousands of conventional and recombinant antibodies including some that recognize cytokines, growth factors, ubiquitin related proteins, kinases, phosphatases and other regulators of cell signaling. These include ~2000 recombinant and ~2500 conventional antibodies including over 300 phospho-specific antibodies and their matched non-phospho-specific counterparts to 21 MAPK, 12 TGF-β and many STAT pathway targets. These arrays can then be incubated with case vs. control plasma or tissue for proteomic comparison or after incubation probed with specific lectins. The specific content of these arrays can be refined throughout the research plan to discover and then select those proteins that are most useful as biomarkers and that are involved in key molecular pathways. Biomarker candidates that pass the statistical threshold on a “discovery” array will be retained and be re-examined using new samples on smaller arrays (to reduce the false discovery rate) – a step we will refer to as “pre-validation” to indicate that a potential biomarker would have shown up as statistically significant in multiple sample sets but was not yet subjected to formal validation. Finally, potential biomarkers that survive these multiple triage steps will be examined via immunoblot, immunohistochemistry and, ultimately, sandwich ELISA. Our filtering procedure, where several thousand biomarkers can be rapidly evaluated and ranked on a first sample set and then re-evaluated and re-ranked on a second sample set, will rapidly triage out those due to chance leaving only the best markers to be tested via conventional low throughput methods as proposed in the final aim.
- high density antibody arrays
- The Lampe lab does protein biomarker discovery via high density antibody arrays.
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