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2009 Scientific Report

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Van Andel Research

Van Andel Research Institute | Scientific Report Brian B. Haab, Ph.D. Laboratory of Cancer Immunodiagnostics Dr. Haab obtained his Ph.D. in chemistry from the University of California at Berkeley in 1998. He then served as a postdoctoral fellow in the laboratory of Patrick Brown in the Department of Biochemistry at Stanford University. Dr. Haab joined VARI as a Special Program Investigator in May 2000, became a Scientific Investigator in 2004, and was promoted to Senior Scientific Investigator in 2007. Staff Students Visiting Scientist John Buchweitz, Ph.D. Yi-Mi Wu, Ph.D. Derek Bergsma, B.S. Steven Kluck, B.S. Andrew Porter, B.S. Amy Nelson Rob Antecki, B.S. Kim Babins, B.S. Carrie Fiebig, B.S. Lee Heeringa, B.S. Kevin Maupin, B.A. Arkadeep Sinha, B.S. Dan Hekman Christopher Madziar Randi VanOcker Tingting Yue, B.S. David Nowack, Ph.D. 26

VARI | 2009 Research Interests All cells secrete molecules that are used to send signals and perform functions in the local and distant spaces of the body. The molecular secretions of cancer cells often are significantly different from those of their normal counterparts. Our lab studies particular proteins and carbohydrates secreted by cancer cells in order to understand their roles in cancer progression, as well as to develop novel clinical tests for the detection and diagnosis of cancer. Glycoprotein biomarkers for pancreatic cancer A great need exists for better tools to detect and diagnose incipient pancreatic cancer. Our laboratory is addressing this problem by taking advantage of a frequently observed molecular feature of pancreatic cancer, i.e., alterations to the carbohydrate side chains of cell-surface and secreted proteins. Most secreted proteins have carbohydrates known as glycans attached to them, and some of the secreted proteins with altered carbohydrates are released into the blood of cancer patients. The measurement of certain secreted glycoproteins, along with their attached glycans, could form the basis of effective diagnostic markers. A particularly valuable platform for probing glycan variants on specific proteins is the antibody-lectin sandwich array (ALSA), developed earlier in our laboratory. The method starts with a microarray of antibodies that target various glycoproteins of interest. A complex biological sample is incubated on the array, resulting in the capture of glycoproteins by the antibodies. Then the array is probed with a lectin (a protein with carbohydrate-binding activity), which binds to the captured glycoproteins that bear the lectin’s glycan target. The amount of lectin binding at each antibody indicates the amount of glycan on the proteins captured by that antibody. Diverse lectins can be used to probe a variety of glycans on a given sample. In addition, the captured proteins can be probed with antibodies targeting the core proteins, as in a “sandwich” immunoassay, to obtain the levels of the proteins in parallel assays. Relative to other technologies, the platform offers a unique combination of capabilities such as reproducible glycan measurements on specific proteins, high-throughput sample processing, and high-sensitivity detection directly from biological samples. These features make the platform ideal for glycoprotein-based biomarker studies. A product based on this technology is now available from GenTel Biosciences (Madison, Wisconsin). Using this tool, we can now explore the hypotheses that particular glycan structures on specific proteins are found uniquely in certain disease states and that their measurement yields effective detection of cancer. We have characterized the prevalence in pancreatic cancer patients of a variety of glycan structures on several types of mucin proteins. Some glycan alterations were found in a high percentage of the cancer patients but not at all in healthy Figure 1 subjects. Furthermore, the glycan levels were altered independently of changes to the protein level, so that measuring both the glycan and protein level gives improved biomarker performance relative to measuring only protein levels as in standard immunoassays. The performance of these initial studies already suggests improvement upon the best current biomarkers for pancreatic cancer. Now we are working to characterize and develop detection methods for both the protein forms that carry cancer-associated glycans and the glycans themselves. Figure 1. Protein and glycan detection using antibody arrays. a) Array-based sandwich assays for protein detection. Multiple antibodies are immobilized on a planar support, and the captured proteins are probed using biotinylated detection antibodies, followed by fluorescence detection using phycoerythrin-labeled streptavidin. b) Antibody-lectin sandwich arrays (ALSA). This format is similar to a), but the detection reagents target the glycans on the capture proteins rather than the core proteins. The glycans on the immobilized antibodies are chemically derivatized to prevent lectin binding to those glycans. c) Example antibody array results for core protein detection (left) and glycan measurement (right). SA-PE, streptavidin-phycoerythrin. 27

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