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Optimization and application of multiplexed Glycan-based Suspension Array (Extension)

Research Project
 | 
01.03.2015
 - 29.02.2016

Due to the poor outcome of ovarian cancer patients and no sensitive and specific early detection test, we have moved our research focus towards the high-throughput screening based on so-called anti-glycan antibody patterns. Research into such glycomic biomarkers requires the understanding of the complex variety of glycan-antibody interactions and their alterations in cancer. We hypothesize that carbohydrate-specific antibodies, directed against tumor-associated carbohydrate antigens (TACAs), appear/alter their activity due to individual stages of malignant transformation. The urgent need for high-throughput analysis of glycan-antibody binding has in recent years led to an increasing variety of glycan-based immunoassays.Recent advances in the field of flow-cytometry enabled a new generation of microbead-based immunoassays, allowing for quantitative simultaneous detection of multiple analytes in a single sample with high sensitivity and reproducibility. We have successfully utilized this technology as the first group world-wide to create a unique novel high-throughput glycan-based suspension array for anti-glycan-antibody profiling in human plasma in ovarian cancer patients and healthy controls [1, 33]. We could hereby demonstrate that the detection of such antibodies is population-independent and could provide a screening tool for early detection of malignancy and assessment of prognosis.The main objective of this current research proposal, which is in direct continuation to a former grant, is the further improvement of our glycan-based flow-cytometric Suspension Array for high-throughput investigation of aberrant glycosylation in ovarian cancer, including the detection of early ovarian cancer biomarkers.Specifically, in this application we are aiming to (A) further optimize the current glycan-based Suspension Array in order to maximally reduce experimental background and exclude false-positive/negative results due to antibody cross-reactivity, (B) design a clinically user friendly version which we will test towards our prior identified anti-glycan antibody candidates, and (C) prospectively design a new double-blind trial for our clinical setting. Additionally, to simplify and shorten the coupling procedure and to broaden the range of array reader techniques, i.e, bring the assay closer to application in clinics, we are aiming to design a novel alternative version of the glyco-suspension array, based on BD Sciences technical platform (BDTM Cytometric Bead Array). In contrast to our current platform, restricted by using Bio-Plex Array Readers only, BD CBA solutions does have an advantage of being available for most BD FACsTM flow cytometers. BD CBA Functional Beads can also be conjugated with antibody or protein of interest using simple protocols, based on sulfo-SMCC chemistry. In our case, glycopolymers or oligosaccharides can be modified for immobilization onto bead surface, using thiol-maleimide activation to create novel and stable multiplex panels.

Funding

Optimization and application of multiplexed Glycan-based Suspension Array (Extension)

SNF Projekt (GrantsTool), 01.2015-12.2016 (24)
PI : Heinzelmann, Viola.

Members (1)

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Viola Heinzelmann

Principal Investigator