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Workpackage 2: Assay development: exosome

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Objectives
  • To optimize exosome isolation techniques, improve normalization methods and sample preparation to facilitate the detection of exosome-based biomarkers
  • To develop in collaboration with sub-contractor Future Diagnostics B.V. a multiplex assay (Luminex) for the validation of 4 proprietary novel exosomal biomarkers to identify patients at risk of developing a secondary cardiovascular event
  • To develop in collaboration with partner Fleet Bioprocessing Ltd an ELISA assay for the validation of an exosomal protein marker specifically identifying females at risk of developing a primary cardiovascular event

Description of Work

Optimization exosome isolation
Cavadis previously developed an in-house buffer for the extraction of exosomes from plasma and serum. This buffer was tested using various incubation times. It appeared that an incubation time of 30 minutes provides an excellent yield. Currently used commercial precipitation buffers require overnight incubation. An accelerated extraction time facilitates sample preparation and decreases processing time making this buffer perfectly suited for the large scale testing of exosomal biomarkers.

4-plex Luminex assay
Initial verfication studies using 4 circulating exosomal proteins demonstrated strong predictive value for secondary cardiovascular events including an impressive 15.5% NRI. In conjunction with clinical evaluation, this assay may help clinicians to classify patients in low, medium or high risk categories and to decide on a type of treatment tailored to the patient's need.Cavadis together with subcontractor Future Diagnostics successfully developed a 4-plex assay on a Luminex platform. Large scale validation with the 4-ple in approx. 8,000 samples of the BiomarCaRE diseased-cohorts is ongoing at the UKE laboratories.

ELISA assay (female marker)
The 4-plex assay was also tested in a longitudinal cohort study (PROSPECT) among 17.357 women who were recruited for breast cancer screening. One of the four markers demonstrated predictive value for primary cardiovascular events in women only. Together with partner Fleet Bioprocessing a separate ELISA assay was developed for this female marker. Currently the ELISA assay is being validated by UKE laboratories in 20.000 samples of the BiomarCaRE case cohort studies

Partners involved

Cavadis (WP coordinator), UMC Utrecht, Fleet Bioprocessing
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Project Description

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Europa-Studien Apr14 webFigure 1: Population cohorts (blue), disease cohorts (red) and clinical trials (green) participating in BiomarCaREBiomarCaRE (Biomarker for Cardiovascular Risk Assessment in Europe) is an EU FP7-funded collaborative research project that integrates clinical, epidemiological and biomarker research, as well as commercial enterprises throughout Europe, North America and Australia. The BiomarCaRE project aims to determine the additional value of multiple (new) biomarkers to improve risk estimation of cardio vascular diseases (CVD) related events in Europe. Ultimately, our BiomarCaRE consortium will develop a "European biomarker panel" for CVD prediction including the classical risk factors and established and novel biomarkers.

The UKE Hamburg is coordinating the collaborative BiomarCaRE consortium which integrates the efforts of 25 academic institutions and five small/medium-sized, research intensive enterprises (SMEs) with a focus on cardiovascular biomarker research across Europe. BiomarCaRE comprises 21 well-established prospective European population-based cohort studies, four cohorts of diseased subjects (disease cohorts, secondary prevention) and five clinical trials, totalling over 300,000 participants with follow-up (Figure 1). The central BiomarCaRE laboratory is directly located at the University Heart Center Hamburg where sample logistics and biomarker measurements but also data analyses has been performed.
Novel, -omics based biomarkers (origination from proteomics, transcriptomics, metabolomics and miRNomics) are disclosed by the academic and SME partners and compared to key established biomarkers such as high-sensitivity assayed troponin I, natriuretic peptides, high-sensitivity C-reactive protein, lipids, and further markers of cardiomyocyte micronecrosis, inflammation, und renal function. The SMEs introduce the technology and guide the development of the innovative assays needed for the measurement of these novel biomarkers.

The BiomarCaRE Project is designed as a multi-modular study including:
  1. biomarker selection based on omics discovery studies as well as literature, and
  2. assay development (Module 1),
  3. data harmonization of large-scale studies and
  4. biomarker determination, analyses and validation (Module 2), and
  5. biomarker assessment in clinical trials and 6) economic evaluation (Module 3) (Figure 2).

The BiomarCaRE project is unique in terms of its dimension, targeting of novel biomarkers based on -omics technology, and the evaluation of the impact of a multiple biomarker score in large prospective population cohorts across different European regions. BiomarCaRE integrates modern, mainly SME driven molecular technologies with epidemiological approaches supplemented by economical assessment. This large individual-based database provides a unique opportunity to investigate the performance of established and novel biomarkers for cardiovascular risk assessment across Europe.

Flow webFigure 2: BiomarCaRE modules.