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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