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Surface Profiling of Extracellular Vesicles from Plasma or Ascites Fluid Using DotScan Antibody Microarrays

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1619)

Abstract

DotScan antibody microarrays were initially developed for the extensive surface profiling of live leukemia and lymphoma cells. DotScan’s diagnostic capability was validated with an extensive clinical trial using mononuclear cells from the blood or bone marrow of leukemia or lymphoma patients. DotScan has also been used for the profiling of surface proteins on peripheral blood mononuclear cells (PBMC) from patients with HIV, liver disease, and stable and progressive B-cell chronic lymphocytic leukemia (CLL). Fluorescence multiplexing allowed the simultaneous profiling of cancer cells and leukocytes from disaggregated colorectal and melanoma tumor biopsies after capture on DotScan. In this chapter, we have used DotScan for the surface profiling of extracellular vesicles (EV) recovered from conditioned growth medium of cancer cell lines and the blood of patients with CLL. The detection of captured EV was performed by enhanced chemiluminescence (ECL) using biotinylated antibodies that recognized antigens expressed on the surface of the EV subset of interest. DotScan was also used to profile EV from the blood of healthy individuals and the ascites fluid of ovarian cancer patients. DotScan binding patterns of EV from human plasma and other body fluids may yield diagnostic or prognostic signatures for monitoring the incidence, treatment, and progression of cancers.

Key words

Exosomes Shed microvesicles Chemiluminescence Chronic lymphocytic leukemia Ovarian cancer CD antigen 

Notes

Acknowledgments

We wish to thank Dr. Pauline Huang (School of Life and Environmental Sciences, University of Sydney, Australia) for making the DotScan antibody microarrays and A/Prof. Philip Beale (Concord Repatriation General Hospital, Concord, NSW, Australia) for supplying blood samples from CRC patients.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.School of Life and Environmental SciencesUniversity of SydneySydneyAustralia
  2. 2.Fiona Elsey Cancer Research InstituteBallaratAustralia
  3. 3.Federation UniversityBallaratAustralia
  4. 4.Department of Obstetrics & Gynaecology, Women’s Cancer Research CentreRoyal Women’s HospitalParkvilleAustralia
  5. 5.Kolling Institute of Medical ResearchRoyal North Shore HospitalSt. LeonardsAustralia
  6. 6.Department of Biochemistry and Genetics, La Trobe Institute for Molecular ScienceLa Trobe UniversityMelbourneAustralia

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