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The EXOTEST is a simple and reliable method based on a double sandwich Elisa kit for detection, quantification and characterization of exosomes from human plasma of people suffering with several diseases. Currently, the test has been developed mainly in the cancer area and proved to be predictive for diagnosing cancer, and more specifically different tumours at different stages. The scope of the test can be enlarged to diagnose diseases other than tumours, such as infectious and chronic inflammatory diseases. To this purpose HBM has been assigned an R&D grant by the Estonian Government, under the European Commission Eureka program umbrella, for further development and marketing.


New technology to characterize and quantify exosomes in human body fluids

 


 

Exosomes are microvesicles of endosomal origin with a size of 30-120 nm, externally released following the fusion of multivesicular body with the plasma membrane. These structures are secreted from different normal cell types, such as dendritic cells (DC), B and T lymphocytes, mast cells, epithelial cells, as well as from tumor cells. In addition to the major histocompatibility complex proteins (MHC I, MHC II) and other proteins involved in antigen presentation, exosomes may carry membrane and cytosolic proteins such as tetraspanins (CD63, CD9, CD81, CD82), heat-shock proteins (HSP70, HSP90), cytoskeletal components (tubulin, actin and actin-binding proteins), proteins involved in fusion and transport of vesicles (Rabs and Lamps), in signal transduction (protein kinases), metabolic enzymes and chaperones.

 

 

Tumor exosomes are proven to be both a good vehicle for the delivery of DC-processed tumor antigens to T cells and for pro-apoptotic molecules able to induce tumor cell death. In fact, we have recently shown that human tumor cells of various origins are able to secrete exosomes expressing death receptors ligands (e.g. FasL, TRAIL) and markers characterizing their cellular source in vitro and in vivo (e.g. Mart-1 or gp100 for melanoma, CEA for colon carcinoma). Some studies suggested that the amount of vesicles released by human cancer cell lines correlated with their in vitro invasiveness and that exosome-like microvesicles may be detected in vivo in physiological fluids, such as bronchoalveolar lavage, malignant pleural and peritoneal effusions, and human plasma from both healthy donors and cancer patients. A central problem in obtaining useful in vivo data on exosomes is the low level of efficiency of the available methods to obtain exosomes in order to quantify and characterize them from human body fluids, particularly from plasma. We have set up a simple a reliable method to detect and quantify exosomes from human plasma. This is an ELISA test that allows exosomes quantification and characterization from human plasma of both healthy donors and tumor patients. Through this test, we were able to characterize exosome-like microvesicles from plasma of both SCID mice engrafted with human melanoma or colon carcinoma cells and tumors patients. We show that plasmatic levels of exosomes are directly related to the tumor size and caveolin-1 is exclusively detectable from exosomes purified from plasma of tumor patients. This disclosure suggests that the detection of tumor exosomes in plasma of human patients is useful in diagnosis and follow up of human tumor malignancies.


Advantages of Exotest:

  1. It allows a non-invasive test useful in clinical practice to diagnosis, follow up and screenings of tumor patients.
  2. It may be used in clinical research of tumors, but also other diseases including viral, transmissible and autoimmune diseases
  3. It may be used to improve existing clinical test based on proteins that are expressed on exosomes (e.g. tumor markers, viral proteins, prion proteins)

We called this assay EXOTEST.

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REFERENCES:

  1. Andreola G et al, J Exp Med 2002
  2. Huber V et al, Gastroenterology 2005
  3. Valenti et al, Cancer Res 2006
  4. Logozzi M et al, Plos One 2009