Quantification of Phosphatidylserine-exposing Extracellular Vesicles in Biofluids


          There are two methods adopted to quantify exosomes: 1). Counting the physical nano-particles using Nanoparticle Tracking Analysis or Tunable-Resistive Pulse Sensing; 2). Measuring exosome-associated protein markers by Western Blot, ELISA and FACS etc. However, counting method does not discriminate between vesicular or non-vesicular particles, functional or non-functional particles, such as viral particles. Those exosome-associated protein markers are proposed to include "universal exosome surface markers" CD63, CD9, CD81, and/or tissue- or disease-specific markers, such as EpCAM, CD24 etc. Given that the protein level on exosome surface is heterogeneous, a suitable pan-exosome surface protein marker does not exist and is not widely accepted yet in extracellular vesicle research community. Furthermore, the affinity of antibody to antigen on exosome surface is weaker than the counterpart, and even the contaminated proteins are always presented in the exosomes isolated by most protocols. Therefore, these methods are not sensitive and accurate enough to quantify exosomes in cell culture media or body fluids.


          In order to overcome the shortcoming of the physical counter and protein-based methods, we developed the lipid-based exosome quantification kits. Briefly, Annexin V or MFGE8 can specifically bind phosphatidylserine (PS)-exposing extracellular vesiclese. These AnnexinV or MFGE8-bound extracellular vesicles represent total extracellular vesicles regardless heterogeneous level of protein antigens or soureces. While total extracellular vesicles can be quantified with annexinV or MFGE8-luciferase, the tissue- or disease- specific extracellular vesiclese subpopulation could be done with tissue- or disease-specific antibody, such as EpCAM, CD24, or directly with exosome enzyme such as Acetyl-CoA Acetylcholinesterase (AChE), human placental alkaline phosphatase (hPLAP). A brief procedure for quantification of extracellular vesicles was outlined below.








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