Improved exosome profiling for therapy and diagnosis

A method of analyzing a plasma or serum sample, comprising:

• a. providing a capture system, comprising at least three populations of distinct fluorescence-labeled magnetic microspheres, wherein each microsphere population displays antibodies directed to a distinct target on the surface of an EV population of a distinct cellular origin;
• b. providing 1-75 μl of an unprocessed plasma or serum sample, or a corresponding amount of intact EV;
• c. incubating the sample with the capture system, under conditions such as to allow specific antigen-antibody binding while substantially maintaining the integrity of the EV membranes, to thereby provide distinct populations of EV-microsphere complexes corresponding to each target;
• d. washing the EV-microsphere complexes using a magnetic device, under conditions enabling selective capturing of said complexes;
• e. incubating said EV-microsphere complexes with a plurality of detection antibodies, each antibody directed to a distinct marker on the surface of the EV, under conditions such as to allow specific antigen-antibody binding while substantially maintaining the integrity of the EV membranes, wherein the plurality of detection antibodies is labeled by the same fluorescent marker;
• f. washing the resulting labeled complexes using a magnetic device to remove excess reagents;
• g. subjecting the resulting complexes to a microfluidic device amenable for simultaneously detecting and quantifying fluorescent emission on a plurality of wave lengths, to thereby quantify the fluorescence emission levels and provide a separate assessment of the level of the surface targets corresponding to each of the EV populations;
• and
• h. comparing the quantified levels to control levels;
• wherein the method is performed using reagents and under conditions so as to retain said EVs in a substantially intact form, wherein said plurality of detection antibodies is directed to a plurality of diagnostic markers or a plurality of tetraspanin markers.

A kit for analyzing EVs from a blood-derived sample, comprising:

• i) a capture system, comprising a first population of magnetic microspheres displaying an antibody directed to GAP43 or CD171, and labeled by a first combination of fluorophores, a second population of magnetic microspheres displaying an antibody directed to CD68, labeled by a second combination of fluorophores, and a third population of magnetic microspheres displaying an antibody directed to P2RY12, labeled by a third combination of fluorophores;
• ii) a plurality of detection antibodies, each antibody directed to a distinct tetraspanin marker, wherein the plurality of detection antibodies is labeled, directly or indirectly, by the same fluorescent marker;
• and optionally
• iii) reagents for performing said evaluation under conditions so as to retain EVs in a substantially intact form.