Methods of measuring exosomes using intrinsic fluorescence

Abstract

Described herein are novel rapid and reliable methods of detection of extracellular vesicles and quantifying extracellular vesicle concentrations and absolute number from various sources, including raw cell harvest. The methods described herein comprise detection of intrinsic fluorescence of extracellular vesicles in biological samples. Extracellular vesicles analyzed by the methods of this application have a stereotypical elution profile distinct from known contaminants. The methods described herein are a significant improvement over the state of the art and fulfills an unmet need in the field of extracellular vesicle manufacturing and quality control.

1 . A method of detecting extracellular vesicles, comprising:
 obtaining a sample comprising extracellular vesicles; and   determining an intrinsic fluorescence emission signal from said sample, wherein said intrinsic fluorescence emission signal is indicative of the presence of said extracellular vesicles within said sample.   
     
     
         2 . The method of  claim 1 , wherein said intrinsic fluorescence emission signal is generated using an excitation wavelength ranging from 450 nm to 650 nm and an emission wavelength that is longer than the excitation wavelength and ranging from 470 nm to 670 nm. 
     
     
         3 . The method of  claim 2 , wherein said intrinsic fluorescence emission signal is determined at an emission wavelength range of 500-600 nm. 
     
     
         4 . The method of  claim 3 , wherein said intrinsic fluorescence emission signal is determined at an emission wavelength range of 550-590 nm. 
     
     
         5 . The method of  claim 3 , wherein said intrinsic fluorescence emission signal is determined at an emission wavelength of 573 nm. 
     
     
         6 . The method of  claim 2 , wherein said intrinsic fluorescence emission signal is determined at an excitation wavelength range of 500-600 nm. 
     
     
         7 . The method of  claim 6 , wherein said intrinsic fluorescence emission signal is determined at an excitation wavelength range of 530-570 nm. 
     
     
         8 . The method of  claim 7 , wherein said intrinsic fluorescence emission signal is determined at an excitation wavelength of 556 nm. 
     
     
         9 . The method of any one of the preceding claims wherein said intrinsic fluorescence emission signal is determined at an excitation wavelength of 550 nm and an emission of 570 nm. 
     
     
         10 . The method any one of the preceding claims, wherein said sample is separated into fractions prior to determining said intrinsic fluorescence emission signal. 
     
     
         11 . The method of  claim 10 , wherein said separation comprises a column chromatography separation step. 
     
     
         12 . The method of  claim 10 , wherein said separation comprises two chromatography separation steps. 
     
     
         13 . The method of  claim 11  or  12 , wherein one or both of said chromatography steps is size-exclusion chromatography. 
     
     
         14 . The method of  claim 11  or  12 , wherein one or both of said chromatography steps is ion-exchange chromatography. 
     
     
         15 . The method of  claim 13 , wherein said ion-exchange chromatography is strong anion-exchange chromatography. 
     
     
         16 . The method of  claim 12 , where said two chromatography steps are anion-exchange chromatography and size-exclusion chromatography. 
     
     
         17 . The method of  claim 16 , wherein said anion-exchange chromatography precedes said size-exclusion chromatography. 
     
     
         18 . The method of any one of  claims 10 - 15 , wherein said intrinsic fluorescence emission signal is determined using a flow cell. 
     
     
         19 . The method of any one of the preceding claims, wherein said sample is subjected to a filtration step prior to determining said intrinsic fluorescence emission signal. 
     
     
         20 . The method of any one of the preceding claims, wherein said sample is subjected to a centrifugation separation step prior to determining said intrinsic fluorescence emission signal. 
     
     
         21 . The method of any one of the preceding claims, wherein said sample is subjected to a sucrose density gradient step prior to determining said intrinsic fluorescence emission signal. 
     
     
         22 . The method of any one of the preceding claims, wherein said sample is a subjected to a separation step comprising use of a density gradient prior to determining said intrinsic fluorescence emission signal. 
     
     
         23 . The method of any one of the preceding claims, wherein said sample is derived from a cell culture. 
     
     
         24 . The method of  claim 23 , wherein said cell culture comprises human embryonic kidney cells, mesenchymal stem cells or neuronal cells. 
     
     
         25 . The method of any one of the preceding claims, wherein said sample is derived from a body fluid of an animal. 
     
     
         26 . The method of any one of the preceding claims, comprising determining an amount of said extracellular vesicles within said sample comprising comparing said intrinsic fluorescence emission signal to the intrinsic fluorescence emission signal of a standard. 
     
     
         27 . The method of any one of the preceding claims, comprising quantifying the amount of said extracellular vesicles within said sample, based upon the area under of the curve of a chromatogram comprising said intrinsic fluorescence emission signal. 
     
     
         28 . The method of any one of the preceding claims, comprising determining an amount of said extracellular vesicles within said sample comprising comparing said intrinsic fluorescence emission signal to the luminescence signal of a standard. 
     
     
         29 . The method of  claim 28 , wherein said standard is measured using a luminescence proximity assay 
     
     
         30 . The method of  claim 29 , wherein said standard is calculated based on the relative abundance of one or more exosome-associated proteins. 
     
     
         31 . The method of  claim 30 , wherein said method is an AlphaScreen assay. 
     
     
         32 . The method of  claims 30 - 31 , wherein said one or more exosome-associated proteins are selected from the group consisting of CD9, CD81, CD63, and combinations thereof. 
     
     
         33 . The method of any one of the preceding claims, wherein said extracellular vesicle is an exosome. 
     
     
         34 . The method of any one of  claims 1 - 33 , wherein said extracellular vesicle is a nanovesicle. 
     
     
         35 . The method of any one of the preceding claims, wherein said extracellular vesicle comprises a therapeutic payload. 
     
     
         36 . A method of detecting extracellular vesicles, comprising:
 obtaining a sample comprising extracellular vesicles; and   determining a luminescence proximity signal from said sample, wherein said luminescence proximity signal is indicative of the presence of said extracellular vesicles within said sample.   
     
     
         37 . The method of  claim 36 , wherein said luminescence proximity signal is determined at an excitation wavelength of 680 nm. 
     
     
         38 . The method of  claim 37 , wherein said luminescence proximity signal is determined at an emission wavelength of 520-620 nm. 
     
     
         39 . The method of  claim 38 , wherein said signal is determined by excitation of ambient oxygen to induce chemiluminescence in a fluorophore. 
     
     
         40 . The method of  claim 39 , wherein said method is an AlphaScreen™ assay. 
     
     
         41 . The method of  claim 40 , wherein said method comprises a donor substrate and an acceptor substrate conjugated to two binding molecules. 
     
     
         42 . The method of  claim 41 , wherein said binding molecules comprise antibodies or fragments thereof. 
     
     
         43 . The method of  claim 42 , wherein said antibodies recognize one or more exosome-associated proteins. 
     
     
         44 . The method of  claim 43 , wherein said one or more exosome-associated proteins is selected from the group consisting of CD9, CD63, CD81, and combinations thereof.