US 12,000,836 B2
Methods for quantitating extra-cellular vesicle surface markers, and compositions for practicing the same
Majid Mehrpouyan, Gilroy, CA (US); and Oleg Guryev, San Jose, CA (US)
Assigned to BECTON, DICKINSON AND COMPANY, Franklin Lakes, NJ (US)
Filed by Becton, Dickinson and Company, Franklin Lakes, NJ (US)
Filed on Dec. 18, 2020, as Appl. No. 17/126,583.
Claims priority of provisional application 62/951,968, filed on Dec. 20, 2019.
Prior Publication US 2021/0190790 A1, Jun. 24, 2021
Int. Cl. G01N 33/58 (2006.01); G01N 21/64 (2006.01)
CPC G01N 33/586 (2013.01) [G01N 21/6428 (2013.01); G01N 21/6486 (2013.01); G01N 33/582 (2013.01); G01N 2496/00 (2013.01)] 14 Claims
 
1. A method of quantitating a surface marker on extra-cellular vesicles of a sample, the method comprising:
comparing:
(i) a mean fluorescence intensity of the surface marker (surface marker MFI) of a labeled extra-cellular vesicle (EV) sample, wherein the labeled EV sample has been labeled with a surface marker label comprising a specific binding member for the surface marker and a fluorophore; with
(ii) a calibration plot obtained from a liposome calibration composition comprising two or more distinct liposome subpopulations each comprising a known amount of the fluorophore that differs from the amount of any other subpopulation in the liposome calibration composition, wherein:
the fluorophore is covalently bound to the surface of a liposome or encapsulated within a liposome, and;
the calibration plot comprises a plot of the known amount of the fluorophore for each of the distinct liposome subpopulations vs. a fluorescence intensity for each of the distinct liposome subpopulations;
to obtain a count of the fluorophores bound to the surface of extracellular vesicles of the EV sample and quantify the surface marker on extra-cellular vesicles of the EV sample.