Compositions, Methods, and Kits for the Isolation of Extracellular Vesicles
The present disclosure relates to compositions, methods and kits for the isolation of extracellular vesicles. The compositions, methods and kits can comprise a high-density liquid reagent that facilitates the sequestration of extracellular vesicles bound to magnetic beads.
1 - 45 . (canceled)
46 . A method of isolating a microvesicle comprising a surface marker from a biological sample, the method comprising:
a) incubating the biological sample with at least one particle that binds the surface marker under conditions sufficient to form a particle-microvesicle complex;
b) contacting the biological sample comprising the particle-microvesicle complex with a high-density matrix; and
c) subjecting the biological sample comprising the particle-microvesicle complex and the high-density matrix to a force such that the particle-microvesicle complex is translocated through the high-density matrix, thereby isolating the microvesicle.
47 . The method of claim 46 , wherein the biological sample comprising the particle-microvesicle complex is contacted with the high-density matrix at a ratio from about 1:1 to about 1:100 (vol/vol) of the biological sample to the high-density matrix.
48 . The method of claim 46 , wherein the force is a magnetic force, gravitational force, centrifugal force, pressure or any combination thereof.
49 . The method of claim 46 , wherein the high density-matrix has a density that is equal to or greater than 1.0 g/ml.
50 . The method of claim 46 , wherein the high-density matrix comprises Optiprep, sucrose, Ficoll, Histopaque, Percoll, or any combination thereof.
51 . The method of claim 46 , wherein the at least one particle comprises an antibody or fragment thereof that binds to the surface marker.
52 . The method of claim 51 , wherein the antibody or fragment thereof is coupled to the at least one particle by perfectly base-paired sense and anti-sense oligonucleotides, a partially double-stranded oligonucleotide, a double-stranded oligonucleotide, a photocleavable linker moiety, a disulfide bond, at least one peptide, or any combination thereof.
53 . The method of claim 46 , wherein the at least one particle comprises an affinity molecule that binds to the surface marker.
54 . The method of claim 53 , wherein the affinity molecule comprises an antibody fragment, an aptamer, an aptamer analog, a lectin, a molecularly imprinted polymer, or any combination thereof.
55 . The method of claim 46 , wherein the at least one particle comprises a ferromagnetic bead.
56 . The method of claim 46 , further comprising d) removing the biological sample and the high-density matrix from the particle-microvesicle complex.
57 . The method of claim 56 , wherein removing the biological sample and the high-density matrix from the particle-microvesicle complex comprises aspiration.
58 . The method of claim 46 , further comprising d) removing at least 80% of the biological sample and at least 80% of the high-density matrix from the particle-microvesicle complex.
59 . The method of claim 46 , wherein the method results in isolation of at least 80% of microvesicles from the biological sample.
60 . The method of claim 46 , wherein the biological sample comprises blood, serum, plasma, urine, cerebrospinal fluid, or any combination thereof.
61 . The method of claim 46 , further comprising extracting a nucleic acid molecule, a carbohydrate molecule, a lipid molecule, a peptide or polypeptide, or any combination thereof, from the isolated microvesicle.
62 . A kit for isolating a microvesicle comprising:
a) a plurality of particles that bind a surface marker on the microvesicle;
b) a high-density matrix; and
c) instructions for isolating the microvesicle from a biological sample, wherein the instructions indicate contacting the biological sample with the high-density matrix at a ratio from about 1:1 to about 1:100 (vol/vol) of the biological sample to the high-density matrix.
63 . The kit of claim 62 , wherein the high density-matrix has a density that is equal to or greater than 1.0 g/ml.
64 . The kit of claim 62 , wherein the high-density matrix comprises Optiprep, sucrose, Ficoll, Histopaque, Percoll, or any combination thereof.
65 . The kit of claim 62 , wherein each particle of the plurality of particles comprises a ferromagnetic bead.