Usages of recombinant extracellular vesicles

A method of determining recovery rate of sample extracellular vesicles, the method comprising:

• mixing a biofluid sample comprising sample extracellular vesicles with a known amount of recombinant extracellular vesicles, wherein the recombinant extracellular vesicles comprise:
• a) a self-assembling protein that directs its own release through vesicles as a luminal membrane-bound protein, and
• b) a heterologous marker,
• to obtain a mixture,
• isolating the extracellular vesicles from the mixture,
• detecting an amount of the recombinant extracellular vesicles among the isolated extracellular vesicles, and
• determining the recovery rate of the sample extracellular vesicles based on a ratio of the amount of recombinant extracellular vesicles after isolation to the known amount of recombinant extracellular vesicles mixed with the biofluid sample before isolation;
• wherein the heterologous marker is a heterologous nucleic acid fused to retroviral packaging signal psi.

A method of determining recovery rate of sample extracellular vesicles, the method comprising:

• mixing a biofluid sample comprising sample extracellular vesicles with a known amount of recombinant extracellular vesicles, wherein the recombinant extracellular vesicles comprise:
• a) a self-assembling protein that directs its own release through vesicles as a luminal membrane-bound protein, and
• b) a heterologous marker,
• to obtain a mixture,
• isolating the extracellular vesicles from the mixture,
• detecting an amount of the recombinant extracellular vesicles among the isolated extracellular vesicles, and
• determining the recovery rate of the sample extracellular vesicles based on a ratio of the amount of recombinant extracellular vesicles after isolation to the known amount of recombinant extracellular vesicles mixed with the biofluid sample before isolation;
• wherein the heterologous marker is a heterologous nucleic acid molecule or a heterologous protein and wherein detecting the amount of the recombinant extracellular vesicles is undertaken by determining amount of any heterologous nucleic acid by real-time PCR, RT-qPCR, digital PCR, RNA sequencing, nanostring, branched DNA amplification or serial analysis of gene expression.

A method of determining recovery rate of sample extracellular vesicles, the method comprising:

• mixing a biofluid sample comprising sample extracellular vesicles with a known amount of recombinant extracellular vesicles, wherein the recombinant extracellular vesicles comprise:
• a) a self-assembling protein that directs its own release through vesicles as a luminal membrane-bound protein, and
• b) a heterologous marker,
• to obtain a mixture,
• isolating the extracellular vesicles from the mixture,
• detecting an amount of the recombinant extracellular vesicles among the isolated extracellular vesicles, and
• determining the recovery rate of the sample extracellular vesicles based on a ratio of the amount of recombinant extracellular vesicles after isolation to the known amount of recombinant extracellular vesicles mixed with the biofluid sample before isolation;
• wherein the self-assembling protein that directs its own release through vesicles as a luminal membrane-bound protein is selected from the group consisting of:
• retroviral group-specific antigen, retroviral group-specific antigen variations, influenza M1 protein, ARRDC1 protein, ARC protein, Ebola virus VP40 protein, and M proteins of vesicular stomatitis virus.

A method of determining recovery rate of sample extracellular vesicles, the method comprising:

• mixing a biofluid sample comprising sample extracellular vesicles with a known amount of recombinant extracellular vesicles, wherein the recombinant extracellular vesicles comprise:
• a) a self-assembling protein that directs its own release through vesicles as a luminal membrane-bound protein, and
• b) a heterologous marker,
• to obtain a mixture,
• isolating the extracellular vesicles from the mixture,
• detecting an amount of the recombinant extracellular vesicles among the isolated extracellular vesicles, and
• determining the recovery rate of the sample extracellular vesicles based on a ratio of the amount of recombinant extracellular vesicles after isolation to the known amount of recombinant extracellular vesicles mixed with the biofluid sample before isolation;
• wherein the recombinant extracellular vesicles are incubated with immunoglobulin proteins IgG and/or IgM before mixing the biofluid sample with a known amount of the recombinant extracellular vesicles so that the recombinant extracellular vesicles and the sample extracellular vesicles are present in a different fraction after the isolation.

A method of determining recovery rate of sample extracellular vesicles, the method comprising:

• mixing a biofluid sample comprising a plasma sample or serum sample with a known amount of recombinant extracellular vesicles, wherein the recombinant extracellular vesicles comprise:
• a) a self-assembling protein that directs its own release through vesicles as a luminal membrane-bound protein, and
• b) a heterologous marker,
• to obtain a mixture,
• treating the mixture with a protease or incubating the recombinant extracellular vesicle with polyethylene glycol (PEG) so that the recombinant extracellular vesicles and the sample extracellular vesicles are present in the same fraction;
• isolating the extracellular vesicles from the mixture comprising and/or combining with a density-based separation step resulting in obtaining a first fraction comprising the recombinant extracellular vesicles and a second fraction comprising the sample extracellular vesicles
• detecting an amount of the recombinant extracellular vesicles among the isolated extracellular vesicles of the first fraction, and
• determining the recovery rate of the sample extracellular vesicles based on a ratio of the amount of recombinant extracellular vesicles after isolation to the known amount of recombinant extracellular vesicles mixed with the biofluid sample before isolation;
• wherein the recombinant extracellular vesicles incubated with PEG and the sample extracellular vesicles isolated in the same fraction after the isolation are incubated with immobilized anti-PEG antibodies so that the recombinant extracellular vesicles incubated with PEG are separated from the sample extracellular vesicles.

A method of determining recovery rate of sample extracellular vesicles, the method comprising:

• mixing a biofluid sample comprising a plasma sample or serum sample with a known amount of recombinant extracellular vesicles, wherein the recombinant extracellular vesicles comprise:
• a) a self-assembling protein that directs its own release through vesicles as a luminal membrane-bound protein, and
• b) a heterologous marker,
• to obtain a mixture,
• treating the mixture with a protease or incubating the recombinant extracellular vesicle with polyethylene glycol (PEG) so that the recombinant extracellular vesicles and the sample extracellular vesicles are present in the same fraction;
• isolating the extracellular vesicles from the mixture comprising and/or combining with a density-based separation step resulting in obtaining a first fraction comprising the recombinant extracellular vesicles and a second fraction comprising the sample extracellular vesicles
• detecting an amount of the recombinant extracellular vesicles among the isolated extracellular vesicles of the first fraction, and
• determining the recovery rate of the sample extracellular vesicles based on a ratio of the amount of recombinant extracellular vesicles after isolation to the known amount of recombinant extracellular vesicles mixed with the biofluid sample before isolation;
• wherein the protease is proteinase K.