METHODS AND KITS FOR EXOSOME ISOLATION AND QUANTIFICATION

• US 20200132682A1
• Filed: 03/20/2018
• Published: 04/30/2020
• Est. Priority Date: 03/24/2017
• Status: Active Grant

• Current Assignee: Biovesicle Inc., Dapi Meng Lin Chiang, Michael W. Pfaffl
• Original Assignee: Biovesicle Inc.
• Inventors: CHIANG, Dapi Meng Lin, PFAFFL, Michael W., BUSCHMANN, Dominik
• Granted Patent: US 11,079,374 B2
• Time in Patent Office: Days
• CPC Class Codes: G01N 15/1404 Handling flow, e.g. hydrodynamic focusing
• CPC Class Codes: G01N 2333/42 Lectins, e.g. concanavalin, phytohaemagglutinin
• CPC Class Codes: G01N 2333/4724 Lectins
• CPC Class Codes: G01N 33/5306 Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
• CPC Class Codes: G01N 33/54326 Magnetic particles
• CPC Class Codes: G01N 33/66 involving blood sugars, e.g. galactose

Abstract

Disclosed herein are methods and kits for isolating and/or quantifying exosomes in a sample. For example, the exosomes are isolating using lectin-modified magnetic beads suspended in an exosome-free buffer, and the exosome-bead complexes are isolated from the remainder of the mixture using a magnetic field. For separating the exosomes from the exosome-bead complexes, an elution buffer having a pH value of 6.8 to 7.6 is used, thereby avoiding the convention elution steps involving acidic or alkaline reagents. For quantification, the exosome-bead complexes, as isolated, are subject to subsequent flow cytometry.

Claims

1. A method for isolating and/or quantifying exosomes in a sample, comprising the steps of,

(a) contacting a plurality of lectin-modified magnetic beads with the sample to obtain a first mixture, wherein the plurality of lectin-modified magnetic beads are suspended in an exosome-free buffer, and each lectin-modified magnetic bead comprises a plurality of lectin molecules that are covalently bonded to the magnetic bead, wherein the exosome-free buffer is an exosome-free bovine serum albumin or exosome-free human serum albumin, and the lectin is galectin-3;

(b) incubating the first mixture at a condition allowing the exosomes to conjugate with the lectin molecules of the lectin-modified magnetic beads thereby obtaining a second mixture comprising a plurality of exosome-bead complexes; and

(c) subjecting the second mixture from the step (b) to a magnetic field, thereby separating the exosome-bead complexes from the remainder of the second mixture.

3. The method according to the claim 1, further comprising a step of separating the exosomes from the magnetic beads by eluting the exosome-bead complexes from the step (c) using an elution buffer having a pH value of 6.8 to 7.6.

4. The method according to the claim 3, wherein the elution buffer comprises a saccharide solution.

5. The method according to the claim 4, wherein the saccharide solution comprises a saccharide selected from the group consisting of lactose, lubricin, fetuin, asialofetuin, maltose, arabinose, glucose, galactose, mannose, xylose, fructose, ribose, sucrose, dextran, and dextrin.

6. The method according to the claim 4, wherein the saccharide solution is 1-10% (wt %) lactose solution.

7. The method according to the claim 1, further comprising the step of quantifying the exosomes by subjecting the exosome-bead complexes from the step (c) to a flow cytometry.

8. The method according to the claim 7, wherein the step of quantifying the exosomes comprises the step of directly adding a primary antibody to the exosome-bead complexes from the step (c).

9. The method according to the claim 8, wherein the primary antibody is a conjugated with a fluorescent dye.

13. The method according to claim 1, wherein the sample is an uncultured biological sample obtained from a subject.

14. The method according to the claim 13, wherein the uncultured biological sample is derived from peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, pre-ejaculatory fluid, female ejaculate, sweat, fecal matter, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, or umbilical cord blood of the subject.

17. The method according to claims 13, wherein the subject is a human.

18. The method according to claim 1, wherein the sample is a cultured sample.

2. (canceled)

10. (canceled)

11. (canceled)

12. (canceled)

15. (canceled)

16. (canceled)

19. A kit for isolating and/or quantifying exosomes in a sample, comprising, a plurality lectin-modified magnetic beads, wherein each lectin-modified magnetic bead comprises a plurality of lectin molecules that are covalently bonded to the magnetic bead;

and an exosome-free buffer, wherein the exosome-free buffer is an exosome-free bovine serum albumin or exosome-free human serum albumin.

21. The kit according to the claim 19, further comprising an elution buffer having a pH value of 6.8 to 7.6.

22. The kit according to the claim 21, wherein the elution buffer comprises 1-10% (wt %) lactose solution.

20. (canceled)