# US 11,079,374 B2 · Methods and kits for exosome isolation and quantification

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- Number: US 11,079,374 B2
- Document ID: US11079374B2
- Status: Granted
- Kind: B2
- Application number: 16/495,833
- Filed: 2018-03-20
- Published: 2020-04-30
- Granted: 2021-08-03
- Earliest claimed: 2018-03-20
- Assignees: Dapi Meng Lin Chiang; Michael W. Pfaffi; Biovesicle Inc.
- Applicants: Dapi Meng Lin Chiang; Michael W. Pfaffl; Biovesicle Inc
- Inventors: Dapi Meng Lin Chiang; Michael W. Pfaffl; Dominik Buschmann
- Web page: https://patentgrep.com/patent/US11079374B2

## CPC

- G01N 33/54326: {Magnetic particles}
- G01N 15/1404: Handling flow, e.g. hydrodynamic focusing
- G01N 33/5306: {Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding}
- G01N 33/66: involving blood sugars, e.g. galactose
- G01N 2333/42: Lectins, e.g. concanavalin, phytohaemagglutinin
- G01N 2333/4724: Lectins
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Disclosed herein are methods and kits for isolating exosomes in a sample. The method comprises contacting and incubating a plurality of galectin-3-modified magnetic beads with the sample, followed by subjecting the mixture to a magnetic field and then isolating the exosomes from the magnetic beads by a lactose solution. The kit of the present disclosure comprises a plurality galectin-3-modified magnetic beads, a lactose solution having a pH value of 6.8 to 7.6, and an exosome-free buffer.

## Claims

### Claim 1

A method for isolating 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;
(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; and
(d) 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, wherein the elution buffer is a lactose solution consisting essentially of lactose.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 1)

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

### Claim 4 (depends on 3)

The method according to the claim 3, 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.

### Claim 5 (depends on 3)

The method according to claims 3, wherein the subject is a human.

### Claim 6 (depends on 1)

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

### Claim 7

A kit for isolating and exosomes in a sample, comprising, a plurality of lectin-modified magnetic beads, an elution buffer having a pH value of 6.8 to 7.6, and an exosome-free buffer, wherein
each lectin-modified magnetic bead comprises a plurality of lectin molecules that are covalently bonded to the magnetic bead, wherein the lectin is galectin-3;
the elution buffer is a lactose solution consisting essentially of lactose; and
the exosome-free buffer is an exosome-free bovine serum albumin or exosome-free human serum albumin.

### Claim 8 (depends on 7)

The kit according to the claim 7, wherein the lactose solution is 1-10% (wt %) lactose solution.