Tagged Ligands For Enrichment of Rare Analytes From A Mixed Sample

• US 20090215088A1
• Filed: 02/25/2008
• Published: 08/27/2009
• Est. Priority Date: 02/25/2008
• Status: Active Grant

• Current Assignee: ZEON Corporation
• Original Assignee: CellPoint Diagnostics, Inc.
• Inventors: Barnes, Helen, Forsyth, Allyn
• Granted Patent: US 8,008,032 B2
• Time in Patent Office: Days
• US Class Current: 435/7.230
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2300/0861 Configuration of multiple channels and/or chambers in a single devices
• CPC Class Codes: B01L 2400/086 using baffles or other fixed flow obstructions
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: G01N 1/405 by adsorption or absorption
• CPC Class Codes: G01N 2333/705 Assays involving receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: G01N 33/54326 Magnetic particles
• CPC Class Codes: G01N 33/574 for cancer
• CPC Class Codes: G01N 33/57492 involving compounds localized on the membrane of tumor or cancer cells
• CPC Class Codes: G01N 33/585 with a particulate label, e.g. coloured latex
• CPC Class Codes: Y10S 435/971 Capture of complex after antigen-antibody reaction
• CPC Class Codes: Y10S 435/973 Simultaneous determination of more than one analyte
• CPC Class Codes: Y10T 436/101666 Particle count or volume standard or control [e.g., platelet count standards, etc.]
• CPC Class Codes: Y10T 436/13 Tracers or tags
• CPC Class Codes: Y10T 436/25125 Digestion or removing interfering materials
• CPC Class Codes: Y10T 436/25375 Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]

Abstract

Method of enriching specific cells from cellular samples are disclosed, comprising contacting in solution a cellular sample with affinity-tagged ligands (ATLs) each comprising a first ligand linked to an affinity tag, wherein the ligand selectively binds a cellular marker of the rare cells and the affinity tag can be selectively captured by a capture moiety, wherein the affinity tags do not comprise a magnetic particle; and flowing the sample through a microfluidic device comprising the capture moiety to selectively retain ATL-bound cells. Methods for enriching circulating tumor cells, and devices for enriching specific cells from cellular samples are also disclosed.

Claims

1. A method for enriching rare cells from a cellular sample comprising:

contacting in solution said cellular sample with affinity-tagged ligands (ATLs) each comprising;

a first ligand linked to an affinity tag, wherein said ligand selectively binds a cellular marker of said rare cells and said affinity tag can be selectively captured by a capture moiety, wherein said affinity tags do not comprise a magnetic particle; and

flowing said sample through a microfluidic device comprising said capture moiety to selectively retain ATL-bound cells.

3. The method of claim 1 or 2, wherein said rare cells are circulating tumor cells or epithelial cells.

4. The method of claim 1 or 2, wherein said ligand is folic acid, epidermal growth factor (EGF), Dolichos Biflorus Agglutinin (DBA), EGF peptide, epi peptide, transforming growth factor-alpha (TGF-alpha), Urokinase Plasminogen Activator (UPA), FasL, MUC1/sec, catenin, ICAM-1, or plasminogen.

5. The method of claim 1 or 2, wherein said ligand is an antibody.

6. The method of claim 5, wherein said antibody is anti-EpCam, anti-ErbB2, or anti-CK19 antibody.

7. The method of claim 1 or 2, wherein said ligand is not an antibody.

8. The method of claim 1 or 2, wherein said affinity tag is biotin, desthiobiotin, histidine, polyhistidine, myc, hemagglutinin (HA), FLAG, fluorescence tag, tandem affinity purification (TAP) tags, FLAG, or glutathione S transferase (GST) or derivatives thereof.

9. The method of claim 1 or 2, wherein said ATLs further comprises a second ligand that selectively binds a second cellular marker and wherein said second ligand is also linked to said affinity tag.

10. The method of claim 1 or 2, wherein said ATL comprises at least 3, 4, 5, 6, 7, or 8 different ligands, each of which selectively binds a different cellular marker.

15. The method of claim 1, 2, or 3 wherein each of said ATL'"'"'s comprises a ratio of Ligand:

Affinity Tag that is less than 1;

5.

16. The method of claim 15, wherein said capture moiety is in a microfluidic device.

17. The method of claim 16, wherein said microfluidic device comprises an array of obstacles.

18. The method of claim 1, 2, or 3 wherein said affinity tag comprises biotin, desthiobiotin, histidine, polyhistidine, myc, hemagglutinin (HA), FLAG, fluorescence tag, tandem affinity purification (TAP) tags, FLAG, glutathione S transferase (GST) or derivatives thereof.

19. The method of claim 1, 2, or 3, wherein said ligand is linked to said affinity tag via a linker comprising one or more of modified dextran, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, and polyvinylpyrrolidone.

20. The method of claim 1 or 2 wherein the cellular marker is a cancer marker for one or more of breast, prostate, liver, ovary, skin, colon, rectum, cervix, esophagus, stomach, brain, lung, or endometrium cancer.

21. The method of claim 1, 2, or 3, wherein the ligand selectively binds epidermal growth factor receptor (EGFR) or folic acid receptor.

22. The method of claim 1, 2, or 3, wherein the capture moiety comprises avidin, streptavidin, Neutravidin™

, nickel, or glutathione.

23. A method of diagnosing a condition in a patient comprising:

enriching rare cells from said patient as provided in claim 1, 2, or 3; and

evaluating, identifying, or quantitating the ATL bound cells.

24. The method of claim 23, wherein said condition is cancer.

2. A method for enriching rare cells from a cellular sample comprising:

contacting in solution said cellular sample with affinity-tagged ligands (ATLs) each comprising;

a ligand linked to a plurality of affinity tags, wherein said ligand selectively binds a cellular marker of said rare cells and said affinity tag can be selectively captured by a capture moiety and wherein the ratio of ligand;

affinity tag of said ATL'"'"'s is less than 1;

1

flowing said sample through a microfluidic device comprising said capture moiety to selectively retain ATL-bound cells.

11. A method for enriching rare analytes from a sample comprising:

contacting in solution said sample with a plurality of affinity-tagged ligands (ATLs) wherein said mixture of ATL'"'"'s comprises;

a first ATL comprising a first ligand that selectively binds a first marker of said rare analytes, wherein said first ligand is linked to a first affinity tag that is selectively captured by a first capture moiety; and

a second ATL comprising a second ligand that selectively binds a second marker of said rare analytes, wherein said second ligand is linked to a second affinity tag, wherein said second affinity tag is selectively captured by said first capture moiety; and

contacting said sample with said capture moiety to selectively enrich said rare analytes.

12. The method of claim 11, wherein said first affinity tag and said second affinity tag are identical.

13. The method of claim 11, wherein said mixture of ATL'"'"'s comprises at least 3, 4, 5, 6, 7, 8, 9, or 10 ATL'"'"'s each of which comprises an affinity tag that can be selectively captured by the first capture moiety.

14. The method of claim 11, wherein said analytes are cells and said mixture is a blood sample.

25. A reagent comprising a plurality of affinity tagged ligands each of which comprises a ligand that selectively binds a different cancer cellular marker linked to an identical affinity tag that is selectively captured by a capture moiety.

26. The reagent of claim 25 wherein the ratio of ligand:

affinity tag per ATL is less than 1;

1.

27. The reagent of claim 25 wherein said reagent comprises at least 5 different ATL'"'"'s.

28. A microfluidic device comprising an array of obstacles, wherein the obstacles are functionalized with one or more capture moieties selected from the group comprising EGF, EGF-peptide, EPI-peptide, folate or DBA.