Magnetic separation of lipoproteins using dextran sulfate

Patent metadata

Field	Value
Publication number	US-9250211-B2
Application number	US-201113340547-A
Country	US
Kind code	B2
Filing date	Dec 29, 2011
Priority date	Dec 30, 2010
Publication date	Feb 2, 2016
Grant date	Feb 2, 2016

Abstract

The invention provides apparatus and methods of preparation of lipoproteins from a biological sample, including HDL, LDL, Lp(a), IDL, and VLDL, for diagnostic purposes utilizing differential charged-particle mobility analysis methods. Further provided are methods for analyzing the size distribution of lipoproteins by differential charged-particle mobility, which lipoproteins are prepared by methods of the invention. Further provided are methods for assessing lipid-related health risk, cardiovascular condition, risk of cardiovascular disease, and responsiveness to a therapeutic intervention, which methods utilize lipoprotein size distributions determined by methods of the invention.

First claim

What is claimed is:

1. A method for purifying lipoproteins suitable for differential charged-particle mobility analysis of lipoprotein class and subclass, said method comprising:
(a) adding one or more cations and dextran sulfate to a sample comprising lipoproteins to form a precipitation mixture;
(b) contacting the precipitation mixture with a microbead under conditions for one or more classes or subclasses of lipoproteins in said mixture to bind to an inert outer layer of the microbead, wherein said inert outer layer comprises carboxylate or NH 2 , but does not include a lipoprotein binding lipoprotein-capture ligand;
(c) separating lipoproteins bound to the microbead from said mixture to obtain a microbead with bound lipoproteins; and
(d) removing said bound lipoproteins from the microbead;
wherein said lipoproteins bound to the microbead comprise HDL and one or more selected from the group consisting of LDL, Lp(a), IDL and VLDL.

2. The method of claim 1 , comprising a step of adding glycine.

3. The method of claim 1 , wherein said cations comprise one or more divalent cations.

4. The method of claim 3 , wherein said one or more divalent cations are selected from the group consisting of Mg 2+ , Ca 2+ , Mn 2+ , and Sr 2+ .

5. The method of claim 3 , wherein said one or more divalent cations comprise Ca 2+ .

6. The method of claim 5 , wherein said precipitation mixture comprises Ca 2+ at a concentration within the range of about 25 mM to about 200 mM.

7. The method of claim 5 , wherein said precipitation mixture comprises Ca 2+ at a concentration of about 150 mM.

8. The method of claim 3 , wherein said one or more divalent cations comprise Mg 2+ .

9. The method of claim 8 , wherein said precipitation mixture comprises Mg 2+ at a concentration within the range of about 25 mM to about 200 mM.

10. The method of claim 8 , wherein said precipitation mixture comprises Mg 2+ at a concentration of about 100 mM.

11. The method of claim 3 , wherein said one or more divalent cations comprise Ca 2+ and Mg 2+ .

12. The method of claim 11 , wherein said precipitation mixture comprises Ca 2+ at a concentration within the range of about 25 mM to about 200 mM and Mg 2+ at a concentration within the range of about 25 mM to about 200 mM.

13. The method of claim 11 , wherein said precipitation mixture comprises Ca 2+ at a concentration of about 25 mM and Mg 2+ at a concentration of about 100 mM.

14. The method of claim 1 , wherein said dextran sulfate is in the size range of about 10 to about 500 kDa.

15. The method of claim 1 , wherein said dextran sulfate is in the size range of about 50 to about 100 kDa.

16. The method of claim 1 , wherein said dextran sulfate has a size of about 50 kDa.

17. The method of claim 1 , wherein said microbeads are in the size range of about 0.2 μm to about 150 μm.

18. The method of claim 1 , wherein said microbeads comprise a magnetic or paramagnetic core.

19. The method of claim 1 , wherein said lipoproteins comprise HDL and LDL.

20. The method of claim 1 , wherein said sample is plasma or serum.

21. The method of claim 1 , wherein said sample is exposed to ethanol prior to step (a) under conditions suitable to precipitate fibrinogen in the sample.

22. A method for analyzing the size distribution of lipoproteins, said method comprising:
(a) adding one or more cations and dextran sulfate to a sample comprising lipoproteins to form a precipitation mixture;
(b) contacting the precipitation mixture with a microbead under conditions for one or more classes or subclasses of lipoproteins in said mixture to bind to an inert outer layer of the microbead, wherein said inert outer layer comprises carboxylate or NH 2 , but does not include a lipoprotein binding lipoprotein-capture ligand;
(c) separating lipoproteins bound to the microbead from said mixture to obtain a microbead with bound lipoproteins;
(d) removing said bound lipoproteins from the microbead; wherein said lipoproteins bound to the microbead comprise HDL and one or more selected from the group consisting of LDL, Lp(a), IDL and VLDL; and
(e) subjecting said lipoproteins to differential charged-particle mobility analysis, thereby determining the size distribution of said lipoproteins.

23. The method of claim 22 , further comprising:
(f) determining said lipoproteins size distribution to conduct an assessment of said individual, said assessment selected from the group consisting of lipid-related health risk, cardiovascular condition, risk of cardiovascular disease, and responsiveness to a therapeutic intervention.

24. A method for purifying lipoproteins for differential charged-particle mobility analysis, said method not including immunopurification, said method comprising:
(a) admixing a solution comprising lipoproteins and non-lipoproteins with one or more polyanionic compounds and one or more divalent cations;
(b) allowing a precipitate containing lipoproteins to form in said admixed solution; and
(c) after step (b), collecting the precipitated lipoproteins by contacting with a microbead under conditions for one or more classes or subclasses of the precipitated lipoproteins to bind to an inert outer layer of the microbead, wherein said inert outer layer comprises carboxylate or NH 2 , but does not include a lipoprotein-capture ligand;
wherein said lipoproteins bound to the microbead comprise HDL and one or more selected from the group consisting of LDL, Lp(a), IDL and VLDL.

25. The method of claim 24 , wherein said polyanionic compounds are not biotinylated and said precipitated lipoproteins are not bound to said solid support by streptavidin.

26. The method of claim 24 , wherein said polyanionic compound is dextran sulfate, and wherein said one or more divalent cations are selected from the group consisting of Mg 2+ and Ca 2+ .

27. The method of claim 24 , wherein said solution is exposed to ethanol prior to step (a) under conditions suitable to precipitate fibrinogen in the sample.

28. The method of claim 24 , wherein said microbeads are in the size range of about 0.2 μm to about 150 μm.

29. The method of claim 24 , wherein said microbeads comprise a magnetic or paramagnetic core.

Assignees

• Caulfield Michael P
• Liu Jackie
• Shalhout Dawn
• Chen Zhihong
• Quest Diagnostics Invest Inc

Inventors

• Caulfield Michael P
• Liu Jackie
• Shalhout Dawn
• Chen Zhihong

Classifications

• G01N27/622 Primary

  Ion mobility spectrometry · CPC title

• C07K1/36

  by a combination of two or more processes of different types · CPC title

• C07K1/30 Primary

  by precipitation · CPC title

• C07K1/22

  Affinity chromatography or related techniques based upon selective absorption processes · CPC title

• C07K1/122

  Hydrolysis with acids different from HF · CPC title