Method and apparatus for protein manipulation

• US 6,649,419 B1
• Filed: 11/28/2000
• Issued: 11/18/2003
• Est. Priority Date: 11/28/2000
• Status: Active Grant

• Current Assignee: Anderson Forschung Group, LLC
• Original Assignee: Large Scale Proteomics Corporation
• Inventors: Anderson, N. Leigh
• Primary Examiner(s): Chin, Christopher L.
• Assistant Examiner(s): Do, Pensee T.
• Application Number:
  US09/722,405
• Time in Patent Office: 1,085 Days
• Field of Search: 436/526, 436/538, 436/535, 436/529, 436/541, 436/805, 436/174, 436/177, 436/806, 436/807, 436/514, 436/516, 204/182.8, 204/299, 435/7.1, 435/7.7, 435/286.7, 435/288.7, 435/287.3, 435/288.6, 435/287.2, 435/287.7, 435/287.9, 210/22, 210/416, 210/234, 210/232, 530/412, 530/413, 428/400
• US Class Current: 436/526
• CPC Class Codes: B03C 1/0332 using permanent magnets
• CPC Class Codes: B03C 1/284 with associated cleaning means, e.g. retractable non-magnetic sleeve
• CPC Class Codes: B03C 1/286 disposed at the inner circumference of a recipient, e.g. magnetic drain bolt
• CPC Class Codes: B03C 2201/18 Magnetic separation whereby the particles are suspended in a liquid
• CPC Class Codes: B03C 2201/26 for use in medical applications
• CPC Class Codes: B03C 2201/28 Parts being easily removable for cleaning purposes
• CPC Class Codes: C07K 1/22 Affinity chromatography or related techniques based upon selective absorption processes
• CPC Class Codes: C07K 1/24 by electrochemical means
• CPC Class Codes: C07K 1/36 by a combination of two or more processes of different types
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10S 436/806 Electrical property or magnetic property
• CPC Class Codes: Y10S 436/807 Apparatus included in process claim, e.g. physical support structures
• CPC Class Codes: Y10S 977/914 Protein engineering
• CPC Class Codes: Y10T 428/298 Physical dimension
• CPC Class Codes: Y10T 436/25 including sample preparation
• CPC Class Codes: Y10T 436/25375 Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]

Abstract

A method and apparatus for extracting, identifying, and manipulating proteins or peptides from a solution uses paramagnetic beads having a coating with an affinity for the target component. In one embodiment, paramagnetic beads coated with C18 are used to adsorb proteins and peptides. The beads can be used to purify, immobilize and assay antibodies. By cycling the beads, many times greater molar amount of binding partner may be separated from a solution. A magnetic probe is used to capture the beads and transfer the beads to selected processing stages.

Claims

1. A method of recovering a target component from a sample, said method comprising the steps of:

providing a solution containing said sample;

contacting said solution with an amount of magnetically responsive beads, said beads having an outer surface with a polymeric coating having pendant hydrophobic groups, said hydrophobic groups having a binding affinity for said target component, said beads being contacted with said solution for sufficient time to bind said target component to said beads; and

recovering said beads from said solution.

2. The method of claim 1, wherein said target component is a protein or peptide, and said coating on said beads has a binding affinity for proteins or peptides.

3. The method of claim 2, further comprising washing said recovered beads with a washing liquid to remove unbound materials from said beads.

4. The method of claim 2, further comprising eluting said bound proteins or peptides from said beads.

5. The method of claim 2, wherein said magnetically responsive beads have a magnetizable core.

6. The method of claim 1, wherein said pendant hydrophobic groups are hydrocarbon groups.

7. The method of claim 1, wherein said pendant hydrophobic groups are C₁₈ hydrocarbon groups.

8. A method of recovering a protein or peptide from a sample, said method comprising the steps of:

providing a solution containing said sample;

contacting said solution with an amount of magnetically responsive beads, said beads having an outer surface with a polymeric coating having a binding partner with a binding affinity for said protein or peptide, said beads being contacted with said solution for sufficient time and under conditions for said binding partner to bind said protein or peptide to said beads, wherein said coating or said binding partner on said beads is reactive with said protein or peptide and forms a covalent chemical bond with said protein or peptide;

separating said beads from said solution; and

cleaving said proteins or peptides from said beads.

9. A method of recovering a plurality of target components from a sample, said method comprising the steps of:

providing a solution of said sample containing first and second target components;

contacting said solution with an amount of first magnetically responsive beads comprising a coating with a specific binding affinity for a first target component, and an amount of second magnetically responsive beads having a coating with a specific binding affinity for a second target component,

simultaneously contacting said beads with said solution for sufficient time to bind said first and second target components to said first and second magnetically responsive beads, respectively; and

recovering said beads from said solution.

10. The method of claim 9, wherein said target component is a protein or peptide.

11. The method of claim 9, further comprising simultaneously eluting said first target component and said second target component from said beads.

12. A method of recovering a target component from a sample, said method comprising the steps of:

providing a solution and a support medium in said solution, said support medium containing said sample;

contacting said solution with an amount of magnetically responsive beads, said beads having an outer surface with a polymeric coating having a binding affinity for said target component,

applying an electric current to said solution to cause said target component to diffuse from said support medium into said solution, said beads being contacted with said solution for sufficient time to bind said target component to said beads; and

recovering said beads from said solution.

13. The method of claim 12, comprising applying a first electrode and a second electrode in said solution, positioning said beads and said support medium between said electrodes, and applying said electric current to cause said target component to diffuse toward said beads and attach to said beads.

14. The method of claim 13, wherein said beads are in the form of a bed, said method comprising positioning said bed of beads between said support medium and at least one of said electrodes.

15. The method of claim 13, comprising providing a permeable membrane between said electrodes, wherein said bed of beads is supported on said permeable membrane.

16. The method of claim 12, wherein said target component is a protein or peptide.

17. The method of claim 12, wherein said support medium is an electrophoresis gel.

18. The method off claim 17, wherein said electrophoresis gel is from a two-dimensional electrophoresis gel.

19. A method of recovering a target component comprising the steps of:

contacting a solution or dispersion of a target component with an amount of magnetically responsive beads having a coating with a binding affinity for said target component, said contacting step being for sufficient time for said target component to bind to said beads;

aspirating said solution or dispersion containing said beads through a tube having a filter device and collecting said beads on said filter device;

washing said beads inside said tube by passing a wash solution through said tube, and

eluting said target component from said beads.

20. The method of claim 19, providing an electromagnetic field around said tube to retain said beads in said tube during said eluting step.

21. The method of claim 20, wherein said tube includes a magnet surrounding said tube for providing said electromagnetic field.

22. The method of claim 21, wherein said magnet is slidable on said tube, said method comprising sliding said magnet proximate said filter device during said eluting step.

23. The method of claim 20, further comprising transferring the tube to a second solution or dispersion of a target component after said eluting step, and deactivating said electromagnetic field to release said beads into said second solution or dispersion.

24. The method of claim 23, further comprising supplying a liquid through said tube to carry said beads into said second solution or dispersion.

25. The method of claim 19, wherein said filter member is a porous glass frit.

26. The method of claim 19, comprising:

inserting a first end of a tube into said solution, wherein said filter member is mounted within said tube; and

aspirating said solution from a second end of said tube to collect said beads on said filter member.

27. The method of claim 26, further comprising subjecting said beads to an electromagnetic field to retain said beads in said tube and removing said electromagnetic field to separate said beads on said frit.

28. The method of claim 27, comprising inserting said tube into a wash liquid, deactivating said electromagnetic field to detach said beads from said tube and to disperse said beads in said wash liquid, activating said tube to induce said electromagnetic field and to reattach said beads to said tube, and removing said beads and said target component from said wash liquid.

29. The method of claim 28, wherein said eluting step comprises eluting said target component from said beads while said beads are attached to said tube.

30. The method of claim 29, wherein said eluting step comprises inserting said tube into an eluting solution, deactivating said tube to discontinue said electromagnetic field and disperse said beads in said eluting solution and elute said target component, activating said tube to produce said electromagnetic field to attached said beads to said tube, and removing said tube and beads from said eluting solution and eluted target component.

31. The method of claim 19, further comprising positioning an open end of said tube in an inlet of an analytical device;

and

eluting said target component from said beads retained in said tube directly into said analytical device and analyzing said target component.

32. A method of continuously recovering a target component from a liquid sample comprising the steps of:

providing a liquid sample containing said target component in a sample vessel and contacting said liquid sample with an effective amount of magnetically responsive beads having a coating with a binding affinity for said target component, said contacting step being for sufficient time to enable said target component to bind to said coating on said beads;

transferring said beads to an eluting vessel containing a liquid eluting agent and eluting said target component from said beads; and

returning said beads to said sample vessel containing said liquid sample.

33. The method of claim 32, wherein said coating on said beads is selected from the group consisting of antibodies, antigens, hydrophobic coating materials, ligands and receptors.

34. The method of claim 32, comprising the step of transferring said beads from said sample vessel to a wash vessel and washing impurities from said beads prior to transferring to said eluting vessel.

35. The method of claim 32, further comprising the step of recovering said target component from said eluting agent in said eluting vessel.

36. The method of claim 32, further comprising the step of inserting a magnetizable probe into said sample vessel, magnetizing said probe to capture said beads, and retracting said probe and said beads from said sample vessel.

37. The method of claim 36, further comprising the step of transferring said probe and beads to said eluting vessel to elute said target component from said beads.

38. The method of claim 37, further comprising demagnetizing said probe to release said beads into said eluting agent.

39. The method of claim 38, further comprising magnetizing said probe after said eluting step to capture said beads from said eluting agent.

40. The method of claim 32, comprising providing a first magnetizable probe and a second magnetizable probe, said first and second probes being coupled to a support member, said support member being rotatable about an axis, wherein said sample contains a first amount of said beads and said eluting vessel contains a second amount of said beads, said method comprising the steps of:

moving said support member to a first position where said first probe is inserted into said sample vessel and said second probe is inserted in said eluting vessel;

magnetizing said first and second probes to capture said first amount of said beads in said sample vessel and said second amount of said beads in said eluting vessel, respectively;

raising said support member to remove said first and second probes from said sample vessel and said eluting vessel, respectively;

rotating said support member and lowering said support member to a second position where said first probe is inserted in said eluting vessel and said second probe is inserted in said sample vessel; and

demagnetizing said probes to release said first amount of said beads in said eluting vessel and to release said second amount of said beads in said sample vessel.

41. The method of claim 32, subsequent to returning said beads to said sample vessel, said method comprising further contacting said liquid with the beads in said sample vessel,

transferring said beads to said eluting vessel again, and

eluting said target component from said beads to said eluting agent in said eluting vessel.

42. The method of claim 32, further comprising repeating at least three times in sequence said steps of contacting said liquid to said beads, said transferring said beads to an eluting vessel, eluting said target component into said eluting vessel, and returning said beads to said sample vessel.

43. A method of claim 32, further comprising repeating said contacting step, said step of transferring said beads to said eluting vessel, said eluting step and said returning step a predetermined number of times, and thereafter recovering said target component from said eluting vessel.

44. A method of recovering a denatured protein from a sample, said method comprising the steps of:

providing a solution containing said denatured protein from said sample;

contacting said solution with an amount of magnetically responsive beads, said beads having an outer surface having a specific binding affinity for native protein, said beads being contacted with said solution for sufficient time to bind said denatured protein to said beads;

recovering said beads from said solution, and

eluting said denatured protein from said beads.

45. The method of claim 44, wherein said beads have a polymeric coating with pendant hydrophobic hydrocarbon groups.

46. The method of claim 44, wherein said beads have a polymeric coating with pendant hydrocarbon groups having about 18 carbon atoms.