Manipulation of colloids for facilitating magnetic separations

• US 5,108,933 A
• Filed: 08/04/1989
• Issued: 04/28/1992
• Est. Priority Date: 09/16/1988
• Status: Expired due to Term

• Current Assignee: Veridex LLC (Johnson & Johnson)
• Original Assignee: Immunicon Corporation
• Inventors: Liberti, Paul A., Gohel, Dhanesh I.
• Primary Examiner(s): Yarbrough, Amelia Burgess
• Assistant Examiner(s): Preston, David R.
• Application Number:
  US07/389,697
• Time in Patent Office: 998 Days
• Field of Search: 435/4, 435/7, 435/4.6, 435/7.15, 435/971, 436/519, 436/520, 436/824, 436/526, 436/525, 436/501, 436/518, 436/524, 436/528, 436/533, 436/534
• US Class Current: 436/501
• CPC Class Codes: B03C 1/01 by addition of magnetic adjuvants
• CPC Class Codes: G01N 2446/20 the magnetic material being present in the particle core
• CPC Class Codes: G01N 2446/30 the magnetic material being dispersed in the polymer composition before their conversion into particulate form
• CPC Class Codes: G01N 33/54333 Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
• CPC Class Codes: Y10S 435/971 Capture of complex after antigen-antibody reaction
• CPC Class Codes: Y10S 436/824 Immunological separation techniques

Abstract

Colloidal particles are converted into magnetic microagglomerates via manipulation of their colloidal properties, thereby facilitating their separation from solution.

Claims

1. A method for magnetically separating a target substance from a test medium suspected of containing said substance said target substance being one member of a specific binding pair, said method comprising:

adding to said test medium agglomerable and resuspendable colloidal particles, said particles when unagglomerated, being capable of stable suspension in said test medium;

forming a magnetic agglomerate comprising said colloidal particles and the product of a biospecific affinity reaction between said target substance and the other member of said specific binding pair, at least one member of said specific binding pair comprising a bioactive protein, said protein, or a bioactive protein capable of binding specifically to at least one member of said specific binding pair, being attached to said colloidal particles; and

separating said agglomerate from said test medium the suspension stability of said particles being substantially undisturbed when subjected to an externally applied magnetic field.

2. A method as claimed in claim 1, wherein the colloidal particles added to said test medium comprise said protein capable of binding specifically to at least one member of said specific binding pair.

3. A method as claimed in claim 2, wherein the colloidal particles added to said test medium comprise a protein capable of binding to said bioactive protein member of said binding pair.

4. A method as claimed in claim 2, wherein said agglomerate is formed by binding of said bioactive protein member of said binding pair to said target pair.

5. A method as claimed in claim 2, wherein an additional binding substance capable of binding specifically to said target substance is contacted with said test medium.

6. A method as claimed in claim 5, wherein said agglomerate is formed by binding of said additional binding substance to said target substance.

7. A method as claimed in claim 5, which includes contacting said test medium with at least one agglomerating agent to promote forming said agglomerate.

8. A method as claimed in claim 7, wherein said agglomerate is formed by contacting said test medium with a non-specific agglomerating agent selected from the group consisting of an alkali metal salt, an alkaline earth metal salt, a colloidal material, ammonium sulfate, an agglomerating solvent, or polyethylene glycol.

9. A method as claimed in claim 7, wherein said agglomerating agent contacted with said test medium comprises magnetic material and is incorporated into said agglomerate.

10. A method as claimed in claim 9, wherein said agglomerating agent contacted with said test medium comprises particulate magnetite having a particle size from about 0.01 to about 0.20 microns.

11. A method as claimed in claim 9, wherein said agglomerating agent contacted with said test medium comprises colloidal material.

12. A method as claimed in claim 7, wherein a specific agglomerating agent is added to said test medium.

13. A method as claimed in claim 2, wherein said agglomerate is formed by contacting said test medium with at least one agglomerating agent.

14. A method as claimed in claim 13, wherein said agglomerate is formed by contacting said test medium with a non-specific agglomerating agent selected from the group consisting of an alkali metal salt, an alkaline earth metal salt, a colloidal material, ammonium sulfate, an agglomerating solvent, or polyethylene glycol.

15. A method as claimed in claim 13, wherein said agglomerating agent contacted with said test medium comprises magnetic material and is incorporated into said agglomerate.

16. A method as claimed in claim 15, wherein said agglomerating agent contacted with said test medium comprises particulate magnetite having a particle size from about 0.01 to about 0.20 microns.

17. A method as claimed in claim 15, wherein said agglomerating agent contacted with said test medium comprises colloidal material.

18. A method as claimed in claim 1, wherein a magnetic field is applied to separate said agglomerate from said test medium.

19. A method as claimed in claim 18, wherein colloidal particles comprising magnetic material are added to said test medium.

20. A method as claimed in claim 19, wherein colloidal particle comprising magnetite and having a particle size from about 0.01 to about 0.20 microns are added to said test medium.

21. A method as claimed in claim 19, wherein colloidal particles added to said test medium are attached to a member of said binding pair comprising said bioactive protein.

22. A method as claimed in claim 21, wherein said agglomerate is formed by binding of said bioactive protein member of said binding pair to said target substance.

23. A method as claimed in claim 21, wherein an additional binding substance capable of binding specifically to said target substance is contacted with said test medium.

24. A method as claimed in claim 23, wherein said agglomerate is formed by binding of said additional binding substance to said target substance.

25. A method as claimed in claim 23 which includes adding to said test medium at least one agglomerating agent to promote forming said agglomerate.

26. A method as claimed in claim 25 wherein there is added to said test medium a non-specific agglomerating agent selected from the group consisting of an alkali metal salt, an alkaline earth metal salt, a colloidal material, ammonium sulfate, a non-solvent for said agglomerate or polyethylene glycol.

27. A method as claimed in claim 25, wherein said non-specific agglomerating agent added to said test medium comprises magnetic material and is incorporated into said agglomerate.

28. A method as claimed in claim 27, wherein said non-specific agglomerating agent contacted with said test medium comprises particulate magnetite having a particle size from about 0.01 to about 0.20 microns.

29. A method as claimed in claim 27, wherein said non-specific agglomerating agent contacted with said test medium comprises colloidal material.

30. A method as claimed in claim 25, wherein a specific agglomerate agent is added to said test medium.

31. A method as claimed in claim 1, wherein colloidal particles added to said test medium comprise said member of said binding pair comprising said bioactive protein.

32. A method as claimed in claim 31, wherein said agglomerate is formed by contacting said test medium with at lest one agglomerating agent.

33. A method as claimed in claim 32, wherein said agglomerate is formed by contacting said test medium with a non-specific agglomerating agent selected from the group consisting of an alkali metal salt, an alkaline earth metal salt, a colloidal material, ammonium sulfate, a non-solvent for said agglomerate or polyethylene glycol.

34. A method as claimed in claim 32, wherein a specific agglomerating agent is added to said test medium.

35. A method as claimed in claim 32, wherein said agglomerating agent contacted with said test medium comprises magnetic material and is incorporated into said agglomerate.

36. A method as claimed in claim 35, wherein said agglomerating agent contacted with said test medium comprises particulate magnetite having a particle size from about 0.01 to about 0.20 microns.

37. A method as claimed in claim 35, wherein said agglomerating agent contacted with said test medium comprises colloidal material.

38. A method for magnetically separating a substance, selected from the group consisting of antigens, antibodies, biological factors, cells, cell components and, viruses, from a biological fluid suspected of containing said substance, each said substance having at least one characteristic determinant associated therewith, said process comprising:

contacting said biological fluid with agglomerable and resuspendable colloidal magnetic particles bearing a proteinaceous receptor capable of binding specifically to a determinant of said substance, said particles, when unagglomerated, being capable of stable suspension in said biological fluid, the suspension stability of said particles being substantially undisturbed when subjected to an externally applied magnetic field;

subjecting said biological fluid to conditions causing binding between said receptor and said determinant;

forming a magnetic agglomerate comprising said particles and any of said substance present in said biological fluid; and

applying a magnetic field to separate said magnetic agglomerate from said biological fluid.

39. A method as claimed in claim 38, wherein said agglomerate is formed as a result of binding between said receptor and said determinant.

40. A method as claimed in claim 38, wherein said agglomerate is formed by adding to said biological fluid at least one non-specific agglomerating agent.

41. A method for magnetically separating a substance, selected from the group consisting of antigens, antibodies, biological factors, cells, cell components and, viruses, from a biological fluid suspected of containing said substance and determining the presence or quantity of said substance in said biological fluid, each said substance having at least one characteristic determinant associated therewith, said process comprising:

adding to said biological fluid agglomerable and resuspendable colloidal magnetic particles bearing a proteinaceous receptor capable of binding specifically to a determinant of said substance, and a labeled binding substance having specific binding affinity for the substance to be separated and determined, said particles, when unagglomerated, being capable of stable suspension in said biological fluid, the suspension stability of said particles being substantially undisturbed when subjected to an externally applied magnetic field;

subjecting said biological fluid to conditions causing binding between said receptor and said determinant;

contacting said biological fluid with an agglomerating agent, thereby to yield an agglomerate comprising said colloidal magnetic particles bound to said substance;

applying a magnetic field to separate said biological fluid into a portion comprising said magnetic agglomerate and a portion substantially free of said magnetic agglomerate; and

detecting or quantitating the labeled binding substance in one of said portions as a measure of the presence of quantity of said substance in said biological fluid.

42. A method as claimed in claim 41, wherein said biological fluid is contacted with at least one non-specific agglomerating agent.

43. A method as claimed in claim 41, wherein agglomerable and resuspendable colloidal magnetic particles comprising a specific binding moiety having binding affinity for said receptor are added to said biological fluid.

44. A method for magnetically separating a substance, selected from the group consisting of antigens, antibodies, biological factors, cells, cell components and, viruses, from a biological fluid suspected of containing said substance, each said substance having at least one characteristic determinant associated therewith, said process comprising:

adding to said biological fluid agglomerable and resuspendable colloidal magnetic particles bearing a proteinaceous receptor capable of binding specifically to a determinant of said substance, said particles, when unagglomerated, being capable of stable suspension in said biological fluid, the suspension stability of said particle being substantially undisturbed when subjected to an externally applied magnetic field;

subjecting said biological fluid to conditions causing binding between said receptor and said determinant;

adding at least one agglomerating agent to said biological fluid to form a magnetic agglomerate comprising said colloidal magnetic particles bound to said substance; and

applying a magnetic field to separate said magnetic agglomerate from said biological fluid.

45. A method as claimed in claim 44, wherein said agglomerate is formed by adding to said biological fluid at least one non-specific agglomerating agent.

46. A method for magnetically separating a substance, selected from the group consisting of antigens, antibodies, biological factors, cells, cell components and viruses, from a biological fluid suspected of containing said substance, each said substance having at least one characteristic determinant associated therewith, said process comprising:

adding to said biological fluid a receptor capable of binding specifically to a determinant of said substance and having one member of a specific binding pair associated therewith;

subjecting said biological fluid to conditions causing binding between said receptor and said determinant to yield an agglomerate comprising said receptor bound to said substance;

contacting said biological fluid with agglomerable and resuspendable protein-bearing colloidal magnetic particles said protein comprising the other member of said specific binding pair, said particles, when unagglomerated, being capable of stable suspension in said biological fluid, the suspension stability of said particles being substantially undisturbed when subject to an externally applied magnetic field, said contacting occurring under conditions causing binding between said members, thereby to render said agglomerate magnetic; and

applying a magnetic field to separate said magnetic agglomerate from said biological fluid.

47. A method as claimed in claim 46, wherein said one member of said specific binding pair is an antigen and said other member of said pair is an antibody which immunospecifically interacts with said antigen.

48. A method as claimed in claim 46, wherein said biological fluid is contacted with at least one non-specific agglomerating agent.

49. A method for magnetically separating a substance, selected from the group consisting of antigens, antibodies, biological factors, cells, cell components and viruses, from a biological fluid suspected of containing said substance, and determining the presence or quantity of said substance in said biological fluid, each substance having at least two characteristic determinants associated therewith, said process comprising:

adding to said biological fluid a receptor capable of binding specifically to a first of said determinants and having one member of a specific binding pair associated therewith, and a labeled binding substance having specific binding affinity for a second of said determinants;

subjecting said biological fluid to conditions causing binding between said receptor and said first determinant and between said labeled binding substance and said second determinant;

contacting said biological fluid with agglomerable and resuspendable, protein-bearing colloidal magnetic particles said protein comprising the other member of said specific binding pair, said particles, when unagglomerated, being capable of stable suspension in said biological fluid, the suspension stability of said particles being substantially undisturbed when subjected to an externally applied magnetic field, said contacting occurring under conditions causing binding between said members;

adding to said biological fluid an agglomerating agent thereby to yield a magnetic agglomerate comprising said colloidal particles bound to said substance;

applying a magnetic field to separate said biological fluid into a portion comprising said magnetic agglomerate and a portion substantially free of said magnetic agglomerate; and

detecting or quantitating the labeled binding substance in one of said portions as a measure of the presence or quantity of said substance in said biological fluid.

50. A method as claimed in claim 49, wherein agglomerable and resuspendable colloidal magnetic particles comprising a specific binding moiety having binding affinity for said receptor are added to said biological fluid.

51. A method as claimed in claim 49, wherein said one member of said specific binding pair is an antigen and said other member of said pair is an antibody which immunospecifically interacts with said antigen.

52. A method as claimed in claim 49, wherein said biological fluid is contacted with at least one non-specific agglomerating agent.

53. A method for magnetically separating a substance, selected from the group consisting of antigens, antibodies, biological factors, cells, cell components and viruses, from a biological fluid suspected of containing said substance, each substance having at lest one characteristic determinant associated therewith, said process comprising:

adding to said biological fluid a receptor capable of binding specifically to a determinant of said substance and having one member of a specific binding pair associated therewith;

subjecting said biological fluid to conditions causing binding between said receptor and said determinant;

contacting said biological fluid with agglomerable and resuspendable, protein-bearing colloidal particles said protein comprising the other member of said specific binding pair, said particles, when unagglomerated, being capable of stable suspension in said biological fluid, the suspension stability of said particles being substantially undisturbed when subjected to an externally applied magnetic field, said contacting occurring under conditions causing binding between said members;

contacting said biological fluid with at least one agglomerating agent, thereby to form an agglomerate comprising said magnetic colloidal particles bound to said substance; and

applying a magnetic field to separate said magnetic agglomerate from said biological fluid.

54. A method as claimed in claim 53, wherein said agglomerate is formed by contacting said biological fluid with a non-specific agglomerating agent.

55. A method as claimed in claim 53, wherein said non-specific agglomerating agent added to said biological fluid comprises colloidal magnetic material.

56. A method for determining the presence of a substance in a biological fluid suspected of containing said substance, in excess of a predetermined level, said substance being selected from the group of antigens, antibodies, biological factors, cells, cell components and, viruses, each said substance having at least one characteristic determinant associated therewith, said process comprising:

contacting said biological fluid with agglomerable and resuspendable colloidal magnetic particles bearing a proteinaceous receptor capable of binding specifically to a determinant of said substance so as to form an agglomerate, said particles, when unagglomerated, being capable of stable suspension in said biological fluid, the suspension stability of said particles being substantially undisturbed when subjected to an externally applied magnetic field, the amount of said particles being sufficient that any said agglomerate so formed is separable from said biological fluid under the influence of an externally-applied magnetic field when the amount of said substance in said biological fluid exceeds said predetermined level;

subjecting said biological fluid to conditions causing binding between said receptor and said determinant;

applying an external magnetic field to said biological fluid; and

determining the occurrence of separated agglomerate as an indication of the presence of said substance in excess of said predetermined amount.

57. A method as claimed in claim 56, wherein the occurrence of separated agglomerate is visually determined.

58. A method for determining the presence of a protein-containing substance in a biological fluid suspected of containing said substance, in excess of a pre-determined level, said substance being selected from the group of antigens, antibodies, biological factors, cells, cell components and, viruses, each said substance having at least one characteristic monovalent determinant associated therewith, said process comprising:

contacting said biological fluid with a receptor capable of binding specifically to said determinant, and with agglomerable and resuspendable colloidal magnetic particles bearing said protein-containing substance in a form providing a plurality of said determinants associated with said particles, said particles, when unagglomerated, being capable of stable suspension in said biological fluid, the suspension stability of said particles being substantially undisturbed when subjected to an externally applied magnetic field, the relative amounts of said receptor and said particles being sufficient to form an agglomerate which is magnetically separable from said biological fluid under the influence of an externally-applied magnetic field when the amount of said substance in said biological fluid is below said pre-determined level;

subjecting said biological fluid to conditions causing binding between said receptor and said determinants;

applying an external magnetic field to said biological fluid; and

determining the occurrence of separated agglomerate as an indication of the presence of said substance in excess of said predetermined level.

59. A method as claimed in claim 58, wherein the occurrence of separated agglomerate is visually determined.

60. A method as claimed in claim 58, wherein determining the occurrence of separated agglomerate is facilitated by labeling said receptor with a detectable label selected from the group consisting of an enzyme, a radioisotope or a fluorescent or phosphorescent substance.