Protein-colloidal metal-aminodextran coated particle and methods of preparation and use

• US 5,945,293 A
• Filed: 10/09/1997
• Issued: 08/31/1999
• Est. Priority Date: 10/09/1997
• Status: Expired due to Fees

• Current Assignee: Coulter International Corp. (Danaher Corporation)
• Original Assignee: Coulter International Corp. (Danaher Corporation)
• Inventors: Gordon, Kristie, Rodriguez, Carlos M., Burshteyn, Alexander, Maples, John A., Siiman, Olavi, Whitesell, James Keller
• Primary Examiner(s): STUCKER, JEFFREY J
• Application Number:
  US08/947,811
• Time in Patent Office: 691 Days
• Field of Search: 435/7.1, 435/7.2, 435/7.21, 435/7.24, 436/172, 436/518, 436/523, 436/524, 436/525, 436/526, 436/534, 436/548, 428/402, 428/403
• US Class Current: 435/7.24
• CPC Class Codes: G01N 33/5094 for blood cell populations red blood cells G01N33/80
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393
• CPC Class Codes: G01N 33/54393 Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
• CPC Class Codes: G01N 33/548 Carbohydrates, e.g. dextran
• CPC Class Codes: G01N 33/553 Metal or metal coated
• CPC Class Codes: Y10T 428/2982 Particulate matter [e.g., sphere, flake, etc.]
• CPC Class Codes: Y10T 428/2991 Coated

Abstract

A stable colloidal particle comprises a colloidal-sized core substrate having amine-reactive functional groups thereon with an aminodextran coating over its peripheral surface and a layer of colloidal-sized metallic solid overlaying the aminodextran coating. A linker comprising aminotrithiolate attached to the metallic solid has a free amino group to which a protein is attached by covalent bonding to the free amino group. Such novel particles are useful in flow cytometry, particularly useful in the simultaneous analyses of subpopulations of leukocytes. Methods of preparation of such particles, as well as methods of use are provided. These conjugates enable the simultaneous analyses of at least two different mutually exclusive subsets of white blood cells, based on the binding affinity of the conjugated protein.

Claims

1. A stable colloidal particle comprising:

(a) a colloidal-sized core substrate having amine-reactive functional groups thereon;

(b) an aminodextran coating over the peripheral surface of said substrate;

(c) a layer of colloidal-sized metallic solid overlaying said aminodextran coating, wherein the metal for said metallic solid is selected from the group consisting of metals which can be reduced from the ionic state to the metal(0) state by the aminodextran coating of said core;

(d) an aminotrithiolate linker attached to said metallic solid, said linker having a free amino group; and

(e) a protein attached to said linker by covalently bonding to said free amino group.

2. The particle according to claim 1, wherein said core substrate (a) is polystyrene.

3. The particle according to claim 2, wherein said core substrate (a) ranges in size from between about 0.2 to about 5.0 microns in diameter.

4. The particle according to claim 1, wherein said aminodextran coating (b) is covalently bonded to said core substrate (a) by covalent bonds between the free amino groups of said aminodextran and said amine-reactive functional groups of said substrate.

5. The particle according to claim 4, wherein said aminodextran coating (b) is selected from the group consisting of aminodextrans in which the degree of diamine substitution in said aminodextran is in the range of 1/40-1/35 compared to a maximum theoretical value of 1/2.5.

6. The particle according to claim 5, wherein said diamine substitution in said aminodextran coating (b) is approximately 1/7 to 1/8.

7. The particle according to claim 1, wherein said metallic solid (c) is selected from the group consisting of metals which form metal ions or metal ion complexes which have a reduction potential of +0.7 volts or higher.

8. The particle according to claim 7, wherein said metallic solid is gold.

9. The particle according to claim 7, wherein said metallic solid is silver.

10. The particle according to claim 1 wherein said linker is tris(3-mercaptopropyl)-N-glycylaminomethane.

11. The particle according to claim 1, wherein said aminotrithiolate linker (d) is covalently bonded to said colloidal metal-aminodextran coated substrate by covalent metal-sulfur bonds.

12. The particle according to claim 11 wherein said colloidal metal is gold.

13. The particle according to claim 1, wherein said protein (e) is an antibody.

14. The particle according to claim 13, wherein said antibody is selected from the group consisting of an anti-CD4 antibody and an anti-CD8 antibody.

15. The particle according to claim 1, wherein said protein (e) is covalently bonded to said aminotrithiolate linker-colloidal metal-aminodextran coated substrate by covalent bonding of the free amino groups on the aminotrithiolate linker which are activated with maleimide in sulfo-SMCC, with sulfhydryl groups on the 2-iminothiolane activated protein.

16. The particle according to claim 1 wherein:

(a) said colloidal-sized core substrate comprises a colloidal-sized polystyrene microparticle;

(b) said aminodextran coating comprises a 1×

-aminodextran coating over the peripheral surface of said polystyrene particle;

(c) said layer of colloidal-sized metallic solid comprises silver or gold; and

(d) said protein comprises an anti-CD4 antibody or an anti-CD8 antibody covalently bonded to said free amino group.

17. The particle according to claim 1, which comprises

(a) a colloidal-sized polystyrene particle;

(b) a 1×

-aminodextran coating over the peripheral surface of said polystyrene particle;

(c) a layer of colloidal-sized silver overlaying said aminodextran coating;

(d) a 5×

-aminodextran linker having a free amino group; and

(e) an anti-CD4 antibody attached to said linker by covalently bonding to said free amino group.

18. A method for the simultaneous quantitative determination of two or more subpopulations of white blood cells in a biological solution/suspension containing both red blood cells and white blood cells, said method comprising the steps of:

(a) mixing with said biological solution/suspension, at least two different stable colloidal particles of claim 1, wherein each different particle contains a different said protein which binds to a different epitope on a subpopulation of said white blood cells, said mixing occurring for a time sufficient to permit the binding of said particles to each said subpopulation of cells;

(b) lysing said red blood cells in said biological solution/suspension, and quenching said cells in said mixture (a);

(c) analyzing said mixture of step (b) in an instrument that distinguishes between subpopulations of white blood cells bound by each said different colloidal particles, thereby simultaneously quantitatively enumerating at least two mutually exclusive subpopulations of said white blood cells.

19. The method according to claim 18 wherein said biological fluid is whole blood.

20. The method according to claim 18 wherein the subpopulations are selected from the group consisting of T lymphocytes, B lymphocytes, granulocytes, and monocytes.

21. The method according to claim 20 wherein said lymphocytes are CD4⁺ lymphocytes and CD8⁺ lymphocytes.

22. The method according to claim 18 wherein each said protein is an antibody.

23. The method according to claim 18 wherein said instrument distinguishes between cells by light scattering, conductivity and volume.

24. A method of preparing a stable colloidal particle which comprises the steps of:

(a) covalently binding an aminodextran to aldehyde functionalized polystyrene particles to provide aminodextran coated polystyrene particles;

(b) crosslinking additional aminodextran to the particles of step (a) by the use of a selected crosslinking agent and reducing the unstable bonds with a reducing agent;

(c) reacting said aminodextran-coated particles with an excess of metal salt in an aqueous medium at an elevated temperature, in the presence of a second reducing agent, resulting in colloidal metal-aminodextran coated particles;

(d) attaching an aminotrithiolate linker having a free amino group to said colloidal metal-aminodextran coated particles, which linker allows conjugation of proteins to the surface of the metal-colloid coated particles;

(e) activating the amino group of the linker on the colloidal metal-coated particles with a bifunctional crosslinking agent;

(f) activating a selected protein with an amino binding reagent which provides a sulfhydryl group;

(g) purifying each said activated component (e) and (f); and

(h) mixing a sufficient amount of an aqueous solution of said activated protein to saturate reactive groups on the particle with an aqueous suspension of said activated particle at room temperature for a sufficient period of time for conjugation between said protein and particle to occur, resulting in a stable colloidal particle.

25. The method according to claim 24, wherein said protein is an antibody.

26. The method according to claim 24, wherein the linker is 5×

aminodextran and said metal salt is silver(I).

27. The method according to claim 24, wherein said metal salt is gold(III).

28. The method according to claim 26, wherein said aminodextran linker is attached to said colloidal metal-aminodextran-polystrene particle by adsorption and crosslinking with glutaraldehyde.

29. The method according to claim 27, wherein said linker forms strong covalent bonds between the sulfur groups on the linker and the gold colloid surface.

30. The method according to claim 24 wherein the temperature of step (d) is in the range of between about 80 to about 100°

C.

31. The method according to claim 24 wherein the bifunctional crosslinking agent of step (e) which links the aminotrithiol-colloidal metal-aminodextran-polystyrene particle to the protein is sulfosuccinimidyl 4- N-maleimidomethyl!cyclohexane-1-carboxylate.

32. The method according to claim 24 wherein the binding reagent of step (f) is selected from the group consisting of naturally occurring sulfhydryl groups of the protein, and an amino-reactive sulfhydryl introducing reagent.