US5945293A
Expired / Lapsed
US
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Protein-colloidal metal-aminodextran coated particle and methods of preparation and use
Assignee: Beckman Coulter Inc
•
Filing: 1997-10-09
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Granted: 1999-08-31
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Expires: 2017-10-09
Abstract
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.
Inventors
- 👤 Olavi Siiman
- 👤 Kristie Gordon
- 👤 Carlos M. Rodriguez
- 👤 Alexander Burshteyn
- 👤 John A. Maples
- 👤 James Keller Whitesell
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-07 02:31
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/5005; G01N33/5094; G01N33/53; G01N33/543; G01N33/54313; G01N33/54353; G01N33/54393; G01N33/544; G01N33/548; G01N33/551; G01N33/553; Y; Y10; Y10T; Y10T428/00; Y10T428/29; Y10T428/2982; Y10T428/2991
Claims Analysis
Total Claims: 34
Claim 1
Independent Claim
103 words
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.
Claim 2
Dependent Claim
103 words
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.
Claim 3
Dependent Claim
13 words
The particle according to claim 1, wherein said core substrate (a) is polystyrene.
Claim 4
Dependent Claim
24 words
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.
Claim 5
Dependent Claim
38 words
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.
Claim 6
Dependent Claim
43 words
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.
Claim 7
Dependent Claim
20 words
The particle according to claim 5, wherein said diamine substitution in said aminodextran coating (b) is approximately 1/7 to 1/8.
Claim 8
Dependent Claim
37 words
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.
Claim 9
Dependent Claim
12 words
The particle according to claim 7, wherein said metallic solid is gold.
Claim 10
Dependent Claim
12 words
The particle according to claim 7, wherein said metallic solid is silver.
Claim 11
Dependent Claim
11 words
The particle according to claim 1 wherein said linker is tris(3-mercaptopropyl)-N-glycylaminomethane.
Claim 12
Dependent Claim
24 words
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.
Claim 13
Dependent Claim
12 words
The particle according to claim 11 wherein said colloidal metal is gold.
Claim 14
Dependent Claim
13 words
The particle according to claim 1, wherein said protein (e) is an antibody.
Claim 15
Dependent Claim
23 words
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.
Claim 16
Dependent Claim
47 words
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.
Claim 17
Dependent Claim
63 words
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.
Claim 18
Dependent Claim
61 words
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.
Claim 19
Dependent Claim
152 words
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.
Claim 20
Dependent Claim
13 words
The method according to claim 18 wherein said biological fluid is whole blood.
Claim 21
Dependent Claim
23 words
The method according to claim 18 wherein the subpopulations are selected from the group consisting of T lymphocytes, B lymphocytes, granulocytes, and monocytes.
Claim 22
Dependent Claim
17 words
The method according to claim 20 wherein said lymphocytes are CD4 + lymphocytes and CD8 + lymphocytes.
Claim 23
Dependent Claim
13 words
The method according to claim 18 wherein each said protein is an antibody.
Claim 24
Dependent Claim
18 words
The method according to claim 18 wherein said instrument distinguishes between cells by light scattering, conductivity and volume.
Claim 25
Independent Claim
214 words
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.
Claim 26
Independent Claim
214 words
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.
Claim 27
Dependent Claim
12 words
The method according to claim 24, wherein said protein is an antibody.
Claim 28
Dependent Claim
18 words
The method according to claim 24, wherein the linker is 5× aminodextran and said metal salt is silver(I).
Claim 29
Dependent Claim
12 words
The method according to claim 24, wherein said metal salt is gold(III).
Claim 30
Dependent Claim
23 words
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.
Claim 31
Dependent Claim
25 words
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.
Claim 32
Dependent Claim
24 words
The method according to claim 24 wherein the temperature of step (d) is in the range of between about 80 to about 100° C.
Claim 33
Dependent Claim
27 words
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.
Claim 34
Dependent Claim
33 words
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.
References Cited (Backward Citations)
GB1561042A
Cited
US5125737A
Cited
US5223398A
Cited
US5231005A
Cited
US5466609A
Cited
US5639620A
Cited
WO1992009682A1
Cited
US5248772A
Cited
US5552086A
Cited
US5492833A
Cited
US5527713A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2002-08-30 | 2003-03-01 | 2003-08-31 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2006-08-30 | 2007-03-01 | 2007-08-31 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2010-08-30 | 2011-03-01 | 2011-08-31 | $7700.00 | Window Closed / Lapsed |