PROTEIN-COLLOIDAL METAL-AMINODEXTRAN COATED PARTICLE AND METHODS OF PREPARATION AND USE

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 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.

Description: PROTEIN-COLLOIDAL METAL-AMINODEXTRAN COATED PARTICLE AND METHODS OF PREPARATION AND USE Field of the Invention This invention generally relates to novel flow cytometry, and cell separation, reagents, and more particularly to novel particles, in which a protein is conjugated to a colloidal-metal coated, aminodextran coated substrate and methods of preparation and use thereof. Background of the Invention The technique of flow cytometry provides investigators with data on physical parameters of cells which can be used to separate various classes of cells, e.g., to classify white blood cells (leukocytes) into three major groups, namely, lymphocytes, monocytes and granulocytes. Other techniques have been described to identify additional leukocyte populations, for example, T-cells, B-cells, cytotoxic cells and suppressor cells, neutrophil, eosinophil, and basophils. See, e.g., Flow Cytometry and Sorting, page 371; edited by Melamed, Mullaney, and Mendelsohn, 1979, John Wiley & Sons, N. Y., N. Y.; U.S. Patent No. 5,125,737; U.S. Patent No. 5,492,833; U.S. Patent No. 5,223,398; U.S. Patent No. - - 5,231,005. The enumeration of subclasses using antibody-coated polystyrene latex beads to target specific white blood cell subpopulations in whole blood, hence, producing a change (a shift) in the targeted cell volume, direct or low frequency current (DC) or high frequency (RF) conductivity, and light scatter (S) has been described. See, e.g., PCT Publication WO92/09682 published June 11, 1992; J.C. Hudson et al, Cytometry, ^2_:150 (1995), U.S. Patent No. 5,639,620 and International Patent application No. W095/24631, published September 14, 1995. In fact, these latter changes can be detected on the commercially available hematology instruments, such as the COULTER ® STKS™ instrument equipped with VCS technology. Such instruments can calculate the percentage of shifted cell population in the total population of white blood cells. All of the above bead-induced shift methods use a single type of polystyrene bead conjugated to an antibody to enumerate subpopulations of lymphocytes one at a time. The use of beads composed of inorganic materials to extend shifts has only recently been described. See, e.g., U.S. Patent No. 5,466,609; International Patent Application No. W09524631; and U.S. Patent No. 5,552,086. Specifically, gold/silver colloid coated polystyrene- aminodextran beads, their preparation and characterization have been described. See, e.g., U.S. Patent No. 5,248,772 and U. S. Patent No. 5,552,086. Current methods of obtaining lymphocyte differentials involve the use of fluorescence markers and measurement of forward light scatter, side light scatter and fluorescence intensity or the use of antibody on beads and measurement of median angle light scatter, DC, and RF. Heretofore, the use of antibody on beads has " involved only a single type of bead coated with antibody at any one time to analyze for a single subpopulation of lymphocytes. See also, P. K. Horan and L.L. Wheeless, Jr., Science, 1_98_: 149-157 (1977); and British Patent No. 1561042, published February 13, 1980. On the other hand, conventionally practiced flow cytometry with fluorescence markers simultaneously uses a number of fluorescent- labeled antibodies (2-6) to analyze for various subpopulations of leukocytes. At present, there is no method that allows a sample of blood to be analyzed using the antibody on bead method, such that subpopulations of lymphocytes may be simultaneously enumerated. Thus, there exists a need in the art for compositions and methods which enable more efficient analysis of blood or other multicellular fluid materials . Summary of the Invention In one aspect, the invention provides 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) a 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. In another aspect, the invention provides methods for preparing the above-described particles, including methods which employ an aminotrithiol linker, to enable the conjugation of protein to the coated particles. In still another aspect, the invention provides methods for performing simultaneous quantitative determinations of two or more subpopulations of white blood cells in a biological solution or suspension, such as whole blood, using the above-described particles. Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof, reference being made to the accompanying figures. Brief Description of the Drawings Fig. 1 is a graph plotting electrophoretic mobility versus pH data for suspensions of aldehyde/sulfate polystyrene latex beads (PS) , uncoated or coated with aminodextran (Amdex) , gold colloid, aminotrithiolate linker, and anti-CD4 (T4) antibody or anti-CD8 (T8) antibody. Fig. 2 is a graph plotting the electrophoretic mobility versus pH data for suspensions of aldehyde/sulfate PS, uncoated or coated with 1,3- diaminopropane (DAP), sulfosuccinimidyl 4-[N- maleimidomethyl] cyclohexane-1-carboxylate (sulfo-SMCC) , and T4 or T8 antibody, all washed with lx phosphate buffered saline (PBS) only. Fig. 3 is a graph plotting the electrophoretic mobility versus pH data for suspensions of aldehyde/sulfate PS, uncoated or coated with sulfo-SMCC- DAP and T4 or T8 antibody, all washed with bovine serum albumin ("BSA") buffer and lxPBS. Fig. 4A is a scanning electron microscope (SEM) photograph of particles of the invention, comprising an T4 antibody linked via an aminotrithiol linker to a gold embedded, aminodextran coated, PS particle (T4- aminotrithiol-Au-Amdex-PS) at magnification X20,000. Fig. 4B is an SEM photograph of the particles of Fig. 4A at magnification X40,000. Fig. 4C is an SEM photograph of the particles of Fig. 4A at magnification X100,000. Fig. 5A is an SEM photograph of the particles similar to that of Fig. 4A, except that the metal colloid is silver (T4-Amdex-Ag-Amdex-PS) at magnification X18,500. Fig. 5B is an SEM photograph of the particles of Fig. 5A at magnification X10,000. Fig. 5C is an SEM photograph of the particles of Fig. 5A at magnification X50,000. Fig. 5D is an SEM photograph of the particles of Fig. 5A at magnification X100,000. Figs. 6A-6F illustrate matrices of the electronic sensing parameter (DC) versus Side Angle Light Scatter (SALS) for whole blood and various mixtures of whole blood with T4-Amdex-Ag-Amdex-PS particles, obtained with 633nm excitation, demonstrating the progressively shifted SALS of targeted T4+ lymphocytes with increasing titers (2, 5, 10, 20, and 40 μL, respectively) of particles. Figs. 7A-7L illustrate matrices of pairs of parameters (DC v. LMALS; DC v. UMALS; LMALS v. PMT1; UMALS v. SALS; DC v. SALS, RF v. SALS, respectively) for mixtures of whole blood and T4-Amdex-Ag-Amdex-PS (7A-7F] or conventional T4-DAP-PS (7G-7L) particles, obtained with 633nm excitation, showing the shifted location of targeted T4+ lymphocytes in each matrix. Figs . 8A-8L illustrate matrices of the same pairs of parameters as for Figs. 7A-7L, for mixtures of whole blood and T4-Amdex-Ag-Amdex-PS or T4-DAP-PS particles, obtained with 780nm excitation, showing the shifted location of targeted T4+ lymphocytes in each matrix. Figs. 9A-9F depict matrices of various pairs of parameters as indicated on the figures, for a mixture of whole blood and T4-DAP-PS particles, obtained with 633nm excitation, showing the shifted location of T4+ lymphocytes. Figs. 10A-10F depict matrices of various pairs of parameters for a mixture of whole blood and T4- aminotrithiol-Au-Amdex-PS particles, obtained with 633nm excitation, showing the shifted location of T4+lymphocytes . Figs. 11A-11F depict matrices of various pairs of parameters for a mixture of whole blood and T4-Amdex-Ag- Amdex-PS particles, obtained with 633 nm excitation, showing the shifted location of T4+ lymphocytes. Figs. 12A-12D illustrate 90° LS versus UMALS matrices for mixtures of four samples of whole blood and T4-DAP-PS particles, obtained with 633nm excitation, showing the shifted location, Rl, of T4+ lymphocytes. Figs. 13A-13E show 90° LS versus UMALS matrices for mixtures of whole blood and five increasing titers of a T4-aminotrithiol-Au-Amdex-PS particle suspension, obtained with 633nm excitation, showing the shifted location, Rl, of T4+ lymphocytes in each matrix. Figs. 14A-14F show 90° LS versus UMALS matrices for mixtures of three samples of whole blood with, left-hand- side, T8-DAP-PS and T4-aminotrithiol-Au-Amdex-PS particle suspensions; and, right-hand-side, T4-DAP-PS and T8- aminotrithiol-Au-Amdex-PS particle suspensions, obtained with 633nm excitation, showing the shifted locations of T8+(R1) and T4+(R2) lymphocytes on LHS and T4+(R1) and T8+(R2) lymphocytes on RHS . Figs. 15A-15F show matrices of various pairs of parameters for a mixture of whole blood with T4-DAP-PS and T8-Amdex-Ag-Amdex-PS particle suspensions, obtained with 633nm excitation, showing the shifted locations of T4+(R1) and T8+(R2) lymphocytes. Detailed Description of the Invention The present invention meets the need in the art by providing novel stable colloidal particles, in which a protein, preferably an antibody, is conjugated to a colloidal-metal coated, aminodextran coated substrate. This particle may be employed in flow cytometry methods which permit the efficient analysis and separation of different cell types in a biological material, provided that each different cell type has at least one unique, non-overlapping antigenic epitope. I. Definitions The following terms as used herein and in the claims may be defined as follows. A "histogram" is a graph of frequency distribution for a single variable, displayed as a two dimensional line graph with the variable plotted on the X axis and the frequency, designated as "number (#)", plotted on the Y axis. A histogram also is the abstract numerical tabulation of such a graph as represented within a computer or some other form of electrical circuit. "Matrix" is a graph of frequency distribution for two independent variables, displayed as a three dimensional contour graph with one variable plotted on the X axis, the second variable plotted on the Y axis, and frequency or count displayed as iso-count contours. For clarity, only one iso-count contour is displayed to illustrate population outlines. Matrix also is defined as the abstract numerical tabulation of such a graph as represented within a computer or some other form of electrical circuit. When describing a matrix herein, the X axis variable is listed first, followed by the Y axis variable . "Parameter" is synonymous with independent variable and refers to any of the simultaneous, independent measurements obtained from particles or cells being analyzed in a flow cytometer. The combination of two or more parameters, by some mathematical function, is defined as yielding another parameter. "Gating" is a filtering process utilized in constructing, from multi-parameter data, a histogram of one parameter, while interrogating one or more of the other parameters. For each event, which is the passage of a single blood cell through the flow cell and the generation of cell measurements by the parameter transducers, the value or measurement corresponding to each parameter that is to be utilized for gating is compared with one or two reference values, or thresholds, and "tested" for being below the threshold, above the threshold, or between the two thresholds. If this test yields a true result for all the gating parameters being considered, then the event is included in the histogram. Gating also can be utilized to construct a matrix. Thus, by utilizing gating, it is possible to simplify the " "* analysis and graphic representation of multi-parameter data. "DC" and "RF" are electronic sensing parameters which refer to the Coulter Principle of aperture impedance cell sensing. "DC" is the pulse peak information obtained from applying a direct or low- frequency current, such that the cell membrane is not penetrated and no current flows through the cell. The peak amplitude of the DC pulse is a function of cell volume. "RF" is the pulse peak information derived from the measurement obtained by applying a high-frequency current, such that the cell membrane is short-circuited and current flows through the cell. RF is a function of cell volume and internal conductivity. "LMALS" is Lower Median Angle Light Scatter, which is light received at the photodetector assembly scattered in or throughout the range of 10° - 20° from the axis of the incident light beam. "UMALS" is Upper Median Angle Light Scatter, which is light received at the photodetector assembly scattered in or throughout the range of 20° - 65° from the axis of the incident light beam. "SALS" or "90° LS" is Side Angle Light Scatter, which is light received at a photodetector assembly located in an approximate angular range of 90° +/- 20° from the axis of the incident light beam. The term "particle", also includes inter alia, microspheres, beads and spheres and such terms are interchangeable . "Antibody" is defined to include polyclonal antibodies from any native source, and native or recombinant monoclonal antibodies of classes IgG, IgM, IgA, IgD, and IgE, hybrid derivatives, humanized or - - chimeric antibodies, and fragments of antibodies including Fab, Fab', and F(ab') 2 . II. Compositions of the Invention The present invention provides a novel 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) a 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. In a preferred embodiment, the particles of the present invention comprise aminodextran coated polystyrene particles, which particles are also coated with a colloidal metal having a linking agent to which a crosslinker is attached. Protein, preferably antibody, is attached to the coated particles through the crosslinker to an amino group of the linker. These conjugates are particularly advantageous in that they enable the simultaneous analyses of at least two different subsets of white blood cells, for example, two subsets of T lymphocytes in a biological material containing white blood cells. A . The Colloidal Metal -Aminodextran Coa ted " ~ Substra te The coated substrate described above preferably has a core substrate (a) which is a polystyrene bead. Preferably the polystyrene bead ranges in size from between about 0.2 to about 5.0 microns in diameter (i.e., colloidal-sized) . Such polystyrene core substrates containing aldehyde and/or sulfate functional groups are commercially available, e.g., from Interfacial Dynamics Corporation, Portland Oregon. Of course, one of skill in the art may select other similar particles from other suppliers . This core substrate has an aminodextran coating over its peripheral surface. Preferably the aminodextran coating (b) is covalently bonded to the core substrate (a) by covalent bonds between the free amino groups of the aminodextran and the amine-reactive functional groups of the polystyrene substrate and further by crosslinking with an agent such as glutaraldehyde . The aminodextran which forms coating (b) may generally be characterized as having a degree of diamine substitution in the range of 1/40-1/35 (lX-aminodextran) compared to a maximum theoretical value of 1/2.5. More preferably, the diamine substitution in the aminodextran coating (b) is approximately 1/7 to 1/8 (5X-aminodextran) . An alternative to this coated substrate employs carboxy functionalized polystyrene particles as the core substrate, coated with aminodextran by EDAC coupling as described in US Patent No. 5,639,620. The coated substrate also contains a layer of colloidal-sized metallic solid (c) overlaying this aminodextran coating. Preferably this layer is uniformly dispersed over the dispersed surface of the aminodextran layer. The colloidal metal useful in forming the coatecl substrate is generally described as a metal which can be reduced from the ionic state to the metal (0) state by the aminodextran coating, or a metal which can form metal ions or metal ion complexes which have a reduction potential of about +0.7 volts or higher. While such metal ions may include: Ag(I), Au(III), Pd(II), Pt(II), Rh(III), Ir(III), Ru(II), Os(II), the preferred metal ions for such use are colloidal gold (III) and colloidal silver (I) . B . The Linker The present invention also provides for a linker which allows conjugation of a selected protein, e.g., an antibody, to the colloidal metal-aminodextran coated substrate described above. The linker generally has a free amino group which can be activated for conjugation to protein. i. Aminodextran Linkers One preferred linker which is introduced to overlay the colloidal metal-aminodextran coated substrate is an aminodextran linker, preferably an aminodextran in which the degree of diamine substitution is in the range of between about 1/40 and about 1/35 compared to a maximum theoretical value of 1/2.5, such as IX aminodextran. Alternatively, the diamine substitution in the aminodextran linker may be between approximately 1/7 to 1/8, such as 5X-aminodextran . Preferably, the 5X- aminodextran linker is used where the colloidal metal is silver . The aminodextrans contain sufficient amino groups to crosslink them to the existing particle surface of silver colloid-coated polystyrene particles as well as to covalently link them to a protein, e.g., antibody. " The crosslinked aminodextran coating prevents the silver colloid from being shed from the polystyrene particles in subsequent chemical reactions needed to activate the particles, conjugate them to monoclonal antibody, and block chemically-reactive functional groups. ii . Aminotri thiol linker Another preferred linker, particularly useful where the colloidal metal is gold, is an aminotrithiolate linker. A particularly useful aminotrithiol linker is tris (3-mercaptopropyl) -N- glycylaminomethane, a spider-like composition having the formula, Cι 2 H 26 N 2 OS 3 , [J.K. Whitesell and H.K. Chang, Science, 261: 73-76 (1993)] and the structural formula: This compound was originally described as forming a layer on a roughened gold film surface, to provide NH 2 initiation sites for polymerization of alanine N-carboxyanhydride to form polyalanine helices that uniformly aligned themselves perpendicular to the surface. The polyalanine helices were shown to remain" attached to the gold surface and retain their conformation even after heating to 180°C for one week. The preferred form of this linker is the freshly deprotected form, which is preferably prepared as described in detail in Example 4A below. Example 4 demonstrates that the aminotrithiol linker functions well as a ligand between gold particles deposited on polystyrene particles and monoclonal antibodies. By coordinating strongly to gold or silver atoms on colloidal metal through its three sulfur atoms, and allowing activation and conjugation of antibody through its amino group, the aminotrithiol linker also serves as a new bifunctional linking agent. The soft base, highly polarizable sulfur atoms of low electronegativity prefer to coordinate to the soft acid, gold or silver atoms. However, as discussed in Example 5B below, the aminotrithiol linker competes too well with aminodextran for binding sites on the surface of deposited silver colloid on polystyrene particles and displaces much of the silver colloid from the surface of silver-aminodextran-polystyrene particles. Thus, aminotrithiol does not work well as a crosslinking agent with silver colloid on polystyrene particles. C. The Cross-Linking Agent The linker as described above is covalently bonded to the colloidal metal-aminodextran- coated substrate or the aminodextran-metal-aminodextran-coated substrate. For activation of the linker on the particles, a heterobifunctional cross-linking agent, such as sulfosuccinimidyl 4- [N-maleimidomethyl] cyclohexane-1- carboxylate ("sulfo-SMCC") is used. Sulfo-SMCC covalently bonds to a linker with reactive amino groups . Various other suitable crosslinking agents and methods " " are cited in texts such as The Pierce Handbook and Chemistry Of Protein Conjugation And Cross-Linking, S.S. Wong, CRC Press, Inc., Boca Raton, FL (1991). D . The Protein The selected activated protein, then attached through the crosslinker to the linking agent on the colloidal metal-aminodextran coated substrate to form the novel, stable colloidal particle of the invention, is preferably an antibody. More specifically, the protein (e) is covalently bonded to the linker-colloidal metal- aminodextran coated substrate by covalent bonding of the free amino groups on the linker through the heterobifunctional crosslinker with sulfhydryl or amino groups on the activated protein. Although any protein may be attached to the substrate in this way, preferably, the protein is a monoclonal or polyclonal antibody or any one of a variety of recombinant antibody constructs. The antibody is desirably directed to an epitope on a cell, which epitope is unique to the cell type, thereby permitting the particle's use in cell separation assays. Polyclonal antibodies may be generated by conventional means, i.e., obtained from sera of animals or humans exposed to a selected antigen. For example, polyclonal antibodies are generated by conventionally stimulating the immune system of a selected animal or human with a selected antigen, allowing the immune system to produce natural antibodies thereto, and collecting these antibodies from the animal or human's blood or other biological fluid. Monoclonal antibodies are obtained by conventional hybridoma methods and purified from ascites fluid by ammonium sulfate (45%) precipitation, centrifugation and affinity chromatography using protein" A. Processes of making monoclonal antibodies are described in G. Kohler and C. Milstein, Nature, 256: 495- 497 (1975) and include the many known modifications thereof, which teachings are incorporated herein by reference. Similarly desirable high titer antibodies are generated by applying known recombinant techniques to the monoclonal or polyclonal antibodies developed to these antigens [see, e.g., PCT Patent Application No. PCT/GB85/00392; British Patent Application Publication No. GB2188638A; Amit et al . , Science, 233:747-753 (1986); Queen et al . , Proc. Nat ' 1. Acad. Sci. USA, 86:10029-10033 (1989); PCT Patent Application No. PCT/WO9007861; and Riechmann et al., Nature, 332:323-327 (1988); Huse et al, Science, 24_6: 1275-1281 (1988)a]. One of skill in the art may generate chimeric, humanized or fully human antibodies for use as the protein of this invention by resort to known techniques by manipulating the complementarity determining regions of animals or human antibodies. See, e.g., E. Mark and Padlin, "Humanization of Monoclonal Antibodies", Chapter 4, The Handbook of Experimental Pharmacology, Vol. 113, The Pharmacology of Monoclonal Antibodies, Springer- Verlag (June, 1994) . Of course, the particular method of making and the type of antibody is not limited to such techniques and it is envisioned that any technique for making such antibodies is within the practice of the invention. In a preferred embodiment, the protein is a monoclonal antibody to the CD3, CD4 or CD8 receptors on T lymphocytes. Examples of such monoclonal antibodies are the following: monoclonal antibody (MAb) "T3" (commercially available from Coulter Corporation, Miami, Florida) is an anti-CD3 antibody of IgGl subclass, ~ "* derived from the hybridization of mouse P3/NS1/1-AG4-1 myeloma cells with spleen cells from BALB/c mice immunized with human infant thymocytes and peripheral blood lymphocytes from a patient with Sezary cell leukemia. MAb "T4" (Coulter Corporation) is an anti-CD4 antibody derived from hybridization of mouse NS/1-AG4 cells with spleen cells of BALB/cJ mice immunized with human peripheral blood T lymphocytes. MAb "T8" is an anti-CD8 antibody, derived as described for T4, but immunized with human thymocytes (Coulter Corporation) . MAbs T4 and T8 are chosen for use in the examples below for the large density of their antigenic receptors (10 4 - 10 5 receptors per targeted cell) and the mutual exclusiveness of their targeted cells. However, as the protein portion of the novel particle, any antibody directed against any targeting receptor site on a cell may be used, as long as the sites per cell are greater than about 10 3 and each such site is exclusive to one cell population in multiple targeted cell populations. E . Embodiments of Particles of the Invention An exemplary embodiment of the stable particle of this invention is the particle comprised of colloidal- sized polystyrene microparticle having a lX-aminodextran coating over the peripheral surface thereof, and a layer of colloidal-sized gold overlaying the aminodextran coating. An aminotrithiolate linker having a free amino group is covalently bound to the gold, and an anti-CD4 antibody is attached to the linker via a heterobifunctional crosslinker. This particle is referred to as the T4-aminotrithiolate-gold-lX- aminodextran-polystyrene particle. Another exemplary particle is made up of a colloidal-sized polystyrene particle having a IX- aminodextran coating over the peripheral surface thereof, with a layer of colloidal-sized silver overlaying the aminodextran coating. A 5X-aminodextran linker having free amino groups is cross-linked around the silver- aminodextran-PS particle, and an anti-CD4 antibody is attached to said linker by covalently bonding to the free amino group through a heterobifunctional crosslinker, such as sulfo-SMCC. This particle is referred to as the T4-5X-aminodextran-silver-lX-aminodextran-polystyrene particle. Another embodiment of a particle according to the invention comprises a colloidal-sized polystyrene particle with a lX-aminodextran coating over the peripheral surface thereof, with a layer of colloidal- sized gold overlaying the aminodextran coating. An aminotrithiolate linker having a free amino group is crosslinked to the gold and an anti-CD8 antibody protein is attached to the free amino group of the linker via a heterobifunctional crosslinker. This particle is referred to as the T8-aminotrithiolate-gold-lX- aminodextran-polystyrene particle. Yet another embodiment of a particle according to this invention comprises a colloidal-sized polystyrene particle having a lX-aminodextran coating over the peripheral surface thereof, with a layer of colloidal- sized silver overlaying the aminodextran coating. A 5X- aminodextran linker having free amino groups is cross- linked around silver-aminodextran PS particle and an anti-CD8 antibody is attached to said linker by covalent bonding to the free amino groups through a heterobifunctional crosslinker, such as sulfo-SMCC. Thds" particle is referred to as the T8-5X-aminodextran-silver- lX-aminodextran-polystyrene particle . These exemplary stable particles of the present invention are particularly desirable as reagents for use in a method of simultaneously analyzing a biological fluid, such as whole blood, for at least two different, mutually exclusive subsets of lymphocytes. III . Methods of Preparing the Particles of the Invention The present invention further provides methods for the preparation of the novel stable colloidal particles described herein. Generally, the particles of the invention are prepared by reacting aminodextran coated polystyrene particles with metal salts, attaching a linker thereto and attaching a protein, e.g., an antibody, to the coated particle through the linker. More specifically, the method for preparing such particles, comprises the first step of reacting commercially available aldehyde functionalized polystyrene particles with aminodextran as described above. Some aminodextran adsorbs onto the surface of, and covalently binds to, a colloidal-sized polystyrene core substrate. Additional aminodextran is covalently bound to the beads by crosslinking with glutaraldehyde . The resulting reaction mixture is treated with a reducing agent to obtain stable, coated beads. This reducing agent can be any conventional reducing agent, such as sodium borohydride . The resulting cross-linked aminodextran coated polystyrene particles are subsequently washed with water, preferably distilled water. The aminodextran is generally capable of reducing a metal ion or metal ion complex to the metal (0) state, i.e., reducing gold(III) to gold(0) or silver(I) to - "* silver (0) . These aminodextran-coated polystyrene particles are then reacted with an aqueous solution of a metal salt, containing an metal ion or metal ion complex which is capable of being reduced by the aminodextran coating. As discussed above, the metal salt is preferably a gold (III) or silver (I) salt. Briefly described, the aqueous suspension of the coated particles is mixed with an excess of aqueous solution of the metal salt at an elevated temperature, in the presence of a predetermined amount of a second reducing agent which reduces the excess metal ion to the metal (0) state, mainly as colloidal metal on the surface of polystyrene particles. The elevated temperature ranges desirably between about 80 to about 100°C. The additional reducing agent is sodium citrate, which is used for rapid formation of colloidal metal. The resulting colloidal metal-aminodextran coated particles are purified by washing, for example, with aqueous sodium citrate solution and distilled water. Further steps involved in preparing the novel particles of the present invention include attaching a linker or linking agent having a free amino group to the colloidal metal-aminodextran coated particles. The linker allows conjugation of proteins to the surface of the metal-colloid coated particles. Preferably, for silver-coated particles or beads the linker is aminodextran, and most preferably 5X-aminodextran. For gold-coated beads the linker is preferably the aminotrithiolate linker described above. Where the linking agent is an aminodextran, it may be attached to the coated particles by adsorption and crosslinking with a bifunctional crosslinking agent, such" as glutaraldehyde. The free amino groups on the aminodextran linker are then activated with a suitable heterobifunctional crosslinking agent which links the colloidal metal particle to the protein, for example, sulfosuccinimidyl 4- [N-maleimidomethyl] cyclohexane-1- carboxylate (sulfo-SMCC) or others known to the art as described above. The aminotrithiol linker may be attached by reaction of thiolate groups with the colloidal gold- surface. More specifically, the method of using the aminotrithiol linker comprises dissolving protected aminotrithiol ligand in anhydrous methanol; adding concentrated hydrochloric acid to the resulting solution; and refluxing the mixture. In one embodiment a preferred time for refluxing is about 5 hours. The components of the mixture are separated, e.g., by size exclusion chromatography on a BioGel P2® gel, and fractions of the first band (representing deprotected aminotrithiol ligand) absorbing at 280nm, are collected. The deprotected aminotrithiol ligand is then mixed with the gold colloid-aminodextran-coated polystyrene beads described above. Thus strong covalent bonds are formed between the sulfur groups on the linker with the gold colloid surface. The free amino group of the linker is then activated with sulfo-SMCC. Once the crosslinker is attached to the particle, a selected protein which is desired to be attached to the colloidal particle is then conjugated to the amino groups of the activated linker on the coated particles by conventional methods, e.g., by activating the protein with an amino binding reagent which provides a sulfhydryl group. A suitable amino binding/activating reagent is-2 " =- iminothiolane. Conventional methods of 2-iminothiolane activation of antibody and maleimide (sulfo-SMCC) activation of amino groups on coated particles to conjugate antibody to particles, are described in Chemistry Of Protein Conjugation And Cross-Linking, S.S. Wong, CRC Press, Inc., Boca Raton, FL, 1991. Still other amino-reactive sulfhydryl introducing reagents may readily be selected by one of skill in the art from available texts [See, also, Pierce Handbook] . Finally, the activated colloidal metal-coated, linker coated particle is purified and the activated protein is purified, both by conventional means. The purified particles in aqueous suspension are then mixed with an amount of an aqueous solution of the activated protein sufficient to saturate the reactive groups on the activated particles. This mixing preferably occurs at room temperature and for a time sufficient to permit conjugation between the protein and particle, resulting in a stable colloidal particle. Selection of the amount of the activated protein solution and the mixing times are clearly within the skill of the art, depending upon the amount of particle to be conjugated to protein, and these parameters are limitations of this method. Preferably, before use, unreacted functional groups on the resulting stable colloidal particles are blocked with appropriate blocking agents, such as L-cysteine and iodoacetamide . For use in biological assays, exemplary particles of the invention, e.g., antibody-conjugated gold- or silver-coated polystyrene particles, may be suspended in suitable buffers, such as bovine serum albumin (BSA) buffer. - - IV. Methods of Use - Simul taneous analysis The above-described colloidal particles of this invention are desirably employed in methods of simultaneous quantitative determination of at least two different subsets of cells. For example, one exemplary method employing the particles of the invention demonstrates 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. Preferably, the subsets of cells are characterized as having some antigenic site which is mutually exclusive and non-overlapping. Thus, a method of the present invention comprises the steps of mixing a biological solution or suspension, e.g., whole blood, with at least two different stable colloidal particles as described above. Each different particle contains a different protein, e.g., a different antibody, conjugated thereto and each protein binds to a different epitope on a subpopulation of white blood cells . The particles and biological material are mixed for a time sufficient to permit the binding of the particles to each subpopulation of cells. Thereafter, the red blood cells in said biological solution/suspension are lysed and quenched. This mixture is analyzed in an instrument that distinguishes between the subpopulations of white blood cells bound by each different colloidal particle, thereby quantitatively enumerating at least two subpopulations of the white blood cells. Preferably, the instrument analyzes the mixture simultaneously and distinguishes between cells by shifting light scatter of targeted cells in VCS (i.e., volume, conductivity and light scatter) technology. In a preferred embodiment, the detection instrument is - ~ equipped with at least two lasers. One particularly desirable instrument for such use is a modified Coulter VCS instrument which is described in U.S. Patent Nos. 5,125,737 and 5,492,833, incorporated herein by reference. By such a method, one may quantitatively detect subpopulations of white blood cells, such as CD4 + T lymphocytes ("T4 cells"), CD8 + T lymphocytes ("T8 cells"), B lymphocytes, granulocytes, basophils, and monocytes, among others . As illustrated in Example 8 below, the method of the present invention further provides for the simultaneous analysis of T4 and T8 cell populations in whole blood using three different colloidal particles. One particle was a commercially available polystyrene bead, the other particles were particles of the invention, i.e., the T4- aminotrithiolate-gold-lX-aminodextran-polystyrene particle and the T8-5X-aminodextran-silver-lX- aminodextran-polystyrene particle . The following examples are provided to illustrate the invention. Specifically, Examples 1-4 illustrate the preparation of particles of the invention. Example 5 provides analytical evaluation of the particles. Examples 6 and 7 provide separate analyses of cell populations, and Example 8 provides an example of simultaneous analysis of cells using the present invention. These examples are illustrative only and do not limit the scope of the invention. Further, one skilled in the art will appreciate that although specific reagents and conditions are outlined in the following examples, modifications can be made which are meant to be encompassed by the spirit and scope of the invention. " - EXAMPLE 1 - COATING OF POLYSTYRENE ALDEHYDE/SULFATE PARTICLES WITH 1X-AMINODEXTRAN OR 5X-AMINODEXTRAN A. Polystyrene microspheres Raw polystyrene aldehyde/sulfate latex particles were obtained from Interfacial Dynamics Corporation (IDC), Portland, Oregon. Initial studies of T4-5X-Amdex-PS and T4-Ag-5X-Amdex-PS particles were carried out with IDC lot 2-300-36 particles of mean diameter 2.15μm, CV = 5.5% as measured by transmission electron microscopy (TEM) . IDC lot 496 particles, which were reported to have a mean diameter of 2.200μm, CV = 5.4% as determined by TEM and found to have a mean diameter of 2.021μm, CV = 1.0% as measured by quasi- elastic light scattering (QELS) on the COULTER ® N4MD™ sub-micron particle analyzer, were later used in preparing T4, T8-PS particles. All later gold or silver- coated PS particles were prepared from IDC lot 540 particles, which had a mean diameter of 2.130μm, CV = 2.7% by TEM and of 2.007μm, CV = 1.1% by QELS. B . Coa ting wi th aminodextran These colloidal particles were prepared in a manner substantially as described in U.S. Patent No. 5,639,620, incorporated by reference. Briefly, 36.6mL of 4.1% w/v solids of these particles were pipetted into a 250mL polypropylene centrifuge tube. These particles were coated with aminodextran as follows . To the above- described suspension was added 3. Og of IX- or 5X- aminodextran, dissolved in 50mL hot, distilled water and filtered through a 250mL 0.2μm filter pack. The IX or 5X-aminodextran was prepared by the procedures described in U.S. Patent No. 5,466,609. Distilled water was then added to the mixture to make a total volume of 150mL, a-nd 18.3μL of 45% w/w potassium hydroxide solution was added to make the mixture ImM in potassium hydroxide. The tube and contents were roller mixed for 16 to 24 hours. C. Crossl inking Crosslinking of the aminodextran was accomplished with 3mg/mL or 1.8mL of 25% glutaraldehyde, reacted with the particle suspension for one hour. A light yellow color in the particle mixture was noted at the end of the mixing period. Sodium borohydride solid at lOmg/mL or 1.531g was added to the reaction tube, which was then roller mixed for an additional three hours. An extra 1.834 mL of 45% w/w potassium hydroxide solution was also added to the tube to reduce the evolution of hydrogen gas. The yellow color of the reaction mixture, due to the Schiffs' base generated in the reaction with glutaraldehyde, changed to a colorless, white particle suspension after about 15 minutes of mixing with sodium borohydride. Gas was released 2-3 times over the three hour mixing period. The particles were then separated by centrifugation for about 15 minutes at 3400g in a Beckman J6-B centrifuge, the clear supernatant was discarded, and the residue was washed four times with 150mL distilled water. The residue was resuspended each time by vigorously vortexing and sonicating for 2 to 5 minutes to redisperse the particles. The resulting aminodextran coated polystyrene particles are referred to as IX-Amdex-PS and 5X-Amdex-PS. EXAMPLE 2 - COATING OF THE IX-AMDEX-PS AND 5X-AMDEX-PS MICROSPHERES WITH COLLOIDAL METAL A . Coa ting of Particles wi th Silver The particles of Example 1 were coated with - - silver colloid substantially as described in U.S. Patent No. 5,552,086. Briefly, 3.12mL of 0.589M silver nitrate solution was added to 540mL of distilled water in a IL, 3-neck, round bottom flask. The solution was stirred with an overhead glass rod sleeve stirrer and heated to about 90°C with a heating mantle (Glas-Col) , top and bottom. 30mL of 1% w/v solids IX-Amdex-PS of Example 1 were added to the colorless, hot silver nitrate solution and the resulting mixture was stirred, with heating, for about 1 to 2 minutes. 48mL of 1.36M sodium citrate solution was then added to the hot, stirred reaction mixture . Reaction progress to form colloidal silver on the particles was noted at various times from the color of the suspension: about 2 minutes; pale yellow; about 4 minutes, dark brown; about 7 minutes, black; about 10 minutes, dark grey; about 15 minutes, dark grey. After 15 minutes, the reaction was quenched by pouring the contents of the flask into a IL beaker and allowing the particle suspension to cool to room temperature. The dark grey particles settled after about 40 minutes, at which time the supernatant was decanted and the residue of about 50mL was resuspended in 0.01M sodium citrate solution to 100ml total volume. The particles were separated and washed by centrifugation for about 5 minutes at 500g, and finally dispersed in 0.01M sodium citrate solution at a total volume of 30mL. B . Scale Up Prepara tion Five times scale-up preparation is similar to that described in Part A above, however, 15.6mL of 0.589M silver nitrate solution in 2600mL distilled water, 150mL of 1% w/v solids IX- or 5X-aminodextran-polystyrene particles, and 240mL of 1.36M sodium citrate solution are needed. About a 15 minute reaction time and about 90°C reaction temperature were still used for reactants in a 5L, 3-neck, round bottom flask with overhead stirring and top and bottom heating mantles. At the end of the reaction period the 5L flask and contents were removed from the mantles and cooled for about 10 minutes, upon which all particles had settled to the bottom of the flask as a black solid. Almost all the supernatant was then discarded by decantation, leaving a dark grey suspension of particles which were resuspended to a total volume of 125mL with O.OIM sodium citrate solution. The particles were separated and washed two times with O.OIM sodium citrate solution by centrifugation for about 5 minutes at 500g, and finally redispersed to a total volume of 150mL with O.OIM sodium citrate solution. C. Analysis of Silver-lX-Amdex-PS and Silver-5X- Amdex-PS Particles Microscopic examination at 1000X magnification of 10 times diluted particles of Part A in 0.01M sodium citrate solution showed mostly single primary particles and small aggregates (2-3 primary particles) with very few large aggregates. The relative populations of singlets (single, primary particles) , doublets (two primary particles stuck together) , higher order multiplets (three or more primary particles stuck together) , and uncoated or lightly-coated PS particles in a colloidal suspension of the metal-coated IX-Amdex-PS and metal-coated 5X-Amdex-PS particles were measured on a flow cytometer (COULTER® Profile II™ with 488nm Ar ion laser excitation for silver-Amdex-PS particles; and COULTER® Elite™ with 633nm He/Ne laser excitation for both gold- and silver-Amdex-PS particles) . The populations of these primary and aggregated particles with different sizes and shape are well-defined in their light scattering ability if the primary particles are monodisperse and the gold or silver coatings are uniform. Serial dilutions of particle concentrates were made with twice-filtered ImM potassium hydroxide solution so that the volume fraction of particles in the final suspension was about 1.3 x 10 "5 . The flow cytometer was optically aligned with COULTER® DNA-CHECK™ particles to give HPCV 2.0% for FS and SS, and Fll and F12 parameters in histograms and calibrated with Standard-Brite particles. Operating parameters were selected as follows: sample aspiration volume of 50μL; sample flow rate of lOμL/min so that total sample running time will coincide with 10,000 events; histogram parameter Y-axis as FS and X-axis as SS; amplifier gain for FS in the middle of the linear gain versus mean channel (Y-axis) region; PMT voltage for SS in the middle of the linear voltage versus mean channel (X-axis) region; histogram 1 (entire region) and 2 (gated on bitmap 1) as 64x64 type, FS-SS, with bitmap 1 encompassing single particles in box 1, doublets in box 2, and all higher order aggregates in box 3, but leaving out amplifier noise; appropriate scaling factor for histograms 1 and 2; bitmap 1 and histogram 2 to count 10,000 events; histogram 2, gated on bitmapl, and set up analysis boxes, 1 for single particles, 2 for doublets, and 3 for multiplets in the same position as in histogram 1. The percent population of each particulate state was read from the extended printout of histogram 2. The analysis boxes on histogram 1 do not reflect the true percentage ratios of particles, since they include electronic noise and any small particles outside of bitmap 1 in histogram 1. Flow cytometry (forward versus side scatter) histograms gave 65% singlets, 32% multiplets, and 3% uncoated polystyrene particles. Microscopic examination of a 10-fold diluted suspension of the scaled up preparations of Part B showed mostly black singlets, many doublets, and some larger clusters. All polystyrene particles of two micron nominal diameter were evenly and densely-coated with black silver particles of diameters ranging from 50 to 200nm. Flow cytometry showed 74% singlets, 22% multiplets, and 4% uncoated polystyrene particles, and a mean shift of silver-polystyrene particles with 488nm laser excitation of 40.1 (3.3) on the side scatter axis compared to polystyrene particles of 15.5 (1.6) . D . Coa ting of Particles wi th Gold The preparation of colloidal gold coated IX- Amdex-PS and 5X-Amdex-PS particles of Example 1 was performed substantially as described in U.S. Patent No. 5,552,086. Briefly, 0.740g of hydrogentetrachloroaurate (III ) , HAuCl 4 .3H 2 θ, were dissolved in 270mL distilled water in a IL, 3-neck, round bottom flask and heated to about 80° C with top and bottom heating mantles, and overhead stirring. 30mL of 1% w/v solids IX- or 5X-aminodextran-polystyrene particles of Example 1 were added to the hot, stirred solution and allowed to mix for about 5 minutes. 1.5mL of 1.36M sodium citrate solution was then pipetted into the stirred, hot mixture. Within 5 seconds the color of the reaction mixture turned from pale yellow to colorless and in about 25 seconds, turned to brown. After one minute of reaction, heating and stirring were stopped and the contents of the flask were transferred to a IL beaker, " " " wherein the particle suspension was sonicated for about 1-2 minutes and allowed to cool to room temperature. Then, the brown particles were separated by centrifugation for about 5 minutes at 500g, the supernatant was discarded, and the residue was washed two times with 0.01M sodium citrate solution and resuspended finally to a total volume of 30mL with 0.01M sodium citrate solution. E . Scale up prepara tion The 5-fold scale-up preparation was similar to that of Part B; however, 3.58g of hydrogentetrachloroaurate (III) in 2800mL of distilled water and 150mL of 1% w/v solids IX- or 5X-aminodextran- polystyrene particles were mixed at about 80°C for 56 minutes in a 5L, 3-neck, round bottom flask, with overhead stirring and top and bottom heating mantles . Then, 7.5mL of 1.36M sodium citrate solution were added to the mixture. In 10 seconds the reaction mixture turned from pale yellow to colorless, then, in 30 seconds, to purple, and in 55 seconds, turned to brown. The reaction was continued at about 80°C for about another 9 minutes. At the end of the reaction period, heating and stirring were stopped and the 5L flask and contents were removed from the mantles and allowed to cool to room temperature. After about one hour, the light purple supernatant was decanted and a heavy brown suspension of about 150mL was separated by centrifugation for 5 minutes at 500g. The supernatant was discarded, and the residue was washed two times with 0.01M sodium citrate solution and finally resuspended in 0.01M sodium citrate solution to a total volume of 150mL. The amount of gold salt that was used per milliliter of 1% w/v particles was chosen so that the ~ ~ volume of metallic gold that could be formed was equivalent to the volume of metallic silver that could be formed in Part B assuming 100% reduction of each metal ion to metal (0) . In the large scale silver coating procedure of Part B, the volume of silver metal that could be formed is 15.6mL/ ( lOOOmL/L) x 0.589M AgN0 3 x 107.87g Ag/mol ÷ 10.49g/cm 3 = 0.0945cm 3 Ag . Thus, the mass of gold salt, HAuCl4.3H 2 0, that is required in the large scale gold coating procedure is 0.0945cm 3 Au x 19.32g/cm 3 x 393.88g HAuCl 4 -3H 2 0/196.97g Au = 3.65g per 150mL of 1% w/v particles. The volume fraction in silver or gold coated on polystyrene particles, 0.05% w/v solids, was 3 x 10 in the reaction mixtures, but was concentrated to 6 x 10 "5 in the final suspension of polystyrene particles. The added metal changes the original 1% w/v solids particle suspensions to about 2.2% w/v solids gold-Amdex-PS particles and to about 1.7% w/v solids silver-Amdex-PS particles, as measured by washing an aliquot of particle suspension exhaustively, 5 or 6 times, with distilled water, freeze-drying the washed sample, and weighing the sample. F. Analysis of Gold-lX-Amdex-PS and Gold-5X-Amdex- PS Particles Microscopic examination of 10-fold diluted particles of Part D showed singlets, small aggregates, and very few large aggregates. Flow cytometry (forward versus side scatter) histograms performed as described above for the silver coated particles showed a mean shift of gold-polystyrene particles with 633nm laser excitation of 42.0 (4.7) on the side scatter axis compared to polystyrene particles of 16.0(2.5). The percentage of gold-Amdex-polystyrene singlets was 43%, aggregates, 53%, and uncoated polystyrene particles, 4%. Flow cytometry performed on the scaled up preparation of Part E showed that the percentage of singlets was 53%; multiplets, 26%; and lightly-coated polystyrene particles, 16%. EXAMPLE 3 - CROSSLINKING AND CONJUGATION OF THE COLLOIDAL SILVER-AMINODEXTRAN-POLYSTYRENE PARTICLES A. Prepara tion of 5X-Aminodextran Cross-Linked Silver-Amdex-PS Particles 25mL of silver-aminodextran-polystyrene particles prepared from 1% w/v solids polystyrene particles and suspended in O.OIM sodium citrate solution as described in Example 2A were separated by centrifugation for 5 minutes at 500g, the supernatant was discarded, and the residue was resuspended in a 2% w/v 5X-aminodextran solution at the original volume. The mixture was roller mixed overnight for 16-24 hours, centrifuged for about 5 minutes at 500g to separate the residue and discard the supernatant. The final residue was resuspended to a total volume of 25mL with ImM potassium hydroxide solution. Thereafter, O.lOmL of 25% w/v glutaraldehyde was added to the particle suspension to make a lmg glutaraldehyde/mL particle suspension, which was roller mixed for about 50 minutes. Next, an equimolar amount, 0.601mg ethylenediamine/mL or 16.9μL, was added to the reaction mixture, which was roller mixed for about 30 minutes further, to block aldehyde groups. A solution of 30mg sodium borohydride in 3mL of ImM potassium hydroxide solution was prepared, and 2. OmL of this solution were pipetted into the particle suspension, which was roller mixed for another 30 minutes. Gas was released after the reaction subsided in about 1 to 2 minutes. At the end of the reaction period the particles were separated by centrifugation for 5 minutes at 500g, the supernatant was discarded, and the residue was washed four times with lxPBS. lxPBS was prepared by dissolving 53.8g K 2 HP0 4 in 1.6L distilled water and adding 12.8g KH 2 P0 4 and stirring until dissolved. Then, 340g NaCl was dissolved in the solution. After all salts are dissolved, distilled water was added to make up to 2L volume and filtered through an 0.2μm filter. The resulting solution is 20xPBS. lxPBS is prepared by dilution of 1 part 20xPBS with 19 parts distilled water. The lxPBS solution has a pH in the range 7.1-7.3, typically 7.2, a conductivity in the range of 13,500 to 15,500 μMho-crrf 1 and is 0.15M in NaCl. The final residue was resuspended to a total volume of 25mL in lxPBS. B . Conj uga tion of T4 T8 an tibody to crossl inked 5 x- aminodextran- coa ted silver -aminodextran-polystyrene particles . 4.5μL of lOmg/mL sulfo-SMCC per mL 1% w/v solids particles or 72.0μL sulfo-SMCC solution was added to 16mL of crosslinked 5X-aminodextran-silver-polystyrene particles in a 50mL polypropylene centrifuge tube. The tube and contents were roller mixed for about one hour, after which the particles were separated by centrifugation for about 5 minutes at 500g, the supernatant was discarded, and the residue was washed four times with lxPBS, as described above. The final residue was resuspended to a total volume of 16mL with lxPBS. lOmg each of T4 (0.225mL) and T8 (0.266mL) concentrate in 0.667mL lxPBS were separately activated with 2-iminothiolane (IT) at a 15:1 = IT:antibody molar ratio, using 65μL of 2mg/mL IT solution. The reaction mixtures in 15mL polystyrene centrifuge tubes were roller mixed for one hour, and then applied to the top of 50mL G-50 Sephadex columns, equilibrated with lxPBS . The columns were eluted with lxPBS, and fractions of the first A 28 o -absorbing band containing IT-T4 or IT-T8 antibody were pooled to yield 2.6mL of 2.351mg/mL IT-T4 antibody and 3. OmL of 2.709mg/mL IT-T8 antibody. Conjugations were run at 0.7mg IT-T4 and 0.8mg IT-T8/mL 1% w/v solids particles, so that 1.191mL IT- T4/1.181mL IT-T8 solution were added to duplicate samples of 4mL of sulfo-SMCC-activated particle suspension and the mixtures were roller mixed for about two hours. At the end of the conjugation period, lmL of each conjugation reaction mixture was pipetted and filtered through an 0.2μm low-protein binding filter disc. The A 280 of the supernatant was measured to yield a 0.504mg/mL IT-T4/0.534mg/mL IT-T8 concentration. By difference, the surface concentration of IT-T4 was 0.107 (0.084)mg/mL or 5.1 (4.0)mg T4 antibody/m 2 of particle surface area and the surface concentration of IT-T8 was 0.391 (0.386) mg/mL or 18.7 (18.5) mg T8 antibody/m 2 of particle surface area, assuming smooth spherical 2μm diameter particles of specific surface area, 2.7082m 2 /g. Both T4- and T8-conjugated particles were divided into two 4.2mL samples, one without blocking and the other with blocking with L-cysteine and iodoacetamide . The with blocking samples were blocked with 0.503mL of 5mg/mL L-cysteine solution in lxPBS (about 15 minutes), and then, 0.563mL of 20mg/mL iodoacetamide solution in lxPBS and 0.105mL of IM borate buffer solution, pH 9.8(30 minutes). At the end of the blocking reactions, all four particle samples were separated by centrifugation for about 5 minutes at 500g, the supernatants were discarded, and the residues were washed two times with BSA buffer, 1% BSA/0.1% sodium azide in lxPBS solution, resuspended in BSA buffer, roller mixed for about one hour, and stored in a refrigerator at about 5°C for 16-24 hours. The samples were then further washed two times with BSA buffer and the total volume of each sample was adjusted to 3.2mL to obtain 1% w/v solids suspensions based on uncoated latex particles . EXAMPLE 4 - CROSSLINKING AND CONJUGATION OF THE COLLOIDAL GOLD-AMINODEXTRAN-POLYSTYRENE PARTICLES A . Prepara tion of the Aminotri thiol Linker The cross-linker, C12H26N 2 OS 3 , was prepared substantially as described in Whitesell & Chang [Science 261, 73 (1993)] and stored as the protected ligand, N- tert-butoxycarbonyl spider triacetate. Before use, lOmg of protected ligand was dissolved in 5mL of anhydrous methanol to give a colorless solution. 2.5mL of concentrated hydrochloric acid (36.5-38.0%) was then added to the solution of protected spider. The mixture was refluxed in a 50mL round bottom flask for about 5 hours to give a yellow solution having the odor of free sulfhydryl groups. The acid-catalyzed hydrolysis mixture (final volume of 3.0mL) of spider ligand was applied to the top of a Bio-Gel P-2 column, 1.1cm x 30cm, and eluted with - - distilled water. Fractions of the first band absorbing at 280nm were collected and tested for free sulfhydryl groups with Ellman' s reagent, 5, 5' -dithio-bis- [2- nitrobenzoic acid], (Pierce Chemical Co.) against a phosphate buffer blank. The reagent reacted with free sulfhydryl groups to form a highly colored chromophore with an absorbance at 412nm [J.C. Ellman, Clin. Chem. Acta, 28: 234-241 (1962)]. About fifteen minutes were allowed for development with Ellman' s reagent. For a quantitative determination of sulfhydryl groups, a standard curve was run for L-cysteine to determine the linear response range for the complex. Twelve deprotected ligand fractions of 56mL total volume gave an A 41 2 reading of 0.549 or sulfhydryl concentration of 0.458mM, representing a yield of 44%. B . Prepara tion of Aminotri thiol Cross-Linked Gold- Amdex-PS Particl es 4.950mL of 0.33M aminotrithiol linker in ethanol solution, was prepared as described above and was added to 15mL of 1% w/v solids gold-lX-aminodextran- polystyrene particle suspension in distilled water in a 50mL polypropylene tube. The tube and contents were roller mixed for 16 to 24 hours, whereupon the particles were separated by centrifugation for about 10 minutes at 200g, the supernatant was discarded, and the residue was washed four times with lxPBS . The final residue was suspended to a total volume of 15mL with lxPBS. C. Conj uga tion of T4/T8 an tibody to crossl inked Aminotri thiola te-coa ted gold-aminodextran-polys tyrene particl es . 1.50μL of lOmg/mL sulfo-SMCC solution in lxPBS per mL of l%w/v solids particles or 22.5μL for 15mL trial of particle suspension were added to particles in 50mL polypropylene centrifuge tube, and roller mixed for about one hour. At the end of the reaction period the particles were separated by centrifugation for about 10 minutes at 200g, the supernatant was discarded, and the residue was washed four times with lxPBS. The final residue was resuspended to a total volume of 15mL with lxPBS, and divided into two 7.5mL portions for each trial. lOmg of each antibody, T4 and T8, were activated with IT and the IT-T4 and IT-T8 derivatives were purified in the same way as described in Example 3 above. Conjugations were run in a similar way with 0.7mg IT-T4/mL 1% particles and 0.8mg IT-T8/mL 1% particles in separate trials. lmL of each trial was pipetted to analyze for IT-Ab in the supernatants: 6.7mg T4 antibody/m 2 and 9.5mg T8 antibody/m 2 , assuming smooth spherical 2μm diameter particles with a specific surface area of 2.7082m 2 /g. Samples were further divided equally for without blocking and with blocking with L-cysteine and iodoacetamide. The particles were then separated by centrifugation for about 15 minutes at 200g, the supernatants were discarded, and the residues were washed two times with 1% BSA/0.1% sodium azide in lxPBS, resuspended in BSA buffer, roller mixed for about one hour, and stored in a refrigerator at about 5°C for 16-24 hours. The T4- and T8-conjugated particles were further washed two times with BSA buffer and resuspended to a total volume of 3.4mL to make 1% w/v solids suspensions based on uncoated latex particles. D. Mul ti -angle Doppler Electrophoretic Light Sca ttering Analysis (DELSA) The presence of aminotrithiolate linker attached to the surface of gold-lX-aminodextran- polystyrene ("IX-Amdex-PS") particles of Example 4B was demonstrated by obtaining the mobilities of these particles and all their parent particles, raw PS aldehyde/sulfate, IX-Amdex-PS (Example 1), and gold-lX- Amdex-PS (Example 2D) particles, as a function of the pH of each suspension of particles using multi-angle Doppler electrophoretic light scattering (DELSA) measurements [Laser Doppler Spectroscopy: Applications To Cell And Particle Electrophoresis, E. E. Uzgiris, Adv. Colloid Interface Sci., 1_4:75-171 (1981)]. DELSA measurements of suspensions of aminotrithiolate-gold-lX-Amdex-PS particles (Example 4B) and their T4- and T8-antibody-conjugated particles (Example 4C) were made on a COULTER® DELSA™ 440. These measurements were used to establish the identity and pH stability range of coated particles layer-by-layer on raw particles . Mobility is directly related to surface charge of particles in aqueous suspension -- the larger the charge, either positive or negative, the higher the mobility. This pH dependence of the mobilities is presented in Fig. 1 for all stages of coated particles. Prior to electrophoretic measurements the raw PS and IX- Amdex-PS particles were suspended in distilled water; the gold-lX-Amdex-PS particles, in 0.01M sodium citrate; the aminotrithiolate-gold-lX-Amdex-PS particles, in lxPBS; and the T4- and T8-aminotrithiolate-gold-lX-Amdex-PS particles, in 1% BSA, 0.1% sodium azide in lxPBS . For measurements, all particles were diluted from either 4 or 1% w/v solids to 4xl0 ~4 percentage w/v solids with O.OΓM -" aqueous potassium nitrate solution. The pH of the diluted particle suspensions was adjusted with 0. IM aqueous potassium hydroxide or 0. IM aqueous nitric acid solutions . DELSA measurements were made by positioning the rectangular sample channel of 1.00mm height at the stationary layer (0.16mm) near the top of the channel. An electrophoretic standard, COULTER EMPSL7, was used to make final adjustments in positioning the sample chamber. A van Gils plot, (H - h) 2 /H 2 versus mobility, where h is the depth in millimeters ( 0 to 1.00) from the top of the channel and H = 0.50mm, of mobilities of the EMPSL7 standard was rectilinear through most of the depth of the channel and was symmetric about the center axis of the channel. Some deviation occurred near the bottom of the channel at h greater than 0.95mm or (H - h) 2 /H 2 greater than 0.8. This showed full development of a parabolic, electroosmotic flow profile of the standard particles in the DELSA sample chamber. Other DELSA instrument parameters were chosen as follows: frequency range, 500Hz; frequency shift, 250Hz; current, 0.7mA unless otherwise noted; on time, 2.0s; off time, 0.5s. Conductivities of the particle suspensions ranged from 1.3 to 1.6 mS/cm. As shown in Fig. 1, aminodextran (lX-Amdex) -coated polystyrene aldehyde/sulfate latex particles show nearly constant mobility of about -1 cm 2 / V-s in the pH 7 to 10 range of measurements, reflecting the presence of positively- charged protonated amino groups that have replaced some neutral aldehyde groups to counter the pre-existing negatively-charged sulfate groups on the surface of the latex particles . The raw polystyrene aldehyde/sulfate latex - "* particles were originally negatively-charged and as shown in Fig 1 have a very high negative mobility of -6 to -7 cm 2 / V-s in the pH 7 to 10 range. After gold colloid was introduced onto the IX-Amdex-PS particles, the steep drop in mobility of the resulting particles from about +2 to - 4 cm 2 /V-s in the pH 4 to 6 range showed the presence of adsorbed citrate on gold-lX-Amdex-PS particles even after dilution of the particles with aqueous sodium nitrate solution. The acid dissociation constants of citric acid as reflected in the stepwise pK a values of 3.14, 4.77, and 6.39 at 20°C, albeit modified for adsorbed citrate, occur in the same pH region as the large drop in mobilities. The electrophoretic mobility profile of aminotrithiolate-gold-lX-Amdex-PS particles showed the mobility of these particles in a range of -0.6 to -3.5 cm 2 /V-s, and also the presence of an artifact at about +0.3 x 10 ~4 cm 2 /V-s throughout the pH 4 to 10 range. This artifact was also present in the same position in mobility profiles of IX-Amdex-PS and gold-lX-Amdex-PS particles, especially at low currents (applied electric field) less than 1mA in the cell. Higher currents of 1 to 2.5mA were needed to obtain electrophoretograms of the latter particle suspensions so that Doppler frequency shifts were greater than +/-10Hz for the lowest scattering angle of 8.6°. The artifact was identified by its constant position in van Gils plots of particle mobility and its constant position near 0. OHz at all four scattering angles in the power spectrum of intensity versus Doppler frequency shift. The mobility of aminotrithiolate-coated gold- lX-Amdex-PS particles was elevated slightly above the mobility of the parent gold-lX-Amdex-PS particles above pH 5.8, but was lowered below that of the parent - - particles at pH less than 5.8. This reflects the lower negative charge that the aminotrithiolate linker can attain by displacing tri-negatively charged citrate on a metal particle surface as a tri-negative trithiolate group. The terminal amino group will still retain a positive charge due to protonation in the pH 4 to 10 region. Also, manifested in the steep rise in mobility from -3.3 cm 2 /V-s to -0.6 cm 2 /V-s in the low pH range of 5.8 to 4.2 may be partial protonation of the thiolate groups. Sulfhydryl groups typically show pK a values in the 7 to 9 range. Whereas as shown in the mobility measurements, single sulfur atoms of thiolate groups attached to surface gold or silver atoms form very strong bonds and are not as easily protonated as either of the carboxylate oxygen atoms of the three carboxylate groups of citrate on colloidal gold or silver. The features in the electrophoretograms of aminotrithiolate-gold-lX-Amdex-PS particles are reversible through several cycles of base addition to pH 10 followed by acid addition to pH 4. The T4- and T8-antibody, conjugated aminotrithiolate-gold-lX-Amdex-PS particles show respective antibody-coated particles with mobilities in a much narrower and more positive +1.2 to -2.3 cm 2 /V-s range than the parent particles. Also, the negative charge of the parent particles throughout the pH 4 to 10 range is counterbalanced but not dominated by the antibody, so that the point of zero charge ("p.z.c") of the antibody- conjugated particles lies between pH 4.5 and 5.0, instead of the pi range of the monoclonal antibodies which is pH 5.4-5.8 for T4 antibody and pH 6.7-7.3 for T8 antibody as determined from the main bands for the respective antibodies by conventional gel electrophoresis. Storage~ of T4- and T8-conjugated particles in 1% bovine serum albumin ("BSA"), 0.1% sodium azide in lxPBS buffer, before dilution with 0.01M potassium nitrate solution may also produce by adsorption of BSA unto the particle surface a p.z.c. range overlapping with the pi of about 4.9 for BSA. The silver-lX-Amdex-PS (Example 2A) particles showed only a slightly decreasing negative mobility (-0.57 to -1.6 cm 2 /V-s) from pH 5 to 10, which is very similar to and only slightly negative of the mobilities of the parent IX-Amdex-PS particles. This compares with mobility data obtained by the moving boundary method for the Carey Lea silver sol (about lOnm mean diameter silver particles) washed with aqueous sodium citrate, which was reported at a zeta potential of -80 +/- 5mV or mobility of -3.7 cm 2 /V-s. [see G. Frens et al, Kolloid Z.Z. Polym. , 233:922 (1969)). Further, microelectrophoresis experiments with several silver sols, for example , silver sol with sodium borohydride (Ag(NaBH 4 ) at pH 8.1), silver sol prepared with ethylenediaminetetraacetic acid (Ag(EDTA) at pH 10.1), Carey Lea silver sol (Ag (Carey Lea) at pH 7.0), and silver sol prepared in aqueous silver sulfate solution (Ag (aqueous AgS0 4 solution) irradiated with 60 Co) at pH of about 5.7 gave mobilities between -4.2 and -5.0 cm 2 /V-s , and with the red gold citrate sol (about 20nm mean diameter gold particles) gave an electrokinetic mobility of -4.5 cm 2 /V-s at pH of about 5.5 [Heard et al, J. Colloid Interface Sci., 93:545 (1983) ] . Although the gold-lX-Amdex-PS particles show a similar mobility of about -4.0 cm 2 /V-s in the pH 6 to 9 range as the mobility of the gold citrate sol at pH of about 5.5, the mobility (-0.6 to -1.6 cm 2 /V-s) of silver= IX-Amdex-PS particles does not resemble that (-4.2 to - 5.0 cm 2 /V-s) of silver citrate sols and suggests that silver-citrate bonding in the silver-lX-Amdex-PS particles was weak so that dilution of the particle suspension in 0.01M potassium nitrate solution for the purpose of electrokinetic measurements released much of the originally adsorbed citrate on silver-lX-Amdex-PS particles . EXAMPLE 5 - ANALYSES OF THE CROSSLINKED AND PROTEIN COATED COLLOIDAL METAL-AMINODEXTRAN COATED PARTICLES The gold- or silver-coated polystyrene particles of Examples 3 and 4 were characterized by SEM micrographs of dried particles on a solid substrate, by flow cytometry histograms of forward scatter (FS) versus side scatter (SS) , and for antibody-conjugated particles by specificity for targeted white blood cell sub-populations in whole blood according to angular light scattering and DC or RF conductivity matrices. Also examined were silver coated particles which were reacted with the aminotrithiol linker of Example 4A. A . Flow Cytometry Analysis of the Cross-linked metal -Amdex-PS Particles and the Protein conj uga ted particles Flow cytometry of the particles of Example 3A showed 41% singlets, 51% multiplets, and 5% uncoated polystyrene particles. Flow cytometry of the protein conjugated particles of Example 3B showed 64% singlets, 33% multiplets, and 1.4% uncoated polystyrene particles for T4-Amdex-Ag-Amdex-PS particles without blocking; 57% singlets, 41% multiplets, and 0.4% uncoated polystyrene particles for blocked T4-Amdex-Ag-Amdex-PS particles; 60 " % singlets, 38% multiplets, and 0.4% uncoated polystyrene particles for T8-Amdex-Ag-Amdex-PS particles without blocking; 56% singlets, 42% multiplets, and 0.3% uncoated polystyrene particles for blocked T8-Amdex-Ag-Amdex-PS particles. However, side scatter shifts in forward versus side scatter histograms obtained by flow cytometry for the silver coated particles cross-linked with the aminotrithioate linker in a manner similar to that described in Example 4B were not as large for silver-PS particles that had been reacted with ligand and tended to revert to the position of uncoated polystyrene particles in the histograms. Flow cytometry of the cross-linked-gold-Amdex- PS particles of Example 4B showed 64% singlets, 23% multiplets, and 8% lightly-coated polystyrene particles. Flow cytometry of the protein conjugated particles of Example 4C showed 82% singlets, 8% multiplets, and 10% lightly-coated polystyrene particles for blocked T4-aminotrithiol-Au-Amdex-PS particles; 75% singlets, 12% multiplets, and 13% lightly-coated polystyrene particles for T4-aminotrithiol-Au-Amdex-PS particles without blocking; 83% singlets, 5% multiplets, and 11% lightly-coated polystyrene particles for blocked T8-aminotrithiol-Au-Amdex-PS particles; and 73% singlets, 12% multiplets, and 15% lightly-coated polystyrene particles for T8-aminotrithiol-Au-Amdex-PS particles without blocking. B . SEM Analyses Scanning electron microscope (SEM) photos of T4-aminotrithiol-Au-Amdex-PS particles dried on a metal stub by evaporation of a small drop of suspension are shown in Figs. 4A-4C. This shows a layer of gold colloid on polystyrene particles, as in original particles of gold deposited on the surface of polystyrene particles without being altered by binding of aminotrithiol linker, activation with sulfo-SMCC, conjugation with antibody, blocking, and washing with BSA buffer. Silver-Amdex-PS particles were coated with aminotrithiol linker and conjugated with antibody in a similar way as for the above-described gold coated particles in Example 4. The aminotrithiol linker, however, competed too well with aminodextran for binding sites on the surface of deposited silver colloid on polystyrene particles and displaced much of the silver colloid from the surface of silver-aminodextran- polystyrene particles. This was seen from the opalescence of the supernatant of particle washings, which contained small silver particles. See the large bare spots on some particles and the presence of large aggregates of small silver particles in the SEM photos displayed in Figs. 5A-5D. Apparently, the aminotrithiolate linker binds too strongly to the silver particles, dislodging them from their embedded positions in the aminodextran coating around the PS particles . Also, the SEM micrographs of samples taken after reaction with ligand showed large aggregates of small silver particles and polystyrene particles with many bare spots, whereas the same particles were uniformly coated with silver colloid before reaction with ligand. Thus, aminotrithiol does not work well as a crosslinking agent with silver colloid on polystyrene particles . EXAMPLE 6 - CONJUGATION OF T4 AND T8 ANTIBODIES TO DIAMINOPROPANE-COATED POLYSTYRENE ALDEHYDE/SULFATE LATEX PARTICLES This example describes coated particles used as controls and as one of a pair of beads in simultaneous experiments to verify the results of the methods and compositions of the present invention. A. Prepara tion of Particles Except for the scale up preparation, coupling of 1, 3-diaminopropane (DAP) to polystyrene latex particles was carried out by methods previously described in International Patent Application No. W09524631, published September 14, 1995. To lOOOmL of 4% w/v solids polystyrene aldehyde/sulfate particles (Interfacial Dynamics Corp., about 2μm diameter) in a 2L polypropylene bottle were added 16.8mg of liquid DAP (0.888g/mL specific gravity) per mL of particle suspension or 19.1mL DAP. The resulting suspension was mixed well by vortexing, sonicated for 30 seconds, and roller mixed for 24 to 72 hours. To reduce Schiff's base groups and unreacted aldehyde on the surface of the particles, 10 mg of solid sodium borohydride per mL of 4% w/v solids particles that were roller mixed with DAP, or 10.4g were added directly to the particle reaction mixture. No excessive effervescence took place in the presence of excess DAP at a pH of about 11.5. The reaction mixture was roller mixed for about three hours with occasional brief 30 second sonication and release of hydrogen gas from the reaction vessel. The particles were then separated by centrifugation at about 2600g for about 25 minutes. The supernatant was discarded and the residue of particles was resuspended in about IL distilled water. Redispersion of the particles was accomplished by vortexing and brief sonication. The washing procedure by centrifugation was repeated four times and the final particle suspension was adjusted to lOOOmL total volume with lxPBS solution. Activation of DAP-coupled particles was accomplished with sulfo-SMCC. In general, 14.2μL of freshly prepared lOmg/mL sulfo-SMCC in lxPBS was used per mL of 4% w/v solids DAP-coated polystyrene particle suspension. In a typical preparation, 14.2mL of the sulfo-SMCC solution was added to lOOOmL of 4% w/v solids particles. The mixture was then roller mixed in a 2L plastic bottle for about one hour, separated by centrifugation, and washed a plurality of times with IxPBS solution. The functionalized, DAP-coated particles resulting from the above series of steps have pendant maleimidyl groups and are suitable for conjugation to a variety of biological molecules. If the substance which is desired to be conjugated to the particles has a sufficiency of active sulfhydryl groups, activation of that substance is not necessary, and the following step may be skipped. Antibodies were activated with 2-iminothiolane (IT) hydrochloride. In a typical run, a 42.77mg/mL concentrate of T4 monoclonal antibody in IxPBS containing 0.1% sodium azide was prepared. For lOOOmg T4 (or T8) antibody and 15mg/mL antibody concentration during coupling, the total reaction volume should be 66.67mL. Using a 15:1::IT:T4 activation ratio, 93.75 μmol (12.9mg) IT (6.45mL of 2mg/mL IT) in IxPBS is required. Therefore, 36.84mL of IxPBS solution was added to 23.38mL of T4 concentrate, to which resulting solution an additional 6.45mL of 2mg/mL IT solution was added. The net resulting solution was roller mixed in a 250mL centrifuge tube for 1 hour. The contents of the reaction tube were then applied to the top of a 2000mL G-50 Sephadex column, equilibrated and washed with IxPBS solution . The derivatized antibody was eluted using IxPBS and a plurality of 15mL fractions were collected with the aid of a UV monitor. Fractions in the middle of the band absorbing at 280nm were pooled and the A2 80 value was used to determine the IT-T4 antibody concentration. Typically, the IT-T4 or IT-T8 concentration was 5-6mg/mL. The IT-T4 or IT-T8 was then conjugated with sulfo-SMCC derivatized particles. In a laboratory scale conjugation, total volume lOOOmL, the concentration of particles was 6.67% w/v solids and the IT-T4 concentration was 0.7mg/mL or the IT-T8 concentration was 0.8mg/mL. In one sample, when the purified IT-T4 solution concentration was 6.236mg/mL, then 112mL of IT- T4 antibody solution in IxPBS and 288mL of IxPBS solution were added to lOOOmL of 4% w/v solids sulfo-SMCC activated particles which had been preconcentrated by the removal of 400mL of supernatant. In another sample, the purified IT-T8 antibody solution concentration was 5.846mg/mL, so that 137mL of IT-T8 antibody solution in IxPBS and 263mL of IxPBS solution were added to lOOOmL of 4% w/v solids sulfo-SMCC activated particles which had been preconcentrated by the removal of 400mL of supernatant. The IT-antibody solution was added to the particles in 25mL increments with vortexing and ultrasonication between additions. The resultant mixture was then roller mixed in a 2L bottle for about two hours. Before blocking, lmL samples of the conjugation mixtures were pipetted, filtered through 0.2μm low- protein binding filter, and the absorbance of the supernatants was measured at 280nm to obtain surface concentration of antibody, 0.44mg/mL T4 and 0.53mg/mL T8, and surface densities of 4.2mg T4 antibody/m 2 and 5. Img T8 antibody/m 2 . Unreacted maleimidyl groups on the sulfo-SMCC activated particles were blocked with L-cysteine after antibody conjugation. Typically, 120mL of 5mg/mL L- cysteine in IxPBS were added to the conjugation mixture of the previous step and the resulting suspension was roller mixed for about 15 minutes. Unreacted sulfhydryl groups were blocked by the addition of 134mL of 20mg/mL iodoacetamide in IxPBS followed by the addition of 25mL of IM, pH 9.8 sodium borate buffer solution. The resulting suspension was roller mixed for about 30 minutes, the blocked conjugation mixture was separated by centrifugation and the particles washed two times with a solution of 1% bovine serum albumin (Pentex fraction V, protease free) and 0.1% sodium azide in IxPBS (BSA buffer solution) . After washing, the particles were resuspended in BSA buffer solution to a total volume of lOOOmL (4% w/v solids), roller mixed for about 1 hour, stored at about 4°C for a time in the range of 24-72 hours, separated by centrifugation and washed three times with BSA buffer solution. B . Analysis of Particles Flow cytometry of particles alone in ImM KOH solution gave 87.2% singlets, 11.6% doublets, and 0.6% multiplets for T4 particles and 94.3% singlets, 5.1% doublets, and 0.1% multiplets for T8 particles. C. DELSA Analysis The T4- and T8-antibody conjugated to diaminopropane (DAP) -coated polystyrene aldehyde/sulfat e~ particles as described above were also investigated for their electrokinetic properties. DELSA measurements of mobility as a function of pH were also made of DAP-PS (IxPBS wash), sulfo-SMCC-DAP-PS (w/blocking and IxPBS wash), T4-DAP-PS (IxPBS wash), and T8-DAP-PS (IxPBS wash) particles. The mobility versus pH profiles of raw particles, DAP-PS particles, sulfo-SMCC-DAP-PS particles, and T4- and T8-DAP-PS particles are compared in Fig. 2, in order to provide evidence for the existence of the specified, coated particles according to expectations of charge on each particle derivative. The results in Fig. 2 are for IxPBS washed particles and in Fig. 3 for BSA buffer and IxPBS washed particles. The raw particles were suspended in distilled water whereas the other derivatized particles were all washed with and suspended in IxPBS before dilution with O.OIM potassium nitrate solution for electrokinetic measurements. The DAP-PS particles showed excellent surface coverage of positively-charged amino groups to compensate for the highly negative charge of raw particles, with mobilities between -2.2 and +4.9 x 10 "4 cm 2 /V-s in the pH 10 to 5.5 range and a p.z.c. of about pH 6.95. The positive charge of DAP-PS particles is somewhat reduced when sulfo-SMCC is used to activate amino groups for antibody conjugation to give mobilities of -3.6 to +2.9 x 10 "4 cm 2 /V-s in the pH 10 to 4 range and a p.z.c. of about pH 5.80. T4- and T8- antibody-conjugated DAP-PS particles assume electrokinetic properties intermediate to DAP-PS and sulfo-SMCC-DAP-PS particles with p.z.c. between 5.9 and 6.2. Particles that had first been washed with BSA buffer and then with IxPBS showed a modified mobility - - versus pH dependence (Fig. 3), with p.z.c. in the pH 5.3 to 5.6 range, closer to the pi of BSA at 4.9. EXAMPLE 7 - INDIVIDUAL ANALYSES OF LYMPHOCYTE SUBPOPULATIONS Each particle was tested singly with whole blood to permit separate determinations of CD4 or CD8 positive cells in white blood cell population of whole blood, i.e., to determine the location of shifted target populations in the various histograms. For both individual and simultaneous analyses of T4 and T8 cell populations in whole blood, the particles used contained T4 and T8 antibodies conjugated to gold-coated PS particles, or silver- coated PS particles, and diaminopropane (DAP) -coated PS aldehyde/sulfate particles. The latter particles are preferred for accuracy as bead controls because they form fewer aggregates. All measurements in Example 7 and Example 8 were made with DAP-PS for comparison and controls. A . Using (T4 or T8) -Amdex-Silver-A dex-PS Particles Separate analyses of the percentage of CD4 and CD8+ cells were made on a modified Coulter VCS instrument using T4 or T8-conjugated, silver-coated polystyrene particles of the invention (Example 3B) or T4 or T8- conjugated polystyrene particles without a metal coating. Coulter VCS technology, which allows measurement of volume, conductivity, and light scatter characteristics of blood cells, was used on the STKS2B hematology analyzer to assay for CD4 and CD8 cell populations in whole blood samples. Daily calibration of the analyzer was performed with COULTER LATRON™ primer and LATRON control. Titers of 1% w/v solids T4-Amdex-Ag-Amdex-PS particles between 2 and 40μL were mixed with 200μL of whole blood for about 2 minutes. The mixtures were analyzed on the modified VCS™ instrument. DC versus SALS (PMT1) matrices are shown in Figs. 6A-6F for the blood sample and various mixtures. Note the successively greater shift of targeted T4 positive lymphocytes to higher SALS intensity as a larger titer of particles was used. The standard 20μL titer appeared to be sufficient to resolve shifted from unshifted lymphocytes with either T4-DAP-PS or T4-Amdex- Ag-Amdex-PS particles. Particle-to-cell ratios for targeted cell populations can be easily calculated and compared with theoretical values. For an average 45% CD4+ lymphocytes out of 2.4-6.8 x 10 5 lymphocytes per 200μL of whole blood, there are 1.1-1.3 x 10 5 CD4+ cells. For 2μm diameter particles at 4% w/v solids, there are 6.83 x 10 9 particles/mL, so that the standard titer of 20μL of 1% w/v solids particles per 200μL of whole blood contains 3.4 x 10 7 particles. The range of particle-to-cell ratio is then 110-310. Thus, Figs. 6A-6F illustrate that the more particles added the greater the shift obtained. It also shows that a minimum of 20μL titer is needed in order to obtain a good separation from shifted to unshifted of targeted cells. A theoretical limit for a monolayer coating of particles around lymphocytes can be estimated for comparison. For lymphocytes of 8-12μm diameter size range, the surface area assuming a spherical shape is 4π r 2 or 201-452μm 2 . Thus, the maximum number of 2μm - " " diameter particles that can be packed around a single lymphocyte in a square lattice will range from 50 to 113. In a closest-packed lattice the area per particle is 3 d 2 /2 instead of d 2 , so that the maximum particle-to-cell ratio will range from 58 to 130. The latter range was borne out almost stoichiometrically by an experiment when T4-DAP-PS particle titer runs showed that lOμL of 1% w/v solids particles, or a range of particle-to-cell ratio from 55 to 155 was sufficient in almost every trial, whereas 5μL was not always enough. Similar results could be derived for T8-PS particle-to-cell ratios. For an average 20% CD8+ lymphocytes, the range of particle-to-cell ratio is 124- 349 for a lOμL titer of l%-2μm diameter particles so that about 2X more particles than theoretically calculated are needed. The titer of particles needed may have to be increased for silver coated PS particles mixed with whole blood to compensate for the substantial numbers of particle aggregates containing more than two particles arranged in a non-linear geometric structure. Also, other factors, such as the immobilized antibody-cell surface antigen affinity constant, the number of specific antigenic receptors per targeted cell, and the accessibility of antigenic receptors on cells to immobilized antibody, can influence the required particle-to-cell ratio. The affinities of T4 and T8 antibodies for their antigenic partners on the surface of T lymphocytes are very high, 10 10 to 10 12 M "1 for T4, and 10 8 to 10 9 M "1 for T8, so that binding is not a limiting factor [Oonishi et al, J. Immunol. Methods, 115:159 (1988); see also, U.S. Patent application 08/624,014, filed March 27, 1996 by Siiman et al] . Also, receptor per cell values range from 10 4 to 10 5 for CD4 and CD8 antigens, which project sufficiently from the surface of cells to offer excellent contact with potential immobilized antibody partners [R.F. Vogt, Jr. et al, Cytometry, 1_2:525 (1991); A.N. Barclay et al, The Leukocyte Facts Book, Academic Press, San Diego, CA, (1993) ] . Comparison of the lymphocyte population shifts due to T4-Amdex-Ag-Amdex-PS or T4-DAP-PS particles were made for various pairs of parameters and for excitation wavelengths of 633nm and 780nm in the matrices shown in Figs. 7A-7L and Figs. 8A-8L. The matrices with the best resolution of T4-Amdex-Ag-Amdex-PS shifted lymphocytes from T4-DAP-PS shifted lymphocytes are SALS (PMTl) versus LMALS or UMALS. Thus, for simultaneous analyses using multiple antibody labels, the best matrices to use are SAL (PMTl) versus LMALS or UMALS. B . Using Protein Conj uga ted Particles of the Invention Separate determinations of the percentage of CD4 or CD8 positive cells in a white blood cell population of whole blood were performed on a modified COULTER VCS instrument using T4 or T8-conjugated, gold- and silver-coated polystyrene particles of the invention or (T4 or T8) -DAP-PS particles not having a metal coating . For percentage CD4/CD8 determination, 40μL or 60μL of a 1% w/v solids particle suspension was mixed with 400μL of EDTA-anticoagulated whole blood for about 2 minutes in a 5mL glass tube on a Coulter model 6705473 single speed mixer. In the manual mode, 150μL of the whole blood-particle mixture were aspirated into the analyzer, which performed an automated acid lyse of red blood cells and quench, immediately after completion of mixing. The mixture was analyzed for various light scatter (SALS, UMALS, LMALS) parameters and DC and RF conductivity of the particle labeled and unlabelled white blood cells. Representative matrices for T4 particles are shown in Figs. 9A-9F, 10A-10F and 11A-11F. T4 (T8) -DAP-PS particles showed the best separation of the targeted cell population in 90° LS versus RF, UMALS, DC, or LMALS and UMALS versus DC matrices. T4 (T8 ) -conjugated, gold- or silver-coated polystyrene particles showed good separation only in 90° LS versus RF, UMALS, DC, or LMALS matrices. Quantitation of percentage CD4 and percentage CD8 from 90° LS versus UMALS matrices was checked with T4 (T8) -DAP-PS particles, 40μL of 1% w/v solids, and 400μL of whole blood from four normal donors in triplicate. Parallel determinations were made with the same particles and blood donors on the COULTER STKS hematology analyzer. Flow cytometry was also used to obtain comparative results for percentage of CD4/CD8 positive cells in lymphocytes with the appropriate fluorescent antibody markers: Mouse IgGl-phycoerythrin/Mouse IgGl Fluorescein Isothiocyanate ("MsIgGl-PE/MsIgGl-FITC") ; or the dual fluorescence markers T3-FITC/T4-PE and T3-FITC/T8-PE (all from Coulter Corporation) for each donor. Table I shows the summarized results as percentages of CD4/CD8 positive cells in whole blood for four donors using T4 (T8 ) -DAP-PS . Table I Percentage CD4, CD8 Positive Cells in Whole Blood Donor: 46848 53522 53960 54152 % in wbc: %CD4 %CD8 %CD4 %CD8 %CD4 %CD8 %CD4 %CD8 Trial 1 10.96 3.88 14.10 5.16 13.59 8.15 13.99 8.64 Trial 2 9.94 3.37 14.33 5.25 13.16 8.33 14.36 8.82 Trial 3 8.82 3.18 14.18 5.66 12.66 8.32 14.40 8.68 Mean 9.91 3.48 14.20 5.36 13.14 8.27 14.25 8.71 %L in wbc: 18.9 27.6 30.1 27.8 % in L, 52.42 18.40 51.46 19.41 43.64 27.46 51.26 31.34 mod. VCS STKS % 54.3 17.3 50.9 19.2 39.8 25.3 49.5 33.1 flow % 56.1 17.6 49.3 20.5 38.6 28.1 50.1 32.6 % diff, 6.56 4.54 4.38 5.32 13.0 2.28 2.31 3.86 mod. VCS- flow Representative 90° LS versus UMALS matrices for each blood donor are shown in Figs. 12A-12D for T4-DAP-PS particle-whole blood mixtures. Donor 53960 appeared to indicate the presence of more cells bordering the gate in the bottom left-hand corner of the matrix between shifted and unshifted white blood cell populations. CD4 positive monocytes with very few particles have sometimes been identified in this region. For this donor, moving the gate slightly to higher 90° LS and higher UMALS gave an average 12.25% CD4 cells in white blood cells or 40.71% CD4 cells in lymphocytes, which gave only a 2.29% difference with the flow cytometry comparator. Further, to establish the optimum amount of particle suspension, titers ranging from 10 to 80μL of 1% w/v solids, T4 (T8 ) -aminotrithiol-Au-Amdex-PS particles were mixed with 400μL of whole blood for three donors and analyzed in the 90° LS versus UMALS matrices for percentage CD4 (percentage CD8) cells in the white blood cell population. Results are tabulated in Table II, showing the particle titer dependence of the percentage of CD4/CD8 positive cells in whole blood for three donors . The titer matrices are shown for representative donor 49070 in Figs. 13A-13E for T4-aminotrithiol-Au- Amdex-PS particles. Table I I Particle Titer Dependence of Percnetage CD4, CD8 Positive Cells in White Blood Cells Donor 49070 5 0184 53608 Particle Titer, μL %CD4 %CD8 %CD4 %CD8 %CD4 %CD8 10, T4(T8)-Au-PS particles 8.40 4.78 3.42 2.25 6.02 5.24 20 9.65 4.29 4.34 2.42 6.49 5.28 40 11.06 4.61 5.18 2.18 7.87 5.09 60 11.36 5.22 5.13 2.32 8.11 5.21 80 11.90 4.98 5.27 1.95 8.22 5.62 60, T4(T8)-PS particles 11.55 5.46 5.54 2.58 8.51 6.78 %L in wbc 24 .5 12.5 19.1 % in L, T4 (T8)-Au-PS 46.4 21.3 41.0 6 4.25 27.3 particles % in L, T4(T8)-PS particles 47.1 22.3 44.3 20.6 44.5 35.5 flow % 45.9 25.9 55.2 24.6 43.2 35.0 STKS % 42.3 22.5 56.2 25.0 41.3 30.4 The percentage CD4 or CD8 generally reached a constant value at a titer of 40μL of 1% w/v solids particles per 400μL of whole blood. This is about twice the minimum titer established previously for 1% w/v solids T4 (T8) -DAP-PS particles of the same size, for which a 20μL per 200μL of whole blood was chosen as a standard titer. Thus, 60μL of 1% w/v solids T4(T8)- aminotrithiol-Au-Amdex-PS particles per 400μL of whole blood was chosen hereafter as the standard titer. For each donor the percentage of lymphocytes in the white blood cell population was obtained on the COULTER STKS hematology analyzer; thereafter, the percentage CD4/percentage CD8 in white blood cell obtained using the respective antibody-aminotrithiol-Au-Amdex-PS and antibody-DAP-PS particles were converted to percentages in L to compare with flow cytometry data with fluorescent markers and STKS data with PS particles. Donors 50184 and 53608 have low percentages of lymphocytes in white blood cell, which are out of the normal range of 20-55%. These donors also showed the largest deviations in percentages of CD4/CD8 determined on the modified VCS versus comparative results obtained on the STKS hematology analyzer for T4/T8 particles mixed with whole blood or obtained by flow cytometry with the __ appropriate fluorescent markers. The most useful matrices in this application were (i) SALS versus LMALS or UMALS, (ii) UMALS versus DC, (iii) SALS versus DC, and (iv) SALS versus RF. Thus, the antibody-conjugated polystyrene particles of this invention can be resolved from antibody-conjugated silver or gold polystyrene particles by a judicious choice of excitation laser wavelength between 450 and 800nm to excite the plasmon resonances of either silver (λ exc = 400-520nm) or gold (λ exc > 520nm) or both colloids (λ exc = 520-800nm) deposited on the surface of polystyrene particles, and choice of the SALS versus UMALS, DC, or RF and UMALS versus DC matrices . The wavelength dependence provides a method of using two- or three-color, antibody-conjugated particles, with or without immobilized silver or gold colloids, and different antibodies targeting mutually exclusive white blood cell subsets, to analyze simultaneously for two or three subset populations of white blood cells. The total light scattering of gold particles of diameter 50 to 200nm, embedded on the surface of spherical 2 micron diameter polystyrene particles, which surround white blood cells specifically targeted by the antibody-coated particles should be a superposition of three main scattering contributions: (1) cell scatter, (2) polystyrene particle scatter, and (3) gold particle scatter. Since the 2μm polystyrene particles are generally smaller than white blood cells, their attachment to the surface of cells perturbs the light scatter of the original cells according to the effect of the particle-to-cell ratio and geometrical arrangement of particles around cells on the shape and size of particle- cell conjugates. The gold particles are much smaller than either the polystyrene particles or the white blood cells; therefore, their effect on the shape and size of particle-cell conjugates is negligible. However, because of their particular wavelength-dependent extinction spectra in the visible-near infrared region, the gold particles show additional scattering and absorption that was not present in polystyrene particle-cell conjugates. Also, the concentration of gold-polystyrene particles on the surface of a cell where the particles become nearest neighbors in close contact to each other can perturb the extinction characteristics of the separated, gold-polystyrene particles. Furthermore, the absorption part of the extinction of gold particles can be substantial and in some angular scattering ranges produces a loss in scattering intensity relative to the scattering from polystyrene particle-cell conjugates. The additional side scatter shift for antibody- aminotrithiol-Au-Amdex-PS particles attached to lymphocyte subsets was small relative to the shift for PS particles attached to the same cells; however, changes in the position of targeted lymphocytes in SALS versus UMALS matrices allowed a clear separation between antibody- aminotrithiol-Au-Amdex-PS and PS shifted lymphocyte populations due to a substantial decrease in UMALS for cells coated with antibody-aminotrithiol-Au-Amdex-PS particles. Antibody-Amdex-Ag-Amdex-PS particles attached to lymphocytes have the same effect as the antibody- " aminotrithiol-Au-Amdex-PS particles themselves, in that forward scatter and UMALS are decreased relative to the scatter of lymphocytes labeled with PS particles. However, side scatter of targeted lymphocytes labeled with antibody-Amdex-Ag-Amdex-PS particles was enhanced about twice as much as the side scatter of lymphocytes labeled with the antibody-aminotrithiol-Au-Amdex-PS particles . EXAMPLE 8 - SIMULTANEOUS ANALYSES OF LYMPHOCYTE SUBPOPULATIONS Simultaneous determination of the percentage of CD4 or CD8+ positive cells in a white blood cell population of whole blood was made on a modified Coulter VCS instrument using T4 or T8-conjugated polystyrene and T8 or T4-conjugated, gold- or silver-coated polystyrene particles of this invention. The particles used contained T4 and T8 antibodies conjugated to gold-coated PS particles, or silver- coated PS particles, and diaminopropane (DAP) -coated PS aldehyde/sulfate particles for the reasons described in Example 7. Briefly, the conjugates were mixed with whole blood, and then white blood cells were analyzed after an acid lyse of red blood cells and quench. Specifically, 40μL of 1% w/v solids T4 (T8 ) -conjugated polystyrene particles and 40-60μL of T8 (T4 ) -conjugated, gold- or silver coated polystyrene particles were mixed with 400μL of whole blood for about 2 minutes . The mixtures were then treated and analyzed as in Example 7. Representative matrices are shown in Figs. 14A-14F. The 90° LS versus UMALS matrices showed the best separation of cells labeled with T4 (T8 ) -conjugated polystyrene particles from cells labeled with T8(T4)- conjugated, gold- or silver-coated polystyrene particles. Only cells labeled with T4 (T8 ) -conjugated polystyrene particles were resolved in UMALS versus DC matrices and cells labeled with T4 (T8 ) -conjugated, gold- or silver- coated particles were sometimes resolved in 90° LS versus RF, DC matrices. Percentage CD4/percentage CD8 in white blood cell were obtained from analyses of 90° LS versus UMALS matrices for mixtures of 60μL T4 (T8 ) -PS/60μL T8(T4)-Au-PS particles with 400μL of whole blood for three donors in triplicate. Table 3 shows the percentage of CD4/CD8 positive cells in whole blood for three donors. Table I I I Percentage CD4, CD8 Positive Cells in Whole Blood Donor 49070 50184 53608 %CD4 %CD8 %CD4 %CD8 %CD4 %CD8 T4-PS/T8-Au- -PS particles Trial 1 12.40 5.10 5.67 2.40 8.84 4.95 Trial 2 12.50 5.43 6.11 2.55 9.67 5.16 Trial 3 (14.16) 5.02 5.61 2.61 9.71 5.30 Mean 12.45 5.18 5.80 2.52 9.41 5.14 4 min 13.48 5.24 5.75 2.76 9.48 4.90 Mean % in L 50.8 21.1 46.4 20.2 49.3 26.9 T8-PS/T4-Au- -PS particles Trial 1 10.05 7.01 6.65 3.18 (19.66) 7.30 Trial 2 11.13 6.72 6.81 2.86 14.43 7.16 Trial 3 (8.80) 7.26 6.55 2.63 14.57 6.98 Mean 10.59 7.00 6.67 2.89 14.50 7.15 4 min 10.29 6.38 6.52 2.96 13.80 7.19 Mean % in L 43.2 28.6 53.4 23.1 75.9 37.4 % in L,T4(T8)-PS 47.1 22.3 44.3 20.6 44.5 35.5 particles flow % 45.9 25.9 55.2 24.6 43.2 35.0 STKS % 42.3 22.5 56.2 25.0 41.3 30.4 For some runs, adjustment of the gate in the bottom left-hand corner of the matrices to higher 90° LS and higher UMALS can improve the agreement between percentage CD4/CD8 obtained on the modified VCS instrument and the results obtained by flow cytometry. For example, after adjustment for donor 49070, percentage CD4 in L = 45.6 with T4-Amdex-PS/T8-aminotrithiol-Au-Amdex-PS particles and percentage CD8 in L = 25.6 with T8-Amdex-PS/T4- aminotrithiol-Au-Amdex-PS particles . For donor 53608, percentage CD4 in L = 35.5 with T4- Amdex-PS/T8-aminotrithiol-Au-Amdex-PS particles, percentage CD4 in L = 49.7 with T8-Amdex-PS/T4- aminotrithiol-Au-Amdex-PS particles, and percentage CD8 in L = 30.6 with T8-Amdex-PS/T4-aminotrithiol-Au-Amdex-PS particles. Similar results, summarized in Table IV for a single donor, were obtained with 40μL T4 (T8 ) -Amdex-PS and 60μL T8 (T4 ) -Amdex-Ag-Amdex-PS particles mixed with 400μL of whole blood and excited with the 633nm He/Ne laser line, except quantitation against flow comparator data was not as good due to the presence of larger numbers of T4 (T8)- Amdex-Ag-Amdex-PS particle aggregates which broaden the non-targeted cell regions in the various matrices. Table IV shows the percentage of CD4/CD8 positive cells in whole blood using the four different particles. Table IV Percentage CD4, CD8 Positive Cells in Whole Blood Matrix % CD4 % CD8 in L in L T4-PS or T8-PS particles 90° LS vs. UMALS 46.1 23.2 UMALS vs. DC 37.4 21.8 T4-Ag-PS or T8-Ag-PS 90° LS vs. DC 20.1 17.2 particles T4-PS/T8-Ag-PS particles UMALS vs . DC 36.9 90° LS vs. UMALS 17.8 T8-PS/T4-Ag-PS particles 90° LS vs. UMALS 53.3 UMALS vs. DC 23.1 90° LS vs. DC 36.9 UMALS vs. DC 23.1 flow comparator 45.6 25.4 Several gating strategies were used to calculate the percentage of targeted cells in the white blood cell population, one of which is illustrated in Figs. 15A-15F, for T4-Amdex-PS/T8-aminotrithiol-Au-Amdex-PS particle mixtures. The percentage of lymphocytes in white blood cell was 18.8 for this donor as determined on the COULTER STKS hematology analyzer. All publications cited in this specification are indicative of the level of skill of those in the art to which this application pertains and are incorporated herein by reference herein. While the invention has been described with reference to a particularly preferred embodiment, it will be appreciated that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims

Claims: WHAT IS CLAIMED IS: 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) a 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 metallic solid (c) and said aminodextran coating (b) are" dispersed on said surface of said polystyrene core substrate . 11. The particle according to claim 1, wherein said linker (d) is an aminodextran linker. 12. The particle according to claim 11, wherein said aminodextran linker (d) is selected from the group consisting of aminodextrans in which the degree of diamine substitution is in the range of between about 1/40 and about 1/35 compared to a maximum theoretical value of 1/2.5. 13. The particle according to claim 12, wherein said diamine substitution in said aminodextran linker is approximately 1/7 to 1/8. 14. The particle according to claim 11, wherein the aminodextran linker is 5X-aminodextran . 15. The particle according to claim 11, wherein said aminodextran (d) is covalently bonded to said colloidal metal-aminodextran coated substrate by cross- linking amino groups with glutaraldehyde. 16. The particle according to claim 1, wherein said linker (d) is an aminotrithiolate linker. 17. The particle according to claim 16 wherein said linker is tris (3-mercaptopropyl) -N-glycylaminomethane . - " * 18. The particle according to claim 16, wherein said aminotrithiolate linker (d) is covalently bonded to said colloidal metal-aminodextran coated substrate by covalent metal-sulfur bonds. 19. The particle according to claim 18 wherein said colloidal metal is gold. 20. The particle according to claim 1, wherein said protein (e) is an antibody. 21. The particle according to claim 20, wherein said antibody is selected from the group consisting of an anti-CD4 antibody and an anti-CD8 antibody. 22. The particle according to claim 1, wherein said protein (e) is covalently bonded to said linker-colloidal metal-aminodextran coated substrate by covalent bonding of the free amino groups on the linker which are activated with maleimide in sulfo-SMCC, with sulfhydryl groups on the 2-iminothiolane activated protein. 23. The particle according to claim 1, which comprises (a) a colloidal-sized polystyrene microparticle; (b) a lX-aminodextran coating over the peripheral surface of said polystyrene particle; (c) a layer of colloidal-sized gold overlaying said aminodextran coating; (d) an aminotrithiolate linker having a free amino group; and - ~* (e) an anti-CD4 antibody attached to said linker by covalently bonding to said free amino group. 24. The particle according to claim 1, which comprises (a) a colloidal-sized polystyrene particle; (b) a lX-aminodextran coating over the peripheral surface of said polystyrene particle; (c) a layer of colloidal-sized silver overlaying said aminodextran coating; (d) a 5X-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. 25. The particle according to claim 1, which comprises (a) a colloidal-sized polystyrene particle; (b) a lX-aminodextran coating over the peripheral surface of said polystyrene particle; (c) a layer of colloidal-sized gold overlaying said aminodextran coating; (d) an aminotrithiolate linker having a free amino group; and (e) an anti-CD8 antibody protein attached to said linker by covalently bonding to said free amino group . 26. The particle according to claim 1, which comprises (a) a colloidal-sized polystyrene particle; (b) a lX-aminodextran coating over the peripheral surface of said polystyrene particle; - "* (c) a layer of colloidal-sized silver overlaying said aminodextran coating; (d) a 5X-aminodextran linker having a free amino group; and (e) an anti-CD8 antibody attached to said linker by covalently bonding to said free amino group. 27. 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. 28. The method according to claim 27 wherein said biological fluid is whole blood. ~ ~ 29. The method according to claim 27 wherein the subpopulations are selected from the group consisting of T lymphocytes, B lymphocytes, granulocytes, and monocytes. 30. The method according to claim 29 wherein said lymphocytes are CD4 + lymphocytes and CD8 + lymphocytes. 31. The method according to claim 27 wherein each said protein is an antibody. 32. The method according to claim 27 wherein said instrument distinguishes between cells by light scattering, conductivity and volume.

Inventors: BURSHTEYN ALEXANDER, GORDON KRISTIE, MAPLES JOHN A, RODRIGUEZ CARLOS M, SIIMAN OLAVI, WHITESELL JAMES KELLER, BURSHTEYN, ALEXANDER, GORDON, KRISTIE, MAPLES, JOHN, A., RODRIGUEZ, CARLOS, M., SIIMAN, OLAVI, WHITESELL, JAMES, KELLER
Applicants: COULTER INT CORP, COULTER INTERNATIONAL CORP.
Application year: 1998
Main classification: G01N33
IPC codes: G01N33