WO2000051814A1 - Simultaneous analysis of an analyte and an interfering substance using flow cytometry - Google Patents

Abstract

The present invention relates to a method of assay and in particular, a method of flow cytometric immunoassay for simultaneous analysis of an analyte and an interfering substance.

Description

SIMULTANEOUS ANALYSIS OF AN ANALYTE AND AN INTERFERING SUBSTANCE USING FLOW CYTOMETRY

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application Serial No.

09/263,399, filed March 5, 1999, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

FIELD OF INVENTION The present invention concerns a method of assay and in particular, a method of flow cytometric immunoassay for simultaneous analysis of an analyte and an interfering substance.

BACKGROUND OF THE INVENTION Immunoassay techniques are based upon known reactions between an antigen and a binding protein, these two binding partners making a binding pair. Often times one of the two binding partners is labeled such as with a fluorescent tag. This label then can be used for the determination of analyte composition or concentration. Using various assay techniques, the determination of specific analytes in a biological sample is possible.

One immunoassay category is the sandwich assay. In sandwich assays, generally there are two binding proteins which can simultaneous attach themselves to the analyte of interest. Usually, one of these binding proteins is attached to a solid phase and the other binding protein is modified with a detectable label, such as, a radioisotope, enzyme or fluorophore. After an incubation period, the amount of label bound to the solid phase is then assessed which permits the determination of the concentration of analyte when compared to a known standard.

Another immunoassay category of particular interest is the competitive binding assay. There are two alternative formats to a competitive binding assay. One of these formats has a binding protein that has affinity for the analyte and attached to a solid phase added to a solution containing the analyte of interest and a known amount of labeled analyte. The analyte of interest and the labeled analyte compete for the binding protein on the solid phase. The concentration of the analyte of interest can thereafter be determined with a previously established standard curve. The other of these competitive assay formats has the labeled analyte-specific binding protein added to a solution containing the analyte of interest and analyte bound to a solid phase. The analyte of interest and the analyte bound to the solid phase compete for the labeled binding protein. The concentration of the analyte of interest can thereafter be determined with a previously established standard curve.

Another of the immunoassay categories is the antibody assay. As in the competitive assay technique, there are two methods of performing these assays; the indirect assay and the antibody-class capture assay. In the indirect assay, the reagents consist of antigen attached to a solid phase and class-specific antibody labeled with some detectable marker (e.g., enzyme, chemiluminescent compound, fluorescent dye, radioactive isotope, etc.). In conducting the assay, sample is first contacted with the antigen-coated solid phase, allowing the antigen-specific antibodies in the sample to become attached. After separation of free from bound, the labeled class-specific antibody is introduced to the bound fraction. Again after separating free from bound, the amount of label on the solid phase is measured. If antigen-specific antibodies of the appropriate class are present in the sample and, therefore, attached to the solid phase, labeled antibody to this class of antibodies will become attached. The amount of antigen-specific class- specific antibodies present in the sample is directly proportional to the signal generated by the label attached to the solid phase.

The second approach is antibody capture. The reagents comprise class specific antibody attached to a solid phase and labeled antigen. Sample is first contacted with the solid phase, allowing the immuno globulin of the appropriate class to become attached thereby. After washing to remove the unbound materials, labeled antigen is introduced. Labeled antigen is captured by the solid phase if antigen-specific antibodies of the appropriate class are present in the sample.

Most immunoassay methods measure one parameter per sample and use this parameter to quantify the analyte. For example, in the ELISA technique, a microtiter well will produce an optical density proportional to the amount of analyte present in the sample. Unique among the immunoassay methods is flow cytometric immunoassay (FCIA) which is capable of multiparameter measurements. In general, flow cytometry is an optical technique that analyzes particles, such as microparticles, in a sample based on the optical characteristics of the particles. The analytical instrument used is a flow cytometer. General background information on flow cytometry techniques is found in Shapiro, Practical Flow Cytometry, Third Ed. (Alan R. Liss, Inc. 1995) and Melamed et al, Flow Cytometry and Sorting, Second Ed. (Wiley-Liss 1990).

In FCIA, a single stream of cells or microparticles passes through a light beam. The light beam acts to probe the optical properties of the microparticles. In addition to the light beam, the stream of particles also passes electro-optical sensors, wherein the signal due to light scattering, refraction, reflection, absorption, phosphorescence and/or fluorescence of each particle is measured. As an example, particles of uniform size will exhibit similar forward and side angle scatter and therefore can be identified from particles of different sizes based on these measured parameters. Similarly, a group of particles uniformly dyed with one or more fluorescent dyes can be identified from other differently dyed particles by their characteristic fluorescence emissions. Thus, it is possible to separate particles as a function of size and fluorescence. Unlike previous immunoassay techniques, FCIA allows these multiparameter measurements.

Simultaneous determination of multiple immunoreactive analytes can be accomplished by using microparticles having a variety of characteristics or detection parameters. For instance, U.S. Patent No. 5,028,545, which issued to Soini, describes multianalyte fluorometric assays performed in a suspension of artificially manufactured microspheres wherein short and long decay time fluorescent dyes are measured. In this assay, short decay time dyes are used for identification of particle category whereas long decay time dyes are used for the detection of the concentration of the analytes by means of biospecific reactions.

In addition, U.S. Patent No. 5,162,863, which issued to Ito, describes a multianalyte analysis method using antigen-antibody reactions. In this method, carriers having latexes that differ in optical properties, such as absorbances are used. The carriers can be inorganic oxides, such as silica, silica-alumina and alumina, mineral powder, metals, blood cells, staphylococci, cell-wall pieces, liposome, etc. By detecting the forward scattered light, the transmitted light and the laterally scattered light of each of the carriers a determination of the analyte can be made.

Since the flow cytometer can discriminate between various particle parameters, such as size, fluorescent properties including intensity and wavelength, and optical properties including absorbances, high and low angle scatter, several different immunoassays can simultaneously be performed on the same sample by using an array of microparticles having different characteristics or parameters. Each microparticle or group of particles can have a variety of characteristics to uniquely identify it in an array of microparticles. Each unique microparticle or group of microparticles can then be used for determination of a different analyte. Discrete detection of multiple analytes can be accomplished by employing an array of multiparameter microparticles.

Frengen et al, (see, Journal of Immunological Methods, 178, (1995) 141- 151) disclose the use of flow cytometric assay for the simultaneous measurement of α- fetoprotein, chorionic gonadotropin and non-specific binding using 7.5, 6.5 and 5.5 μm size microparticles respectively. For the α-fetoprotein and chorionic gonadotropin, the particle is coated with a monoclonal antibody specific for that analyte. The 5.5 μm particle was coated with mouse IgG of irrelevant specificity. The measurement of nonspecific binding is included to identify sera, which gives irrelevant binding resulting in falsely high concentrations of analyte. Irrelevant binding interferes with the accurate determination of analyte concentration. Drawbacks of this assay technique for nonspecific binding are at least twofold. First, this detection method will only detect those interferences that will cause the binding of labeled antibody to the mouse IgG-coated solid phase. For instance, human anti-mouse antibodies, which will cause an artificially depressed signal, are not detected. In addition, using this method it was impossible to identify the interfering substances bound to the non-specific binding particles. An interfering substance is any substance that causes the analyte concentration to be incorrectly reported in an assay. For instance, rheumatoid factor (R_f) is an autoantibody that typically binds to the Fc portion of antibodies, normally of the IgG class, and is generally cross-reactive to various species. R can interfere with immunological assays for specific analytes by a variety of mechanisms. For example, in a sandwich assay, R can bridge between the first antibody and the second labeled antibody, producing an artificially elevated signal for the analyte. In a competitive assay, R_f can block the binding of labeled analyte to the analyte specific antibody and give falsely low signals. Various interferents include, but are not limited to, R , lipids, bilirubin, antimouse antibodies and anti-nuclear antibodies. In view of the foregoing discussion, what is needed in the art is a flow cytometric immunoassay method to simultaneously measure both an analyte of interest as well as known interfering substances. The present invention fulfills this and other needs.

SUMMARY OF THE INVENTION Interfering substances, or interferents, are substances that cause an analyte concentration to be incorrectly reported in an immunological assay. Interferents can give falsely high concentration amounts of an analyte of interest or falsely low concentration amounts. A flow cytometric assay method is needed wherein the method can simultaneously and discretely identify the analyte of interest as well as identify the presence of an interfering substance. By determining that an interferent exists, precautions can be taken to rid the sample of the interfering substance and its adverse effect on analyte concentration measurements or at least notify the analyst that the results may be questionable.

As such, the present invention relates to a method of individually measuring at least one analyte and at least one interferent in a sample comprising:

(a) contacting the sample with an array of microparticles wherein, i) a first member of the array of microparticles comprises an analyte specific-member capable of binding to an analyte partner; ii) a second member of the array of microparticles comprises an interferent specific-member capable of binding to an interferent partner, thereby forming a mixture of the sample and first and second specific members;

(b) contacting the mixture of the sample and first and second specific members with an array of labeled agents, the labeled agents capable of detection wherein, i) a first member of the array of labeled agents is capable of selectively binding to the analyte-specific member to form an analyte complex; ii) a second member of the array of labeled agents is capable of selectively binding to the interferent-specific member to form an interferent complex, wherein the analyte-complex and interferent-complex are capable of individually producing a specific signal; and (c) detecting individually an array of signals, the array of signals being separately distinguishable from each other by flow cytometry techniques, and thereby separately measuring the at least one analyte and the at least one interferent in the sample.

As used herein the term "measuring" can mean identifying, quantitating, detecting, characterizing, recognizing, measuring and combinations thereof. In certain embodiments, the analyte-specific members are coated with an analyte-specific binding protein, an analyte, an analyte-analog or mixtures thereof. The interferent-specific members are coated with an interferent-specific binding protein, an interferent, an interferent-analog or mixtures thereof. The analyte partner can be an analyte-specific binding protein, an analyte, an analyte-analog or mixtures thereof. The interferent partner can be an interferent-specific binding protein, an interferent, an interferent-analog or mixtures thereof.

The microparticles of the present invention each have at least one distinguishing physical characteristic that produces correspondingly unique forward or side angle scatter or single or multiple fluorescences or a combination thereof. In certain embodiments, the microparticles are multi-parametered, i.e., they possess a plurality of distinguishing characteristics.

The microparticles of the present invention consist of a material capable of being coated by antibodies, binding proteins, antigens, analytes, haptens, interferents, analogs or combinations thereof. The microparticles consist of a variety of materials, such as polymers, rubbers, biomolecules, polysaccharide polymers (e.g., agar), glass, biological cells (e.g., erythrocytes), polystyrene, lipids, biological materials, magnetically responsive materials, metals or combinations thereof. In some embodiments, the distinguishable parameters or characteristics of the microparticles of the present invention can be introduced to the surface of the particle, embedded in the particle or can be bound to the molecules of the particle material. In the multianalyte identification methods of the present invention, the microparticles are preferably coated with binding proteins, antibodies, antigens, haptens or analogs thereof, interferents, etc., and participate in immunological assays such as sandwich assays, competitive assays or antibody assays. In certain other aspects, the present invention relates to a kit for use in diagnostic assay applications. The kits of the present invention comprise an array of microparticles and an array of labels in order to simultaneously and discretely identify analytes of interest as well as identify interfering substances. The kit also contains instructions for use. In certain aspects, the kits are assembled for a panel of diagnostic tests that can be run on a mammal to diagnose certain causative agents for illness and syndromes. Preferably, the mammal is a human being.

These aspects and further embodiments and advantages will be more fully understood when read with the accompanying description and examples set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows an assay using the methods of the present invention.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED

EMBODIMENTS A. Immunological Assays

Interfering substances, can give falsely high concentration amounts or falsely low concentration amounts when determining the concentration of analytes of interest by flow cytometric immunoassays. In order to respond appropriately to the presence of interfering substances in the sample, they first must be identified. The present invention relates to a method of simultaneously identifying at least one analyte and at least one interferent in a sample. By identifying that an interferent exists, the analyst can either take the necessary precautions to ensure accurate analytical determinations or report the results with a cautionary note.

The present method can be used with a variety of immunological assay techniques, such as sandwich assays, competitive assays and antibody assays which include the indirect antibody assay and the antibody class capture assay and combinations thereof. In certain embodiments, each of the analytes being detected will use the same assay technique, such as a sandwich assay. In other embodiments, the analyte(s) will be detected using a combination of techniques, such as sandwich assays, competitive assays and antibody assays. Similar to the analyte(s) of interest, the interferent(s) being detected can be detected using the same assay technique, such as a competitive assay, or a combination of techniques, such as sandwich assays, competitive assays and antibody assays.

As explained previously, in the sandwich assay, the microparticle has bound thereto a binding protein having affinity to the analyte or interferent that is of current interest. The substance to be identified (e.g., analyte or interferent) will bind to the binding protein that is bound to the solid support. After the solid support is placed in contact with the sample, a second binding protein, such as an antibody, having a detectable label is then added either simultaneously or sequentially. In a preferred embodiment, the binding site of the first binding protein has an affinity for a different epitope on the substance to be determined than the second binding protein. In this assay, the substance to be measured (e.g., analyte or interferent of interest), binds to both binding proteins in a non-interfering manner. In certain instances, the 'analyte-specific member', comprises a member of a distinct identifiable sub-population of particles coated with a first analyte-specific binding protein, whereas the 'first member of the array of labeled agents' comprises a labeled second analyte-specific binding protein. Likewise, in certain embodiments, the 'interferent-specific member' comprises a member of a subgroup of particles coated with a first interferent-specific binding protein, whereas the 'second member of the array of labeled agents', comprises a labeled second interferent- specific binding protein. In certain aspects, the sample having the substance to be determined is contacted with a mixture of the array of microparticles and thereafter the sample is removed prior to the addition of the array of labeled agents.

In one embodiment, the microparticles of the present invention can be a magnetically responsive material. In a sandwich assay using magnetically responsive microparticles, after a suitable incubation period, the sample having the microparticle therein is exposed to a magnetic field. The magnetic field causes the microparticles to adhere to the walls of the sample vessel and the sample is removed. The microparticles are then washed to remove excess amounts of the labeled antibody that has not been immobilized on the microparticle. The microparticle can then be resuspended in a carrier liquid to be analyzed by flow cytometry.

Another assay of importance is the competitive assay. In this technique, a known amount of the substance to be identified in a labeled form (e.g., analyte or interferent or both) and a relatively small amount of a binding protein which is specific for the analyte or interferent are incubated with the sample. The labeled analyte or interferent and the analyte or interferent present in the sample then compete for the binding protein. It is then possible to determine the amount of labeled substance to be identified that is bound. The substance to be determined bears an inverse concentration relationship to the amount of labeled substance that is bound.

In one competitive assay embodiment, a binding protein, such as an antibody, is immobilized on a microparticle. The antibody is specific for the substance to be determined (analyte or interferent of interest). During the assay, the sample, which contains the exogenous substance or is suspected of containing the substance of interest, and the labeled substance (analyte or interferent of interest) are mixed with the microparticles with the immobilized binding protein. Because there are relatively few binding sites, there is a competition between the labeled substance and the substance endogenous to the sample. After a suitable incubation period, the unbound label is removed by washing, and the sample is introduced into a flow cytometer to determine the amount of label and thereby the analyte or the interferent of interest.

In certain embodiments of the competitive assay, the 'analyte-specific member' comprises a member of a distinct identifiable sub-population of particles coated with an analyte-specific binding protein, whereas the 'first member of the array of labeled agents' comprises a labeled analyte or analog thereof. Likewise, in certain embodiments, the 'interferent-specific member' comprises a member of a sub-group of particles coated with a interferent-specific binding protein, whereas a 'second member of the array of labeled agents', comprises a labeled interferent or analog thereof.

In another competitive assay embodiment, the microparticle has bound thereto an analyte or interferent to be identified. During the assay, the sample, which is suspected of containing the substance of interest and a labeled binding protein specific for the substance are admixed with the microparticles with the immobilized analyte or interferent, causing the bound analyte (or interferent) and the endogenous analyte (or interferent) to compete for the labeled binding protein in a competitive assay. In certain aspects, the microparticle array is added to a mixture containing the sample having the analyte to be determined and the array of labeled agents.

In another embodiment of the competitive assay, the 'analyte-specific member', comprises a member of a distinct identifiable sub-population of particles coated with an analyte or analog thereof , whereas the 'first member of the array of labeled agents' comprises a labeled analyte-specific binding protein. In addition, the 'interferent- specific member' comprises a member of a sub-group of particles coated with the interferent or analog thereof, whereas a 'second member of the array of labeled agents', comprises a labeled interferent-specific binding protein.

Additional assay techniques that can be used in the methods of the present invention are antibody assays, such as the indirect antibody assay and the antibody class capture assay. In the indirect technique, the sample is added to a solid support that has bound thereto an antigen that is specific to the analyte or interferent that is of current interest. The substance to be determined (e.g., analyte or interferent) will bind to the antigen that is bound to the solid support. After the solid support is placed in contact with the sample, a labeled antibody to the class of antibody being detected is added either simultaneously or sequentially. In this assay, the analyte or interferent which itself is an antibody, binds to the antigen on the solid support and is bound by the labeled antibody. Excess label is removed and the sample is then ready to be analyzed in a flow cytometer. In certain aspects, the sample is added to a mixture of an array of microparticles and an array of labeled agents. In other aspects, the array of microparticles is removed from the sample prior to the addition of the array of labeled agents.

In certain instances of this indirect antibody assay, the 'analyte-specific member' comprises a member of a distinct identifiable sub-population of particles coated with an analyte-specific antigen, whereas the 'first member of the array of labeled agents' comprises a labeled binding protein selective for the class of the antibody analyte. In certain embodiments, the 'interferent-specific member' comprises a member of a subgroup of particles coated with interferent-specific antigen, whereas the 'second member of the array of labeled agents' comprises a labeled binding protein selective for the class of the antibody interferent.

In the antibody class capture assay, the solid support has bound thereto a binding protein, such as an antibody, specific to the class of antibody being detected. After the solid support is placed in contact with the sample, a labeled antigen to the antibody being detected is added either simultaneously or sequentially. In this assay, the analyte or interferent which itself is an antibody, is bound by the binding protein on the solid support while binding the labeled antigen in an non-interfering manner. The sample is then ready to be analyzed in a flow cytometer. In certain aspects, the sample is added to a mixture of an array of microparticles and an array of labeled agents. In other aspects, an array of microparticles is added to a mixture of the sample and an array of labeled agents. In certain aspects, the array of microparticles is removed from the sample prior to the addition of the array of labeled agents.

In certain instances of the class capture antibody assay, the 'analyte- specific member' comprises a member of a distinct identifiable sub-population of particles coated with a binding protein selective for the class of the antibody analyte, whereas the 'first member of the array of labeled agents' comprises a labeled analyte- specific antigen. In certain embodiments, the 'interferent-specific member' comprises a member of a sub-group of particles coated with a binding protein selective for the class of the antibody interferent, whereas a 'second member of the array of labeled agents' comprises a labeled interferent-specific antigen.

B. Analytes, Interferents and Labels

The methods of the present invention can be used to simultaneously detect a wide variety of analytes and interferents. For instance, analytes of interest include, but are not limited to, thyroid stimulating hormone, T₄, human chorionic gonadotropin, luteinizing hormone, an antibody specific to a microorganism, such as virus, bacteria or protozoa, vitamin B₁₂, folate, digoxin and carbamazepine. Antigenic viruses, bacterii or protozoas include, but are not limited to, Toxoplasma gondii, Rubella virus, Cytomegalo virus, Herpes Simplex Virus Type 1, E. coli, Herpes Simplex Virus Type 2 and Rabies virus. An interfering substance is any substance that causes an analyte concentration to be incorrectly reported in an assay. The present invention can be used to identify a variety of interferents. These interferents include, but are not limited to, cross- reacting substances, hemoglobin, rheumatoid factor (R ), lipids, bilirubin, heterophilic antibodies, antimouse antibodies and anti-nuclear antibodies. Using the methods of the present invention and assay techniques detailed above, a plurality of analytes and a plurality of interferents can be determined simultaneously using an array of microparticles. In addition, a combination of assay types can be used. For example, using an array of four microparticles and an array of four labels, two analytes and two interferents can be determined. In this example, a first analyte that is readily detected is free-thyroxine (FT4). In a competitive assay, the particle bound assay reagent for this analyte can be an antibody specific for thyroxine, and a label-bound analyte can be thyroxine (or analog) covalently linked to phycoerythrin. While using a competitive assay for FT4, a sandwich assay can concurrently be used to detect a second analyte, thyroid-stimulating hormone (TSH). The label on the second antibody specific for TSH can again be phycoerythrin. As described hereinbelow, the binding reactions can be identified with the same label because the microparticles themselves can be distinguished with unique characteristic parameters.

The third member of the microparticle array in this example, is an interferent that is simultaneously identified and detected in the same sample as the proceeding analytes. A heterophilic antibody is a potential interferent and in some instances, can be an anti-animal antibody. Anti-animal antibodies are often found in the sera of cancer patients that have undergone therapy with anti-tumor monoclonal murine antibodies (human anti -mouse antibody (HAMA)). To detect HAMA, a particle is coated with non-specific mouse IgG or a fragment thereof. The label can be bound to anti- human IgG. The presence of HAMA in a sample can then be detected by the increase in fluorescence caused by the binding of HAMA to the IgG coated particle and the binding of the labeled anti-human IgG to the HAMA.

In conjunction with detection of the preceding two analytes and one interferent, the array of microparticles can have reagents bound thereto which are specific for the interferent R_f. This fourth member of the microparticle array is coated with an IgG or fragment thereof of any one of a variety of mammals (e.g., goat, rabbit etc.) other than human. Human IgG cannot be used for coating because the labeled anti-human IgG used for the HAMA interferent assay would bind to the particle. The label, such as phycoerythrin, can be bound to anti-human IgM. The presence of R_f in a sample can then be detected by the increase of fluorescence caused by the simultaneous binding of R_f to the IgG coated particle and the labeled IgG.

The labels of the present invention are typically fluorescent moieties. The fluorochrome preferably employed results in a change in the emission of the assay medium when excited by energy of an appropriate wavelength. Preferred fluorochromes include, but are not limited to, fluorescein isocyanate, B-phycoerythrin, R-phycoerythrin, allophycocyanin, rhodamine, PE Tandem, fluorescently dyed microparticles and Texas red. All of the aforementioned fluorochromes are commercially available.

In other aspects, the present invention relates to labels that are energy emitting moieties other than fluorochromes that include, but are not limited to, phosphorescent labels, and bioluminescent labels. When these labels are used, the cytometer employed must have the necessary fluidics device and a light detector with the appropriate electronics, or other suitable means that allows detection. C. Solid Supports and Microparticles

Simultaneous determination of multiple immunoreactive analytes and interferents of the present invention can be accomplished by using microparticles having a variety of characteristics or detection parameters. The microparticles of the present invention each have at least one distinguishing physical characteristic that includes, but is not limited to, size, fluorescence, phosphorescence, absorbance, reflectivity, refractivity, composition or a combination thereof. Preferably, an optical characteristic, such as forward angle light scatter, side angle light scatter, fluorescence, phosphorescence, or a combination thereof can be used to distinguish the microparticle. In certain embodiments, the microparticles are multi-parametered, i.e., they possess a plurality of distinguishing characteristics.

In certain aspects, the particles are distinguished by size. In this aspect, particle types are conveniently distinguished by size because conventional flow cytometers can determine particle size on the basis of the intensity of light scattered by the particles. A wide range of types of monosized particles having different compositions, diameters, reactive surface groups etc., are commercially available and can be used in accordance with the present invention. These particles are highly monosized and thus a substantial number of particles with various sizes can be mixed and easily identified as non-overlapping populations in a flow cytometric light scatter histogram. Preferably, the microparticles for use in the present invention are approximately spherical and are about equal in size, wherein the nominal size ranges from about 1 μm to about 100 μm. Preferably, the size ranges from between from about 1 μm to about 20 μm. In another aspect, the present invention relates to microparticles that contain fluorescent materials and can be distinguished by their fluorescence decay times. In one embodiment, a short decay time is used for identification of the individual microparticle or group of microparticles in the array. A long decay time label can then be used for detecting the concentration of the particular analyte on the microparticle by means of a binding assay reaction discussed above (see, U.S. Patent No. 5,028,545 and WO 94/01774). In one aspect, the fluorescent labels associated with the binding reaction at the microparticle surface is based on the use of long decay time fluorescent labels e.g., lanthanide chelates of europium, terbium, etc. The short decay time fluorescent labels are preferably incorporated into the microparticle for particle/assay identification. These fluorescent dyes with short decay times include, but are not limited to, l,4-bis(5- phenyloxazol-2-yl)benzene (POPOP), l,4-bis(2-methylstyryl)benzene (bisMSB), fluorescent inorganic microcrystals, fluorescein and rhodamine. A device for time- resolved fluorescence detection is used that typically incorporates a pulsed light source and a gated detector, which is activated after a certain delay time from the excitation pulse. Thus, one detector registers the short decay time fluorescence of the dye that is in the particle because its lifetime is much shorter than the time delay and another detector detects only long decay time emission that is specific to the long decay time fluorescent label in the sample.

In one preferred embodiment, the array of microparticles are distinguished using light scatter and emission and combinations thereof (see, WO/9714028). Light scatter correlates with particle size, granularity, absorbance and surface roughness. Forward angle light scatter is mainly affected by size and refractive index, while side angle scatter is generally influenced by size particle granularity, surface roughness and absorbance. Intensity of fluorescence is correlated with the amount of fluorescent label present. It is possible to have a plurality of fluorescent emissions at various wavelengths. In this embodiment, the wavelength of the emission(s) from the fluorescent dye(s) within the microparticle is distinct from the wavelength of the emission(s) from the binding reactions associated with reactants on the microparticle surface.

In a preferred embodiment, the microparticles will have two or more fluorochromes incorporated within them so that each microparticle in the array will have at least four distinguishable parameters associated with it, forward and side scatter and fluorescent emissions at two separate wavelengths. For example, the microparticle can be made to contain a red fluorochrome, such as Cy5, which emits light at one wavelength and also contain another fluorochrome, such as Cy5.5 that emits at a different wavelength. Additional fluorchromes can be used to further expand the system. Using this embodiment, it is possible that each microparticle can have a plurality of fluorescent dyes at varying wavelengths thereby enabling the methods of the present invention to determine panels of analytes and panels of interferents simultaneously. In certain preferred embodiments, using light scatter and variation in wavelength, the microparticles in the array of the present invention contain at least 4 distinguishing characteristics or parameters including, but not limited to, i) forward light scatter; ii) side angle light scatter; iii) fluorescence at a first wavelength and iv) fluorescence at a second wavelength. In this embodiment, the fluorescence at a first and second wavelength is preferably red. Preferably, the fluorochromes associated with the binding reactions are orange wavelength emitters.

In another embodiment, microparticles that differ in absorbances are used (see, U.S. Patent No. 5,162,863). Again, when a light is applied to the microparticles the information of the absorbance of the particles is chiefly included in the strength of the laterally scattered light and the information of the particle size is chiefly included in the strength of the forward-scattered light. Consequently, the difference in absorbance between various latexes (colored dyes) associated with the microparticles appears in the strength of the laterally scattered light. In yet another embodiment, it is possible to distinguish the microparticle group by counting. For example, it is known within the art that assay specific sub-groups of particles can be differentiated by their relative populations (see, U.S. Patent No. 5,351,118, which issued to Spinell).

As discussed above, the microparticles of the present invention possess one or more distinguishable parameters or characteristics. For example, the microparticles possessing excitable fluorescent dyes or colored dyes possess certain different emission spectrums and/or scattering characteristics. In addition, the microparticles of the present invention can also be distinguished by a predetermined different concentration of one or more fluorescent dyes. Moreover, the distinguishable microparticle parameter(s), such as a fluorescent dye or color, can be introduced to the surface of the particle, or embedded in the particle or can be bound to the molecules of the particle material. For instance, fluorescent microparticles can be manufactured combining the polymer material with the fluorescent dye. Alternatively, the fluorescent dye or colored dye can be impregnated into the microparticle. Microparticles suitable for use in the present invention are also commercially available, from manufactures such as Spherotech (Libertyville, IL) and Molecular Probes (Eugene, OR). A list of vendors of flow cytometric products can be found on the Internet at www.molbio.princeton.edu/facs/FCMsites.html.

In general, the microparticles of the present invention consist of a material appropriate for the coating of binding proteins, antibodies, antigens, analytes, interferents or combinations thereof. Various microparticle materials can be used including, but not limited to, polymers, rubbers, biomolecules, polysaccharide polymers (e.g., agar), glass, biological cells (e.g., erythrocytes), polystyrene, lipids, biological materials, metals or combinations thereof. The microparticles can be functionalized or non-functionalized, preferably functionalized for covalent bonding to the binding moiety. When the linking element is a polymeric material, various procedures are known in the art for the activation of polymer surfaces and the attachment of immunoglobulins, glycoproteins, saccharide- containing organic molecules, analytes, interferents and polynucleotides (see, U.S. Pat. Nos. 4,419,444; 4,775,619; 3,956,219; and 3,860,486 as well as European Patent Application No. 84308143.1, Scouten, W. H. (ed.) Solid Phase Biochemistry, Analytical and Synthetic Aspects (1983), Wiley & Sons, New York, page 779 and Griffin, C. et al, Microparticle Reagent Optimization (1994), Seradyn Inc., Indiana.) Examples of suitable functional groups are amines, ammonium groups, hydroxyl groups, carboxylic acids, and isocyanites.

D. Instrumentation

Flow cytometry and methods for its use are known in the art. In general, flow cytometry is an optical technique that analyzes particles, such as microparticles, in a sample based on the characteristics of the particles. The analytical instrument used is a flow cytometer. General background information on flow cytometry techniques is found in Shapiro, Practical Flow Cytometry, Third Ed. (Alan R. Liss, Inc. 1995) and Melamed et al, Flow Cytometry and Sorting, Second Ed. (Wiley-Liss 1990).

In the flow cytometric immunoassay, a single stream of microparticles passes through a beam of light. The light beam can then excite any fluorescent labels present on or within the microparticles. In addition to the light beam, the stream of particles also passes electro-optical sensors, wherein each particle produces a signal due to light scattering. The amplitude of the signal varies primarily with particle size. Measurements of the scattered light are obtained for each illuminated microparticle by a plurality of optical detectors. In addition, if the microparticle contains at least one appropriate fluorescent compound, it will fluoresce when illuminated at the appropriate wavelength. A plurality of optical detectors within the flow cytometer measure fluorescence at a plurality of wavelengths. Typically microparticle characteristics include, but are not limited to, forward light scatter, side light scatter, red fluorescence, green fluorescence and orange fluorescence. As will be apparent to those of skill in the art, additional detectors and laser beams are possible to allow for excitation and detection at additional wavelengths.

E. Kits

In one embodiment, the present invention relates to a kit for use in diagnostic assay applications. The kits of the invention comprise an array of microparticles and an array of labels in order to simultaneously and discretely identify analytes of interest as well as identify interfering substances. The kit also comprises instructions for use.

In certain aspects, the kits are assembled for a panel of diagnostic tests that can be run on a mammal to diagnose certain causative agents for illness and syndromes. Kits can be assembled for the detection of infectious agents or disease markers such as bacteria, viruses, fungi, microorganisms, Mycoplasma, rickettia, Chlamydia, protozoa, autoimmune disease, sexually transmitted disease, prostate-specific antigen, carcinoembryonic antigen (CEA), tumor markers, pollen, allergens, α-fetoprotein, chorionic gonadotropin, hepatitis B surface antigen, thyroid stimulating hormone, T₄, luteinizing hormone, an antibody specific to a virus, vitamin B_]2, folate, digoxin, carbamazepine, antigenic viruses include, but are not limited to, rubella virus, cytomegalovirus, herpes simplex virus Type 1, herpes simplex virus Type 2 and rabies virus. Moreover, kits can be assembled for the detection of interfering substances such as, rheumatoid factor (R_f), lipids, bilirubin, heterophilic antibodies, antimouse antibodies, hemoglobin and anti-nuclear antibodies.

Examples EXAMPLE 1

This example illustrates the attachment of human IgG to magnetic particles.

12.5 mg of particles (500 μL, 25 mg/mL, SPHERO™ Carboxyl Magnetic particles, from Spherotech, Inc., Libertyville, Illinois, USA; poly(styrene/acrylic acid particles), 7.10 micrometers (μm) in diameter, density 1.10 g/cc, containing 5% magnetite (by weight)) were placed into a test tube and washed multiple times with 25 niM 2-[N- morpholinojethanesulfonic acid (MES) buffer, pH 6.1. To the washed particles was added: 735 μL of deionized water, 100 μL of 0.5M MES, 115 μL of 50 mg/mL N- hydroxysuccimide (NHS) in deionized water and 50 μL of 50 mg/mL l-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC) in deionized water. The tube was placed on a vortexer for 30 minutes at ambient temperature. After this time, the particles were washed multiple times with 25 mM MES. To the washed particles was added: 190 μL deionized water, 50 μL of 0.5M MES and 200 μL of 5 mg/mL human IgG in deionized water (human IgG from Sigma Chemical Co., St. Louis, Missouri, USA). The tube was placed on a vortexer for 3 hours at ambient temperature. After this time, 50 μL of 0.25M hydroxylamine in 25mM MES, pH 6.1 was added. The tube was placed on a vortexer for 30 minutes at ambient temperature. After this time, the resulting beads were washed multiple times with wash buffer consisting of 50 mM phosphate buffer, pH 7.4, 0.1% Tween 20, 1% bovine serum albumin, 0.1% sodium azide, 150 mM sodium chloride and then washed multiple times with storage buffer consisting of 50 mM phosphate buffer, pH 7.4, 5% glycerol, 1% bovine serum albumin, 0.1% sodium azide, 150 mM sodium chloride. The final particle preparation was suspended in 2 mL of storage buffer and stored at 4°C until needed.

EXAMPLE 2 This example demonstrates a rheumatoid factor (R_f) flow immunoassay. In the assay a magnetic particle coated with human IgG (prepared in Example 1) is brought into contact with the sample. During the subsequent incubation period, the R_f contained in the sample binds to the human IgG on the particle surface.

After the unbound material is removed by washing, a conjugate composed of anti-human IgM linked to phycoerythrin (anti-human IgM-PE) is added. During this second incubation period the conjugate binds to the R_f (i.e., human IgM) bound to the human IgG attached to the surface of the particle. After the particles are washed to remove unbound materials, the particles are resuspended and analyzed by a flow cytometer. The following procedure was used: 1. 100 μL of sample (diluted 1:101 in wash buffer) is added to 12x75 mm polypropylene test tubes.

2. To each tube is added 100 μL of a particle mixture in wash buffer composed of human IgG coated particles (described in Example 1, 1:200 dilution).

3. The tubes were vortexed at ambient temperature for 15 minutes. 4. Afterwards, 750 μL of wash buffer was added to each tube and the tubes vortexed.

5. The tubes were placed in a magnetic separator for 3 minutes and the liquid phase removed.

6. Steps 4 and 5 were repeated twice more with 1000 μL of wash buffer.

7. To each tube was added 200 μL of a solution containing a 1 :300 dilution of anti human IgM-phycoerythrin conjugate in wash buffer (Chemicon International Inc.,

Temecula, California, USA, catalog number AQ188E).

8. The tubes were vortexed at ambient temperature for 15 minutes.

9. Afterwards, 750 μL of wash buffer was added to each tube and the tubes vortexed.

10. The tubes were placed in a magnetic separator for 3 minutes and the liquid phase removed.

11. Steps 9 and 10 were repeated twice more with 1000 μL of wash buffer.

12. The particles were resuspended in 125 μL of wash buffer.

13. The particles were injected into the flow cytometer (Bryte HS, Bio-Rad Laboratories, Inc., Hercules, California, USA) equipped with a Mercury/Xenon arc lamp.

The data are shown in Table 1. A 4-parameter logistic regression was performed using the signal of the standards and their assigned values. The R_f concentration of the standards and patients were calculated from this equation. The patients were assayed using a commercially available R_f Kit (Behring Diagnostics Inc., Somerville, New Jersey, USA). There is strong correlation between the flow immunoassay (FCIA) R values for the patient samples and the Behring results.

Table 1 Summary of Results

*rlfu = relative linear fluorescence units

EXAMPLE 3

This example illustrates the attachment of rabbit IgG to magnetic particles.

5 mg of particles (200 μL, 25 mg/mL, SPHERO™ Carboxyl Magnetic particles, from Spherotech, Inc., Libertyville, Illinois, USA; poly(styrene/acrylic acid particles), 4.35 micrometers (μm) in diameter, density 1.165 g/cc, containing 12%) magnetite (by weight)) were placed into a test tube and washed multiple times with 25 mM MES buffer, pH 6.1. To the washed particles was added: 294 μL of deionized water, 40 μL of 0.5M MES, 46 μL of 50 mg/mL NHS in deionized water and 20 μL of 50 mg/mL EDC in deionized water. The tube was placed on a vortexer for 30 minutes at ambient temperature. After this time, the particles were washed multiple times with 25 mM MES. To the washed particles was added: 435 μL deionized water, 50 μL of 0.5M MES and 14.6 μL of rabbit IgG (618 μg, rabbit IgG from Chemicon International Inc., Temecula, California, USA, 42.3 mg/mL in 40mM phosphate buffer). The tube was placed on a vortexer for 3 hours at ambient temperature. After this time, 20 μL of 0.25M hydroxylamine in 25mM MES, pH 6.1 was added. The tube was placed on a vortexer for 30 minutes at ambient temperature. After this time, the resulting beads were washed multiple times with wash buffer and then multiple times with storage buffer. The final particle preparation was suspended in 2 mL of storage buffer and stored at 4°C until needed.

EXAMPLE 4

This example illustrates the attachment of mouse IgG (F(ab')₂ fragment) to magnetic beads. 5 mg of particles (200 μL, 25 mg/mL, SPHERO™ Carboxyl Magnetic particles, from Spherotech, Inc., Libertyville, Illinois, USA, poly(styrene/acrylic acid particles), 7.10 micrometers (μm) in diameter, density 1.10 g/cc, containing 5%> magnetite (by weight)) were placed into a test tube and washed multiple times with 25 mM 2-[N- morpholino]ethanesulfonic acid (MES) buffer, pH 6.1. To the washed particles was added: 294 μL of deionized water, 40 μL of 0.5M MES, 46 μL of 50 mg/mL N- hydroxysuccimide (NHS) in deionized water and 20 μL of 50 mg/mL EDC in deionized water. The tube was placed on a vortexer for 30 minutes at ambient temperature. After this time, the particles were washed multiple times with 25 mM MES. To the washed particles was added: 365 μL deionized water, 50 μL of 0.5M MES and 85.1 μL of mouse IgG (400 μg, F(ab')₂ fragment, 4.7 mg/mL in phosphate buffered saline from Jackson ImmunoResearch Inc., West Grove, Pennsylvania, USA). The tube was placed on a vortexer for 3 hours at ambient temperature. After this time, 20 μL of 0.25M hydroxylamine in 25mM MES, pH 6.1 was added. The tube was placed on a vortexer for 30 minutes at ambient temperature. After this time, the resulting beads were washed multiple times with wash buffer and then washed multiple times with storage buffer. The final particle preparation was suspended in 2 mL of storage buffer and stored at 4°C until needed.

EXAMPLE 5 This example demonstrates a HAMA flow immunoassay with simultaneous detection of R_f as an interferent.

In the assay two identifiable, monodispersed, magnetic particle groups (i.e., identifiable by the forward and side scatter characteristic of the particle diameter) coated with rabbit IgG (prepared by Example 3) and with mouse IgG (F(ab')₂ fragment, prepared by Example 4) are brought into contact with the sample. After an incubation period in which R_f contained in the sample binds to the rabbit IgG coated particles and HAMA binds to the mouse IgG coated particles, the unbound material is removed by washing. Next a mixture of conjugates composed of anti-human IgM-PE and anti-human IgG linked to phycoerythrin (anti-human IgG-PE) is added. During this second incubation period, the anti -human IgM-PE binds to the R_f bound to the rabbit IgG coated particles and the anti-human IgG-PE binds to the HAMA bound to the mouse IgG coated particles. After the particles are washed to remove unbound materials, they are resuspended in wash buffer and read in a flow cytometer. In this system of analyte measurement (i.e., HAMA) and interferent detection (i.e., R_f), rabbit IgG is used instead of human IgG for the R_f detecting particle because the labeled anti -human IgG for the HAMA assay would bind to a human IgG-coated particle. In a similar way, the HAMA detecting particle, the anti-human IgG-PE and anti-human IgM-PE all use the F(ab')₂ fragment of the respective IgG so as to minimize R_f binding and the production of a false positive signal. The following procedure was used:

1. 100 μL of sample (diluted 1:31 in wash buffer) is added to 12x75 mm polypropylene test tubes.

2. To each tube is added 100 μL of a particle mixture in wash buffer composed of rabbit IgG coated 4.35μm particles (described in Example 3, 1/150 dilution) and mouse IgG coated 7.10μm particles (F(ab')₂ fragment, described in Example 4,

1/35 dilution). 3. The tubes were vortexed at ambient temperature for 15 minutes.

4. Afterwards, 750 μL of wash buffer was added to each tube and the tubes vortexed.

5. The tubes were placed in a magnetic separator for 3 minutes and the liquid phase removed. 6. Steps 4 and 5 were repeated twice more with 1000 μL of wash buffer.

7. To each tube was added 200 μL of a solution containing anti-human IgG-PE and anti-human IgM-PE both diluted 1/200 with wash buffer (Jackson ImmunoResearch Inc., West Grove, Pennsylvania, USA, catalog numbers 709- 106-149 and 709-106-073, respectively). 8. The tubes were vortexed at ambient temperature for 15 minutes.

9. Afterwards, 750 μL of wash buffer was added to each tube and the tubes vortexed.

10. The tubes were placed in a magnetic separator for 3 minutes and the liquid phase removed.

11. Steps 9 and 10 were repeated twice more with 1000 μL of wash buffer. 12. The particles were resuspended in 125 μL of wash buffer.

13. The particles were injected into the flow cytometer (Enhanced Bryte, Bio-Rad Laboratories, Inc., Hercules, California, USA) equipped with a Mercury/Xenon arc lamp.

TSH zero standard (TSH 0, Bio-Rad Laboratories, Inc., Hercules,

California, USA), normal human serum (NHS, Western States Plasma Co., Fallbrook, California, USA) and 9 HAMA-positive patient samples were analyzed by FCIA. The patient samples were obtained commercially with assigned HAMA values (Fitzgerald Industries International, Inc., Concord, Massachusetts, USA). A 4-parameter logistic regression was performed using the signals of the patient samples and their assigned values. The HAMA concentration of the patient samples were calculated from this equation.(The data are shown in Table 2. There is strong correlation between the flow immunoassay (FCIA) HAMA values and the concentrations supplied by the vendor. Table 2. Summary of Results

*rlfu = relative linear fluorescence units

EXAMPLE 6

This example illustrates the attachment of mouse IgG (F(ab')₂ fragment) to magnetic beads. 3.75 mg of particles (150 μL, 25 mg/mL, SPHERO™ Carboxyl Magnetic particles, from Spherotech, Inc., Libertyville, Illinois, USA; poly(styrene/acrylic acid particles), 3.18 micrometers (μm) in diameter, density 1.165 g/cc, containing 12% magnetite (by weight)) were placed into a test tube and washed multiple times with 25 mM 2-[N-morpholino]ethanesulfonic acid (MES) buffer, pH 6.1. To the washed particles was added: 258 μL of deionized water, 40 μL of 0.5M MES, 70.8 μL of 94.3 mg/mL sodium N-hydroxysulfosuccinimide (NHSS) in deionized water and 30.8 μL of 50 mg/mL EDC in deionized water. The tube was placed on a vortexer for 30 minutes at ambient temperature. After this time, the particles were washed multiple times with 25 mM MES. To the washed particles was added: 316 μL deionized water, 50 μL of 0.5M MES and 134.3 μL of mouse IgG (631 μg, F(ab')₂ fragment, 4.7 mg/mL in phosphate buffered saline from Jackson ImmunoResearch Inc., West Grove, Pennsylvania, USA). The tube was placed on a vortexer for 3 hours at ambient temperature. After this time, 20 μL of 0.25M hydroxylamine in 25mM MES, pH 6.1 was added. The tube was placed on a vortexer for 30 minutes at ambient temperature. After this time, the resulting beads were washed multiple times with wash buffer and then washed multiple times with storage buffer. The final particle preparation was suspended in 2 mL of storage buffer and stored at 4°C until needed.

EXAMPLE 7

This example illustrates the attachment of viral antigen (rubella (RUB)) to magnetic beads.

The 5 mg of particles (200 μL, 25 mg/mL, SPHERO™ Carboxyl Magnetic particles, from Spherotech, Inc., Libertyville, Illinois, USA; poly(styrene/acrylic acid particles), 7.10 micrometers (μm) in diameter, density 1.10 g/cc, containing 5% magnetite (by weight)) were placed into a test tube and washed multiple times with 25 mM 2-[N- morpholino]ethanesulfonic acid (MES) buffer, pH 6.1. To the washed particles was added: 294 μL of deionized water, 40 μL of 0.5M MES, 46 μL of 94.33 mg/mL NHSS in deionized water and 20 μL of 50 mg/mL EDC in deionized water. The tube was placed on a vortexer for 30 minutes at ambient temperature. After this time, the particles were washed multiple times with 25 mM MES, to the washed particles was added: 96.4 μL deionized water, 50 μL of 0.5M MES and 353.6 μL of rubella viral antigen (antigen from Microbix Biosystems Inc., Toronto, Ontario, Canada, 200 μg/mL in lOmM PBS). The tube was placed on a vortexer for 3 hours at ambient temperature. After this time, 20 μL of 0.25M hydroxylamine in 25mM MES, pH 6.1 was added. The tube was placed on a vortexer for 30 minutes at ambient temperature. After this time, the resulting beads were washed multiple times with wash buffer and then multiple times with storage buffer. The final particle preparation was suspended in 2 mL of storage buffer and stored at 4°C until needed.

EXAMPLE 8 This example illustrates the use of rubella antigen, mouse IgG (F(ab')₂ fragment) and rabbit IgG coated magnetic particles prepared by the procedures of Examples 7, 6 and 3, respectively, in a flow cytometric immunoassay (FCIA) for the measurement of the analyte: rubella IgG, and the detection of two interferents: R_f and HAMA.

In the assay three identifiable, monodispersed, magnetic particle groups (i.e., identifiable by the forward and side scatter characteristic of the particle diameter of each group) coated with rubella antigen (7.10μm, prepared in Example 7), rabbit IgG (4.35μm, prepared in Example 3) and mouse IgG (3.18μm, F(ab')₂ fragment, prepared in Example 6) are brought into contact with the sample. During the subsequent incubation period, assay and interferent components of the sample become attached to their respective particles; the rubella antibodies become attached to the rubella antigen coated particles, R_f binds to the rabbit IgG coated particles and HAMA binds to the mouse IgG coated particles. After incubation, the unbound material is removed by washing. Next a mixture of conjugates composed of anti-human IgM-PE and anti -human IgG-PE is added. During the subsequent incubation period the anti-human IgM-PE binds to the R_f bound to the rabbit IgG coated particle and the anti-human IgG-PE binds to the HAMA bound to the mouse IgG coated particle and the human anti-rubella antibodies bound to the rubella antigen coated particle. After the particles are washed to remove unbound materials, they are resuspended in wash buffer and analyzed by a flow cytometer. In this system of analyte measurement (i.e., human anti-rubella IgG antibodies) and interferent detection (i.e., HAMA and R_f), rabbit IgG is used instead of human IgG for the R_f detecting particle because the labeled anti-human IgG for the HAMA assay would bind to a human IgG- coated particle. In a similar way, the HAMA detecting particle, the anti-human IgG-PE and IgM-PE all use the F(ab') fragment of the respective IgG so as to minimize R binding and the production of a false positive signal. In addition, the anti-human IgG-PE must be species specific so that it does not react with the other IgG components. The following procedure was used:

1. 100 μL of sample (diluted 1:31 in wash buffer) is added to 12x75 mm polypropylene test tubes. 2. To each tube is added 100 μL of a particle mixture in wash buffer composed of rubella antigen coated particles (described in Example 7, 1 :50 dilution), mouse IgG (F(ab')₂ fragment) coated particles (described in Example 6, 1 :200 dilution) and rabbit IgG coated particles (described in Example 3, 1/150 dilution). 3. The tubes were vortexed at ambient temperature for 15 minutes.

4. Afterwards, 750 μL of wash buffer was added to each tube and the tubes vortexed.

5. The tubes were placed in a magnetic separator for 3 minutes and the liquid phase removed.

6. Steps 4 and 5 were repeated twice more with 1000 μL of wash buffer. 7. To each tube was added 200 μL of a solution containing a 1 :200 dilution of anti human IgG-PE and anti-human IgM-PE in wash buffer (both conjugates use F(ab')₂ fragments to reduce binding by R_f and are manufactured by Jackson ImmunoResearch Laboratories Inc., West Grove, Pennsylvania, USA, catalog numbers 709-106-149 and 709-106-073, respectively). 8. The tubes were vortexed at ambient temperature for 15 minutes.

9. Afterwards, 750 μL of wash buffer was added to each tube and the tubes vortexed.

10. The tubes were placed in a magnetic separator for 3 minutes and the liquid phase removed.

11. Steps 9 and 10 were repeated twice more with 1000 μL of wash buffer. 12. The particles were resuspended in 150 μL of wash buffer.

13. The particles were injected into the flow cytometer (Enhanced Bryte, Bio-Rad Laboratories, Inc., Hercules, California, USA) equipped with a Mercury/Xenon arc lamp.

The data are shown in Table 3. A 4-parameter logistic regression was performed using the signal of the standards and their assigned values. The rubella concentration of the standards, controls and patients were calculated from this equation. These same standards, controls and patients were assayed using a commercially available ELISA Rubella IgG Kit (Catalog 536-3000, Bio-Rad Laboratories, Inc., Hercules, California, USA). There is strong agreement between the flow immunoassay (FCIA) rubella IgG values for the controls and patient samples and the ELISA results. Table 3. Summary of Results

*rlfu = relative linear fluorescence units EXAMPLE 9

This example illustrates a Thyroid Stimulating Hormone (TSH) and Free T4 (FT4) panel of assays with simultaneous detection of the potential interferents HAMA and R_f (see, Figure 1). The assay kit comprises standards, a mixture of particles, a mixture of labeled reagents and wash buffer. The standards will contain known amounts of the analytes (i.e., TSH and FT4) from which a standard curve can be generated. These standard solutions will also contain known amounts of interferents (i.e., HAMA and R_f) from which a cutoff value will be generated, such that, interferent amounts higher than this value might cause a significant deviation in the measured analyte concentration from its actual concentration. For this assay, the particle mixture will be composed of four identifiable particle groups: TSH, FT4, R_f and HAMA. The TSH particle will have TSH- specific antibody (F(ab')₂ fragment) bound to its surface. The process for attachment of the antibody is analogous to that detailed in Example 1. The FT4 particle will have FT4- specific antibody (F(ab')₂ fragment) bound to its surface. As with the TSH particles, the attachment is accomplished by analogy with Example 1. The procedure followed to prepare the particles in Examples 3 and 4 will be used in this example for the preparation of the R_f and HAMA particles for this example. The commercially available conjugates, anti-human IgG-PE and anti -human IgM-PE (both F(ab')₂ fragments), utilized in step 7 of Example 5 will also be used in this example. Anti-TSH (F(ab')₂ fragment)-phycoerythrin and thyroxine (T4)-phycoerythrin will be prepared by analogy to published methods for protein-protein and hapten-protein conjugation (c.f: R. P. Haugland in Methods in Molecular Biology, volume 45, pages 205-221, W. C. Davis editor, Humana Press, Totowa, New Jersey, 1995 and G. T. Hermanson, Bioconjugate Techniques, Academic Press, San Diego, California, 1996). F(ab')2 fragments are used for the immunoglobulin components of the reagents so as to minimize R binding. Rabbit IgG, instead of human IgG, is used for coating the R_f detecting particle because anti-human IgG-PE is required for HAMA detection and will react with this particle. In addition, the anti-human IgG-PE should be species specific so that it does not react with the other IgG components. The first step of the assay will bring the anti-TSH, anti-T4, rabbit IgG and mouse IgG coated identifiable particle groups into contact with the sample to be assayed. During the subsequent incubation period, TSH, T4, R_f and HAMA, if present in the sample, will become attached to the particles of the identifiable groups, that is, the anti- TSH, anti-T4, rabbit IgG and mouse IgG coated particles, respectively. After a wash step to remove unbound materials, the labeled reagents will be added (i.e., anti-TSH-PE, T4- PE, anti-human IgG-PE and anti-human IgM-PE). During this second incubation period, the anti-TSH-PE will become bound to the TSH attached to the anti-TSH coated particles, the asti-T4-PE will be captured by the unoccupied sites of the anti-T4 coated particles, the anti-human IgM-PE will become bound to the R_f attached to the rabbit IgG coated particles and the anti-human IgG-PE will become bound to the HAMA attached to the mouse IgG coated particles.

This mixture of analyte and interferent particles are is then analyzed by a flow cytometer. The identity of each particle is ascertained by the optical parameter used to label that particle group. For instance, if each particle group is of a different size, with the particles within each group being monodispersed, then when a particle passes through the illumination region of the flow cytometer, the resulting forward and side scattered light produced and detected will be uniquely characteristic of a particular particle group and thereby identifies the particle as belonging to that group. The concomitant fluorescence of that identified particle will be related to the amount of label attached to its surface that in turn is related to the amount of analyte present in the sample. In this manner a statistically significant population of each analyte or interferent-specific group of particles is identified and its corresponding analyte or interferent-specific signal measured. For the analytes, samples of known multi-analyte concentrations (i.e., standards) are measured and functions relating the median fluorescent signal each of the analyte-specific particle groups to analyte concentration developed. Using these functions and the median fluorescent signal of each of the analyte-specific particle group of the sample, the multi-analyte concentrations of each sample can be calculated. Concomitantly, the signal of each interferent of each sample will be compared against a cutoff value generated for each interferent by the standards. If an interferent concentration falls above this value, the analyst is notified which of the analytes results can be adversely affected by the interferents presence. In some cases, a sample can be treated to remove an interferent and then re-assayed. All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification in their entirety for all purposes. Although the invention has been described with reference to preferred embodiments and examples thereof, the scope of the present invention is not limited only to those described embodiments. As will be apparent to persons skilled in the art, modifications and adaptations to the above-described invention can be made without departing from the spirit and scope of the invention, which is defined and circumscribed by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method of individually measuring at least one analyte and at least one interferent in a sample, said method comprising: (a) contacting said sample with an array of microparticles wherein, i) a first member of said array of microparticles comprises an analyte specific-member that selectively binds to an analyte partner; ii) a second member of said array of microparticles comprises an interferent specific-member that selectively binds to an interferent partner, thereby forming a mixture of said sample and first and second specific members; (b) contacting said mixture of the sample and first and second specific members with an array of labeled agents, said labeled agents capable of detection wherein, i) a first member of said array of labeled agents that selectively binds to said analyte-specific member to form an analyte complex; ii) a second member of said array of labeled agents that selectively binds to said interferent-specific member to form an interferent-complex, wherein said analyte-complex and said interferent-complex each individually produces a specific signal; and (c) individually detecting each array of signals, said array of signals being separately distinguishable from each other by flow cytometry techniques, and thereby separately measuring said at least one analyte and said at least one interferent in the sample.

2. The method in accordance with claim 1, wherein said at least one analyte and said at least one interferent independently participate in an assay selected from the group consisting of a sandwich assay, a competitive assay, an indirect antibody assay and an antibody class capture assay.

3. The method in accordance with claim 1, wherein said analyte- specific member comprises a bound said at least one analyte or an analog thereof, said first member of said array of labeled agents is a labeled binding protein, causing said analyte-specific member and said at least one analyte to compete for said labeled binding protein in a competitive assay.

4. The method in accordance with claim 3, wherein said array of microparticles is added to a mixture containing said sample and said array of labeled agents.

5. The method in accordance with claim 1, wherein said analyte- specific member comprises a binding protein and said first member of said array of labeled agents is a labeled said at least one analyte or analog thereof, causing said at least one analyte and said labeled at least one analyte to compete for said binding protein in a competitive assay.

6. The method in accordance with claim 5, wherein said array of microparticles is added to a mixture containing said sample and said array of labeled agents.

7. The method in accordance with claim 1, wherein said analyte- specific member is a first binding protein and said first member of said array of labeled agents is a second binding protein which is specific for said at least one analyte, causing said at least one analyte to bind to both said first binding protein and said second binding protein in a sandwich assay.

8. The method in accordance with claim 7, wherein said sample is added to a mixture of said array of microparticles and said array of labeled agents.

9. The method in accordance with claim 1, wherein said at least one analyte is an antibody, said analyte-specific member is an antigen specific to said antibody and said first labeled agent is a labeled binding protein selective for the class of antibody to which said at least one analyte is a member, causing an indirect antibody assay.

10. The method in accordance with claim ^'9, wherein said sample is added to a mixture of said array of microparticles and said array of labeled agents.

11. The method in accordance with claim 9, wherein said array of microparticles is added to a mixture of said sample and said array of labeled agents.

12. The method in accordance with claim 9, wherein said sample is removed from said array of microparticles, comprising said analyte-specific member prior to the addition of said array of labeled agents.

13. The method in accordance with claim 1, wherein said at least one analyte is an antibody, said analyte-specific member is an antibody class specific binding protein capable of capturing the class of antibody of said antibody and said first labeled agent is a labeled antigen specific for said antibody, causing an antibody class capture assay.

14. The method in accordance with claim 13, wherein said sample is added to a mixture of said array of microparticles and said array of labeled agents.

15. The method in accordance with claim 13, wherein said array of microparticles is added to a mixture of said sample and said array of labeled agents.

16. The method in accordance with claim 13, wherein said sample is removed from said array of microparticles, comprising said analyte-specific member prior to the addition of said aπay of labeled agents.

17. The method in accordance with claim 1, wherein said interferent- specific member comprises a bound at least one interferent or an analog thereof and said second member of said array of labeled agents is a labeled binding protein, causing said interferent-specific member and said at least one interferent to compete for said labeled binding protein in a competitive assay.

18. The method in accordance with claim 17, wherein said array of microparticles is added to a mixture containing said sample and said array of labeled agents.

19. The method in accordance with claim 1, wherein said interferent- specific member comprises a binding protein and said second member of said array of labeled agents is a labeled said at least one interferent, causing said at least one interferent and said labeled at least one interferent to compete for said binding protein in a competitive assay.

20. The method in accordance with claim 19, wherein said array of microparticles in added to a mixture containing said sample and said array of labeled agents.

21. The method in accordance with claim 1, wherein said interferent- specific member is a first binding protein and said second member of said array of labeled agents is a second binding protein which is specific for said at least one interferent, causing said at least one interferent to bind to both said first binding protein and said second binding protein in a sandwich assay.

22. The method in accordance with claim 21, wherein said sample is added to mixture of said array of microparticles and said aπay of labeled agents.

23. The method in accordance with claim 1, wherein said at least one interferent is an antibody, said interferent-specific member is an antigen specific to said antibody and said second labeled agent is a labeled binding protein selective for the class of antibody to which said antibody is a member, causing an indirect antibody assay.

24. The method in accordance with claim 23, wherein said sample is added to a mixture of said array of microparticles and said array of labeled agents.

25. The method in accordance with claim 23, wherein said aπay of microparticles is added to a mixture of said sample and said array of labeled agents.

26. The method in accordance with claim 23, wherein said sample is removed from said aπay of microparticles comprising an interferent-specific member prior to the addition of said aπay of labeled agents.

27. The method in accordance with claim 1, wherein said at least one interferent is an antibody, said interferent-specific member is an antibody class specific binding protein capable of capturing the class of antibody of said antibody, said second labeled agent is a labeled antigen specific for said antibody, causing an antibody class capture assay.

28. The method in accordance with claim 27, wherein said sample is added to a mixture of said aπay of microparticles and said aπay of labeled agents.

29. The method in accordance with claim 27, wherein said array of microparticles is added to a mixture of said sample and said aπay of labeled agents.

30. The method in accordance with claim 27, wherein said sample is removed from said aπay of microparticles comprising an interferent-specific member prior to the addition of said aπay of labeled agents.

31. The method in accordance with claim 1, wherein said at least one analyte is a plurality of analytes.

32. The method in accordance with claim 1, wherein said at least one interferent is a plurality of interferents.

33. The method in accordance with claim 1, wherein each member of said aπay of microparticles comprises at least one distinguishable parameter.

34. The method in accordance with claim 33, wherein said at least one distinguishable parameter is a member selected from the group consisting of a fluorescent dye or a colored dye.

35. The method in accordance with claim 33, wherein each individual said at least one distinguishable parameter is a different emission spectrum.

36. The method in accordance with claim 33, wherein each individual said at least one distinguishable parameter is a predetermined different concentration of one or more fluorescent dyes.

37. The method in accordance with claim 33, wherein said at least one distinguishable parameter is attached to the surface of the particle, is embedded in the particle or is bound to the molecules of the particle material.

38. The method in accordance with claim 33, wherein said at least one distinguishable parameter is selected from the group consisting of forward light scatter, side angle light scatter, fluorescence at a first wavelength and fluorescence at a second wavelength.

39. The method in accordance with claim 38, wherein said fluorescence at said first wavelength is red and said fluorescence at said second wavelength is orange.

40. The method in accordance with claim 1, wherein said microparticle is approximately spherical.

41. The method in accordance with claim 40, wherein said microparticles have a diameter from about 1 μm to about 100 μm.

42. The method in accordance with claim 33, wherein said aπay of microparticles has several discreet sizes within said aπay.

43. The method in accordance with claim 33, wherein said aπay of microparticles has a plurality of sub-populations having different diameters enabling a member of one of said sub-populations to be identified to its specific sub-population by characteristic high and low angle scatter.

44. The method in accordance with claim 33, wherein said aπay of microparticles has a plurality of sub-populations having various amounts of microparticles enabling said sub-populations to be identified to its specific sub-population by said amount of microparticles.

45. The method in accordance with claim 1, wherein said microparticles consist of a material appropriate for the coating of antibodies or antigens.

46. The method in accordance with claim 45, wherein said material is selected from the group consisting of a polymer, a rubber, a biomolecule, a polysaccharide polymer, a glass, a biological cell, a polystyrene, a lipid, a biological material, a metal and combinations thereof.

47. The method in accordance with claim 33, wherein each member of said aπay of microparticles is coated with a different analyte, antigen or antibody.

48. The method in accordance with claim 1, wherein said aπay of microparticles is comprised of a combination of a polymer and a paramagnetic substance.

49. The method in accordance with claim 1, wherein said at least one analyte is a member selected from the group consisting of thyroid stimulating hormone, T₄, human chorionic gonadotropin, luteinizing hormone, an antibody specific to a microorganism, vitamin B₁₂, folate, digoxin and carbamazepine.

50. The method in accordance with claim 49, wherein said microorganism is selected from the group consisting of Toxoplasma gondii, Rubella virus, Cytomegalovirus, Herpes Simplex Virus Type 1, Herpes Simplex Virus Type 2 and Rabies virus.

51. The method in accordance with claim 1, wherein a plurality of analytes and a plurality of interferents are simultaneously measured using an aπay of microparticles, each member of said array of microparticles being separately distinguishable from each other by said flow cytometer.

52. The method in accordance with claim 1, wherein said interferent is a member selected from the group consisting of, hemoglobin, rheumatoid factor, heterophilic antibodies, anti-animal antibodies and anti-nuclear antibodies.

53. The method in accordance with claim 1, wherein said detecting individually an aπay of emission signals in a flow cytometer wherein the fluorescence or light scatter data of each microparticle is ascertained and measured.

54. The method in accordance with claim 53, wherein said emission signals are produced by through laser-generated iπadiation.

55. A diagnostic kit for individually measuring at least one analyte and at least one interferent in a sample, comprising: (a) an array of microparticles; (b) an aπay of labeled agents; and (c) instructions for use.

56. A kit in accordance with claim 55, wherein said analyte or said interferent is a member selected from the group consisting of thyroid stimulating hormone, T₄, human chorionic gonadotropin, luteinizing hormone, an antibody specific to a virus, vitamin B₁₂, folate, digoxin, carbamazepine, rheumatoid factor (R_f), heterophilic antibodies, antimouse antibodies and anti-nuclear antibodies.