FLUORESCENCE-LABELED SUBSTRATES AND THEIR USE TO ANALYZE ENZYME ACTIVITY USING FLOW CYTOMETRY

Abstract: The present invention involves a reagent suitable for determining the presence or absence of enzymatic activity in a test sample using flow cytometry, comprising an enzyme substrate immobilized on a solid support. The invention also involves a method of determining the presence or absence of enzymatic activity in a test sample using a fluorophore-labeled enzyme substrate immobilized on a solid support under such conditions as to allow enzymatic digestion of the labeled substrate, washing the digested immobilized substrate, passing the digested immobilized substrate through a flow cytometer; and identifying the amount of net enzyme activities in the test sample based on the measured signals.

Description: FLUORESCENCE-LABELED SUBSTRATES AND THEIR USE TO ANALYZE ENZYME ACTIVITY USING FLOW CYTOMETRY FIELD OF THE INVENTION The present invention relates to compositions and methods of assaying enzymatic activity using flow cytometry. BACKGROUND OF THE INVENTION Analysis of Enzyme Activity: Enzymes are catalytic proteins produced by living cells. Enzymes catalyze the chemical reactions involved in the body, including the digestion of foods, the biosynthesis of macromolecules, and the controlled release and utilization of chemical energy. One characteristic of enzymes is their high degree of specificity: the majority of enzymes catalyze only one type of reaction and act on only one substrate or on a group of closely related substrates. Enzymes are divided into six main classes. Class 1 contains the oxidoreductases, which catalyze reactions involving electron transfer and play an important role in cellular respiration and energy production. Class 2 comprises the transferases, which catalyze the transfer of a particular chemical group from one substrate to another. The hydrolases of Class 3 catalyze the cleavage of many substrates, including proteins, nucleic acids, starch, fats, and phosphate esters, by the addition of water (hydrolysis). Class 4 enzymes comprise the lyases which catalyze the the nonhydro lyric cleavage of their substrates by the formation of double bonds as well as the reverse reactions. Class 5 contains the isomerases which transfer groups within molecules to yield isomeric forms. The ligases or synthetases of Class 6 catalyze the formation of C-C, C-S, C-O, and C-N bonds by condensation reactions coupled to ATP cleavage. Enzyme assays typically measure the amount of enzymatic activity in a sample to determine the quantity of enzyme present and/or its level of activity. For example, in the field of medicine, detection of enzymatic activity in biological and chemical samples is useful for obtaining information about metabolism, diseases state, the identity of microorganisms, or the success of genetic manipulations. In order to detect a number of diseases, for example, samples of a body fluid, such as blood or spinal fluid, can be taken from a patient and tested to determine the presence of certain enzymes known to be present only during or after the occurrence of a specific disease. Some enzymatic activities are more amenable to the development of rapid, sensitive, and reproducible assays than others. Given the importance of enzymatic analysis in research and medicine, there is a constant need for improvement and advances in the field. Flow cytometry is a useful technique for identifying the presence of certain analytes or particles of interest in a sample, enumerating those particles and, in some instances, providing a sorting capability so as to be able to collect those particles of interest. (See McHugh In: Darzynkiewycz, Robinson, and Crissman (eds.) Methods in Cell Biology (New York: Academic Press, 1994) 42:575-595). Flow cytometry apparatuses rely upon the flow of particles in a liquid flow stream in order to determine one or more characteristics of the particles under investigation. In a typical flow cytometry apparatus, a fluid sample containing particles is directed through the flow cytometry apparatus in a rapidly moving liquid stream so that each particle passes serially, and substantially one at a time, through a sensing region. A focused light beam illuminates the particles in this region, and the instrument measures optical interactions of the light with each moving particle; for example, multiple wavelength absoφtion, scatter as a function of angle, and fluorescence as a function of either wavelength or polarization may be measured. After particle analysis is performed by the flow cytometry apparatus, those particles that have been identified as having the desired properties may be sorted if the 5 apparatus has been designed with such capability. Representative flow cytometry apparatuses are described in U.S. Patent Nos. 3,826,364 and 4,284,412, and in the publication by Herzenberg et al, (1976) Sci. Am. 234(3):108. The use of colloidal particles and magnetic particles to bind a compound has long been known and used in industrial and laboratory procedures. For example, cross linked o polystyrene-divinylbenzene beads, among the earliest and most widely used particles, have been used in organic synthesis, catalysis and the biotechnical arts, especially immunology. In combination with the appropriate reagents, the particles have been used to remove specific cells from a sample containing a plurality of cell types or to enhance the results of instrumental biomedical assays. Unless specified otherwise, the terms "particles", s "spheroids", "spheres", "microspheres" and "beads" as used herein, are interchangeable. Such microspheres can be coated with different capture reagents or substrates that react with specific analytes in a sample. The fluorescence associated with each microsphere class can be quantitated in order to assay for different analytes in a sample. The flow cytometer can accurately detect different classes of microspheres based upon a physical o characteristic such as size or color. The use of different microsphere classes, each coated with a different capture reagent, allows for the rapid and simultaneous detection of multiple analytes. This provides the potential to perform multiple assays in the same reaction mixture reducing cost and hands-on time, as well as generating results using the same method between analytes. 5 Microspheres have been used with a variety of capture reagents, including antigens (from infectious agents, cell surfaces, or other soluble proteins) to capture antibodies, antibodies to capture soluble antigens, receptors to capture immunoglobulins, oligonucleotides to capture products from the polymerase chain reaction, and proteins for competitive immunoassays with soluble protein or DNA. Usually after the reaction of the test sample with the microspheres, a fluorescent detection reagent is added to the microspheres and allowed to react. The microsphere classes are then analyzed using flow cytometry and the different classes separated by size or color. Each microsphere class can then be analyzed independently: the fluorescence associated with each microsphere class is quantitated and used to indicate the presence or absence of the test substance. This method has been used to detect and separate antigens and antibodies in biological samples (U.K. Patent No. 1,561,042). Flow cytometry has been used as a method of detecting antibodies to specific enzymes in biological samples. As an example, an immunoassay has been developed that uses pyruvate dehydrogenase enzyme complex as a specific antigen for diagnosis of primary biliary cirrhosis (PBC) (Elkhalifa et al, (1992) Am. J. Clin. Pathol 97(2):202-208). In this assay, pyruvate dehydrogenase enzyme complex was attached to polystyrene microbeads, incubated with sera from PBC patients, incubated with a fluorescein isothiocyanate conjugated goat anti-human immunoglobulin, then analyzed by flow cytometry. Flow cytometry and monoclonal antibodies have also been used in the detection of the human enzyme monocytic lysozyme in patients suffering from acute myeloid leukemia (Leculier et al, (1992) Blood 79(3):760-764). These assays, however, do not measure enzymatic activity. Flow cytometry has also been used to detect enzyme activity in cells. For example, fluorescent substrates have been used to determine beta-galactosidase activity in viable gram-negative bacteria (Flovins et al, (1994) Applied and Environmental Microbiology 60( 12) :4638-4641 ); to detect enzymatic activity in microbial colonies (Sahar et al, (1994) Cytometry 15(3):213-221); and to measure cellular dehydrogenase activity (Severin and Seidler (1992) Cytometry 13(3):322-326). These assays measure in vivo enzymatic activity using flow cytometry and fluorescently-labeled substrates that are permeable to cytoplasmic membranes. Flow cytometry has also been used to show an augmentation of cell surface expression of MMPs (Leppert et al, (1995) FASEB J. 9(14):1473-1481). This review of enzyme assays currently available indicates that a need remains for a direct method of determining net enzymatic activity that is sensitive, reproducible, rapid, easy to use, and suitable for large-scale use. It is therefore an object of the present invention to provide an easy, reliable assay to determine qualitative and quantitative measurements of enzyme activity in biological samples on a routine basis. Analysis ofMMP and Gelatinase B Activity: One example of a group of enzymes for which an improved assay is required is the matrix metalloproteinase family. The matrix metalloproteinase (MMP) family of proteinases is thought to play a crucial role in extracellular matrix (ECM) degradation. The integrity of connective tissues is determined by the delicate balance between the synthesis and reabsorption of ECM molecules. Breakdown of this balance is associated with a variety of pathological conditions, such as joint degeneration in rheumatoid arthritis, demyelination in multiple sclerosis, and dissemination of malignant tumors (Liotta et al, (1991) Cell 64:327-336; McCachren (1991) Arthritis. Rheum. 34:1085-1093; Opdenakker and Van Damme (1992) Cytokine 4:251-258; and Opdenakker and Van Damme (1994) Immunol Today 15:103- 107). The ability of MMPs to degrade collagen and other components of the ECM allows circulating leukocytes and tumor cells to progress through the basement membrane underlying the blood vessel wall, and invade the surrounding tissue. The activity of MMPs also plays a pivotal role in the degradation of matrix components in degenerative diseases. It is well established that during inflammatory processes such as rheumatoid arthritis, cytokines stimulate macrophages and fibroblasts to secrete MMPs (Opdenakker and Van Damme (1994) Immunol. Today 15:103-107; Feldmann et al, (1996) Annu. Rev. Immunol. 14:397-440). The elevated levels of MMPs in serum or in synovial fluid (SF) have been shown to reflect the inflammatory conditions of the joints of patients with arthritis [Opdenakker et al, (1991) Lymphokine and Cytokine Research 10:317-324; Yoshihara et α/., (1995) Arthritis and Rheumatism 7:969-975; Koolwijk etα/., (1995) J. Rheumatol 22:385-393; Ahrens et al, (1996) Arthritis Rheum. 39:1576-1587; Ishiguro et al, (1996) J. Rheumatol. 23:1599-1604). MMPs are secreted as inactive proenzymes by macrophages and fibroblasts. They are activated by proteases such as plasmin, and also by other members of the MMP family, such as MMP-2, MMP-3, and MT-MMP. MMP activation is orchestrated by a cysteine switch mechanism: the clipping of an aminoterminal fragment of which the cysteine sulfhydryl group is replaced by the catalytic water molecule bound to the Zinc atom in the active site (Springman et al, (1990) Proc. Natl Acad. Sci. USA 87:364; Knauper et al, (1996) J. Biol Chem. 271(29):17124-17131; Nagase and Okada (1997) In: Kelley et al, (ed.) "Textbook of Rheumatology" (W.B. Saunders) 323-241). The members of the MMP family can be divided into four groups, based on protein domain structure and substrate specificity: i) the collagenases (e.g. MMP-1, MMP-5, MMP-8 and MMP- 13), which degrade interstitial collagen during what is considered the rate limiting step in connective tissue degradation; ii) the stromelysins (MMP-3, MMP- 10 and MMP- 11), all of which can degrade a variety of substrates in addition to proteoglycans; iii) the gelatinases A and B (MMP-2 and MMP-9, respectively), which are responsible for the degradation of basement membrane collagen and denatured collagen (gelatin) generated by the action of the collagenase; and iv) the membrane-type MMPs (MT-MMP), which are anchored by a transmembrane domain and are crucial in the activation of other MMPs. Recent studies have demonstrated that members of the MMP family are not restricted exclusively to one type of substrate as they have overlapping specificity for many of the different components of the ECM. One member of the MMP family is gelatinase B, also known as MMP-9. Gelatinase B (Class 3, E.C. 3.4.24.35) is a proteolytic enzyme which expresses a high degree of homology between species (Masure et al, (1993) Euro. J. Biochem. 218:129-141; Tanaka et al, (1993) Biochem. Biophys. Res. Com. 190:732-740). Accumulating evidence demonstrates a causal relationship between gelatinase B activity and the invasive behavior of tumor cell lines. Gelatinase expression by cancer cell lines has been correlated with the invasive and metastatic potential of these cells in vivo (Bernhard et al, (1990) Cancer Res. 50:3872-3877; Bernhard et al, (1994) Proc. Natl. Acad. Sci. USA 91 :4293-4297; Ura et al, (1989) Cancer Res. 49:4615-4621). In vivo, the activity of MMPs is regulated by a family of natural inhibitors known as tissue inhibitors of metalloproteinases (TIMPs). These compounds form a tight stoichiometric noncovalent complex with the activated MMPs and block their proteolytic activity (Woessner (1991) FASEB J. 5:2145-2154). The equilibrium between the MMPs and their inhibitors appears to be altered in inflamed synovia, suggesting that degradation of ECM proteins may result from a positive balance toward excessive proteolytic activity (McCachren (1991) Arthritis Rheum. 34:1085-1093). Such excess activity could result from increased expression of the MMPs, from the conversion of the proenzyme to its active form, and from a relative decrease in inhibitory activity by TIMPs. For example, increased expression of MMPs in serum and in synovial fluids (SF) of patients diagnosed with various inflammatory diseases has been noted at the gene level using RT-PCR on synovial cells ( McCachren (1991) Arthritis. Rheum. 34:1085-1093; DiBattista et al, (1995) J. Rheumatol Suppl. 43:123-128; Tsuchiya et al, (1996) Biotechnology. Histochem. 71 :208-213; Kikuchi et al, (1996) Clin. Exp. Pharmacol Physiol. 23:885-889), and at the protein level, using either capture assays (Clark et al, (1993) Athritis Rheum. 36:372-379; Cawston et al, (1995) Clin. Exp. Rheumatol. 13:431- 437; Yoshihara et al, (1995) Arthritis and Rheumatism 7:969-975; Sopata et al, (1995) Rheumatol. Int. 15:9-14; Ahrens et al, (1996) Arthritis Rheum. 39:1576-1587; Ishiguro et al, (1996) J. Rheumatol. 23:1599-1604), or gelatin-zymography [Opdenakker et al, (1991) Lymphokine and Cytokine Research 10:317-324; Tetlow et al, (1993) Rheumatol Int. 13:53-59; Ahrens t α/., (1996) Arthritis Rheum. 39:1576-1587). The latter measures proteolytic activity of MMPs following dissociation from their natural inhibitors (TIMP) by SDS-PAGE electrophoresis. Administration of recombinant TIMPs to mice has been shown to inhibit colonization of ras-transfected malignant cells (Alvarez et al, (1990) J. Natl Cancer Inst. 82:589-595), while suppression of TIMP activity by antisense technology enhances the invasive phenotype (Khokhe et al, (1989) Science 243:947-950). Development of specific inhibitors of MMPs is of crucial importance in new therapeutic approaches designed to block tumor cell dissemination and ECM degeneration during o inflammation. Key to developing such inhibitors is the need for an assay that can measure, on a routine basis, the net activity resulting from the balance between active MMPs and the presence of inhibitors. Currently, there are several assays to measure MMP activity (for a review of these 5 methods, see Muφhy and Crabbe In Barrett (ed.) Methods in Enzymology. Proteolytic Enzymes: Aspartic Acid and Metallopeptidases (New York: Academic Press, 1995) 248:470). One method, the gelatinolytic assay, is based on the degradation of radiolabeled type I collagen. Although this method is relatively sensitive, it requires the use of radiolabeled specific substrates. o Another widely-used technique is the zymography assay. In this assay, MMP activity is detected by the presence of negatively-stained bands following electrophoresis in substrate-impregnated SDS polyacrylamide gels. The zymography assay is a sensitive and quantitative method for the detection of various MMPs in biological samples; nonetheless, it is labor intensive and has a low dynamic range. Zymography, moreover, is not suitable 5 to measure the intrinsic net activity in biological samples: SDS dissociates MMP-TIMP complexes and activates latent enzyme forms. This is particularly important since matrix degradation ultimately depends on the ratio of free active gelatinase to latent proenzyme or TIMP-complexed forms. A microtiteφlate assay has been developed recently (Pacmen et al, (1996) Biochem. Pharm. 52: 105-111). This assay provides measurement of net biological enzymatic activity of MMP, does not require a radioisotope safety environment, and could be used efficiently for routine measurement of inhibitory activity of MMP; however, it is not likely to be highly efficient as a diagnostic test since the incubation times are long and the sensitivity is much lower than that obtained by standard zymography and radio-labeled substrate assays. Although many fluorogenic substrates have been designed for the quantitation of MMPs (reviewed by Nagase and Fields (1996) Biopolymers 40:399-416), their use for measurement of MMP activities in biological fluids has been hampered by the optical interactions of the fluorophore with the medium. Separation of the degradation products by chromatography has been used to solve this problem. The fluorogenic substrate TN0211, for instance, has been used to deterrnine the "pan-MMP activity" in SF [Beekman et al, (1996) FEBS Letters 390:221-225). Unfortunately, this process is laborious and is not sensitive. Given the important roles of MMPs in disease, one skilled in the art could easily appreciate the utility of a method of routine MMP analysis in the fields of medicine and research. It is therefore an object of the present invention to provide a rapid, easy, sensitive, and reproducible assay to measure the net biological activity present in samples. This background information is provided for the puφose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention. Publications referred to throughout the specification are hereby incoφorated by reference in their entireties in this application. SUMMARY OF THE INVENTION The problems noted above have been overcome by the use of a new method that allows qualitative and quantitative measuring of specific enzymatic activity in a sample using flow cytometry, immobilized substrates, and fluorescence-labeling. In one embodiment, there is provided a reagent suitable for determining the presence or absence of enzymatic activity in a test sample using flow cytometry, comprising an enzyme substrate immobilized on a solid support. In another embodiment, there is provided a method of determining the presence or absence of enzymatic activity in a test sample which comprises the following steps: (a) adding a test sample to a reagent suitable for determining the presence or absence of enzymatic activity in a test sample using flow cytometry, comprising an fluorophore-labeled enzyme substrate immobilized on a solid support, under such conditions as to allow enzymatic digestion of the labeled substrate; (b) washing the digested immobilized substrate; (c) passing the digested immobilized substrate through a flow cytometer; and (d) identifying the amount of net enzyme activities in the test sample based on the measured signals. In yet a further embodiment, there is provided a method of determining the presence or absence of enzymatic activity in a test sample which comprises the following steps: (a) adding a test sample to a solution containing a reagent suitable for determining the presence or absence of enzymatic activity in a test sample using flow cytometry, comprising a fluorophore-labeled enzyme substrate immobilized on a solid support and a second substrate under such conditions as to allow enzymatic conversion of the labeled substrate; (b) washing the digested immobilized substrate; (c) passing the digested immobilized substrate through a flow cytometer; and (d) identifying the amount of net enzyme activities in the test sample based on the measured signals. In still a further embodiment, there is provided, a method of determining the presence or absence of enzymatic activity in a test sample which comprises the following steps: (a) adding a test sample to a solution containing a reagent suitable for determining the presence or absence of enzymatic activity in a test sample using flow cytometry, comprising an enzyme substrate immobilized on a solid support, and a second substrate that has been labeled, under such conditions as to allow enzymatic conversion of the labeled substrate; (b) washing the digested immobilized substrate; (c) passing the digested immobilized substrate through a flow cytometer; and (d) identifying the amount of net enzyme activities in the test sample based on the measured signals. A further embodiment provides a method of determining the presence or absence of enzymatic activity in a test sample, which comprises the following steps: (a) selecting an appropriate enzyme/substrate pair; (b) a step selected from: (i) labeling the substrate with a fluorophore and immobilizing the labeled substrate on a solid support suitable for use in flow cytometry, or (ii) immobilizing the substrate on a solid support suitable for use in flow cytometry, and labeling the immobilized the substrate with a fluorophore; (c) adding a test sample to a portion of the immobilized labeled substrate under such conditions as to allow enzymatic digestion of the labeled substrate; (d) washing the digested immobilized substrate; (e) passing the digested immobilized substrate through a flow cytometer; and (f) identifying the amount of net enzyme activities in the test sample based on the measured signals. Another embodiment provides a method of determining the presence or absence of enzymatic activity in a test sample, which comprises the following steps: (a) selecting an appropriate enzyme/substrate group; (b) a step selected from: (i) labeling one substrate of the enzyme/substrate group with a fluorophore and immobilizing the labeled substrate on a solid support suitable for use in flow cytometry, or (ii) immobilizing the labeled substrate on a solid support suitable for use in flow cytometry and labeling one substrate of the enzyme/substrate group with a fluorophore; (c) adding a second substrate of the enzyme/substrate group and a test sample to a portion of the immobilized labeled substrate under such conditions as to allow conversion of the immobilized substrate to occur; (d) washing the converted immobilized substrate; (e) passing the converted immobilized substrate through a flow cytometer; and (f) identifying the amount of net enzyme activity in the test sample based on the measured signals. Another embodiment provides a method of determining the presence or absence of enzymatic activity in a test sample, which comprises the following steps: (a) selecting an appropriate enzyme/substrate group; (b) immobilizing an unlabeled substrate of the enzyme/substrate group on a solid support suitable for use in flow cytometry; (c) labeling a second substrate of the enzyme/substrate group with a fluorophore; (d) adding the labeled second substrate and a test sample to a portion of the immobilized unlabeled substrate under such conditions as to allow conversion of the immobilized substrate to occur; (e) washing the converted immobilized substrate; (f) passing the converted immobilized substrate through a flow cytometer; and (g) identifying the amount of net enzyme activities in the test sample based on the measured signals. Another embodiment provides a kit for the preparation of a reagent suitable for determining the presence or absence of enzymatic activity in a test sample using flow cytometry, comprising an enzyme substrate immobilized on a solid support. Various other objects and advantages of the present invention will become apparent from the detailed description of the invention. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Fluorescence- Activated Substrate Conversion (FASC). Figure 2. Analysis of substrate conversion. Figure 3. Comparison of relative fluorescence of uncoated microspheres (A) and microspheres coated with FITC-labeled gelatin (B). On the left, forward-light scatter (FS) vs side scatter (SS) histograms of the respective microsphere preparations are shown. Figure 4. Specificity of the enzymatic degradation of FITC-labeled gelatin. FITC-labeled gelatin (A) and FITC-labeled casein (B) were coated on microspheres and incubated with 200 ng of purified gelatinase B for 16 h at 37°C. The histograms shown are representative of at least ten independent experiments with five different microsphere preparations for each substrate. Similar results were obtained with different FITC to protein ratios and different concentrations of substrates during coating. Figure 5. Inhibition of gelatin degradation by blocking with monoclonal antibody. FITC- labeled gelatin-coated microspheres were incubated with 200 ng of gelatinase B in the presence of various amounts of inhibitory monoclonal antibody specific for gelatinase B. The final volume of the reaction was 100 μl. The results were expressed as the percentage of gelatin degraded in the presence of the blocking monoclonal antibody. Controls included microspheres without gelatinase B (MCF239.4 ± 1.3), and microspheres with gelatinase B without monoclonal antibody (MCF:21.0 ± 2.4). Maximal degradation in the absence of blocking monoclonal antibody was calculated as 91.2 ± 1.0%. Figure 6. Inhibition of gelatin degradation in the presence of low molecular weight inhibitors. FITC-labeled gelatin-coated microspheres were incubated with 200 ng of gelatinase B in the presence of the indicated concentrations of inhibitors. Inhibitors were preincubated for 15 min. at room temperature with gelatinase B prior to the addition of microspheres. The enzymatic reaction was carried out at 37 °C for 16 h. The results are expressed as the mean channel of fluorescence (MCF) of the microsphere populations obtained after treatment and are representative of two independent experiments. Figure 7. Sensitivity of the assay. Five different microsphere preparations were obtained by varying the FITC concentrations (from 2.0 μM to 1.25 mM) during fluoresceination of gelatin substrate. (A): fluorescence of the five microsphere preparations obtained. The five right end peaks correspond to five-fold increases in FITC concentrations. The left end peak corresponds to uncoated microspheres. (B) and (C): degradation of FITC-labeled gelatin using the lowest (B) and the highest (C) fluorescent microsphere preparations, as shown in (A). The results are representative of two independent experiments. Figure 8. Dose-dependent degradation of gelatin by purified human neutrophil gelatinase B (MMP-9). (A) Increasing concentrations of gelatinase B were incubated with gelatin- FITC-coated microspheres in a final volume of 100 mL and incubated for periods of 90 min or 18h. Degradation of gelatin-FITC was analyzed on a Coulter XL-MCL. The results are representative of three independent experiments done in triplicate (±S.D.). (B) Gelatin- zymography was used to analyze decreasing concentrations of neutrophil gelatinase B. The lanes 1 to 7 indicate the reactions of 200 ng, 100 ng, 50 ng, 25 ng, 12.5 ng, 6.3 ng and 3.3 ng of purified enzyme, respectively. Figure 9. Kinetics of proteolytic activity against gelatin-FITC coated microspheres in SF. Gelatin-FITC coated microspheres were incubated with serial dilutions of SF in a final volume of 100 mL for the indicated times. Results are representative of two independent experiments. Figure 10. Reproducibility of the FASC analysis of SF. Serial dilutions of SF were submitted to three freeze-thaw cycles. After each cycle, a FASC analysis was performed with gelatin-FITC coated microspheres using an incubation period of 90 min. Figure 11. Zymographic analysis of serum from patients with rheumatoid arthritis (RA). A panel of individual serum samples was analyzed by zymography on (A) a gelatin- impregnated gel using 2 mL of undiluted serum; and on (B) a casein-impregnated gel using 5 mL of undiluted serum. Each lane represents an individual sample. Figure 12. Titration of SF activity by FASC. Gelatin-FITC coated micropheres were incubated with serial dilutions of SF in a final volume of 100 mL for a period of 18 h. Figure 13. Correlation between FASC titer and zymography. The titer of activity for all SF of RA patients tested (234 samples) deterrnined by FASC analysis was plotted against arbitrary scanning units obtained by zymography. Figure 14. Effect of inhibitors on the net proteolytic activity of SF. The dilution of SF that caused 50% degradation over a period of 18 h, determined in a preliminary experiment as described in Figure 12, was used. (A) Samples were pretreated 30 min with 1 mM of PHEN, and then analyzed by FASC with gelatin-FITC coated microspheres for an incubation period of 18 h. (B) SF samples were pretreated for 18 h with 2 mg of REGA- 3G12 at 4°C. Afterward, FASC analysis was performed with gelatin-FITC coated microspheres for a further incubation of 18 h. (C) The ratio of percentage of inhibition obtained with monoclonal antibody to the percentage of inhibition obtained with PHEN is shown for each sample tested. Figure 15. Gelatin-zymography of SF. Undiluted SF samples (2 μl) were analyzed by gelatin zymography. (A) FASC-negative samples. (B) FASC-positive samples. (C) The FASC titer for each of the samples analyzed in (B). Figure 16. Casein-zymography of SF. SF samples (5 μl) were analyzed by casein- zymography. DETAILED DESCRIPTION OF THE INVENTION The following terms are used throughout the specification and in the claims: "enzymatic reaction" means a specific chemical reaction catalyzed by an enzyme; "enzyme/substrate pair" means an enzyme and the corresponding substrate specific to that enzyme; for example, gelatinase B and gelatin are an enzyme/substrate pair; and "enzyme/substrate group" means an enzyme and the corresponding substrates specific to that enzyme; for example, guanidinoacetate methyltransferase, S-adenosylmethionine, and guanidinacetate are an enzyme/substrate group. "substrate" means a molecule that can be chemically modified by enzymatic catalysis, and includes a synthetic polypeptide. "reagent" means a substance to produce a chemical reaction so as to detect, measure, produce, etc. other substances; in the present invention, reagent is used to connote the particle generated by attaching a substrate, labeled or unlabeled, to an appropriate solid surface for use with the method described herein employing flow cytometry. The present invention resides in the discovery that net enzymatic activity of a sample can be quantitatively measured by monitoring enzymatic activity using fluorophore-labeled substrates, immobilization of substrates on a solid support, and flow cytometry. Briefly, the reagents and methods of this invention entail the selection of an appropriate enzyme/substrate pair or group. In one embodiment wherein the reagent comprises an enzyme/substrate pair, a fluorophore can be attached to the substrate, which is then immobilized on a solid support such as a microsphere. A test sample is added to a portion of the immobilized labeled substrate (reagent) under such conditions as to allow enzymatic digestion of the labeled substrate, which is then washed. Finally, the immobilized substrate and controls are passed through a flow cytometer and the amount of enzyme activity in the test samples (eg. net enzyme activity) and controls are measured. In another embodiment, wherein the reagents comprise an enzyme/substrate group, one substrate is labeled with a fluorophore while another is left unlabeled. Either the labeled or unlabeled substrate can be immobilized on a solid support. A test sample is then added to a portion of the immobilized substrate along with the second substrate under such conditions as to allow the enzymatic reaction to occur. The immobilized substrate is then washed. Finally, the immobilized substrate and controls are passed through a flow cytometer and the amount of enzyme activity in the test samples and controls are measured. The enzymatic reaction can be allowed to proceed to completion or it can be stopped at a particular point in the reaction, and the enzyme activity determined for that time point. Selection of Enzyme/Substrate Pair or Group The regents of this invention and method of use can be generated with many different enzyme/substrate pairs or groups. It is particularly adaptable to transferases (Class 2), hydrolases (Class 3), lyases (Class 4), and ligases (Class 6). Within Class 3, the following enzymes are particularly adaptable to the method of this invention: the esterases including exo- and endonucleases active on ribo- and deoxyribonucleic acids (e.g., DNAse, RNAse, and restriction enzymes) and carboxylic ester hydrolases (e.g., phospholipase Al); glycosidases including hydrolyzing glycosyl compounds such as xylanase, insulinase, lysozyme, and hyaluronidase; and peptidases including exo- (amino- and carboxypeptidases) and endopeptidases (proteinases such as gelatinases). Any enzyme/substrate pair can potentially work in the reagents and methods of this invention if the functional group on the substrate is one that can be labeled by a fluorophore and removed by the action of the enzyme. Any enzyme/substrate group can potentially work in this invention if the functional group on one substrate can be labeled by a fluorophore and be transferred to a second substrate by the action of the enzyme, or if one substrate, which is joined to a second substrate by the action of an enzyme, can be labeled by a fluorophore. An enzyme/substrate pair comprises the enzyme to be assayed and an appropriate natural or synthetic substrate, which is labeled with a fluorophore. Examples of suitable pairs of enzyme/substrate are gelatinase B/gelatin; DNAse/DNA; RNAse/RNA; amylase/starch; various glycosidases/their polysaccharide substrates; and peptidases/polypeptides. Other substrates that can be employed include the MHC class I heavy chain (Demaria et al, (1994) J. Biol Chem. 269:6689-694), the folate receptor (Elwood et al, (1991) J. Biol Chem. 266: 2346-2353); the tumor necrosis factor-alpha precursor (Gearing et al, (1994) Nature 370:555-557; McGeehan et al, (1994) Nature 558-560), and the fas ligand (Tanaka, M., et al., (1996) Nature Medicine 2: 317-322). Additional substrates include the inflammatory cytokine IL-I (Ito et al, (1996) J. Biol Chem. 271 :14657-14660) and myeline basic protein (Proost et al, (1993) Biochem. Biophys. Res. Commun.192:1175- 1181; Gijbels et al, (1993) J. Neurosci. Res. 36:432-440). Additionally, any synthetic polypeptide can be used that has a specific recognition sequence for a particular enzyme. Nagase and Fields (1996) Biopolymers 40:399-416 describe a list of synthetic peptides that can be used to determine the specific activity of a particular enzyme or of a group of functionally-related enzymes, such as stromelysins or gelatinases. Linkers or spacers could be employed to minimize steric hindrance and allow the catalytic domain of the enzyme to reach the cleavage site. As shown in the following working example, gelatin, a component of the extracellular matrix (ECM) is an enzymatic substrate for the enzyme gelatinase-B, a metalloproteinase. Other members of the metalloproteinase family can degrade in vitro other components of the ECM, including collagens, fibronectin, laminin, elastin, proteoglycans, and entactin (Matrisian (1992) BioEssays 14:455-463), as well as synthetic decapeptides with a sequence identical to that derived from soluble β-amyloid sequence isolated from Alzheimer's disease patients (Miyazaki et al, (1993) Nature 362:839-841). These are all examples of potential substrates for detecting metalloproteinase activity. An enzyme/substrate group comprises the enzyme to be assayed and appropriate natural or synthetic substrates, one of which is labeled with a fluorophore. For example, transferases, such as guanidinoacetate methyltransferase or serine hydroxymethyltransferase, catalyze the transfer of a chemical group from one substrate (donor) to another (acceptor). They can be assayed using the method of the present invention by labeling the chemical group on the donor to be transferred and immobilizing the donor on microspheres. Following the transfer of the labeled chemical group to the acceptor, there will be a decrease in fluorescence of the microspheres, indicating the presence of transferase activity. As another example, ligases, such as peptide synthases, DNA ligases, and RNA ligases, catalyze the joining of two substrates (an acceptor and a donor) with the concomitant hydrolysis of a pyrophosphate bond in ATP. They can be assayed using the method of the present invention by immobilizing the unlabeled acceptor on microspheres. Measurement of ligase activity is indicated by an increase in fluorescence of the microspheres following ligation of the fluorophore-labeled donor compound to the immobilized acceptor. Generating Fluorescence-Labeled Substrates It will be appreciated that any fluorescence-emitting detectable label may be used. Fluorescent substances (fluorophores) used for labeling proteins are well known in the art. There are many constraints on the choice of fluorophore. One constraint is the absoφtion and emission characteristics of the fluorophore, since materials in the sample under test will fluoresce and interfere with an accurate determination of the fluorescence of the label. This phenomenon is called autofluorescence or background fluorescence. Another consideration is the ability to conjugate the fluorophore to substrate and the effect of this conjugation on both the fluorophore and the substrate. A third consideration is the quantum efficiency of the fluorophore; this should be high for sensitive detection. A fourth consideration is the light absorbing capability or extinction coefficient of the fluorophore, which should be as large as possible. The choice of fluorophore depends upon the assay configuration, reagent availability, and excitation/emission possibilities in the flow cytometer. Fluorophores that are available include fluorescein isothiocyanate, Texas Red, AMCA, phycobiliproteins such as allophycocyanin, cyanine derivatives, and rhodamine. Rhodamine, a conventional red fluorescent label, has proved to be less effective. Texas Red is a useful labeling reagent that can be excited at 578 nm and fluoresces maximally at 610 nm. Phycobiliproteins, such as phycoerythrin, have a high extinction coefficient and high quantum yield. Cyanine dyes are described in U.S. Patent No. 5,486,616. In a preferred embodiment, fluorescein isothiocyanate (FITC) is chosen as a fluorophore for the following practical and theoretical reasons: 1) FITC is a small molecule; thus it minimizes the steric hindrance around putative cleavage sites; 2) it is easily conjugated to substrates; and 3) it has spectral properties compatible with most flow cytometers. Generally, FITC has been used most often because of its widespread availability in a variety of conjugates, its ease of excitation at 488 nm with argon ion lasers, and the availability of appropriate optical filters to detect emission at 525 nm. Although other fluorophores, such as phycoerythrin or allophycocyanin have a better quantum yield than FITC, they may cause significant stearic hindrance because of their high molecular weight. Texas Red or AMCA, which have low molecular weights and are easily conjugated to proteins, are viable alternatives. These fluorophores require alternative laser sources and related optics to obtain adequate excitation. Excitation of Texas-Red can be achieved with He/Ne lasers or diode lasers, whereas AMCA requires UV excitation, which can be provided by He/Cd lasers, UV lamps, or diode lasers. To label substrates with FITC, the protein substrates of interest are dissolved in carbonate buffer (pH 9.2) to a final concentration of 2 mg/ml. Fluorescein isothiocyanate (FITC; Sigma; dissolved in MDSO at 5 mg/ml) is added to the appropriate concentrations. Labeling is carried out for 24 h at 4°C. Free FITC molecules are removed by chromatography on PD-10 columns (Pharmacia, Uppsala, Sweden) using PBS, pH 7.4 as eluent buffer. Immobilization of Substrates The solid support used in the claimed methods can be of variable but limited dimensions, generally ranging from 0.5 to 100 micrometers in diameter (most preferably 0.5 to 50 micrometers) and made of any substance provided that an enzymatic substrate can be either adsorbed onto or covalently bound to its surface. The support may be porous or hollow, or solid and non-porous. For reasons of cost, availability, and uniform size and shape, polymeric materials are preferred. Such polymers include polystyrene, polystyrene- divinylbenzene, polymethacrylate, and polyphenylene oxide. Polystyrene and polystyrene latex supports are optimum because of their availability as various sized microspheres or beads, inexpensiveness, compatibility with most biological systems, and familiarity to those skilled in the art. The polymeric support may contain amine-reactive surface functional groups; for example, aldehydes, aldehyde/sulfate, carboxylic acids and esters, and tosyl groups. In a preferred embodiment, substrate is immobilized on polystyrene microspheres. The use of polystyrene allows efficient noncovalent adsoφtion of most proteins. Although noncovalent adsoφtion to polystyrene is based only on electrostatic interactions and/or van der Waals forces, this coating is stable for months, provided the microspheres are kept in the dark at 4°C with 0.05% sodium azide as a preservative. Smaller proteins, such as synthetic peptides, often do not attach well to polystyrene; in these cases, attachment to polystyrene may be achieved using covalent coupling techniques or an intermediate reagent (McHugh (1994) In: Darzynkiewycz, Robinson, and Crissman (eds.) Methods in Cell Biology (New York: Academic Press, 1994) 42:575-595). Other techniques for attaching substrates to microspheres, which are well known in the art, can also be used in the present invention. In a preferred embodiment, polystyrene microspheres of 15 μm diameter are used to allow for maximal available surface in order to capture the greatest amount of substrate and to generate an optimal signal-to-noise ratio. Polystyrene microspheres of 15.5 μm (± 1.919) diameter (Polysciences, Warrington, PA) are incubated for 2 h at 37 °C with substrates (1 mg/ml in PBS, pH 7.4) to allow noncovalent adsoφtion of the substrates to the surface of the microspheres. The microspheres are then washed twice in phosphate buffer (pH 7.4) containing 0.5% BSA and 0.05% sodium azide (PBA). Microspheres are kept at 4°C in PBA (10 6 beads/ml) in the dark and resuspended by gentle vortexing before use. The number of microspheres per reaction mixture may be varied. Enough microspheres must be collected to allow gating of single populations or to separate distinct microsphere classes and to produce an accurate fluorescent peak for measurement (McHugh (1994) In: Darzynkiewycz, Robinson, and Crissman (eds.) Methods in Cell Biology (New York: Academic Press, 1994) 42:575-595). Enzymatic Reaction: Incubation with the enzyme of interest is carried out for variable periods of time, usually from 60 to 90 minutes, under the appropriate conditions. Analysis Using a Flow Cytometer In the present invention, enzymatic activity is determined by a change in the signal of label as detected by a flow cytometer. It is understood that the present method can use many different types of flow cytometry apparatuses. In a preferred embodiment of the present invention, the flow cytometer is a laser flow cytometer with standard optics for collection of fluorescent signals. Although the assay has been developed on a Coulter XL-MCL flow cytometer equipped with an air-cooled argon laser emitting at 488 nm, analysis can be carried out with other commercially available bench-top flow cytometers found in most university hospitals. The light source in the flow cytometer used in the present invention is not limited to the afore-mentioned argon ion laser; any other light source can be employed, such as a mercury arc lamp, a xenon arc lamp, a He-Cd laser, a He-Ne laser, a diode laser, or a Krypton ion laser. The assay in the presently described form requires minimal flow cytometer capabilities. It can be clearly upgraded through multicolor analyzers to allow simultaneous measurements of multiple enzymatic reactions using different fluorophores. Alternatively, simultaneous enzymatic reactions can be monitored using microspheres having multiple diameters. In this case, a FS-SS histogram can be used to distinguish between different microsphere populations . Analysis of Samples and Controls Samples and controls are passed through the flow cytometer. Figure 2 illustrates an example of how substrate conversion is detected using flow cytometry. In certain instances, the microspheres or other solid support will require a reading. The unreacted substrate immobilized on the solid support will also be read, as will the enzyme-reacted sample. For a net enzyme analysis, the enzyme-reacted sample will be allowed to digest to completion. Time course studies can be performed on samples arrested at certain time points of interest. Someone skilled in the art will be able to determine the appropriate controls and time points appropriate to the design of the experiment. MMPs and Gelatinase B The steps of this invention and its principles are demonstrated by the following general description of the use of the method to determine gelatinase B activity. It is to be understood that while this discussion is directed largely to the utility of the method for studying the regulation of gelatinase activity, for developing new anti-metastatic agents against matrix metalloproteinases, and for monitoring the activity of other proteolytic enzymes, the utility of this invention is not so limited; rather, the following discussion is provided merely for exemplification of the invention's versatility and usefulness. In this particular embodiment of the present invention, the substrate gelatin, labeled with fluorescein isothiocyanate, is immobilized on polystyrene microspheres. A biological sample is then added to a portion of the immobilized labeled gelatin under such conditions as to allow digestion of the gelatin by active gelatinase B in the sample. Control samples are added to another portion of the immobilized labeled gelatin under similar conditions. Following washing of the microspheres, samples are analyzed using flow cytometry and net gelatinase-B activity is determined. It is important to note that the method of the present invention determines net gelatinase B activity: inactive proenzyme forms and inhibited forms are not detected. This is in contrast to most conventional assays that monitor the presence of all forms of the enzyme. For example, the standard zymography assay detects both active and latent forms of the enzyme. Additionally, it dissociates inactive gelatinase-TIMP complexes, and this enzyme is also detected. The assay of the present invention, however, measures the net activity of gelatinase B in biological samples. Uses: The reagents and method of the present invention will have many applications for use in medical diagnosis and treatment, in research, and in industrial use. These reagents and method can be used clinically to diagnose and monitor disease states. For example, they can be used to determine enzyme levels in order to diagnose disease states in which enzymatic degradation of biological substrates is involved. In particular, the reagents and assay will be useful for monitoring enzyme activity in various degenerative diseases forms of arthritis, autoimmune diseases, and cancer metastasis. For example, rheumatoid arthritis and other arthritis-related diseases, which are characterized by the degradation of proteins of the extracellular matrix (ECM), can be monitored by determining the levels of MMPs and other proteinases in synovial fluids. This method can also be used to determine levels of gelatinase B in Alzheimer's patients. Alzheimer's disease is characterized by the presence of beta-amyloid peptides that form amyloid plaques. The active form of gelatinase B is known to cleave these peptides (Backstrom et al, (1996) J. Neurosci. 16:7910-7919); however, the latent inactive form of gelatinase B accumulates in the brain. One embodiment of the reagents and method of the present invention can be used to distinguish between active and inactive forms, and to determine net gelatinase B activity. 5 The reagents and assay methods of the present invention are also useful for the development of new enzymatic inhibitors for therapeutic uses; for example, it can be used to develop anti-metastatic reagents for new therapeutic approaches designed to block tumor cell dissemination. The invention can also be used to develop anti-inflammatory reagents designed to inhibit ECM degeneration during inflammation. This method can be o used to search for inhibitors of gelatinase B activity for use in the treatment of amyotrophic lateral sclerosis (ALS), which involves gelatinase B-mediated degradation (Lim et al, (1996) J. Neurochem. 67:251-259), or for use in wound healing (Moses et al, (1996) J. Cell Biochem. 60:379-386). In a specific embodiment, demonstrated in Example II, the method of the present invention 5 can be used to monitor objectively and rapidly the therapeutic efficiency of inhibitors on the net proteolytic activity in biological fluids of patients with degenerative diseases. Additionally, this method can be used to measure intracellular proteolytic activity in phagocytic cells, such as macrophages. Macrophages phagocyte bacteria and other parasites and digest them by proteolysis. In a particular embodiment of the present o invention, a sample containing phagocytic cells could be added to immobilized labeled substrate under such conditions as to allow the phagocytic cells to engulf and degrade the substrate. The ability of the phagocytic cells to digest the substrate could be measured by flow cytometry. This method can be used further for routine screening procedures during quality control 5 assays for recombinant and naturally occurring enzymes. This is particularly useful for industrial settings. The method of the present invention can also be used in research to identify enzymes that are involved in a particular disease. For example, the cerebrospinal fluids of patients with neuro-degenerative diseases could analyzed to determine which enzymes are present. Once particular enzymes are identified as being involved in a disease, the method of the present invention could be used to determine specific inhibitors of the enzymes. Additionally, this method is useful to study the regulation of enzymatic activity, such as mechanisms of inhibition and activation of enzymatic products. It can also be used for the identification and characterization of new enzymatic substrates. Advantages: The present invention provides a rapid, sensitive, and reproducible assay to measure the net biological activity present in samples. In certain applications, the method of the present invention constitutes a significant improvement over curcent methods of enzymatic analysis. It is characterized by high specificity, sensitivity, and reproducibility. One of the advantages of the present invention is its ability to measure net enzymatic activity. Many biological samples contain enzyme inhibitors. It is important to determine the net activity of enzymes in the presence of these inhibitors since biological activity often depends on the ratio of free active protein to inactive protein. The methods of the present invention allow the determination of net enzyme activity whereas other assays may not. The method can also take advantage of the rapidity and reproducibility of laser flow cytometric analysis. The assay can be automated to require a minimum of handling, and can thus be applied to large-scale screenings of antagonist reagents or biological samples for diagnostic use. Routine screening procedures could also take advantage of flow cytometers equipped with an autoloader. Up to 300 samples can be analyzed per hour with minimal handling of samples. Flow cytometry also allows for the rapid and simultaneous detection of multiple analytes. This provides the potential to perform multiple assays in the same reaction mixture reducing cost and hands-on time as well as generating results using the same method between analytes. The method of the present invention presents many advantages as compared to other available approaches. The FASC assay in its present design is almost as sensitive as the standard zymography, and it is much less time-consuming; it has the potential to evaluate hundreds of specimens per day for net proteolytic activity. For many, if not the majority of samples, a net proteolytic activity could be detected within 90 min, which compares favorably with the period required to perform an ELISA test. In a preferred embodiment, fluorescence-activated substrate conversion (FASC) is used to take advantage of the high sensitivity obtained by fluorescence-activated signals (using a 488 nm laser excitation wavelength) (see Figure 1). FASC is characterized by its high specificity, sensitivity, and reproducibility. It is also environmentally safe. In most cases, the signal-to-noise ratio between autofluorescent microspheres and those coated with the FITC-labeled substrate is near 500. This allows for accurate measurements of enzyme activity in the presence of chemical or biological inhibitors. The present invention is described in further detail in the following non-limiting examples. It is to be understood that the examples described below are not meant to limit the scope of the present invention. It is expected that numerous variants will be obvious to a person skilled in the art to which the present invention pertains, without any departure from the spirit of the present invention. The appended claims, properly construed, form the only limitation upon the scope of the present invention. EXAMPLE I Flow Cytometric Analysis of Gelatinase B (MMP-9) Activity using Immobilized Fluorescent Substrate on Microspheres Reagents: Gelatin (300 Bloom) casein, fluorescein isothiocyanate (FITC), and 1,10-phenanthroline were obtained from Sigma (St. Louis, MO). Bovine albumin was purchased from ICN Pharmaceuticals (Montreal, PQ, Canada). Purified gelatinase B was prepared as described 5 (Masure et al, (1993) Eur. J. Biochem. 218:129-141). The monoclonal antibody REGA- 3G12 has been described by Paemen et al, (1995) Eur. J. Biochem. 234: 759-765). This monoclonal antibody binds to gelatinase B (Kd: 2.1 x 10 "9 ) and inhibits the enzymatic activity. Fluorescent Labeling of Substrates: o Casein and gelatin were dissolved at a final concentration of 2 mg/ml in carbonate buffer (pH 9.2). FITC (dissolved in DMSO at 5 mg/ml) was added to the indicated concentrations. Labeling was carried out for 24 h at 4 ° C. Free FITC molecules were removed by chromatography on PD- 10 columns (Pharmacia, Uppsala, Sweden) using PB S , pH 7.4 as eluent buffer. 5 Protein Assay: Protein concentrations were determined by the BCA protein assay (Pierce, Rockford, IL) with bovine serum albumin (BSA) to construct a standard curve. Microsphere Coating: Polystyrene microspheres of 15.5 μm (± 1.919) diameter (Polysciences, Warrington, PA) o were incubated for 2 h at 37°C with FITC-conjugated substrates (1 mg/ml in PBS, pH 7.4) to allow noncovalent adsoφtion. This method was chosen for its simplicity and for the minimal conformational change it might induce (McHugh (1994) In: Darzynkiewycz, Robinson, and Crissman (eds.) Methods in Cell Biology (New York: Academic Press, 1994) 42:575-595). The microspheres were then washed twice in phosphate buffer (pH 5 7.4) containing 0.5% BSA and 0.05% sodium azide (PBA). Microspheres were kept at 4°C in PBA (10 6 beads/ml) in the dark and resuspended by gentle vortexing before use. Gelatinase B digestion of FITC-Conjugated Substrates Immobilized on Microspheres: The enzymatic reactions were carried out in a final volume of 100 μl at 37 °C for 16 hours in eppendorf tubes using RPMI medium (without FCS) as the reaction buffer. The samples contained 10 μl gelatinase B-containing solutions, 10 μl 10X solution of inhibitors or monoclonal antibodies, and 10 μl FITC-labeled substrate-coated microspheres. The volume was completed with RPMI medium. The enzymatic reaction was stopped by adding 1 ml PBA, followed by two washes of the microspheres (1 min. at 8000 φm). The pellets were then resuspended in 500 μl PBA. Flow Cytometric Analysis: Samples were analyzed on a Coulter XL-MCL (Coulter Electronics, Hialeah, FL) using standard optics for detection of FITC fluorescence. A neutral density filter was used to lower the signal to the forward light scatter detector. Voltage of the photomultiplier tube used to detect FITC (FL1) was adjusted to allow visualization within the same histogram of the autofluorescence of the microspheres and the signal given by the microspheres coated with the FITC-labeled substrate. This voltage was kept constant for all analysis. Fluorescence was measured on a single parameter histogram using a log scale allowing for the discrimination of microspheres with a wide range of diameter. Between 1000 to 5000 events were analyzed for each histogram. Determination of Gelatinolytic Activity using FITC-labeled Substrate Immobilized on Microspheres: Figure 3 shows a typical one-parameter histogram illustrating the clear separation between the (auto)fiuorescence of uncoated microspheres and the fluorescence of FITC-gelatin- coated microspheres. The two-parameter histogram with the forward-angle light scatter (FS) and side scatter (SS) on the x and y axes respectively was used to position the window on microspheres and to minimize interference with debris. Although the noise discriminator could be increased up to the levels of the microspheres, the present invention opted to display the "noise" so as to better monitor the quality of the samples. In most of the experiments, the present invention obtained a signal-to-noise ratio near 500 between autofluorescent beads and those coated with the FITC-labeled substrate. To determine the specificity of the enzymatic cleavage in immobilized FITC-conjugated gelatin on microspheres, the present invention involved the incubation of the FITC-gelatin- and control FITC-casein-coated microspheres with 200 ng purified gelatinase B from human neutrophil (Masure et al, (1993) supra). The digestion temperature (37°C) and incubation time (16 hours) used were determined in preliminary experiments designed to establish the conditions necessary to achieve maximal sensitivity. Gelatinase B induced a 95% decrease of the fluorescent signal on gelatin-coated beads but not on the casein- coated beads (Figure 4). To further confirm the specificity of the enzymatic reaction, a recently described gelatinase B-specific mouse monoclonal antibody with blocking activity was used (Paemen et al, (1995) supra). The results of a representative experiment (Figure 5) show the dose- dependent blocking effect of inhibitory monoclonal antibody REGA-3G12. No significant differences were observed using mouse IgG control antibody (data not presented). Inhibition of Gelatinase B Activity by Pharmacological Reagents: Gelatinase B activity has been shown to be inhibited by 1,10-phenanthroline, a specific inhibitor of zinc-dependent metalloproteinases. It is also inhibited by EDTA, since calcium ions are necessary to maintain a catalytically active conformation (Masure et al, (1990) Biochem. Biophys. Acta. 1054:317-325). To determine whether the gelatinase B activity observed was sensitive to these agents, the activity of gelatinase B was measured in the presence of these inhibitors. Figure 6 shows that EDTA and phenanthroline inhibit more than 90% of gelatinase B activity. No inhibition was noted with sodium azide, but a significant inhibition was observed with DMSO. Sensitivity and Reproducibility of the Assay: The amount of FITC chemically linked to a protein can be controlled by varying the amount of FITC molecules added during conjugation. The FITC:protein (F:P) ratio may theoretically interfere with the enzymatic activity of gelatinase B due to steric hindrance or conformational changes of the substrate; thus, five different stocks of microspheres were prepared, keeping the gelatin concentration constant during the coating, but varying the F:P ratio on the gelatin molecules. The resulting stocks of FITC-gelatin-coated microspheres had a mean channel of fluorescence (MCF) ranging from 8 to 300 arbitrary units of fluorescence (Figure 7A). The data show that the sensitivity of the assay was not affected by the F:P ratio (Figures 7B and 7C). Therefore, the FITC does not interfere with enzymatic activity, either through the hydrophobicity generated by the labeling, or through stearic hindrance around the cleavage sites. Using an F:P ratio that approaches the plateau of maximal MCF therefore generates a higher dynamic range, and precludes the possibility of quenching between FITC molecules. Although it was expected that the FITC might interfere with the coating through the hydrophobicity generated by the labeling, the F:P ratio did not affect the sensitivity of the assay. The linear range of the assay extended from 1 to 200 ng of gelatinase B. The variation between samples was tested and the results obtained were highly reproducible. For example, in most experiments, the variation was between 0.5 to 1% among samples (Figures 5 and 7). This high homogeneity among samples was also evident when the same samples were measured with different protocols of acquisition (Table 1). In the present invention, the MCF of a microsphere sample with approximately 50% FITC-gelatin degraded on its surface was measured; the sample was then run in different conditions of acquisition. Varying the flow rate and the total number of events analyzed had no significant impact on the MCF. This is consistent with previous observations using microspheres coated with capture reagents in flow microsphere immunoassays (McHugh (1994) In: Darzynkiewycz, Robinson, and Crissman (eds.) Methods in Cell Biology (New York: Academic Press, 1994) 42:575-595). Table 1. Data Reproducibility Number of Events Flow Rate MCF'(s.d.) 10,000 500-700 events/s 69.9 (0.2) 1,000 500-700 events/s 70.1 (0.4) 10,000 50 events/s ND 2 1,000 50 events/s 69.7 (0.8) 'Mean Channel of Fluorescence: average of triplicates. 2 Not doable since the beads pellet at the bottom of the tube. EXAMPLE II Evaluation of Net Gelatinase B Activity in Biological Samples of Patients with Rheumatic Diseases The method of the present invention was used to evaluate the net proteolytic activity of MMPs contained in the biological fluids of patients suffering from various degenerating diseases. Reagents: Gelatin (300 Bloom), casein, fluorescein isotiocyanate (FITC), and 1,10-phenanthroline (PHEN) were obtained from Sigma (St. Louis, MO). Bovine albumin was obtained from ICN Pharmaceuticals (Montreal, PQ). Polystyrene microspheres were obtained from Polyscience (Warrington, PA). Purified gelatinase B (MMP-9, E.C. 3.24.4.35) was prepared as described (Masure et al, (1991) Eur. J. Biochem. 198:391-398). The gelatinase B specific blocking monoclonal antibody REGA-3G12 has been described by Paemen et al. (1995) Eur. J. Biochem. 234: 759-765, which is herein incoφorated by reference. Patients: Patients were selected from the out- and in-patient clinic. Whenever there was an indication for athrocenthesis, as judged by a senior rheumatologist, a fraction of the sample was withheld for further analysis. Clinical data, including age, sex, and diagnosis, were collected at bedside and from patient records. Synovial fluid was collected in dry tubes and stored immediately at -20 °C until analysis. Detection of MMPs by Zymographic Analysis: The activity of MMPs in SF and serum samples was determined by SDS-PAGE zymography using gelatin or casein as substrate as described by Tremblay et al. (1995) Cytokine 7:130-136, with minor modifications. Briefly, the samples were run without prior denaturation on a 8% acrylamide gel containing 1% of substrate for 18 h at 50 V, at room temperature. After electrophoresis, the gels were washed to remove SDS and incubated for 18 h at 37°C in a renaturing buffer (50 mM Tris, 5 mM CaCl 2 , 0.02% NaN 3 , 1% Triton X- 100). Subsequently, the gels were stained with Coomassie Brilliant Blue R-250, then destained in methanol/acetic acid. The enzymatic activity was detected as unstained bands on a blue background. The activity was quantitated by computerized image analysis (BioRad, model GS-670 Densitometer, Missauga, ON). Results were expressed as arbitrary scanning units. FASC and Flow Cytometric Analysis: Gelatin-FITC or casein-FITC coated microspheres were prepared as described previously (St-Pierre et al, (1996) Cytometry 25:374-380). Serial dilutions of the samples were made in serum-free RPMI- 1640. 10 mL of substrate-coated micropheres were added to the samples for a final volume of 100 mL. The samples were incubated for the time indicated and analyzed on a Coulter XL-MCL (Coulter Electronics, Hialeah, FL) using standard optics for detection of FITC fluorescence. PHEN was added 30 min before the addition of the microspheres. For the study using the monoclonal antibody REGA-3G12 (Paemen et al, (1995) Eur. J. Biochem. 234: 759-765) (or isotypic control), dilutions of SF were incubated with 2 mg of antibody at 4°C, 18 h prior to the addition of the microspheres, then incubated at 37°C for a further 18 h. Time dependent cleavage: The conditions for measuring gelatinolytic activity in biological fluids were established. Dose-response curves of enzymatic substrate conversion using purified human gelatinase B were determined for various incubation periods (90 min and 18 h) using the FASC and flow cytometric analysis (Figure 8A). The response obtained showed a broad range dose- dependency for the cleavage of gelatin-FITC on the coated microspheres. As little as 3 ng of gelatinase B was reproducibly detected after an 18 h incubation. The sensitivity of the assay decreased to 20 ng if a 90 min incubation time was used. The sensitivity obtained by FASC and flow cytometric analysis using an 18h incubation time was similar to that obtained by standard gelatin-zymography (Figure 8B). Analysis of Enzyme Activity in Synovial Fluids: In synovial fluids (SF), a significant net gelatinase B activity was detected using FASC and flow cytometry as early as 30 min after the start of incubation with gelatin-coated microspheres (Figure 9). The amount of converted substrate by the SF activity steadily augmented with incubation time (data not shown); for instance, after a period of 180 min, an increase in substrate conversion of one order of magnitude was observed. The reproducibility and the stability of the response obtained by FASC and flow cytometry was ascertained by multiple measurements following repeated freeze-thaw cycles (Figure 10). Similar results were obtained using casein as a substrate on the micropheres. Determination of Proteolytic Activity In Sera ofRA Patients: The presence of gelatinolytic and caseinolytic activity in 80 serum samples obtained from patients for which a diagnosis of RA had been established was also determined, following the criteria of the American Rheumatism Association (ARA). As previously disclosed [Masure et al, (1991) Eur. J. Biochem. 198:391-398; Opdenakker et al, (1991) Lymphokine and Cytokine Research 10:317-324), most of the sera contained significant but variable amounts of gelatinase A (MMP-2, 64 kDa) and gelatinase B (MMP-9, 92.5 kDa) as detected by gelatin-zymography (Figure 11 A). When tested by casein- zymography, other enzyme species were detected of which the molecular weights (MW) corresponded to those reported for neutrophil collagenase (MMP-8, latent 85 kDa and activated 65 kDa) (Woessner (1991) FASEB J. 5:2145-2154), or plasminogen activators (t-PA, 65-70 kDa) (Bunning et al, (1987) Biochim. Biophys. Acta 924: 473-482) (Figure 1 IB). When tested by FASC and flow cytometry, however, no detectable gelatinolytic nor caseinolytic activity was found in any of the sera (data not shown). Determination of Proteolytic Activity in Synovial Fluids of Patients: Net proteolytic activity was tested in 254 SF samples from patients suffering from various joint diseases. The distribution for the presence of gelatinase A, gelatinase B, and both gelatinase A and B was determined for patients with rheumatoid arthritis, rheumatoid factor positive (RAp) and rheumatoid arthritis, rheumatoid factor negative (RAn) as assessed by zymography or by FASC-flow cytometry, using either gelatin- or casein- coated microspheres (Table 2). The samples were found to contain gelatinase A (MMP-2) and/or gelatinase B (MMP-9) as assayed by gelatin zymography. Net enzyme activity was also evaluated in the SFs from patients suffering from RA, Reiter syndrome, Lyme disease, and other arthritis-related diseases (Table 3). Table 3 Comparative Study of the Proteolyt ic Activities of Synovial Fluids from Patients Diagnosis Number of Zymography FASC FASC FASC Samples gelatin casein gelatin A 1 B A+B +casein Rheumatoid Arthritis RF 2 + 110 3 12 95 23 4 4 Rheumatoid Arthritis RF 2 - 54 1 6 45 11 1 3 Spondylarthropathy 16 3 1 12 5 2 0 Gout/pseudogout 10 2 3 5 4 0 0 Psoriatic Arthritis 9 0 2 7 2 2 0 Lyme Disease 8 0 1 7 2 0 0 Osteoarthritis 6 nd 1 nd 0 0 0 Polymyalgia Rheumatica 5 0 1 4 0 0 0 Juvenile Chronic Arthritis 4 0 0 4 1 1 0 Systemic Lupus 2 nd 0 1 0 0 0 Erythematodes Adult Onset Still Disease 3 nd 0 0 0 0 0 Septic Arthritis 1 nd 1 0 0 0 0 Charcot Joint 1 0 1 0 0 0 0 Behcet Disease 1 0 0 1 0 1 0 Total 230 9 29 181 48 11 7 1 Presence of Gelatinase A, B, or A+B 2 Rheumatoid Factor The results demonstrate that a significant number of samples contain net gelatinolytic or caseinolytic activity. Most of the samples, 91% (63/69), scored positive with gelatin- coated microspheres as the substrate, while only 14.5% (10/69) were positive for both caseinolytic and gelatinolytic activities. In 9% (6/69) of the samples, only caseinolytic activity was detected. No significant difference in the frequency of positive samples was observed in patients who were found to be positive or negative for the presence of rheumatoid factor (28 and 31%, respectively). To quantitate accurately the net proteolytic activity in SFs, the activity of the positive samples was further evaluated using serial dilutions of the SFs. Examples of typical titration curves obtained are shown in Figure 12. One FASC unit was defined as the reciprocal dilution that resulted in 50% cleavage of the fluorescent substrate. The titer of 5 net activity varied greatly among the 69 different positive samples, ranging from 10 to 89,125 units. The titer of FASC units of all samples was plotted against the corresponding titer of gelatinase B as measured by gelatin zymography (Figure 13). There was no significant correlation between the net proteolytic activity present in the SF and the corresponding titer determined by zymography. l o Net Proteolytic Activity ofSF Treated with Inhibitors: Since proteolytic degradation of ECM components had previously been attributed to MMPs, the contribution of MMPs in the total gelatinolytic activity found in SF of our series of patients was evaluated. For this puφose, the net proteolytic activity in 30 FASC- positive SFs was evaluated in the absence and in the presence of PHEΝ, an inhibitor of 15 MMPs. We found that PHEΝ had a significant inhibitory effect on all tested SF samples (Figure 14A). In almost all cases, at least 50% of the net proteolytic activity was inhibited by PHEΝ. In more than half of the samples, more than 80% of the activity was inhibited by PHEΝ. Since gelatinase B has been associated with degenerative activity and visualized in situ by 20 immunohistochemistry in various joint diseases (Grillet et al, (1997) Brit. J. Rheumatol 36:(7):744-747), the present invention examined the use of specific gelatinase B-blocking monoclonal antibodies. It was found that in about half of the SFs, the proteolytic activity was significantly inhibited by the blocking antibody (Figure 14B), but rarely more than 40%) of the total activity. The ratio of gelatinase B versus other degradative enzyme 25 activity, however, showed that in some samples the gelatinase B activity predominated, whereas in others the proteolytic activity was almost totally mediated by other MMPs (Figure 14C). This demonstrates that the participation of other members of the MMP family can overwhelm that of gelatinase B. Comparison of FASC-positive and negative SF by zymographic analysis: No quantitative relationship was observed between the net gelatinase B activity obtained by FASC-flow cytometry and that obtained by zymography. Therefore zymographic analysis of FASC-negative (Figure 15 A) and FASC-positive (Figure 15B) SF samples was performed to distinguish between the latent and activated forms of MMPs. It was found that all SFs, independent of net proteolytic activity, had identical molecular species for the gelatinase A. The pro-gelatinase A form migrated at a MW of 64 kDa and an activated form at 59 kDa. The patterns observed in the case of gelatinase B, however, were different. In the FASC-negative samples, the major monomer form migrated at 89.5 kDa, while in the FASC-positive samples, a lower MW activation form of 79.4 kDa was observed. This demonstrates that only the FASC-positive samples contained activated gelatinase B. No relationship was found between the FASC titer and the amount of active gelatinase activity contained in these samples as quantitated by scanning densitometry of the zymography analysis. (Figure 15 B and C), which is consistent with the findings of Koolwijk et al, (1995) J. Rheumatol. 22:385-393. Casein zymography was used to visualize the enzyme species that contribute to the casein FASC assay (Figure 16). When the SF samples were tested by FASC analysis using casein-coated microspheres, only 9% (15/173) of the samples showed a net enzymatic casein-specific activity. In contrast, the zymographic analysis on casein-impregnated gels demonstrated the presence of proteases in all samples tested. Based on the molecular weight forms, MMP-3 and MMP-8 were present in all samples, whereas variable amounts of other caseinolytic activities were detected in more than 50% of the tested samples. Two major caseinolytic activities were detected in parallel, which migrated at approximately 55 and 35 kDa. As in the case of the gelatin FASC and zymography assays, no correlation was found between the net FASC caseinolyic activity and the casein zymography (data not shown). These experiments show again how the FASC-flow cytometry assay of the present invention was able to determine net enzymatic activity, while the zymography recorded latent and inhibited forms as well. Although zymography revealed that all the biological fluids tested showed the presence of at least one MMP, this occurrence was not directly associated with a net proteolytic activity in the sample, as evaluated by FASC analysis; thus, the use of zymography is not a reliable assay for determining net proteolytic activity. Only direct measurement of net proteolytic activity in SF is reliable. This is an important issue since the efficacy of therapeutic regimens depends on the inhibition of the net local proteolytic activity. Accordingly, the method of the present invention will be useful in clinical protocols. From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications to the invention to adapt it to various usages and conditions. Such changes and modifications are properly, equitably, and intended to be within the full range of equivalence of the following claims.

Claims: We claim: 1. A reagent suitable for determining the presence or absence of enzymatic activity in a test sample using flow cytometry, comprising an enzyme substrate immobilized on a solid support. 5 2. A reagent as in claim 1 , wherein the solid support is a stable colloidal particle. 3. A reagent as in claim 2, wherein the colloidal particle is a polymeric material. 4. A reagent as in claim 3, wherein the polymeric material is selected from the group consisting of: (a) polystyrene; 0 (b) polystyrene-divinylbenzene; (c) polymethacrylate; (d) polyphenylene oxide; or (e) polystyrene latex. 5. A reagent as in claim 4, wherein the polystyrene microspheres are in the size range s of 0.5 to 50 micrometers in diameter. 6. A reagent as in claim 1, 2, 3, 4, or 5, wherein a fluorophore has been covalently attached to the substrate. 7. A reagent as in claim 6, wherein the fluorophore is selected from the group consisting of: o (a) fluorescein isothiocyanate; (b) Texas Red; (c) AMCA; or (d) a phycobiliprotein. 8. A reagent as in claim 7, wherein said phycobiliprotein is selected from the group consisting of: (a) phycoerythrin; (b) allophycocyanin; (c) a cyanine derivative; or (d) rhodamine. 9. A method of determining the presence or absence of enzymatic activity in a test sample which comprises the following steps: (a) adding a test sample to a reagent suitable for determining the presence or absence of enzymatic activity in a test sample using flow cytometry, comprising an fluorophore-labeled enzyme substrate immobilized on a solid support, under such conditions as to allow enzymatic digestion of the labeled substrate; (b) washing the digested immobilized substrate; (c) passing the digested immobilized substrate through a flow cytometer; and (d) identifying the amount of net enzyme activities in the test sample based on the measured signals. 10. A method of determining the presence or absence of enzymatic activity in a test sample which comprises the following steps: (a) adding a test sample to a solution containing a reagent suitable for determining the presence or absence of enzymatic activity in a test sample using flow cytometry, comprising a fluorophore-labeled enzyme substrate immobilized on a solid support and a second substrate under such conditions as to allow enzymatic conversion of the labeled substrate; (b) washing the digested immobilized substrate; (c) passing the digested immobilized substrate through a flow cytometer; and (d) identifying the amount of net enzyme activities in the test sample based on the measured signals. 11. A method of determining the presence or absence of enzymatic activity in a test sample which comprises the following steps: (a) adding a test sample to a solution containing a reagent suitable for determining the presence or absence of enzymatic activity in a test sample 5 using flow cytometry, comprising an enzyme substrate immobilized on a solid support, and a second substrate that has been labeled, under such conditions as to allow enzymatic conversion of the labeled substrate; (b) washing the digested immobilized substrate; (c) passing the digested immobilized substrate through a flow cytometer; and o (d) identifying the amount of net enzyme activities in the test sample based on the measured signals. 12. A kit to conduct an assay based on the method of claims 9, 10, and 11. 13. A method of determining the presence or absence of enzymatic activity in a test sample, which comprises the following steps: 5 (a) selecting an appropriate enzyme/substrate pair; (b) a step selected from: (i) labeling the substrate with a fluorophore and immobilizing the labeled substrate on a solid support suitable for use in flow cytometry, or (ii) immobilizing the substrate on a solid support suitable for use in flow o cytometry, and labeling the immobilized the substrate with a fluorophore; (c) adding a test sample to a portion of the immobilized labeled substrate under such conditions as to allow enzymatic digestion of the labeled substrate; (d) washing the digested immobilized substrate; (e) passing the digested immobilized substrate through a flow cytometer; and 5 (f) identifying the amount of net enzyme activities in the test sample based on the measured signals. 14. The method according to claim 13, wherein the enzyme is selected from the group consisting of hydrolases and lyases. 15. The method according to claim 14, wherein the enzyme/substrate pair is gelatinase B/ gelatin. 16. A method of determining the presence or absence of enzymatic activity in a test sample, which comprises the following steps: (a) selecting an appropriate enzyme/substrate group; (b) a step selected from: (i) labeling one substrate of the enzyme/substrate group with a fluorophore and immobilizing the labeled substrate on a solid support suitable for use in flow cytometry, or (ii) immobilizing the labeled substrate on a solid support suitable for use in flow cytometry and labeling one substrate of the enzyme/substrate group with a fluorophore; (c) adding a second substrate of the enzyme/substrate group and a test sample to a portion of the immobilized labeled substrate under such conditions as to allow conversion of the immobilized substrate to occur; (d) washing the converted immobilized substrate; (e) passing the converted immobilized substrate through a flow cytometer; and (f) identifying the amount of net enzyme activity in the test sample based on the measured signals. 17. The method according to claim 16, wherein the enzyme is a transferase. 18. A method of determining the presence or absence of enzymatic activity in a test sample, which comprises the following steps: (a) selecting an appropriate enzyme/substrate group; (b) immobilizing an unlabeled substrate of the enzyme/substrate group on a solid support suitable for use in flow cytometry; (c) labeling a second substrate of the enzyme/substrate group with a fluorophore; (d) adding the labeled second substrate and a test sample to a portion of the immobilized unlabeled substrate under such conditions as to allow conversion of 5 the immobilized substrate to occur; (e) washing the converted immobilized substrate; (f) passing the converted immobilized substrate through a flow cytometer; and (g) identifying the amount of net enzyme activities in the test sample based on the measured signals. o 19. The method according to claim 18, wherein the enzyme is selected from the group consisting of ligases and lyases. 20. The method according to claims 13 , 16, or 18, wherein the test sample is a human or veterinary biological sample. s 21. The method according to claim 13, wherein the use is to measure intracellular proteolytic activity in phagocytic cells. 22. The method according to claim 13, 16 or 18, wherein the use is for medical diagnosis. 23. The method according to claim 13, 16, 18, wherein the diagnosis is for one of the o disorders selected from the group consisting of: (a) rheumatoid arthritis; (b) Reiter syndrome; (c) Lyme disease; or (d) an arthritis-related disease. 5 24. The method according to claim 13, 16, or 18, wherein the human or veterinary biological sample is whole blood, plasma, serum, or spinal fluid. 25. A kit for the preparation of a reagent suitable for determimng the presence or absence of enzymatic activity in a test sample using flow cytometry, comprising an enzyme substrate immobilized on a solid support.

Inventors: ST-PIERRE YVES, ST-PIERRE, YVES
Applicants: FRAPPIER ARMAND INST, ST PIERRE YVES, INSTITUTE ARMAND-FRAPPIER, ST-PIERRE, YVES
Application year: 1997
Main classification: C12Q1
IPC codes: C12Q1