WO1998020153A1 - Fluorescence-labeled substrates and their use to analyze enzyme activity using flow cytometry - Google Patents
Fluorescence-labeled substrates and their use to analyze enzyme activity using flow cytometry Download PDFInfo
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- WO1998020153A1 WO1998020153A1 PCT/CA1997/000823 CA9700823W WO9820153A1 WO 1998020153 A1 WO1998020153 A1 WO 1998020153A1 CA 9700823 W CA9700823 W CA 9700823W WO 9820153 A1 WO9820153 A1 WO 9820153A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
- G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
- G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
- G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
- G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
- G01N2333/96486—Metalloendopeptidases (3.4.24)
Definitions
- the present invention relates to compositions and methods of assaying enzymatic activity using flow cytometry.
- 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.
- a body fluid such as blood or spinal fluid
- 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.
- 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.
- 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 abso ⁇ tion, scatter as a function of angle, and fluorescence as a function of either wavelength or polarization may be measured.
- those particles that have been identified as having the desired properties may be sorted if the 5 apparatus has been designed with such capability.
- colloidal particles and magnetic particles to bind a compound has long been known and used in industrial and laboratory procedures.
- 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.
- the particles 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.
- the terms "particles”, s "spheroids”, “spheres”, “microspheres” and “beads” as used herein, are interchangeable.
- 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.
- 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.
- antigens from infectious agents, cell surfaces, or other soluble proteins
- 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.
- 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).
- PBC primary biliary cirrhosis
- 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 has also been used to detect enzyme activity in cells.
- 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)
- One example of a group of enzymes for which an improved assay is required is the matrix metalloproteinase family.
- MMP matrix metalloproteinase
- ECM extracellular matrix
- MMPs 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
- 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,
- 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.
- the collagenases e.g. MMP-1, MMP-5, MMP-8 and MMP- 13
- 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.
- MMPs tissue inhibitors of metalloproteinases
- TIMPs tissue inhibitors of metalloproteinases
- TIMP proteolytic activity of MMPs following dissociation from their natural inhibitors (TIMP) by SDS-PAGE electrophoresis.
- TIMP proteolytic activity of MMPs following dissociation from their natural inhibitors
- zymography Another widely-used technique is the zymography 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 microtite ⁇ late 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.
- fluorogenic substrates 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.
- 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.
- 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;
- a method of determining the presence or absence of enzymatic activity in a test sample which comprises the following steps:
- a method of determining the presence or absence of enzymatic activity in a test sample which comprises the following steps:
- 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:
- 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;
- Another embodiment provides a method of determining the presence or absence of enzymatic activity in a test sample, which comprises the following steps:
- kits 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.
- FIG. 1 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.
- FS forward-light scatter
- SS side scatter
- FIG. 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.
- FIG. 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 ⁇ l. The results were expressed as the percentage of gelatin degraded in the presence of the blocking monoclonal antibody.
- FIG. 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.
- MCF mean channel of fluorescence
- FIG 7. Sensitivity of the assay.
- Five different microsphere preparations were obtained by varying the FITC concentrations (from 2.0 ⁇ M to 1.25 mM) during fluoresceination of gelatin substrate.
- Figure 8. Dose-dependent degradation of gelatin by purified human neutrophil gelatinase B (MMP-9).
- 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.
- FIG. 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.
- FIG. 11 Zymographic analysis of serum from patients with rheumatoid arthritis (RA).
- RA rheumatoid arthritis
- 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-
- FIG. 15 Gelatin-zymography of SF. Undiluted SF samples (2 ⁇ l) 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).
- enzyme 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.
- the reagents and methods of this invention entail the selection of an appropriate enzyme/substrate pair or group.
- 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.
- 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.
- 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.
- 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.
- 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).
- the esterases including exo- and endonucle
- 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.
- 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
- 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.
- gelatin a component of the extracellular matrix (ECM) is an enzymatic substrate for the enzyme gelatinase-B, a metalloproteinase.
- ECM extracellular matrix
- Other members of the metalloproteinase family can degrade in vitro other components of the ECM, including collagens, fibronectin, laminin, elastin, proteoglycans, and entactin
- An enzyme/substrate group comprises the enzyme to be assayed and appropriate natural or synthetic substrates, one of which is labeled with a fluorophore.
- 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.
- 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.
- Fluorescent substances used for labeling proteins are well known in the art.
- fluorophore There are many constraints on the choice of fluorophore.
- One constraint is the abso ⁇ tion 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.
- fluorescein isothiocyanate 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.
- FITC Fluorescence Activated Cell Sorting
- 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.
- FITC Fluorescein isothiocyanate
- 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.
- 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.
- substrate is immobilized on polystyrene microspheres.
- polystyrene allows efficient noncovalent adso ⁇ tion of most proteins. Although noncovalent adso ⁇ tion 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.
- polystyrene microspheres of 15 ⁇ m 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 ⁇ m ( ⁇ 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 adso ⁇ tion 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.
- 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:
- Incubation with the enzyme of interest is carried out for variable periods of time, usually from 60 to 90 minutes, under the appropriate conditions.
- enzymatic activity is determined by a change in the signal of label as detected by a flow cytometer.
- the flow cytometer is a laser flow cytometer with standard optics for collection of fluorescent signals.
- 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 .
- FIG. 2 illustrates an example of how substrate conversion is detected using flow cytometry.
- 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.
- 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.
- 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.
- 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.
- 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 measures the net activity of gelatinase B in biological samples.
- 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.
- reagents and method can be used clinically to diagnose and monitor disease states.
- the reagents and assay will be useful for monitoring enzyme activity in various degenerative diseases forms of arthritis, autoimmune diseases, and cancer metastasis.
- rheumatoid arthritis and other arthritis-related diseases which are characterized by the degradation of proteins of the extracellular matrix (ECM)
- ECM extracellular matrix
- 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.
- 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).
- ALS amyotrophic lateral sclerosis
- 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.
- 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.
- 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.
- 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.
- the present invention provides a rapid, sensitive, and reproducible assay to measure the net biological activity present in samples.
- 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.
- 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.
- 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.
- FITC dissolved in DMSO at 5 mg/ml
- 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.
- Protein concentrations were determined by the BCA protein assay (Pierce, Rockford, IL) with bovine serum albumin (BSA) to construct a standard curve.
- Polystyrene microspheres of 15.5 ⁇ m ( ⁇ 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 adso ⁇ tion. 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
- 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.
- 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.
- 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).
- 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.
- 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 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).
- 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.
- 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.
- Gelatin 300 Bloom
- casein fluorescein isotiocyanate
- PHEN 1,10-phenanthroline
- 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 inco ⁇ orated by reference.
- 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.
- MMPs 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.
- 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.
- gelatinase B 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
- 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.
- Casein zymography was used to visualize the enzyme species that contribute to the casein FASC assay (Figure 16).
- 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.
- 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.
Abstract
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EP97911088A EP0951562A1 (en) | 1996-11-04 | 1997-11-04 | Fluorescence-labeled substrates and their use to analyze enzyme activity using flow cytometry |
AU48595/97A AU4859597A (en) | 1996-11-04 | 1997-11-04 | Fluorescence-labeled substrates and their use to analyze enzyme activity using flow cytometry |
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CA 2189486 CA2189486A1 (en) | 1996-11-04 | 1996-11-04 | Analysis of enzyme activity using immobilized fluorescence-labeled substrates |
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Cited By (11)
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EP1394270A2 (en) * | 2002-08-22 | 2004-03-03 | Bioarray Solutions Ltd | Molecular constructs and methods of use for detection of biochemical reactions |
US20110306035A1 (en) * | 2008-07-28 | 2011-12-15 | Dorit Arad | Methods and Compositions for Detection of a Pathogen, Disease, Medical Condition, or Biomarker Thereof |
US8691594B2 (en) | 1996-04-25 | 2014-04-08 | Bioarray Solutions, Ltd. | Method of making a microbead array with attached biomolecules |
US8691754B2 (en) | 2003-09-22 | 2014-04-08 | Bioarray Solutions, Ltd. | Microparticles with enhanced covalent binding capacity and their uses |
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US9251583B2 (en) | 2002-11-15 | 2016-02-02 | Bioarray Solutions, Ltd. | Analysis, secure access to, and transmission of array images |
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US9709559B2 (en) | 2000-06-21 | 2017-07-18 | Bioarray Solutions, Ltd. | Multianalyte molecular analysis using application-specific random particle arrays |
US10415081B2 (en) | 2001-10-15 | 2019-09-17 | Bioarray Solutions Ltd. | Multiplexed analysis of polymorphic loci by concurrent interrogation and enzyme-mediated detection |
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1997
- 1997-11-04 WO PCT/CA1997/000823 patent/WO1998020153A1/en not_active Application Discontinuation
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- 1997-11-04 EP EP97911088A patent/EP0951562A1/en not_active Withdrawn
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EP0329190A2 (en) * | 1988-02-19 | 1989-08-23 | Showa Denko Kabushiki Kaisha | Filler for measuring enzyme activity, column packed with the filler, and method of measuring enzyme activity using the column |
WO1989011101A1 (en) * | 1988-05-11 | 1989-11-16 | Dynal A.S. | Method of assay |
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CA2189486A1 (en) | 1998-05-04 |
AU4859597A (en) | 1998-05-29 |
EP0951562A1 (en) | 1999-10-27 |
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