WO2009047489A1 - Procédés de mesure de l'activité dépolymérase - Google Patents

Procédés de mesure de l'activité dépolymérase Download PDF

Info

Publication number
WO2009047489A1
WO2009047489A1 PCT/GB2008/003391 GB2008003391W WO2009047489A1 WO 2009047489 A1 WO2009047489 A1 WO 2009047489A1 GB 2008003391 W GB2008003391 W GB 2008003391W WO 2009047489 A1 WO2009047489 A1 WO 2009047489A1
Authority
WO
WIPO (PCT)
Prior art keywords
gel
amount
sample
activity
depolymerase
Prior art date
Application number
PCT/GB2008/003391
Other languages
English (en)
Inventor
James Fawcett
Jessica Kwok
Original Assignee
Cambridge Enterprise Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cambridge Enterprise Limited filed Critical Cambridge Enterprise Limited
Publication of WO2009047489A1 publication Critical patent/WO2009047489A1/fr

Links

Classifications

    • 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
    • C12Q1/527—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving lyase
    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • 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)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
    • 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
    • G01N2333/988—Lyases (4.), e.g. aldolases, heparinase, enolases, fumarase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/105—Osteoarthritis, e.g. cartilage alteration, hypertrophy of bone
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy

Definitions

  • This invention relates to assays for determining the activity of depolymerases, such as GAG lyases and proteases, in a sample.
  • Depolymerases cleave chemical linkages within polymers.
  • Depolymerases may include proteases, nucleases, glycosidases and glycosaminoglycan (GAG) lyases, such as chondroitinase, hyaluronidase, keratanase, and heparinase.
  • GAG lyase assays generally require a long (>7 days) purification of glycans followed by analysis by high performance liquid chromatography (HPLC) . A large amount of pure disaccharide must be purified from the test sample because the level of GAG can only be measured when the molecules are extracted and purified from mixed solutions.
  • the present inventors have developed an assay for measuring the activity of a range of depolymerases and which offers advantages over known techniques .
  • One aspect of the invention provides a method of measuring depolymerase activity in a sample comprising; providing a gel which incorporates a polymer substrate, incubating the gel with a sample, and determining the amount of polymer substrate in the gel after the incubation, the amount of polymer substrate in the gel after the incubation being indicative of the activity of depolymerase in the sample .
  • the depolymerase in the sample converts the polymer substrate within the gel into depolymerised products.
  • the polymer substrate has a high molecular weight and is retained within the gel.
  • the depolymerised products produced by the action of the depolymerase have a low molecular weight and are not retained within the gel . Depolymerised products are therefore able to diffuse out of the gel, leaving only undegraded polymer substrate within the gel .
  • the amount of polymer substrate which remains in the gel after the incubation with the sample is therefore inversely related to the amount of depolymerase activity in the sample .
  • An assay may be calibrated using standard samples containing predetermined amounts of depolymerase.
  • Gel incorporating a polymer substrate may be incubated with one or more standard samples under the same conditions as the incubation with the tissue sample.
  • the amount of polymer substrate in the gel following incubation with the one or moire standards may be measured.
  • the relationship between the amount of polymer substrate in the gel and the amount of depolymerase activity in the one or more standard samples may be determined. For example, the relationship may be determined graphically, e.g. in a calibration curve, or numerically as an equation or algorithm.
  • the use of standard samples to calibrate an assay is routine in the art.
  • the amount of depolymerase in the tissue sample may be determined by comparing the amount of polymeric substrate which remains in the gel after the incubation with the tissue sample with the amounts of polymeric substrate which remain in the gel after the incubation with the control samples. For example, the amount of depolymerase in the tissue sample may be determined from the amount of polymeric substrate which remains in the gel after the incubation with the tissue sample using the relationship between depolymerase activity and the amount of remaining polymer substrate established using standard samples. Depolymerases cleave chemical linkages within the substrate polymer to produce products which are able to diffuse out of the gel.
  • Suitable depolymerases include glycosaminoglycan (GAG) lyases (EC 4.2.2) which cleave polysaccaride linkages, glycosidases (EC 3.2.1) which cleave O and S glycosyl linkages, proteases (EC 3.4) which cleave peptidyl linkages, and nucleases (3.1.11 to 3.1.31) which cleave ester linkages.
  • GAG glycosaminoglycan
  • EC 4.2.2 glycosaminoglyases
  • EC 4.2.2 glycosaminoglyases
  • EC 3.2.1 glycosidases
  • proteases EC 3.4
  • nucleases 3.1.11 to 3.1.31
  • the depolymerase is a glycosaminoglycan (GAG) lyase and the polymeric substrate is a glycosaminoglycan (GAG) .
  • GAG within the gel is degraded by the GAG lyase into disaccarides which diffuse from the gel.
  • the depolymerase may be hyaluronidase (EC 4.2.2.1; EC 3.2.1.35; EC 3.2.1.36) and the substrate hyaluronan
  • the depolymerase may be chondroitinase (EC 4.2.2.5, EC 4.2.2.20; EC4.2.2.21) and the substrate chondroitin sulphate or dermatan sulphate
  • the depolymerase may be heparinase (EC 4.2.2.7; EC 4.2.2.8) and the substrate heparin or heparan sulphate
  • the depolymerase may be keratanase (EC 3.1.2.103) and the substrate keratan sulphate.
  • GAG lyase substrates such as hyaluronan, chondroitin sulphate, dermatan sulphate, heparin, heparan sulphate, or keratan sulphate may be readily obtained from commercial sources (e.g. Sigma-Aldrich, Poole UK) . GAG lyase substrates may be incorporated into an insoluble gel as described below.
  • the depolymerase may be a protease and the substrate is a polymer containing peptidyl bonds, such as a polypeptide.
  • Polypeptide within the gel is degraded by proteases within the sample into amino acids or short peptide fragments which diffuse from the gel.
  • proteases may be specific for a particular substrate or nonspecific .
  • Suitable proteases include serine proteases, such as chymotrypsin, and matrix metalloproteinases, such as collagenases and gelatinases.
  • Suitable substrates may be natural or synthetic polypeptides and may include casein.
  • the depolymerase may be a nuclease and the substrate is a nucleic acid, such as an RNA or DNA molecule. Nucleic acid within the gel is degraded by nucleases within the sample into nucleotides or oligonucleotide fragments which diffuse from the gel.
  • Nucleases may be specific for a particular nucleotide sequence or non-specific. Suitable nucleases include exo- and endo-nucleases. Suitable substrates include natural or synthetic nucleic acids and may be produced using conventional techniques .
  • the depolymerase may be a glycosidase (EC 3.2.1) and the substrate is a polysaccharide.
  • the substrate is a polysaccharide.
  • Polysaccharides within the gel are degraded by glycosidases within the sample into monosaccarides, disaccarides or short oligosaccaride fragments which diffuse from the gel.
  • Glycosidase may be specific for a particular substrate or nonspecific.
  • Suitable substrates include natural or synthetic polysaccharides which may be isolated and/or synthesised using standard techniques .
  • Suitable gels comprise an insoluble matrix of polymer fibrils which form a continuous network around an interstitial fluid.
  • the polymer fibrils may be of any suitable material, including polyacrylamide .
  • Agarose gels may be useful for nuclease assays.
  • a polyacrylamide gel may be produced by dissolving acrylamide and bisacrylamide monomers in buffer and then inducing them to polymerise into polyacrylamide by adding N 7 N 7 N',N'- tetramethylethylenediamine (TEMED) and ammonium persulfate.
  • TEMED N 7 N 7 N',N'- tetramethylethylenediamine
  • ammonium persulfate typically, for 10ml gel of 10% polyacrylamide, 3.3 ml of 30% acrylamide may be mixed with 2.5 ml 1.5M Tris pH8.8, 0.1 ml 10% ammonium persulfate, 0.005ml TEMED and 4.095 ml water.
  • the polymeric substrate includes a high proportion of linkages which are cleavable by the depolymerase whose activity is being measured and may be a natural or synthetic polymer.
  • polymeric substrate depends on the depolymerase activity which is being measured.
  • a glycosaminoglycan may be employed in a method to determine glycosaminoglycan (GAG) lyase activity.
  • the substrate may be hyaluronan.
  • the substrate may be a chondroitin sulphate or dermatan sulphate.
  • the substrate may be heparin or heparan sulphate .
  • the substrate may be keratan suphate.
  • the polymeric substrate is sufficiently large to be held within the insoluble gel by the network of polymerised fibrils and unable to diffuse freely out of the gel.
  • the polymeric substrate is held within the gel by non-covalent interactions and/or steric effects and is not covalently bonded to the gel.
  • Cleavage of the polymeric substrate by the depolymerase produces depolymerised products which have a lower molecular weight than the substrate. These products are sufficiently small to diffuse out of the insoluble gel during the sample incubation, while the polymeric substrate is retained within the gel .
  • Polymeric substrate may be incorporated into the gel by any convenient technique.
  • the polymeric substrate may be incorporated into the gel by admixing it with a monomer solution which is subsequently polymerised into a gel.
  • a method may comprise: introducing a polymeric substrate to a solution of monomers; and polymerising the monomers in the solution to produce a gel which incoporates the polymeric substrate .
  • a method may comprise: introducing glycosaminoglycan (GAG) polymers to a solution of acrylamide and bisacrylamide; and polymerising the acrylamide and bisacrylamide in the solution to produce a polyacrylamide gel which incorporates the GAG polymers .
  • GAG glycosaminoglycan
  • Suitable GAG polymers are described elsewhere herein.
  • the gel incorporating the polymeric substrate may be processed.
  • the gel may be divided into segments, such as discs, strips, tablets or blocks. This may be performed using conventional cutting techniques, for example using a blade.
  • the gel may be dehydrated before, during or after processing. Dehydration may be achieved using conventional techniques, for example air or oven drying, or dessication. Gel dryers suitable for dehydrating gels are commercially available (e.g. Bio-Rad CA USA; Perkin-Elmer MA USA; Thermo Fisher Scientific MA USA) .
  • the gel or segments thereof may be stored prior to use.
  • the dehydrated gel may be incorporated into an assay device, such as a multiwell plate, a reaction vessel, such as a microfuge tube, or other support or holder. This may facilitate manipulation of the gel and/or detection or measurement of the polymeric substrate in the gel .
  • an assay device such as a multiwell plate, a reaction vessel, such as a microfuge tube, or other support or holder. This may facilitate manipulation of the gel and/or detection or measurement of the polymeric substrate in the gel .
  • the dehydrated gel may be rehydrated before incubation with the tissue sample or standards. Typically, the gel may be rehydrated in sample buffer. Alternatively, the dehydrated gel may be rehydrated by contact with the reaction buffer during incubation with the tissue sample or controls
  • the sample which is tested for depolymerase activity may be obtained from an individual .
  • the individual may be healthy or may have or be suspected of having a disease condition.
  • the sample may be obtained from any biological fluid, tissue, or other body part in which it is desired to determine depolymerase activity.
  • Biological fluid samples may include blood, synovial fluid or urine samples.
  • biological fluid samples may be concentrated using standard liquid concentration techniques (e.g. by SpeedVacTM) before use in the methods described herein.
  • a tissue sample may be a tissue biopsy obtained from an individual or an extract or fraction thereof.
  • the cells in the tissue sample may be disrupted, for example by homogenisation.
  • Protease inhibitors may be added to a tissue sample to prevent digestion of enzyme by proteases released during tissue disruption.
  • disrupted tissue may be fractionated or extracted using standard techniques before use in the methods described herein.
  • the gel incorporating the substrate may be incubated with the tissue sample or control for a predetermined period of time, for example, 1 min or more, 10 mins or more, 60 mins or more, or 120 mins or more, preferably 15-60 minutes.
  • the optimal duration of the incubation will depend on the enzyme and its concentration in the sample and may be determined using routine experimentation.
  • the incubation is preferably performed at 37°C.
  • the gel may be washed after incubation or otherwise treated to remove the depolymerase and depolymerised products.
  • the gel is preferably subjected to constant agitation or shaking. This facilitates the diffusion of the deploymerised products out from the gel.
  • the gel may be fixed before the detection procedure, for example with methanol/acetic acid.
  • the amount of polymeric substrate remaining in the gel may be determined . This may be determined by any convenient method. For example , staining, scanning or plate -reading may be employed.
  • the reporter molecules may bind to the polymeric substrate remaining within the gel and directly or indirectly generate detectable, and preferably measurable, signals.
  • the amount of binding of reporter to the substrate in the gel may then be determined by measuring the detectable signals. The amount of binding indicates the amount of substrate which is left in the gel and is therefore indicative of the depolymerase activity in the sample .
  • the reporter molecule may comprise a signal moiety, which generates a detectable signal , for example a colourmetric , fluorescent or bioluminescent signal .
  • the amount of signal is indicative of the amount of substrate within the gel .
  • Reporters include fluorochromes such as fluorescein, rhodamine, phycoerythrin, Europium, Alexa 488, Alexa 647 and Texas Red, chromogenic dyes such as diaminobenzidine, alcian blue and Coomassie blue, macromolecular colloidal particles or particulate material such as latex beads that are coloured, magnetic or paramagnetic, and biologically or chemically active agents that can directly or indirectly cause detectable signals to be visually observed, electronically detected or otherwise recorded.
  • fluorochromes such as fluorescein, rhodamine, phycoerythrin, Europium, Alexa 488, Alexa 647 and Texas Red
  • chromogenic dyes such as diaminobenzidine, alcian
  • Bioly or chemically active agents include enzymes, which catalyse reactions that develop or change colours or cause changes in electrical properties, for example. They may be molecularly excitable, such that electronic transitions between energy states result in characteristic spectral absorptions or emissions. They may include chemical entities used in conjunction with biosensors. Biotin/avidin or biotin/streptavidin and alkaline phosphatase detection systems may be employed. Further examples include horseradish peroxidase and chemiluminescence.
  • reporter molecule will depend on the substrate. Suitable reporters for the polymeric substrates described herein are readily apparent to the skilled person. For example, glycosminoglycan polymers may be detected using Alcian blue, nucleic acid may be detected using an intercalating agent, such as ethidium bromide or SYBR green, and protein may be detected using Coomassie blue or standard silver staining.
  • an intercalating agent such as ethidium bromide or SYBR green
  • protein may be detected using Coomassie blue or standard silver staining.
  • the detectable reporter may further comprise a binding moiety, which binds to the polymeric substrate.
  • Suitable binding moieties may include antibodies, antibody fragments and lectins .
  • Linkage of a signal moiety to a binding member may be direct or indirect, covalent, e.g. via a peptide bond, or non-covalent .
  • Linkage via a peptide bond may be as a result of recombinant expression of a gene fusion encoding the binding member (e.g. antibody) and moiety.
  • Linkage via a non-covalent bond may be a result of a binding between a biotinylated specific binding member and a streptavidin/avidin linked reporter molecule.
  • the polymeric substrate within the gel may comprise a detectable reporter which is released on depolymerisation by the depolymerase .
  • a detectable reporter may be connected to the polymeric substrate through a linkage which is cleaved by the depolymerase. The amount of detectable reporter which remains in the gel is thus indicative of the amount of substrate remaining in the gel following incubation with the sample. Depolymerase activity in the sample may thus be determined by measuring the amount of detectable reporter in the gel after incubation.
  • suitable reporters include biotin, which may detected using streptavidin-linked detectable labels.
  • the mode of determining binding is not a feature of the present invention and those skilled in the art are able to choose a suitable mode according to their preference and general knowledge.
  • Suitable approaches include colormetric assays, chemical staining, immunofluorescence, enzyme-linked immunosorbent assays (ELISA) , radioimmunoassays (RIA) , immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA) , including sandwich assays using monoclonal and/or polyclonal antibodies. All of these approaches are well known in the art.
  • the methods of measuring depolymerase activity described herein may have a diagnostic purpose, for example in determining whether or not an individual has a disease condition or is at risk of a disease condition, or for assessing the efficacy of a particular treatment.
  • a method of assessing an medical condition in an individual or assessing the efficacy of a depolymerase treatment, for example a GAG lyase treatment, in an individual may comprise; providing an insoluble gel which incorporates a polymeric substrate , incubating the gel with a tissue sample obtained from the individual, and determining the amount of polymeric substrate in the gel after the incubation, the amount of polymeric substrate in the gel after the incubation being indicative of the activity of the depolymerase in the sample .
  • the depolymerase activity which is measured depends on the disease condition or therapy to be assessed.
  • Chondroitinase-ABC may be used in the treatment of neural damage and the promotion of neural regeneration. Methods described herein may be useful in assessing the activity of chondroitinase-ABC after administration and may therefore be useful in designing chondroitinase-ABC treatment regimens, as well as assessing the efficacy of chondroitinase-ABC treatment for such conditions.
  • a method of assessing the efficacy of chondroitinase-ABC treatment in an individual may comprise; providing an insoluble gel which incorporates a chondroitin sulphate , incubating the gel with a tissue sample obtained from the individual, and determining the amount of chondroitin sulphate in the gel after the incubation, the amount of chondroitin sulphate in the gel after the incubation being indicative of the activity of the chondroitinase- ABC in the sample.
  • the sample may be a tissue sample which is obtained from the individual to which the GAG lyase was administered. Suitable tissue includes central or peripheral nervous system tissue. In some embodiments, the tissue sample may be homogenized. In order to achieve optimal regeneration, the enzyme has to remain active for a certain period of time after injection. The result of this assay helps to determine the duration which the enzyme remains active in the animal and therefore helps to determine the dosage required to achieve optimal results.
  • the methods described herein may also be used to assess the efficacy of a GAG lyase preparation prior to administration to the individual.
  • a method of assessing or diagnosing osteoarthritis in an individual may comprise; providing an insoluble gel which incorporates a glycosminoglycan polymer, incubating the gel with a synovial fluid sample obtained from the individual , and determining the amount of glycosminoglycan in the gel after the. incubation, the amount of glycosminoglycan in the gel after the incubation being indicative of the glycosminoglycan lyase activity in the sample .
  • the glycosminoglycan polymer may be hyaluronan or a chondroitinase sulphate .
  • glycosminoglycan lyase activity such as hyaluronidase or chondroitinase activity
  • Suitable controls include glycosminoglycan lyase activity in synovial fluid from healthy individuals or predetermined standard levels of glycosminoglycan lyase activity. The individual may then undergo appropriate therapeutic intervention. This may include, for example, dietary glycoaminoglycan supplements, which are readily available from commercial sources.
  • a method of assessing renal damage or kidney function in an individual may comprise; providing a gel which incorporates an N-acetyl- ⁇ -D-glucosamine polymer, incubating the gel with a urine sample obtained from the individual , and determining the amount of polymer in the gel after the incubation, the amount of polymer in the gel after the incubation being indicative of N-acetyl- ⁇ -D-glucosaminidase activity in the sample.
  • Suitable controls include N-acetyl- ⁇ -D-glucosaminidase activity in urine from healthy individuals or pre-determined standard levels of N-acetyl- ⁇ -D-glucosaminidase activity.
  • An increased amount of acetyl- ⁇ -D-glucosaminidase activity in the sample relative to controls is indicative that the individual is suffering from or at risk of renal damage or loss of kidney function.
  • Individuals identified as suffering from or at risk of renal damage or loss of kidney function may be investigated further and/or subjected to appropriate dietary, lifestyle or therapeutic interventions .
  • MPS Mucopolysaccharidosis
  • a method of assessing mucopolysaccharidosis (MPS) and lysosomal storage disorders in an individual may comprise; providing a gel which incorporates a GAG polymer, incubating the gel with a sample obtained from the individual, and determining the amount of GAG polymer in the gel after the incubation, the amount of GAG polymer in the gel after the incubation being indicative of GAG lyase activity in the sample.
  • the GAG polymer which is incorporated into the gel depends on the type of class of MPS to be assessed. For example, dermatan sulfate and/or heparan sulphate may be incorporated into a gel to assess MPS 1 or MPS II. N-acetylglucosamine and/or heparan sulphate may be incorporated into a gel in the assessment of MPS III . Keratan sulfate may be incorporated into a gel in the assessment of MPS IV, chondroitin sulphates may be incorporated into a gel in the assessment of MPS VI and hyaluronan may be incorporated into a gel in the assessment of MPS IX.
  • Suitable samples for assessment of MPS include tears and blood leukocytes .
  • kits and packaged products for use in performing the methods described herein.
  • a kit for measuring depolymerase activity in a sample may comprise; an insoluble gel comprising a polymer substrate and a sample buffer suitable for depolymerase activity.
  • Insoluble gels are described in more detail elsewhere herein.
  • the insoluble gel may be in the form of a disc, tablet, strip, block or bead.
  • the gel may be incorporated into an assay device, for example a multiwell plate .
  • the gel is in a dehydrated form.
  • the gel is a polyacrylamide gel.
  • the polymer substrate may be a polypeptide, nucleic acid, glycosaminoglycan or polysaccharide as described above depending on the depolymerase activity which is to be measured.
  • kits may comprise more than one gel.
  • Each gel in the kit may comprise a different polymeric substrate to allow the assessment of a different depolymerase activity.
  • the kit may further comprise a detectable reporter for determining the amount of polymer substrate in the gel . Suitable detectable reporters are described in more detail above.
  • the kit may also comprise reagents, substrates and buffers for detecting reporter which is bound to substrate within the gel .
  • the detectable reporter may be separate from the insoluble gel in the kit or may be incorporated into the polymer substrate within the gel, such that it is released upon depolymerisation of the polymer substrate .
  • kits may be provided in a suitable container such as a vial in which the contents are protected from the external environment .
  • the kit may include instructions for use of the components in a method for determining depolymerase activity in a test sample as described herein.
  • a kit for use as described herein may include one or more articles and/or reagents for performance of the method, such as means for providing and storing the test sample itself, e.g. a swab for removing cells from the buccal cavity, a syringe for removing a blood sample (such components generally being sterile) , and one or more vessels or vials.
  • the kit may be adapted for measuring depolymerase activity in a sample by a method described herein.
  • kits related to methods of producing kits, reagents and packaged products for use in performing the methods described herein.
  • a method of producing a kit for measuring depolymerase activity in a sample may comprise; incorporating a polymer substrate in an insoluble gel and dehydrating the insoluble gel .
  • the gel may be processed, for example by shaping or cutting, before or after dehydration.
  • the dehydrated gel incorporating the substrate may be positioned in an assay device, such as a microwell plate
  • the dehydrated gel and/or assay device may be packaged in the kit along with a sample buffer.
  • FIG. 1 shows ChABC protein and activity in vivo.
  • ChABC protein was labelled with anti-chABC antibody in immunoblots of protein extracts from tissue dissected from the injury and injection site. ChABC protein remained detectable for over 10 days in vivo.
  • FIG. 2 shows tissue dissected from around the injection site retained ChABC activity for 10 days after injection.
  • Discs containing CS-A were stained after incubation with the brain homogenates or the standard enzyme solution.
  • Brain extracts from the chABC treated brain at 3 , 7 and 10 days after injection digested the CS on the discs and therefore give no staining.
  • Control Penicillinase treated brains showed a similar undetectable chondroitinase activity to OmM control enzyme digestion.
  • Figure 3 show the steps in a method described herein.
  • Figure 3A shows the incorporation of chondroitin sulphate A into polyacrylamide gel discs .
  • Figure 3B shows the rehydration of gel discs containing CS-A with enzyme buffer and then incubation with either cell homogenate or standard enzyme solution. The enzyme digests the CS-A into disaccarides which diffuse out of the gel.
  • Figure 3C shows the staining of discs with alcian blue, which stains undigested CS-A.
  • Figure 3D shows the drying of gel discs incorporating CS-A in a standard gel dryer.
  • a Scouten wire knife (Kopf Instruments, Harvard Apparatus, UK) was used to transect the tract .
  • a dental drill was used to expose a hole over the skull at -3 mm anterior (A) and -3 mm lateral (L) relative to the bregma.
  • the dura was referenced, gently pierced and the Scouten knife lowered through the hole to -8 mm ventral (V) below the dura.
  • the blade was extruded 3 mm, raised 3 mm, and retracted before being extruded again and lowered 1.5 mm back to the original starting point.
  • Each animal was perfused through the heart with 200 ml cold phosphate-buffered saline prewash (pH 7.4) followed by 200 ml cold 4% paraformaldehyde (pH 7.4) .
  • Brains were post- fixed for 24 h at 4 0 C then transferred to 30% sucrose (4 0 C) overnight.
  • the right hemisphere of each brain was sliced into 10 series of 40 ⁇ m sagittal sections using a sledge microtome, and stored at 4 0 C in Trizma (Sigma) -buffered saline (TBS) with 0.05% sodium azide.
  • chABC activity 3mm cubes of brain tissue were dissected from the chABC- or penicillinase-treated area at 3 , 7 and 10 days after injury and injection. The tissue was gently homogenized in a Potter-Elvehjem homogenizer with cold 0. IM acetate buffer, pH 8.0 on ice. The homogenates were then centrifugated at 500Og for 5 min. The supernatant was used for the activity assay. 3.5mm discs cut from a polyacrylamide gel, each containing 7.5ug of chondroitin sulphate (CS-A) were incubated with the supernatant for 30 min at 37 0 C. Fresh enzyme was used as standards to compare the activity with the homogenates. After incubation, the discs were rinsed briefly with water and stained with 0.2% Alcian blue for the undigested CS-A for 30 min until a standard colour was achieved.
  • CS-A chondroitin sulphate
  • the stained gel discs were dried and immobilized on filter paper. The discs were scanned and the image saved. The intensity of the staining in each disc was analysed with density measurement software (e.g. ImageJTM) .
  • density measurement software e.g. ImageJTM

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Cette invention porte sur des procédés de mesure de l'activité d'une dépolymérase, telle que la glycosaminoglycan (GAG) lyase, la hyaluronidase, la chondroïtinase, l'héparinase ou la kératanase, dans un échantillon, par incubation de l'échantillon avec un gel insoluble qui incorpore un substrat polymère de la dépolymérase, et de mesure de la quantité de substrat polymère laissée dans le gel après incubation. Ceci peut être utile, par exemple, dans l'évaluation et le traitement d'états de maladies, tels que l'ostéoarthrite, un dommage neuronal, un dommage rénal et une mucopolysaccharidose (MPS). L'invention porte également sur des coffrets à utiliser dans la réalisation des procédés.
PCT/GB2008/003391 2007-10-12 2008-10-08 Procédés de mesure de l'activité dépolymérase WO2009047489A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0720030.6 2007-10-12
GB0720030A GB0720030D0 (en) 2007-10-12 2007-10-12 Depolymerase assay

Publications (1)

Publication Number Publication Date
WO2009047489A1 true WO2009047489A1 (fr) 2009-04-16

Family

ID=38788123

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2008/003391 WO2009047489A1 (fr) 2007-10-12 2008-10-08 Procédés de mesure de l'activité dépolymérase

Country Status (2)

Country Link
GB (1) GB0720030D0 (fr)
WO (1) WO2009047489A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019011856A2 (fr) 2017-07-11 2019-01-17 Basf Se Procédé de fabrication de plaques de mousse au moins bicouches par collage par l'application d'au moins une plaque de mousse thermodurcissable plus mince avec des couches de revêtement de métal

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0693499A1 (fr) * 1994-07-22 1996-01-24 Seikagaku Kogyo Kabushiki Kaisha (Seikagaku Corporation) Dérivé de glycosaminoglycane; gel à base d'un copolymère d'acrylamide et de ce dérivé; procédé d'identification des enzymes
EP0884393A1 (fr) * 1996-02-29 1998-12-16 Fuji Photo Film Co., Ltd. Procede d'analyse de proteases et membrane fine utilisee dans ce procede

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0693499A1 (fr) * 1994-07-22 1996-01-24 Seikagaku Kogyo Kabushiki Kaisha (Seikagaku Corporation) Dérivé de glycosaminoglycane; gel à base d'un copolymère d'acrylamide et de ce dérivé; procédé d'identification des enzymes
EP0884393A1 (fr) * 1996-02-29 1998-12-16 Fuji Photo Film Co., Ltd. Procede d'analyse de proteases et membrane fine utilisee dans ce procede

Non-Patent Citations (14)

* Cited by examiner, † Cited by third party
Title
BOULWARE KEVIN T ET AL: "Protease specificity determination by using cellular libraries of peptide substrates (CLiPS)", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 103, no. 20, May 2006 (2006-05-01), pages 7583 - 7588, XP002505351, ISSN: 0027-8424 *
BROWN R L ET AL: "Variation in in vitro alpha-amylase and protease activity is related to the virulence of Aspergillus flavus isolates", JOURNAL OF FOOD PROTECTION, DES MOINES, IO, US, vol. 64, no. 3, 1 March 2001 (2001-03-01), pages 401 - 404, XP008098747, ISSN: 0362-028X *
EICKHOFF HOLGER ET AL: "Protein array technology: the tool to bridge genomics and proteomics", ADVANCES IN BIOCHEMICAL ENGINEERING, BIOTECHNOLOGY, SPRINGER, BERLIN, DE, vol. 77, 1 January 2002 (2002-01-01), pages 103 - 112, XP008084652, ISSN: 0724-6145 *
FREDERIKS WILMA M ET AL: "Metabolic mapping of proteinase activity with emphasis on in situ zymography of gelatinases: Review and protocols", JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY, vol. 52, no. 6, June 2004 (2004-06-01), pages 711 - 722, XP002504610, ISSN: 0022-1554 *
HIROTA YASUSHI ET AL: "Possible roles of thrombin-induced activation of protease-activated receptor 1 in human luteinized granulosa cells.", JOURNAL OF CLINICAL ENDOCRINOLOGY AND METABOLISM, vol. 88, no. 8, August 2003 (2003-08-01), pages 3952 - 3957, XP002504611, ISSN: 0021-972X *
KABERDIN VLADIMIR R ET AL: "Expanding the use of zymography by the chemical linkage of small, defined substrates to the gel matrix.", GENOME RESEARCH, vol. 13, no. 8, August 2003 (2003-08-01), pages 1961 - 1965, XP002504614, ISSN: 1088-9051 *
KIYONAKA SHIGEKI ET AL: "Semi-wet peptide/protein array using supramolecular hydrogel.", NATURE MATERIALS, vol. 3, no. 1, January 2004 (2004-01-01), pages 58 - 64, XP002504612, ISSN: 1476-1122 *
KONISHI HIROYUKI ET AL: "Identification of peripherin as a Akt substrate in neurons.", THE JOURNAL OF BIOLOGICAL CHEMISTRY 10 AUG 2007, vol. 282, no. 32, 10 August 2007 (2007-08-10), pages 23491 - 23499, XP002505349, ISSN: 0021-9258 *
LAUER-FIELDS JANELLE L ET AL: "Substrate conformation modulates aggrecanase (ADAMTS-4) affinity and sequence specificity - Suggestion of a common topological specificity for functionally diverse proteases", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 282, no. 1, January 2007 (2007-01-01), pages 142 - 150, XP002505350, ISSN: 0021-9258 *
MARTINEZ TOMAS F ET AL: "Improved detection of amylase activity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with copolymerized starch", ELECTROPHORESIS, vol. 21, no. 14, August 2000 (2000-08-01), pages 2940 - 2943, XP002504616, ISSN: 0173-0835 *
NEMORI R AND TACHIKAWA T: "A review for in situ zymography - method for localization of protease activities in a tissue", THE TISSUE CULTURE ENGINEERING, vol. 25, no. 9, 1999, pages 29 - 32, XP002504609 *
OETTL MARTIN ET AL: "Comparative characterization of bovine testicular hyaluronidase and a hyaluronate lyase from Streptococcus agalactiae in pharmaceutical preparations.", EUROPEAN JOURNAL OF PHARMACEUTICAL SCIENCES : OFFICIAL JOURNAL OF THE EUROPEAN FEDERATION FOR PHARMACEUTICAL SCIENCES MAR 2003, vol. 18, no. 3-4, March 2003 (2003-03-01), pages 267 - 277, XP002504615, ISSN: 0928-0987 *
SNOEK-VAN BEURDEN P A M AND VON DEN HOFF J W: "Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors", BIOTECHNIQUES, vol. 38, no. 1, January 2005 (2005-01-01), pages 73 - 83, XP008098711 *
WANG R-F ET AL: "DRIED GEL HYBRIDIZATION IN PLACE OF SOUTHERN HYBRIDIZATION FOR DETECTION OF LISTERIA-MONOCYTOGENES DNA FRAGMENTS", LETTERS IN APPLIED MICROBIOLOGY, vol. 12, no. 6, 1991, pages 224 - 227, XP002504617, ISSN: 0266-8254 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019011856A2 (fr) 2017-07-11 2019-01-17 Basf Se Procédé de fabrication de plaques de mousse au moins bicouches par collage par l'application d'au moins une plaque de mousse thermodurcissable plus mince avec des couches de revêtement de métal

Also Published As

Publication number Publication date
GB0720030D0 (en) 2007-11-21

Similar Documents

Publication Publication Date Title
Tomatsu et al. Newborn screening and diagnosis of mucopolysaccharidoses
Ciesielski et al. Citrullination in the pathology of inflammatory and autoimmune disorders: recent advances and future perspectives
Ricciardelli et al. Extracellular matrix of ovarian tumors
KR101680392B1 (ko) 장기간 투석을 예측하기 위한 소변 바이오마커
Hasmann et al. Novel peptidoglycan-based diagnostic devices for detection of wound infection
Li et al. Dramatic regulation of heparanase activity and angiogenesis gene expression in synovium from patients with rheumatoid arthritis
US9267946B2 (en) Biomarkers, methods and kits for the diagnosis of rheumatoid arthritis
Morita et al. Activity-dependent remodeling of chondroitin sulfate proteoglycans extracellular matrix in the hypothalamo–neurohypophysial system
Avenoso et al. Hyaluronan fragments produced during tissue injury: A signal amplifying the inflammatory response
Tomatsu et al. Newborn screening and diagnosis of mucopolysaccharidoses: application of tandem mass spectrometry
Junker et al. Expression and regulation patterns of hyaluronidases in small cell lung cancer and glioma lines
Srivastava et al. Dysregulation of hyaluronan homeostasis during white matter injury
WO2010133589A1 (fr) Procédé de détection de l'infection d'une plaie
Chanzu et al. Platelet α‐granule cargo packaging and release are affected by the luminal proteoglycan, serglycin
JPS63119498A (ja) 新規なモノクロナル抗体、その製造及び使用
Paulsson et al. Structure and function of basement membrane proteoglycans
GB2418145A (en) Wound treatment system
WO2009047489A1 (fr) Procédés de mesure de l'activité dépolymérase
Theocharis et al. High‐performance capillary electrophoretic analysis of hyaluronan and galactosaminoglycan‐disaccharides in gastrointestinal carcinomas. Differential disaccharide composition as a possible tool‐indicator for malignancies
Paschos et al. Enzymatic function of multiple origins regulates the progression of colorectal cancer and the development of metastases
Oke et al. Assessment of three variations of the 1, 9-dimethylmethylene blue assay for measurement of sulfated glycosaminoglycan concentrations in equine synovial fluid
CN100595281C (zh) 一种腺癌标志物及其应用
Collatos et al. Fibrinolytic activity in plasma from horses with gastrointestinal diseases: changes associated with diagnosis, surgery, and outcome
CN106501523A (zh) 基质蛋白酶3测定试剂盒
US6630295B2 (en) High throughput assay for monitoring polycation or polyanion molecular weight, degradation or synthesis

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08806531

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08806531

Country of ref document: EP

Kind code of ref document: A1