WO2004027421A2 - Dosage adp - Google Patents

Dosage adp Download PDF

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Publication number
WO2004027421A2
WO2004027421A2 PCT/GB2003/004015 GB0304015W WO2004027421A2 WO 2004027421 A2 WO2004027421 A2 WO 2004027421A2 GB 0304015 W GB0304015 W GB 0304015W WO 2004027421 A2 WO2004027421 A2 WO 2004027421A2
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adp
atp
assay
binding protein
enzyme
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PCT/GB2003/004015
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WO2004027421A3 (fr
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Nick Gee
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Innova Biosciences Limited
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Publication of WO2004027421A3 publication Critical patent/WO2004027421A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • 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
    • C12Q1/008—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions for determining co-enzymes or co-factors, e.g. NAD, ATP
    • 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
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G01N33/5735—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes co-enzymes or co-factors, e.g. NAD, ATP

Definitions

  • This invention relates to an assay for ADP.
  • the invention also relates to an assay for reactions that generate ADP.
  • this invention relates to an assay for enzymes catalysing ADP-forming reactions such as protein kinases.
  • Further embodiments of the invention include a method for screening for modulators of the activity of ADP-forming enzymes and a method of screening for substrates of ADP- forming enzymes.
  • a number of assays for ADP have been described. In many of these assays, ADP is detected following enzymatic transformation of ADP into another molecule that is more easily measured. Assays for ADP are known that employ the reaction of pyruvate kinase (PK). PK catalyses a reaction between ADP and phosphoenolpyruvate (PEP) to generate two products, ATP and pyruvate. In one assay, the amount of pyruvate is determined indirectly by reducing pyruvate to lactate in a reaction catalysed by lactate dehyrogenase (LDH), followed by measuring the NAD generated in this reaction (Tanzer and Gilvarg, J. Biol. Chem.
  • LDH lactate dehyrogenase
  • the ATP generated is detected indirectly by following the production of NADPH in a coupled spectrophotometric assay using hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6PDH) (Rosalki, J. Lab. Clin. Med. 69: 697, 1967).
  • HK hexokinase
  • G6PDH glucose-6-phosphate dehydrogenase
  • ADP is converted to ATP and then measured in a bioluminescent assay with firefly luciferase (Summerfield et ah, Clin. Lab. Haematol. 3: 257, 1981).
  • ADP assays that involve the measurement of ATP formed by phosphorylation of ADP are more difficult if there is contaminating ATP present in the sample.
  • AMP -forming enzymes have been used to remove contaminating ATP.
  • ATP is often a precursor of ADP in enzymatic reactions, samples consisting of the products of enzymatic reactions are often contaminated with ATP.
  • ATP sulphurylase is an AMP-forming enzyme that has been used to remove ATP, followed by phosphorylation of ADP to ATP (Schultz et ah, Anal. Biochem. 215: 302, 1993). The ATP sulphurylase is inactivated prior to the phosphorylation step to prevent the ATP generated from ADP from being degraded.
  • Luciferase is an AMP-forming enzyme that has been used to measure ATP in cell extracts followed by further measurement of nascent ATP generated from ADP in the same sample. This approach has been exploited in a commercially available adenine nucleotide ratio assay (Lumitech).
  • nucleoside diphosphate kinase (NDPK) labelled with an environmentally sensitive fluorescent reporter has been used as a nucleotide diphosphate sensor (Brune et ah, Biochemistry 40: 5087, 2001).
  • the sensor detects subtle changes in the conformation of NDPK as ADP is converted into ATP.
  • ADP is converted into ATP.
  • only a four-fold change in fluorescence is observed under optimum conditions. In the presence of a 12-fold excess of ATP the change in fluorescence is reduced to just 25% of the maximum because of the competing reverse reaction.
  • ADP present in samples containing ATP is physically separated from ATP using hplc methods (Samizo et ah, Anal. Biochem. 293: 212, 2001).
  • ADP may be measured using a variety of assay systems. Coupled assays are prone to interference when used for high-throughput drug screening as the test compounds may inhibit the accessory enzymes thus giving rise to false positives. ADP assay methods involving the destruction of ATP and the generation of ATP from ADP are laborious and impractical when large numbers of tests need to be performed.
  • the PK/LDH assay of ADP is more direct than assays involving ATP destruction and generation, but still involves two enzymatic steps. Both the HK/G6PDH assay and the PK/LDH assay involve the interconversion of nicotinamide adenine dinucleotide cofactors.
  • kinases and ATPases are important therapeutic targets and are the focus of many high-throughput drug screens.
  • ATPases may be assayed by measuring the release of inorganic phosphate (Sudo et al, J Chromatogr B Biomed Sci Appl 744: 19, 2000; Ohnishi et al, Anal. Biochem. 69: 261, 1975) or by using PK/LDH coupled assays (Olland and Wang, J. Biol. Chem. 274: 21688, 1999) as described above.
  • An hplc method to measure ADP generated by ATPases has also been reported (Sudo et al, J Chromatogr B Biomed Sci Appl 744: 19, 2000).
  • the radioactively labelled product is then separated from the unutilised radiolabelled ATP and measured by scintillation counting.
  • the separation step commonly involves the capture of a basic (positively charged) substrate on cation-exchange paper, such as phosphocellulose (Casnellie, Methods Enzymol. 200: 115, 1991) though a variety of other separation methods have also been used including gel electrophoresis, precipitation, hplc and biotin-streptavidin capture. While the development of heterogeneous radioactive assays is relatively straightforward the laborious separation and/or wash steps limit the utility of this type of assay in the HTS environment.
  • Scintillation proximity assay methods eliminate the need to separate the labelled product from the labelled substrate.
  • the kinase substrate is tagged with biotin to facilitate the capture of the radioactive product of the kinase onto a surface that is impregnated with scintillant.
  • the radioactive product excites the scintillant to emit light.
  • the solvent absorbs the energy from molecules that undergo radioactive decay in solution.
  • SPA technology works optimally with 3 H- and 125 I-labelled molecules.
  • SPA assays for kinases using immobilised substrate and 33 P-ATP have been developed, but the energy of 33 P is sufficiently high that radioactive molecules in solution may activate the scintillant. Wash steps or other methods for separating SPA beads from the bulk fluid have been used to reduce the background (Park et al, Anal. Biochem. 269: 94, 1999) though these steps negate the potential advantages of the technology.
  • kinase assays have been developed using phosphorylation state specific antibodies (PSSAs), which can discriminate between the phosphorylated product of a kinase reaction and the unphosphorylated substrate.
  • PSSAs phosphorylation state specific antibodies
  • the principle drawback of the PSSA approach is that it can take many months to develop a suitable antibody.
  • the final reagent is often highly specific for the phospho-epitope used as the immunogen, the application of the antibody is often limited to assays that use one particular substrate.
  • PSSAs have been used in the development of fluorescence polarisation (FP), fluorescence resonance energy transfer (FRET) and ELISA assays for kinases, as described below.
  • FP measures the rotational diffusion of fluorescent molecules and is based on the principle that a rotating fluorescent molecule in solution depolarises plane-polarised light (Owicki, J. Biomol. Screen 5: 297, 2000). Since the speed of rotation is inversely related to molecular size, the depolarising effect of a small fluorescently labelled ligand when bound to a large biomolecule is small compared with that of the free ligand.
  • a fluorescent phosphorylated analogue of the peptide substrate (a 'tracer') is incubated with the unlabelled PSSA, together with the substrate and kinase.
  • a direct assay configuration may also be used in which the substrate itself is labelled with a fluorescent group.
  • large protein substrates are not suitable for direct FP assays since there is little change in rotational diffusion when the PSSA binds to the phosphorylated product.
  • both the PSSA and the product of the reaction carry fluorophores.
  • the absorption spectrum of one fluorophore overlaps with the emission spectrum of the other (the acceptor).
  • the two fluorophores are in close proximity specific excitation of the donor leads to radiationless energy transfer to the acceptor, which then emits light of a characteristic wavelength.
  • one fluorophore is on the PSSA and the other is located on the substrate.
  • the epitope for the PSSA is present only following phosphorylation of the substrate by the kinase, and thus the magnitude of the FRET signal is a measure of kinase activity.
  • lanthanides such as Eu 3+ are popular donors because of their relatively long-lived light emission. This allows temporal separation of the specific signal from the more rapidly decaying background fluorescence (Diamandis, Clin. Biochem. 21: 139, 1988).
  • FRET assays are safe compared with radioactive assays but they have other drawbacks.
  • the amount of time required to produce a suitable PSSA is a significant disadvantage, as is the need for covalent modification of both the substrate and antibody, which may lead to impaired biological activity.
  • ELISA assays have also been employed to measure kinase activity (Schraag et al, Anal. Biochem. 211: 233, 1993; Farley et al, Anal. Biochem. 203: 151, 1992; Lazaro et al, Anal. Biochem. 192: 257, 1991; Yu et al, J. Biochem. (Tokyo) 129: 243, 2001; Angeles et al, Anal. Biochem. 236: 49, 1996; Forrer et al, Biol. Chem. 379: 1101, 1998; Cleaveland et al, Anal. Biochem. 190: 249, 1990; Lehel et al, Anal. Biochem.
  • the substrate for the kinase is often passively adsorbed onto the ELISA plate prior to the addition of the kinase. Phosphoepitopes are then detected with a PSSA that is conjugated to an enzyme label or detected indirectly with a secondary antibody conjugate (Schraag et al, Anal. Biochem. 211: 233, 1993).
  • the readout of the assay may be colorimetric (Lazaro et al, Anal. Biochem. 192: 257, 1991) or chemiluminescent (Mosier et al, Methods Enzymol. 305: 410, 2000) depending on the particular detection method that is used.
  • the substrate may be biotinylated and captured on a streptavidin-coated plate (Mosier et al, Methods Enzymol. 305: 410, 2000).
  • a fluorescently labelled antibody may be used instead of an enzyme label (Braunwalder et al, Anal. Biochem. 238: 159, 1996).
  • the principal disadvantage of ELISA and related assays in the HTS environment is the need for multiple incubation and wash steps.
  • no suitable product- specific antibodies are available.
  • ADP is a common product of all kinase reactions, it is a very attractive marker of kinase activity.
  • Other enzymes such as ATPases and some ligases also generate ADP.
  • the assays presently available for measuring ADP are unattractive for high-throughput screening. There is thus a need for a generalised ADP assay that does not suffer from the drawbacks of the presently known ADP assays.
  • the development of a generic ADP assay that can be used on samples containing ATP presents significant technical challenges owing to the structural similarity of ADP and ATP.
  • the present invention describes a generic ADP assay that does not suffer from the drawbacks of the presently known assays for ADP. Accordingly, the present invention provides an assay for ADP, which assay comprises a step of detecting ADP present using an ADP-binding protein.
  • an ADP-binding protein is defined as any protein that is capable of binding to ADP.
  • the binding protein does not bind to other compounds present.
  • the ADP-binding protein is incapable of binding to ATP.
  • the ADP-binding protein may also be labelled. Any suitable label may be used such as a fluorophore, an enzyme, biotin, H, C S, P, P or I. The label must not prevent the binding of the binding protein to ADP.
  • Suitable ADP binding proteins for use in the present invention include hsp90 (J. Biol. Chem. 272:23843-20850, 1997), ADP-dependent glucokinase (J. Biol. Chem. 270: 30453-30457, 1995) and platelet membrane ADP-binding protein (J. Biol. Chem. 254: 3866-3872, 1979). It will be apparent to one skilled in the art that these binding proteins may be modified post-translationally or through engineering the DNA that encodes the proteins. Thus the binding protein may contain tags at either terminus or internally to facilitate purification or interactions with other components or surfaces in the detection method. The binding protein may be modified by in vitro or in vivo biotinylation.
  • Antibodies recognising ADP may also be employed as ADP binding proteins in the present invention. These antibodies may also recognise 3'-dephosphocoenzyme A (DPCoA; an analogue of ADP).
  • DCoA 3'-dephosphocoenzyme A
  • the invention provides a process for producing an antibody capable of binding to ADP.
  • This antibody may also recognise 3'-dephosphocoenzyme A.
  • the process utilises an immunogen comprising ADP, DPCoA or another ADP analogue.
  • Antibodies raised using the method of the invention are also provided.
  • the invention provides a method of isolating a non-antibody ADP- binding protein using a resin comprising ADP, 3'-dephosphocoenzyme A or another ADP analogue.
  • ADP binding proteins isolated using such affinity resins are also provided.
  • ADP does not undergo any chemical transformation when bound to the ADP binding protein.
  • ADP is not phosphorylated to form ATP when bound to the ADP binding protein.
  • Any method of detecting the ADP present may be used.
  • Direct assay formats may be used in which a labelled analogue of ADP is a product of the reaction. Any suitable label may be used such as a fluorophore, an enzyme, biotin, 3 H, 14 C 35 S, 32 P, 33 P or 125 I. The label must not prevent binding of ADP to the ADP-binding protein.
  • Indirect assays formats may also be used.
  • a labelled compound that competes with ADP for binding to the ADP-binding protein is added. Any labelled compound that competes with ADP for binding to the ADP-binding protein may be used as a competitor of ADP. Suitable competitors include labelled ADP analogues.
  • 3'- dephosphocoenzyme A may be used as a competitor. Any suitable label may be used such as a fluorophore, an enzyme, biotin, 3 H, 14 C 35 S, 32 P, 33 P or 125 I. Again, the label must not prevent binding of the competitor to the ADP-binding protein.
  • the assay may also comprise a step of modifying the ATP.
  • the method by which ATP is modified is not particularly limited, but the modification must prevent the modified ATP from binding to the ADP-binding protein.
  • the assay comprises a step of adding to the sample one or more enzymes that selectively remove the ATP via pathways that do not involve ADP as an intermediate. The step of adding the enzyme or enzymes may take place prior to, or be simultaneous with, the addition of the components of the detection step.
  • the amount of ADP is determined by measuring the magnitude of a signal generated in the detection step.
  • the signal may be generated by the labelled ADP analogue, by the labelled competitor, by the labelled binding protein.
  • this invention provides an assay for ADP in which ADP is measured without either chemical transformation or separation by liquid chromatography from other assay components.
  • the assay system provided by the present invention is also extremely flexible and may be configured for most instrument platforms and detection principles.
  • the ADP assayed is produced in an ADP-forming reaction.
  • the ADP-forming reaction may be enzymatically catalysed.
  • the ADP-forming reaction may be catalysed by a kinase, an ATPase, or a ligase.
  • Kinases suitable for use in the present invention include protein kinase A, protein kinase C, CAM kinase, a cyclin dependent kinase, a MAP kinase, a SAP kinase, lck protein tyrosine kinase and protein histidine kinase.
  • the ADP assay may take place following termination of the ADP- forming reaction.
  • the components of the ADP assay may also contain substances that terminate the ADP-forming reaction.
  • ADP may be assayed during the ADP-forming reaction. If the ADP-forming reaction requires ATP, addition of the components of the ATP modification step may terminate the ADP-forming reaction.
  • the precursor of ADP in the ADP-forming reaction may be labelled with fluorophore, an enzyme, biotin, 3 H, 14 C, 35 S 32 P, 33 P or 125 I in such a manner that the ADP produced in the ADP-forming reaction is also labelled with a fluorophore, an enzyme, biotin, 3 H, 14 C, 35 S 32 P, 33 P or 125 I.
  • a precursor of ADP is a substrate of the ADP-forming reaction which contributes at least one atom to the ADP product.
  • the rate of the ADP-forming reaction may be determined by measuring the rate of production of ADP. Where the ADP-forming reaction is enzymatically catalysed, the rate of this reaction is a measure of the activity of the enzyme on a substrate under the reaction conditions used.
  • This invention also provides a method of screening for a compound capable of modulating the activity of an ADP-forming enzyme, which method comprises (a) measuring the activity of said ADP-forming enzyme on a substrate in the presence and absence of a test compound (b) comparison of the activity of the ADP-forming enzyme on a substrate in the presence and absence of the test compound; and (c) identifying a compound capable of modulating the activity of said ADP-forming enzyme as one in which the activity of said ADP-forming enzyme with a substrate is substantially different in the presence and absence of the test compound.
  • This invention also provides a compound capable of modulating the activity of an ADP- forming enzyme, a pharmaceutical composition comprising this compound and a pharmaceutically acceptable diluent or excipient, a method of manufacturing the pharmaceutical composition and a method of treating a patient having a condition caused by aberrant activity of the ADP-forming enzyme with the pharmaceutical composition.
  • the invention also provides for the use of a compound capable of modulating the activity of the ADP-forming enzyme in the manufacture of a medicament for the treatment of a condition caused by aberrant activity of said ADP-forming enzyme.
  • Conditions which may be treated by a medicament or pharmaceutical composition comprising a compound capable of modulating the activity of an ADP-forming enzyme include rheumatoid arthritis, asthma, stroke, diabetes and cancers such as chronic myelogenous leukaemia.
  • the invention thus provides a method of identifying a substrate for an ADP-forming enzyme, which method comprises (a) contacting the putative substrate with the ADP- forming enzyme under conditions suitable for enzyme activity (b) identifying a substrate of the enzyme as one in which ADP is detected in the reaction mixture.
  • the invention also provides for a kit capable of carrying out the invention that comprises (a) an ADP-binding protein, and (b) a labelled competitor of ADP.
  • the competitor may be labelled with a fluorophore, an enzyme, biotin, 3 H, 14 C, 35 S 32 P, 33 P or 125 I.
  • the ADP-binding protein may also be labelled with a fluorophore, an enzyme, biotin, 3 H, 14 C, 35 S 32 P, 33 P or 125 I.
  • the invention also provides for a kit capable of carrying out the invention that comprises (a) an ADP-binding protein, and (b) a labelled precursor of ADP.
  • the precursor may be labelled with a fluorophore, an enzyme, biotin, H, C, S P, P or 125 I.
  • the ADP-binding protein may also be labelled with a fluorophore, an enzyme, biotin, 3 H, 14 C, 35 S 32 P, 33 P or 125 I.
  • the invention also provides a method of removing ATP from a sample via a pathway that does not comprise ADP.
  • the invention provides for the use of Methionine adenosyl transferase (MAT) enzymes MAT I, MAT II or MAT III for the purpose of degrading ATP in a sample, wherein the sample is to undergo an assay for ADP.
  • MAT Methionine adenosyl transferase
  • the sample is not particularly limited.
  • the sample comprises the products of an ADP-forming reaction.
  • Figures 1 to 7 show the range of methods available for detection of ADP. However, they are not intended to be limiting and any detection method may be utilised.
  • Figures 8 to 13 shows the results of experiments demonstrating that the invention is capable of reduction to practice.
  • FIG. 1 is a schematic diagram showing a generic indirect detection method for ADP.
  • ADP is generated from an ADP precursor via the action of the ADP-forming reaction.
  • the ADP generated competes with a labelled analogue of ADP (X) for binding to an ADP-binding protein.
  • the signal generated is related to the amount of the labelled competitor that is bound to the binding protein and thus inversely related to the amount of ADP generated by the ADP-forming reaction.
  • ATP if present, is extensively degraded so as to avoid unwanted cross-reaction of the ADP-binding protein with ATP or AMP.
  • the assay may be configured for different instrument platforms and detection methods.
  • FIG 2 is a schematic diagram showing one embodiment of an indirect detection method for ADP.
  • An indirect fluorescence polarisation detection method is employed.
  • the competitor (X) of ADP is labelled with a fluorescent label.
  • the competitor competes with ADP generated in an ADP-forming reaction for binding to the ADP- binding protein.
  • a large FP signal is observed when the fluorescently-labelled competitor is bound to the ADP-binding protein.
  • the competitor is displaced leading to a loss of polarisation of plane polarised light.
  • Figure 3 is a schematic diagram showing one embodiment of an indirect detection method for ADP.
  • An indirect FRET detection method is employed.
  • the competitor of ADP (X), and the ADP-binding protein are labelled with different fluorophores.
  • the absorption spectrum of one of the fluorophores overlaps with the emission spectrum of the other fluorophore.
  • the competitor competes with ADP generated in an ADP- forming reaction for binding to the ADP-binding protein.
  • FRET occurs and light of a characteristic wavelength is released.
  • the competitor is displaced and the FRET signal is diminished.
  • FIG 4 is a schematic diagram showing one embodiment of an indirect detection method for ADP.
  • An indirect scintillation proximity method is employed.
  • the competitor of ADP (X) is radioactively labelled.
  • the unlabelled ADP generated by an ADP-forming reaction competes with the radiolabelled competitor for binding to an ADP-binding protein that is immobilised on a surface that is coated or impregnated with scintillant. If the radiolabelled competitor is bound to the ADP-binding protein, a scintillation proximity signal is observed. The signal is diminished when ADP is bound to the ADP-binding protein.
  • FIG. 5 is a schematic diagram of one embodiment of the invention in which ADP is detected using an indirect heterogeneous method.
  • the labelled competitor of ADP (X) competes with ADP for binding to the ADP-binding protein which is immobilised on a surface. After washing, the amount of label is determined either by direct measurement or, where the label is biotin, after detection of biotin using a labelled streptavidin conjugate.
  • FIG. 6 is a schematic diagram of another embodiment in which ADP is detected using an indirect heterogeneous method.
  • a competitor of ADP (X) is immobilised on a surface.
  • the ADP-binding protein is labelled.
  • ADP formed in an ADP-forming reaction competes with the immobilised competitor for binding the binding protein.
  • the assay signal is inversely related to the amount of the ADP produced by the ADP-forming reaction.
  • FIG. 7 is a schematic diagram of another embodiment in which ADP is detected using a direct scintillation proximity method.
  • Radiolabelled ADP is generated in an ADP- forming reaction.
  • the radiolabelled ADP binds to an ADP-binding protein that is immobilised on a surface that is coated or impregnated with scintillant. When bound, a scintillation proximity signal is observed.
  • Figure 8 shows the results of an ELISA assay to determine the affinity of various sera for the ADP analogue 3'-dephosphocoenzyme A, and for ATP.
  • Figure 8(a) relates to serum 22, which was raised using DPCoA conjugated through its sulphydryl group to maleimide activated KLH as an immunogen.
  • Figure 8(b) relates to serum 30, which was raised using a 50:50 mixture of ADP directly conjugated to KLH and ADP conjugated via a cysteamine linker to maleimide-activated KLH as an immunogen.
  • Figure 8(c) relates to serum 23, which was raised using a 50:50 mixture of ATP conjugated directly to KLH and ATP conjugated via a cysteamine linker to maleimide- activated KLH as an immunogen.
  • Figure 8(d) is a control assay in which no serum was added. In each experiment (except the control), serum (the primary antibody) was added to an ELISA plate coated with biotin-DPCoA and an ELISA plate coated with biotinylated ATP. The plates were then washed and a secondary antibody conjugated to horseradish peroxidase was added.
  • Figure 9 shows the results of an ELISA assay to determine the ability of various metabolites to interfere with antibody binding to a DPCoA ELISA plate.
  • Figure 9(a) relates to antibody 24, which was raised using a 50:50 mixture of ATP directly conjugated to KLH and ATP conjugated via a cysteamine linker to maleimide activated KLH as an immunogen.
  • Figure 9(b) relates to antibody 30, which was raised using a 50:50 mixture of ADP directly conjugated to KLH and ADP conjugated via a cysteamine linker to maleimide activated KLH as an immunogen.
  • Each antibody was added to an ELISA plate coated with DPCoA in the presence of competitor ATP, ADP, AMP, adenosine, S-adenosyl methionine, pyrophosphate, ribose 5-phospate, or in the absence of competitor.
  • the plate was then washed and a secondary antibody conjugated to horseradish peroxidase was added.
  • the substrate for horseradish peroxidase was added to the plate and the degree of binding of the secondary antibody (and hence the primary antibody) was assessed by measuring the absorbance of the plates at 405 nm.
  • Figure 10 shows the results of an ELISA assay to determine the ability of various additives commonly found in drug screening assays to interfere with the binding of antibody 30 to a DPCoA ELISA plate.
  • Antibody 30 was raised using a 50:50 mixture of ADP directly conjugated to KLH and ADP conjugated via a cysteamine linker to maleimide activated KLH as an immunogen.
  • the antibody was added to a DPCoA- coated ELISA plate in the presence of each of 10% DMSO, 12.5 mM EDTA, 2.5 mM DTT, 5 mM N-ethylmaleimide, 5 mM ATP, and in the presence of no additives.
  • the plates were then washed and a secondary antibody conjugated to horseradish peroxidase was added.
  • the substrate for horseradish peroxidase was added to the plates and the degree of binding of the secondary antibody (and hence the primary antibody) was assessed by measuring the absorbance of the plates at 405 nm.
  • the results shows that DMSO and N-ethylmaleimide have no effect on the binding of antibody 30 to DPCoA. ATP abolishes binding, and DTT and EDTA partially inhibit antibody 30 binding to DPCoA.
  • Figure 11 shows the results of a series of competition ELISA assays in which immobilised biotinylated-DPCoA and varying amounts of ADP are in competition for antibody 30 (raised using a 50:50 mixture of ADP directly conjugated to KLH and ADP conjugated via a cysteamine linker to KLH as an immunogen). A range of concentrations of ADP was used and antibody 30 (1/200 dilution of serum) was added to each well. The wells were then washed and a secondary antibody conjugated to horseradish peroxidase was added.
  • Recombinant human MAT III enzyme was refolded by rapidly diluting the unfolded enzyme in the following buffers, 50mM Tris pH 8.0 (Tris), 50mM Tris pH 8.0/10% DMSO, 50mM Tris pH 8.0/ 10 mM DTT/10% DMSO, 50mM Tris pH 8.0/lOmM DTT/10% DMSO/30 mM KC1, 50mM Tris pH 8.0/10 mM DTT/10% DMSO/10 mM MgCl 2 and 50mM Tris pH 8.0/10 mM DTT/10% ethylene glycol.
  • Tris Tris pH 8.0
  • 50mM Tris pH 8.0/10% DMSO 50mM Tris pH 8.0/10% DMSO
  • 50mM Tris pH 8.0/ 10 mM DTT/10% DMSO 50mM Tris pH 8.0/lOmM DTT/10% DMSO/30 mM KC1
  • the refolded enzymes were assayed in buffer B (50 mM Tris, 250 mM KC1, 1 mM DTT, 1 mM ATP, 1 mM methionine, 10 mM MgCl 2 , 10% DMSO, pH 8.0). Released phosphate was measured as a phosphomolybdate complex using malachite green according to the method of Geladopoulous and colleagues (Geladopoulos et. al, Anal. Biochem. 192, 112-116, 1991).
  • Figure 13 shows the results of a study to determine the factors that affect the activity of human recombinant MAT 111.
  • the activity of refolded MAT III enzyme was assayed in buffer B (50 mM Tris, 250 mM KC1, 1 mM DTT, 1 mM ATP, 1 mM methionine, 10 mM MgCl 2 , 10% DMSO, pH 8.0), and in modified buffer B lacking certain components. Where ATP was omitted, this was replaced by 1 mM ADP. Released phosphate was measured as a phosphomolybdate complex using malachite green according to the method of Geladopoulous and colleagues (Geladopoulos et. al., Anal. Biochem. 192, 112-116, 1991). The results show that all the components of buffer B are required for activity, although a low level of activity is still observed in the absence ofKCl.
  • All embodiments of the invention comprise a step of detecting ADP in which an ADP- binding protein is employed.
  • the ADP-binding protein is not particularly limited. Any protein which binds to ADP may be used.
  • the ADP-binding protein may be modified post-translationally or through engineering the DNA that encodes the protein. Thus the binding protein may contain tags at either terminus or internally to facilitate purification or interactions with other components of surfaces in the detection method.
  • the binding protein may be modified by in vitro or in vivo biotinylation.
  • ADP binding proteins for use in the present invention include hsp90, ADP dependent glucokinase and platelet membrane ADP binding protein.
  • Antibodies recognising ADP are also suitable binding proteins for use in the detection step. Methods for generating antibodies are well known to one skilled in the art. In this application, the word antibody encompasses polyclonal antibodies, monoclonal antibodies, single chain antibodies, chimeric antibodies, fragments derived from proteolysis of whole antibodies and/or by reduction of disulphide bonds, or antibodies generated by means of expression libraries.
  • Suitable antibodies for use in the invention may be raised using an immunogen comprising ADP or DPCoA.
  • DPCoA is an intermediate in the biosynthesis of acetyl co-enzyme and comprises an ADP moiety joined to a pantotheine moiety.
  • Other analogues of ADP or other molecules structurally related to ADP may also be used as immunogens.
  • the immunogen comprises ADP linked to a carrier protein via its ⁇ -phosphate group, or DPCoA linked to a carrier protein via its sulphydryl group.
  • the immunogen is injected into a host animal such as a rabbit, sheep, goat or mouse.
  • antibodies for use in the present invention include antibodies generated following immunisation with an ADP-ribose conjugate.
  • IC 50 values for inhibition of binding of [ 3 H] ADP -ribose to the antibody by 5 '-AMP, ADP and ADP-ribose were 26 nM, 33 nM and 133 nM, respectively indicating that 5'- AMP binds most strongly, followed by ADP and then ADP-ribose (Breskyt et al, Eur. J. Biochem. 82: 105, 1978).
  • Antibodies raised against an AMP -bovine serum albumin conjugate also recognised ADP, ATP and (2':3')AMP (Drocourt and Leng, Eur. J. Biochem. 56: 149, 1975).
  • Antibodies recognising ADP or DPCoA may be isolated from immune sera by affinity chromatography on resins containing ADP, resins containing DPCoA or resins containing other ADP analogues. These affinity resins may also be used to isolate non- antibody ADP-binding proteins from cells and tissue extracts.
  • An ADP affinity resin may be produced by linking the ⁇ -phosphate of ADP to a commercially available resin, using standard carbodiimide chemistry.
  • a DPCoA affinity resin may be produced by linking sulphydryl group of DPCoA to a commercially available activated resin.
  • DPCoA contains an ADP moiety linked by a phosphoryl group, corresponding to the ⁇ phosphoryl group of ADP, to a pantotheine moiety. Where DPCoA is coupled to Sepharose resin via its sulphydryl group, the pantotheine moiety acts as a 'spacer' between the ADP moiety and the Sepharose matrix.
  • nucleotide-binding proteins are not necessarily particularly selective, and that ADP-binding proteins may be developed or purified using analogues of ADP, ATP or related molecules.
  • the present invention addresses the potential lack of nucleotide selectivity of ADP-binding proteins and allows ADP to be measured in samples that contain ATP.
  • the specificity of binding proteins for DPCoA may be determined using ELISA techniques. DPCoA is reacted with iodoacetyl-LC-biotin (Pierce) and the biotinylated analogue of DPCoA is captured on a streptavidin-coated plate.
  • a streptavidin plate coated with a biotinylated molecule that is structurally unrelated to DPCoA is used as a control.
  • the interaction of the binding protein on the two plates is then assessed using a standard ELISA detection system.
  • the selectivity of DPCoA-binding proteins is assessed by co-incubation with a variety of nucleotides and nucleosides, including ATP, ADP, AMP and adenosine.
  • a wide range of labels or tags may be attached to ADP, the precursor of ADP, the ADP-binding protein and the competitor of ADP for the ADP-binding protein, if present. This permits considerable flexibility in assay design. Assays may be configured for a number of different instrument platforms and in a variety of formats, including homogeneous and heterogeneous formats, each of which can involve direct or indirect detection of ADP. In direct assay formats (see figure 7), the ADP is labelled and the assay signal is directly related the amount of labelled ADP present in the sample. By contrast, indirect assays (see figure 1) enable unlabelled ADP to be measured. Typically, a labelled competitor of ADP for the ADP-binding protein is added to the sample. In some indirect assays, the ADP-binding protein may be labelled rather than the competitor. In all indirect assays, the assay signal diminishes with increasing concentration of unlabelled ADP.
  • Detection methods suitable for use with the present invention are given below. These are not intended to be limiting and any suitable detection method may be used.
  • FIG 4 An indirect (competition) scintillation proximity detection method is illustrated in figure 4.
  • a labelled competitor of ADP for the ADP-binding protein is present.
  • the label is either 3 H or 125 I.
  • the binding protein is immobilised either covalently or non-covalently on a surface that has been impregnated or coated with scintillant.
  • the binding protein may be incubated with the sample comprising ADP and the radiolabelled competitor before or after attachment to the surface.
  • the radiolabelled competitor competes with the unlabelled ADP for the ADP- binding protein, and the radioactivity associated with the binding protein is determined by scintillation counting.
  • the signal is inversely related to the amount of unlabelled ADP present in the sample.
  • An 'inverted' indirect scintillation proximity detection method may also be used to detect ADP.
  • the unlabelled competitor of ADP for the ADP- binding protein is coupled to a surface that is coated or impregnated with a scintillant.
  • the ADP-binding protein is labelled directly with 3 H or 125 I by covalent modification or indirectly by non-covalent interaction with a labelled molecule.
  • the unlabelled ADP present in the sample competes with the immobilised competitor for the radiolabelled ADP-binding protein.
  • the radioactivity associated with the surface is determined by scintillation counting.
  • the signal is inversely related to the amount of unlabelled ADP present in the sample.
  • a direct scintillation proximity detection method is shown schematically in figure 7.
  • radiolabelled ADP is generated in an ADP-forming reaction from a 3 H- or 33 P-labelled precursor.
  • the ADP-binding protein is attached to a surface that has been impregnated or coated with scintillant.
  • the linkage of the binding protein to the surface may be covalent or non-covalent and the ADP-binding protein may be incubated with the sample prior to or after its attachment to the surface.
  • Figure 2 illustrates an indirect fluorescence polarisation (FP) detection method for ADP.
  • a fluorescently labelled competitor of ADP for the ADP-binding protein is added to the sample. Labelling may be carried out with a sulphydryl-reactive fluorescein derivative such as iodoacetamidofluorescein or with an amine-reactive derivative depending on the functional groups available on the competitor.
  • a sulphydryl-reactive fluorescein derivative such as iodoacetamidofluorescein or with an amine-reactive derivative depending on the functional groups available on the competitor.
  • the binding protein binds the labelled competitor, the rotational motion of the fluorescent label is reduced and the polarisation signal is high.
  • the plane polarised light becomes depolarised when the unlabelled ADP present in the sample displaces the labelled competitor from the ADP- binding protein.
  • Small ADP-binding proteins which rotate relatively quickly, may be coupled to other macromolecules in order to exagger
  • the detection system can be configured for non-isotopic FRET assays.
  • An indirect FRET detection method is shown in figure 3.
  • a fluorescently labelled competitor of ADP for the ADP-binding protein is added to the sample.
  • the ADP-binding protein is labelled with a second fluorophore and upon binding of the labelled competitor, FRET may occur.
  • Suitable pairs of fluorphores include Eu 3+ with an allophycocyanin or with a small molecule such as Cy 5. However, this is not intended to be limiting and other FRET pairs may be used instead of the ones described above.
  • the FRET signal is suppressed when unlabelled ADP present in the sample displaces the labelled competitor from the ADP-binding protein.
  • a heterogeneous direct binding assay takes the form of that shown in figure 7, except the surface in this case is not impregnated with scintillant.
  • the precursor of ADP is labelled with 3 H, 14 C, 125 I, 32 P or 33 P and the ADP-binding protein is linked to a surface that does not contain scintillant.
  • the ADP-binding protein is immobilised either covalently or non-covalently to the surface which may be composed of nitrocellulose, paper or other fibrous materials, including glass fibre, or a plastic surface (e.g. a multiwell plate).
  • the ADP-binding protein may be incubated with the sample before or after attachment of the ADP-binding protein to the surface. Following several wash steps the radioactivity associated with the ADP-binding protein is determined by addition of liquid scintillant followed by scintillation counting.
  • An indirect heterogeneous detection method is shown in figure 5. Where the label is an isotope the method is similar to that described for indirect SPA, except that the surface is not impregnated with scintillant. A broader range of isotopes may be used than in the case of SPA, and in a preferred embodiment the competitor is labelled with 3 H, 14 C, 125 I, S, P or P.
  • the ADP-binding protein is immobilised either covalently or non- covalently to the surface.
  • the surface may be nitrocellulose, glass fibre, plastic (e.g. multiwell plate) or some other material.
  • the ADP-binding protein may be incubated with the sample and the radiolabelled competitor before or after attachment to the surface. Following several wash steps the radioactivity associated with the binding protein is determined by addition of liquid scintillant followed by scintillation counting. The signal is inversely related to the amount of the direct product generated in the conversion reaction.
  • An indirect ELISA detection method is also possible.
  • a labelled competitor of ADP for the ADP-binding protein is added to the sample and the ADP- binding protein is attached to a surface.
  • the ADP-binding protein may be incubated with the sample either before or after attachment to the surface, which may be by covalent or non-covalent means.
  • the attachment to the surface may be by passive adsorption or may exploit specific structural features on the binding protein or tags introduced translationally or post-translationally.
  • immobilisation may be via immobilised protein A or protein G or another antibody. Following several wash steps the amount of labelled competitor associated with the binding protein is determined.
  • the label is peroxidase or alkaline phosphatase, which allows colorimetric and fluorimetric readouts depending on the particular substrate used.
  • the label on the competitor may also be biotin to allow labelled streptavidin conjugates to be used for detection, or a fluorescent label. It will be obvious to those of ordinary skills that other variations are possible.
  • the ADP-binding protein is precipitated by the addition of a precipitating agent.
  • a precipitating agent may include but is not limited to polyethylene glycol or antibodies that recognise the binding protein.
  • beads coated with protein A or protein G may also be used precipitate the binding protein.
  • precipitation reactions may exploit the chemical and physical properties of the tag rather than the properties of the binding protein per se. The extent of binding of labelled ADP or the labelled competitor to the precipitated binding protein is determined by measuring the amount of label remaining in the supernatant or in a washed precipitate.
  • the ADP assay will often comprise a step of ATP modification.
  • Present methods for measuring ADP in the presence of ATP often involve conversion of the triphosphate to AMP. This allows enzymatic transformation of residual ADP into molecules (ATP or pyruvate) that may then be measured using auxiliary enzymes. These enzymatic methods are both tedious and time consuming. The method used by Schultz et al, Anal. Biochem. 215: 302, 1993) requires centrifugation and heat inactivation steps.
  • ATP may be modified by enzymatic transformation such that the modified compound cannot bind to the ADP-binding protein.
  • ATP may be extensively modified by a series of enzymatic reactions to prevent binding of the modified compound to the ADP-binding protein. In a preferred embodiment of the assay, however, only a single enzyme is required.
  • ATP present in the sample is modified via a pathway that does not involve the production of ADP to generate metabolites that have little or no affinity for the ADP- binding protein. Pathways that do not involve the production of ADP are described below.
  • ATP is removed using methionine adenosyl transferase (MAT) (EC 2.5.1.6).
  • MAT is attractive because it displaces the whole triphosphate chain of ATP thus abolishing, in one step, the reactivity of binding proteins that require both the adenosine moiety and either the ⁇ or ⁇ / ⁇ phosphate groups of DPCoA/ADP.
  • MAT itself also degrades the tripolyphosphate chain to pyrophosphate (PPi) and inorganic phosphate (Pi).
  • MAT is found in all species and catalyses the reaction between ATP and L-methionine to generate S-adenosylmethionine (AdoMet), which is an important source of methyl groups for a variety of in vivo reactions.
  • AdoMet S-adenosylmethionine
  • a number of different isozymes have been described which differ with respect to tissue distribution and kinetic properties (Okada et al, Biochemistry 20: 934, 1981; Liau et al, Cancer Res. 37: 427, 1977).
  • Three forms of MAT are known and three different systems of classification are in use.
  • MAT I, MAT II and MAT III correspond to ⁇ , ⁇ , and ⁇ and, with reference to the substrate L- methionine, to the 'intermediate Km' isozyme, 'low Km' isozyme and 'high Km' isozyme, respectively (for review see Tabor and Tabor Adv. Enzymol 56:251, 1984).
  • MAT I and MAT m are strikingly different it is now known that both represent different oligomeric states of the same subunit (Suma et al, J. Biochem 100: 67, 1986; Cabrero et. al., Eur. J. Biochem. 170: 299, 1987).
  • MAT I may be converted into MAT III by treatment with LiBr and the properties of the form generated by treatment with LiBr are identical to that of the MAT III purified from liver (Cabrero and Alemany, Biochim. Biophys. Acta 952: 277, 1988).
  • MAT II has about 65% sequence homology to MAT I/MAT III and is distinguished kinetically by its sensitivity to product inhibition by AdoMet and PPi, and to inhibition by DMSO.
  • MAT III is only weakly inhibited by AdoMet and PPi, and is markedly stimulated by DMSO.
  • MAT I is weakly stimulated by DMSO. Since DMSO is the solvent invariably employed to dissolve test compounds for HTS assays, MAT III is a particularly preferred form of MAT for eliminating ATP.
  • MAT III has been purified from a number of sources including rat liver (Cabrero et al, Eur. J. Biochem. 170: 299, 1987; Suma et al, J. Biochem. 100: 67, 1986), sheep liver (Xue et al, Biochem t 18: 525, 1989), bovine brain (Mitsui et al, J Biol. Chem. 263, 11211, 1988), human lymphocytes (Kotb and Kredich, J. Biol. Chem. 260: 3923, 1985), Bakers' yeast (Chiang and Cantoni, J. Biol. Chem.
  • MAT I/MAT III The gene encoding MAT I/MAT III is termed MAT1A and the genes encoding the two subunits of MAT II are designated MAT2A and MAT2B (Kotb et al, Trends Genet. 13: 51, 1995). Nucleotide sequences for MAT isozymes have been obtained for over twenty species (see Chiang et al, Biochem. J. 344: 571, 1999, and references contained therein).
  • the liver enzyme has been recombinantly expressed in E. coli (Lopez-Nara et al, Protein Exp. Purif. 19: 219, 2000; Alvarez et al, Biochem. J. 301: 557, 1994) and crystal structures of the recombinant E. coli enzyme have also been obtained (Gilliland et al, J. Biol. Chem. 258: 6963, 1983; Fu et al, J. Biomol. Struct. Dyn. 13: 727, 1996). It will be evident to one of normal ability that MAT enzymes may be purified from other sources and the gene cloned and expressed from other species and tissues for use in the present invention.
  • a MAT enzyme may be used in combination with pyrophosphatase (PPase) to degrade PPi.
  • PPase pyrophosphatase
  • This is particularly useful in the case of MAT II if high concentrations of ATP are present because inhibitory amounts of PPi might otherwise be produced.
  • the product of the PPase reaction, Pi is a relatively weak inhibitor at concentrations likely to be generated from unutilised ATP (usually 0.01-1 mM) in most enzyme catalysed ADP-forming reactions.
  • Enzymes that degrade ATP to AMP may be used in the present invention, especially in combination with an enzyme or enzymes that further degrade AMP. Some of these enzymes are described below, although it will be apparent to one skilled in the art that any enzyme capable of degrading AMP may be used in an embodiment of the invention where ATP is degraded to AMP.
  • ATP sulphurylase catalyses a reaction between ATP and inorganic sulphate in which adenosine phosphosulphate and Pi are produced.
  • molybdate a molbdolysis reaction occurs in which PPi is formed together with an unstable intermediate, AMP-Mo, which immediately breaks down non-enzymatically to give AMP and molybdate (Wilson and Bandurski J. Biol. Chem. 233: 975, 1958).
  • ATP sulphurylase from Bakers' yeast is commercially available (Sigma) and the enzyme has been purified from a variety of sources, including rat liver (Yu et al, Arch. Biochem. Biophys., 269: 156, 1989). Human and mouse ATP sulphurylase have been cloned and recombinantly expressed (Yanagisawa et al, Biosci. Biotechnol. Biochem. 62: 1037, 1998; Li et al, J Biol. Chem,. 270: 29453, 1995). It will be obvious to those of normal ability that the enzyme from other sources may also be used in the present invention.
  • the E.coli enzyme has been overexpressed and purified (Sugiyama et al, Biosci. Biotechnol. Biochem. 56: 376, 1992).
  • AMP generated by an AMP-forming enzyme may be further metabolised using one of three different pathways (see below) to adenosine and PPi, inosine and Pi, or adenine and ribose-5-phosphate.
  • adenosine PPi and Pi
  • PPi and Pi which is the end point achieved with a single MAT enzyme.
  • Inclusion of PPase may again be used to degrade PPi further to Pi.
  • a large number of 5'-nucleotidase enzymes have been purified, characterised, cloned and expressed, and some are commercially available (e.g. enzymes from bovine liver and Crotalus atrox venom; Sigma).
  • the nucleotide specificity may vary markedly between tissues and species and a 5'-nucleotidase that acts efficiently on AMP is preferred for this particular route of degradation.
  • the adenosine thus formed may be converted into inosine by the action of adenosine deaminase (ADA), which is also commercially available.
  • ADA adenosine deaminase
  • AMP deaminase (EC 3.5.4.6) catalyses the reaction of AMP to IMP and has been used to remove AMP from rabbit reticulocyte lysates (Mosca et al, Biochemistry, 22: 346, 1983).
  • the amine on the 6-position of the adenine moiety is converted to a ketone with the release of ammonia.
  • the same modification occurs in the ADA reaction described above.
  • the enzyme from Aspergillus is commercially available (Sigma).
  • a 5' nucleotidase that acts on IMP as found in and purified from yeast (Itoh, Biochem. J. 298: 593, 1994), inosine and Pi are generated.
  • AMP nucleosidase splits the C-N bond releasing ribose 5-phosphate and adenine and provides an alternative means of completing the modification of ATP to abolish antibody reactivity.
  • AMP nucleosidase is the major enzyme of AMP catabolism in E. coli, from which it has been purified (Leung and Schramm, J. Biol. Chem. 255: 10867, 1980).
  • the E.coli enzyme has also been cloned, sequenced and overproduced in Azotohacter vinelandi and E.coli (Leung et al, Biochemistry 28: 8726, 1989).
  • the yeast enzyme has been cloned and expressed in E.coli (Meyer et al, Biochemistry 28: 8734, 1989).
  • the step of modifying ATP such that the modified compounds are incapable of binding the ADP-binding protein may also be used to terminate an ADP-forming reaction that requires ATP.
  • the amounts of MAT I, MAT II or MAT III and L-methionine added to the sample may be optimised to ensure rapid conversion of ATP to Adomet, PPi and Pi.
  • the concentration of DMSO may be increased at the end of the ADP- forming reaction to accelerate the removal of ATP and/or to inhibit the ADP-forming enzyme.
  • DMSO may contribute to terminating the ADP-forming reaction. It is also possible to dilute the components of the ADP-forming reaction and/or adjust the composition of the buffer to aid in rapidly halting the ADP-forming reaction. The time needed to stop the ADP-forming reaction is preferably short in comparison with the duration of the reaction.
  • DPCoA 3'-Dephosphocoenzyme A
  • KLH maleimide-activated keyhole limpet haemocyanin
  • KLH maleimide-activated keyhole limpet haemocyanin
  • the resulting KLH conjugate is dialysed against two changes of 2L of 0.9% NaCl over a period of 48 hours at 4°C.
  • the conjugate may be used as an immunogen for generating an anti-ADP antibody.
  • ATP or ADP is directly coupled to KLH via lysine residues using standard carbodiimide chemistry.
  • nucleotides are reacted with cystamine, followed by reduction and purification of the thiolated product, which is then coupled to maleimide-activated KLH.
  • Coupling with cystamine is presumed to be mediated via the gamma-phosphate (where ATP is used) or beta-phosphate (where ADP is used) as the products are resistant to alkaline phosphatase.
  • a 50/50 mixture of the ATP/KLH conjugate generated by direct coupling and the ATP/KLH conjugate generated by reaction with cystamine is used as an immunogen for generating an anti-ATP antibody.
  • a 50/50 mixture of the ADP/KLH conjugate generated by direct coupling and the ADP/KLH conjugate generated by reaction with cystamine is used as an immunogen for generating an anti-ADP antibody.
  • the immunogens are used in a 77-day regimen for the production of specific antibodies in rabbits with immunisations on days 0, 14, 28, 42, 57 & 70.
  • the antigen is emulsified with Freund's complete adjuvant for the first immunisation and incomplete adjuvant for subsequent immunisations.
  • the serum is harvested on day 77 and either used without purification or used after purification of the specific antibody fraction on affinity columns (Example lb).
  • Antibodies generated by the method disclosed in Example 1(a) are isolated from sera using a DPCoA affinity column.
  • the column is producing by coupling 3-30 ⁇ mole DPCoA to 3 ml of epoxy-activated Sepharose (Amersham) according to the manufacturer's recommended procedure.
  • the crude antibody preparation/serum or an IgG fraction isolated on protein A-Sepharose is applied at 3 ml/h to the DPCoA affinity column, after which the column is washed with 50 mM Tris/150 mM NaCl, pH 7.5, prior to elution with 50 mM glycine pH 2.3.
  • the material emerging from the column is immediately returned to pH 7.5 by addition of IM Tris base. Peak fractions are located using protein assays and are pooled and dialysed against 100 mM sodium phosphate, pH 7.5.
  • antibodies generated by the method of Example 1(a) may be isolated from sera using commercially available ATP Sepharose resin (Upstate). 5 ml serum is mixed with 5 ml of 50 mM Tris/150 mM NaCl/ 10 mM MgCl 2 , pH 7.5 and 1 ml of ATP Sepharose resin. After 1 h of constant mixing the beads are poured into a column and washed with 60 ml buffer. Fractions containing bound antibody may be eluted with 50 mM glycine, pH 2.3 and neutralised with 1/10 volume of 0.5 M Tris base.
  • mercaptoethanol is added in excess with respect to the iodoacetyl groups to quench the reaction.
  • Streptavidin-coated ELISA plates are incubated with 50 ⁇ l of 1/1000 diluted unpurified biotinylated DPCoA per well in 50 mM Tris/150 mM NaCl pH 7.5 (buffer A) for 30-60 min at 22°C. Capture by streptavidin orients the ADP moiety of DPCoA for subsequent binding reactions. Finally, the plate is flicked or aspirated to remove excess liquid and then blocked for 30 min with buffer A containing 0.1% BSA (blocking buffer). An ATP-coated plate is prepared similarly using a commercially available biotinylated analogue of ATP (Affinity Labelling Technologies Inc).
  • Example 1(c) The blocked ELISA plates generated as described in Example 1(c) are washed and incubated with a test serum serially diluted in blocking buffer for 30 min at 22°C.
  • the test serum was produced by a method disclosed in Example 1(a). After the primary incubation, the plates are washed and processed using standard ELISA methodology with a secondary HRP-conjugate. HRP activity is detected using 50 ⁇ l of 1 mg/ml 2,2'- azino-bis-(3-ethylbenzthiazoline sulfonate) (ABTS) substrate in 50 mM sodium acetate, pH 5, containing 1 ⁇ l H 2 O 2 per ml.
  • ABTS 2,2'- azino-bis-(3-ethylbenzthiazoline sulfonate
  • the extent of specific binding of the serum to ADP is determined by comparing the signal at 405 nm for ELISA plates coated with DPCoA, ELISA plates coated with ATP, and control ELISA assay in which no antibody or excess competing ligand was used. The results showed that different sera produced from different host aninals had different degrees of selectivity. Representative results are shown in figure 8.
  • Figure 8 shows the results of such an ELISA assay using three different sera.
  • Serum 22 was raised using DPCoA linked via its sulphydryl group to maleimide-activated keyhole limpet haemocyanin
  • serum 30 was raised using a 50/50 mixture of ADP/KLH conjugates produced by direct conjugation of ADP with keyhole limpet haemocyanin and by conjugation via a cysteamine linker to maleimide activated keyhole limpet haemocyanin
  • serum 23 was raised using a 50/50 mixture of ATP conjugate prepared by direct conjugation of ATP with keyhole limpet haemocyanin and by conjugation via a cysteamine linker to maleimide activated keyhole limpet haemocyanin.
  • the ELISA assay shows that serum 22 preferentially binds to the DPCoA-coated plate ( Figure 8(a)). Accordingly such an antibody is a selective for ADP/DPCoA.
  • Serum 30 binds to both the DPCoA-coated plate and the ATP-coated plate ( Figure 8(b)).
  • Serum 23 binds preferentilly to the ATP-coated plate ( Figure 8(c)).
  • the control shows that there is a low level of HRP activity where no serum is added (Figure 8(d)).
  • the selectivity of antibodies may be further analysed using the DPCoA ELISA method described above with minor modification.
  • An anti-ADP antibody and an anti-ATP antibody produced according to the methods disclosed in Example 1(a) and 1(b) was incubated in the presence of ATP and various metabolites of ATP, including ADP, AMP, adenosine, ribose-5-phosphate, s-adenosyl methionine and pyrophosphate (Ppi).
  • ELISA assays were then conducted according to the method disclosed in Example 1(d). IC 50 values for these metabolites may be obtained by testing a range of dilutions. In this way chemicals that are likely to cause interference with particular anti-DPCoA antibodies or with other binding proteins are readily identified and a procedure to remove the interference can be implemented (see example 4).
  • Figure 9 shows the results of an ELISA assay using an anti-ADP antibody and an anti- ATP antibody in the presence of metabolites.
  • Ab24 was raised using the ATP/KLH immunogen described in example 1(a).
  • Ab30 was raised using the ADP/KLH immunogen described in example 1(a).
  • Ab24 is displaced from the DPCoA plate with approximately equal efficiency by ATP, ADP, AMP, Adenosine and S-adenosyl methionine with IC 50 values in excess of 100 ⁇ M ( Figure 9(a)). Ribose -5-phosphate and pyrophosphate do not displace Ab24 ( Figure 9(a)).
  • S-adenosyl methionine, pyrophosphate, and inorganic phosphate will not interfere with ADP assays using Ab30. This is advantageous since these are products of the MAT III enzyme, which may be used to remove contaminating ATP.
  • DPCoA immobilised on a column may be used to identify DPCoA/ ADP- binding proteins present in cell and tissue extracts using affinity chromatography.
  • a separate column coupled with an unrelated thiol compound is used as a control.
  • Binding proteins eluted from the DPCoA and control affinity columns using ADP or low pH are characterised by microsequencing and mass spectrometry. Biomolecules that are likely to show superior performance in ADP assays may be positively selected by mimicking the actual conditions that will be used in assays. For example, selection of binding proteins that will perform well in conjunction with Mat III is achieved by carrying out chromatography in the presence of 10% DMSO.
  • ADP-forming reaction which may be catalysed for example by kinases, ATPases or ligases, and the nature of the detection method employed the basic competitive assay procedure is the same.
  • the analogue of ADP which may be DPCoA, is labelled with radioactivity, fluorescent groups or other tags and used in conjunction with an ADP-binding protein in competition assays.
  • ADP generated by a reaction competes with the labelled analogue and inhibits the interaction of the labelled analogue with the binding protein. Nucleotides that interfere with the system are removed using enzymatic methods.
  • the primary consideration therefore is the compatibility of the buffer used in the ADP-forming reaction with the subsequent ADP detection step, and not the nature of the ADP-forming enzyme or its substrate.
  • the sensitivity of the detection system is also important and this may need to be tuned (see example 2b) to suit each set of conditions.
  • the ELISA assay disclosed in Example 1(d) can be used to identify assay components that may interfere with an ADP assay coupled to a kinase assay or other ADP forming assay.
  • Figure 10 shows the results of an assay for determining the effects of additives commonly found in drug screening assays for kinases, or which are required for degradation of ATP using MAT III. These include 10% DMSO (enhances MAT m activity), 12.5 mM EDTA, 2.5 mM DTT, 5 mM N-ethylmaleimide and 5 mM ATP.
  • Figure 10 shows that DMSO and N-ethylmaleimide have little or no effect on antibody binding. Partial inhibition is observed with EDTA and DTT. Excess ATP abolishes the signal to the level of the control with no antibody.
  • Ab30 is a polyclonal antibody, it may contain subpopulations of antibodies that are sensitive to certain agents. A sub-population that requires metal ions for binding might explain the effect of EDTA and DTT observed in this experiment. Clearly, certain populations may be purified if required, and the effects of DTT may be overcome by the addition of N-ethylmaleimide to the antibody.
  • SPA assays may be carried out using [ 3 H]ADP, which is commercially available (Amersham or NEN) as an exogenous competing agent.
  • the ADP-forming reaction is carried out in a white microplate (e.g. Optiplate) and the detection cocktail which comprises protein A-SPA beads, anti-DPCoA antibody and [ 3 H]ADP is then added.
  • the sensitivity of the assay may be adjusted by altering the amount of competing radioligand or the volume of the assay. If an enzyme such as MAT III is required (example 4) the buffer also contains an appropriate amount of DMSO (taking account of any solvent used to dissolve test compounds in the ADP-forming reaction), L- methionine, and other essential components.
  • EDTA in excess over Me 2+ ions
  • EDTA excess over Me 2+ ions
  • this SPA assay has significant advantages over other assays because the emission spectrum of [ 3 H] is far better suited to SPA bead technology than 33 P.
  • the assay is set up using the competition assay format described above.
  • a fluorescently labelled analogue of DPCoA is required, together with a soluble ADP- binding protein in free solution.
  • the binding protein is tested over a range of concentrations using different fixed amounts of the fluorescein analogue to determine suitable conditions for FP.
  • Fluorescein-DPCoA is prepared by reacting DPCoA with a thiol-reactive analogue of fluorescein, 6-iodoacetamidofluorescein (Pierce).
  • the fluorescently labelled analogue is isolated using standard purification methods.
  • the FP technique is highly sensitive and ideal for reactions in which the level of ADP production is expected to be relatively low.
  • the concentrations of fluorescent ligand cannot be raised in this assay to reduce sensitivity since excess ligand causes depolarisation.
  • DPCoA is coupled to a maleimide-activated plate (Pierce) and the purified DPCoA/ADP-binding protein is labelled with HRP using standard methods.
  • the primary ADP-forming reaction is performed in the plate (or subsequently transferred to it) and the HRP-labelled binding protein is added.
  • ADP generated in a primary reaction inhibits the interaction of the HRP-labelled binding protein with the immobilised DPCoA.
  • the assay may also be inverted such that an unlabelled antibody or other ADP-binding protein is immobilised in microwell plates.
  • the interaction with the plate surface may be passive.
  • the binding protein may be oriented on the surface by protein A or with an antibody that recognises a specific tag on the binding protein.
  • the plate is blocked and the primary ADP-forming reaction is reaction is either performed in the plate or subsequently transferred to it.
  • Biotinylated DPCoA (example lc) is added and competes with ADP for the immobilised binding protein. After a period to allow equilibration to occur the plate is washed and biotin-DPCoA is detected using a streptavidin-HRP conjugate, with detection of HRP as noted in example Id.
  • the sensitivity of the assay may be increased or decreased by adjusting the amount of biotinylated DPCoA.
  • Figure 11 shows the results of a competition ELISA assay.
  • Ab30 is used as the ADP binding protein and varying amount of ADP are added to wells of a 96-well plate.
  • ADP is in competition with immobilised biotinylated DpCoA for the antibody.
  • the concentration of ADP added increases, the Ab30 is displaced from the DPCoA and the absorbance observed decreases. There is almost complete displacement of the antibody at 5 ⁇ M ADP and the IC 50 value is ⁇ 1 ⁇ M.
  • [ 3 H]ADP is generated by the ADP-forming reaction using a radiolabelled precursor, such as [ 3 H]ATP.
  • a radiolabelled precursor such as [ 3 H]ATP.
  • SPA beads coated with the DPCoA/ADP-binding protein are added to the assay and the signal generated is directly related to the amount of [ 3 H] ADP formed.
  • MAT-III is the preferred form of enzyme for degrading ATP
  • Rat liver supernatant is obtained after homogenisation of 5g of tissue in 5 volumes of 50mM Tris/lOOmM KCl/lOmM EDTA/lmM DTT, pH 8.0. The supernatant is applied directly to a 2ml (packed volume) Phenyl-Sepharose column which is then washed with lOmM Tris/lmM EDTA/lmM DTT, pH 8.0. This buffer elutes most of the total protein, including MAT-I.
  • Bound proteins are eluted with wash buffer containing 0.5% Tween 20 and immediately captured on or applied to a 1ml Q Sepharose column equilibrated inlOmM Tris/lmM DTT, pH 8.0 (equilibration buffer). A linear salt gradient 0-400 mM in equilibration buffer is applied and MAT-III elutes at around 300 mM salt.
  • PCR primers (all written 5' to 3') were: forward, ATG AAT GGA CCG GTG GAT GGC TT.
  • Two reverse primers were synthesised so as to generate PCR products encoding MAT proteins either with or without a C-terminal His 6 tag. These primers were CTA ATG ATG ATG ATG ATG ATG AAA TAC AAG CTT CCT GGG AAC and CTA AAA TAG AAG CTT CCT GGG AAC respectively.
  • PCR products were cloned into the EcoRN site of PetBlue-1 ( ⁇ ovagen) and ligated products were transformed into ⁇ ovaBlue bacteria ( ⁇ ovagen). Plasmid D ⁇ As from clones in the correct orientation were transformed into Tuner (DE3) pLacI cells ( ⁇ ovagen) for induction of expression with 1 mM IPTG.
  • MAT proteins both with and without a his 6 tag, formed inclusion bodies in the induced Tuner (DE3) pLacl cells.
  • the inclusion bodies were prepared and washed essentially as described by Lopez-Nara et al. (Protein Expression and Purification 19, 219-226, 2000) except that unfolding was carried out using 50 mM Tris/10 mM DTT/8M urea/lOmM glycine, pH 8.0.
  • the volume of unfolding buffer used was 1-2% of the volume of the original bacterial culture.
  • the optimum refolding buffer and dilution factor was then determined on a 50 ⁇ l-lml scale prior to refolding of the bulk mixture.
  • the enzyme was refolded by rapid dilution in 50mM Tris pH 8.0, 50mM Tris pH 8.0/10% DMSO, 50mM Tris pH 8.0/10 mM DTT/10% DMSO, 50mM Tris pH 8.0/lOmM DTT/10% DMSO/30 mM KC1, 50mM Tris pH 8.0/10 mM DTT/10% DMSO/10 mM MgCl 2 and 50mM Tris pH 8.0/10 mM DTT/10% ethylene glycol.
  • the refolded samples were assayed for activity in buffer B (50 mM Tris/250 mM KC1, 1 mM DTT, 1 mM ATP, 1 mM L methionine 10 mM MgC12 10% DMSO pH 8.0).
  • buffer B 50 mM Tris/250 mM KC1, 1 mM DTT, 1 mM ATP, 1 mM L methionine 10 mM MgC12 10% DMSO pH 8.0.
  • the release of inorganic phosphate (Pi) was measured as a phosphomolybdate complex using malachite green according to the method of Geladopoulos and colleagues (Geladopoulos et. al., Anal. Biochem. 192, 112-116, 1991).
  • the refolded recombinant MAT III enzyme has similar properties to the MAT enzyme produced from rat liver (see Tabor and Tabor, Adv. Enzymol., 56: 251, 1984). Thus, it is hypothesised that the refolded recombinant MAT III enzyme has a similar mechanism of action to the characterised rat enzyme.
  • MAT III purified from rat liver or recombinantly expressed using the methods described in Examples 3(a) and 3(b) may be used to remove ATP present in ADP-forming reactions, ifthe ATP interferes with the detection system.
  • the ADP-forming reaction is terminated by the addition of a detection cocktail containing DPCoA/ADP -binding protein, MAT III, L-methionine and DMSO.
  • MAT III is stimulated by potassium ions and inclusion of KC1 may therefore be required.
  • competition assay formats a labelled analogue of either DPCoA or ADP is also included.
  • the components of the cocktail must not inhibit the binding of ligands to the ADP-binding protein, but preferably the composition is unfavourable for the primary ADP-forming reaction. Since a substantial volume addition is possible at the last step of the procedure there is considerable scope to adjust the reaction conditions as necessary.
  • the amount of MAT III required for any new set of assay conditions must be determined empirically.
  • the assay buffer (excluding the ADP-forming enzyme and ATP) is mixed with the detection cocktail in the appropriate ratio and the resulting buffer is used to prepare a set of solutions containing [ 3 H]ATP, [ 3 H] ADP or [ 3 H]AMP with varying amounts of the appropriate unlabelled nucleotide.
  • the range of nucleotide concentrations reflects the range likely to be found in assays of ADP-forming enzymes.
  • One complete set of tubes is required for each concentration of MAT III to be tested.
  • the efficiency with which the detection cocktail to stops the ADP-forming reaction may be determined by including [ 3 H]ATP as a tracer.
  • the detection cocktail is added or an agent known to stop the ADP-forming reaction is added. After 60 minutes the amount of [ 3 H]ADP present is quantified as described above. If the amount of [ 3 H]ADP produced in both cases is the same then the detection cocktail is effective at stopping the ADP-forming reaction. If the amount of [ 3 H]ADP is significantly greater using the detection cocktail the dilution of the ADP-forming enzyme (and thus duration of the assay) may be increased and/or the amount of Mat III may be increased.
  • the production of ADP by the ADP-forming enzyme may be measured in the presence and absence of the test compound.
  • the test compound may interfere with the enzyme(s) used to degrade ATP.
  • the risk of this is reduced because of the significant dilution upon addition of the detection cocktail.
  • Compounds that show up as 'hits' in the HTS screen may be re-screened using a detection cocktail containing a different enzyme system.
  • the initial screen might be carried out with MAT III followed by a counterscreen with ATP- sulphurylase and a 5'-nucleotidase.
  • Compounds that register as hits in both assays are likely to be targeting the ADP-forming enzyme.
  • MAT isozymes from different tissues and species may be combined. If each type alone is present in sufficient quantity to eliminate ATP the probability that false positives will be obtained is significantly reduced.

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Abstract

La présente invention a trait à un dosage ADP dans lequel l'ADP est détecté au moyen d'une protéine de liaison à l'ADP. On peut utiliser un format de dosage direct dans lequel l'ADP étiqueté est généré à partir d'un précurseur étiqueté. Le signal généré par l'ADP étiqueté est détecté lors de la liaison de l'ADP à la protéine de liaison à l'ADP. En variante, dans un mode de réalisation préféré, on peut utiliser un format de dosage indirect dans lequel un compétiteur de l'ADP est présent. Le compétiteur de l'ADP ou la protéine de liaison à l'ADP est étiqueté(e). Un signal généré lors de la liaison du compétiteur à la protéine de liaison à l'ADP, et le signal est réduit en présence de l'ADP. Si l'ATP est présent dans l'échantillon, le dosage peut également inclure une étape de modification de l'ATP de sorte qu'il ne puisse se lier à la protéine de liaison à l'ADP.
PCT/GB2003/004015 2002-09-17 2003-09-17 Dosage adp WO2004027421A2 (fr)

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EP1592813A2 (fr) * 2003-01-30 2005-11-09 BellBrook Labs, LLC Procede relatif a des essais de reactions a transfert de groupe
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US7355010B2 (en) 2003-01-30 2008-04-08 Bellbrook Labs, Llc Assay method for group transfer reactions
WO2010011607A1 (fr) * 2008-07-22 2010-01-28 Promega Corporation Dosage luminescent de la phosphotransférase ou de l’atp hydrolase basé sur la détection de l’adp
WO2010032001A1 (fr) * 2008-09-19 2010-03-25 Medical Research Council Capteur
US8088897B2 (en) 2003-01-30 2012-01-03 BellBrook Labs, Inc. Assay method for group transfer reactions
US9677117B2 (en) 2014-10-08 2017-06-13 Promega Corporation Bioluminescent succinate detection assay

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US7847066B2 (en) 2003-01-30 2010-12-07 Bellbrook Labs, Llc Antibody that binds uridine diphosphate
EP1592813A4 (fr) * 2003-01-30 2006-06-14 Bellbrook Labs Llc Procede relatif a des essais de reactions a transfert de groupe
EP1592813A2 (fr) * 2003-01-30 2005-11-09 BellBrook Labs, LLC Procede relatif a des essais de reactions a transfert de groupe
US8088897B2 (en) 2003-01-30 2012-01-03 BellBrook Labs, Inc. Assay method for group transfer reactions
US7332278B2 (en) 2003-01-30 2008-02-19 Bellbrook Labs, Llc Assay methods for group transfer reactions
US7355010B2 (en) 2003-01-30 2008-04-08 Bellbrook Labs, Llc Assay method for group transfer reactions
WO2006127008A1 (fr) * 2005-05-26 2006-11-30 Bellbrook Labs, Llc Procede d’analyse destine aux reactions a transfert de groupe
EP1869083A1 (fr) * 2005-05-26 2007-12-26 BellBrook Labs, LLC Procede d analyse destine aux reactions a transfert de groupe
WO2010011607A1 (fr) * 2008-07-22 2010-01-28 Promega Corporation Dosage luminescent de la phosphotransférase ou de l’atp hydrolase basé sur la détection de l’adp
US8183007B2 (en) 2008-07-22 2012-05-22 Promega Corporation ADP detection based methods using adenylate cyclase and bioluminescence
CN102159948B (zh) * 2008-07-22 2014-06-25 普罗美加公司 基于adp检测的发光磷酸转移酶或atp水解酶测定
US8802411B2 (en) 2008-07-22 2014-08-12 Promega Corporation ADP detection based luminescent phosphotransferase or ATP hydrolase assay
CN103983634A (zh) * 2008-07-22 2014-08-13 普罗美加公司 基于adp检测的发光磷酸转移酶或atp水解酶测定
CN102159948A (zh) * 2008-07-22 2011-08-17 普罗美加公司 基于adp检测的发光磷酸转移酶或atp水解酶测定
WO2010032001A1 (fr) * 2008-09-19 2010-03-25 Medical Research Council Capteur
US20110294137A1 (en) * 2008-09-19 2011-12-01 Medical Research Council Sensor
US8703913B2 (en) 2008-09-19 2014-04-22 Medical Research Council Sensor
US9677117B2 (en) 2014-10-08 2017-06-13 Promega Corporation Bioluminescent succinate detection assay

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