WO2001096862A2 - Method for increasing luminescence assay sensitivity - Google Patents
Method for increasing luminescence assay sensitivity Download PDFInfo
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- WO2001096862A2 WO2001096862A2 PCT/US2001/018363 US0118363W WO0196862A2 WO 2001096862 A2 WO2001096862 A2 WO 2001096862A2 US 0118363 W US0118363 W US 0118363W WO 0196862 A2 WO0196862 A2 WO 0196862A2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/90241—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/968—High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/975—Kit
Definitions
- the present invention relates generally to the fields of cell biology and molecular biology.
- this invention relates to methods, compositions and kits for increasing the sensitivity of a luminescence assay measurement.
- Reporter molecules are routinely used to monitor molecular events in the fields of biology, biochemistry, immunology, cell biology and molecular biology.
- reporter molecules are employed in assays where the levels of the reporter molecule are due to transcription from a specific promoter linked to the reporter molecule. These assays can be used to study eukaryotic gene expression, receptor activity, transcription factors, intracellular signaling, mRNA processing, protein folding, and the like.
- Reporter molecules that are typically used in such assays include radioactive isotopes, fluorescent agents, enzymes, and luminescent agents. See for example, Akhavan-Tafti, et al, in: Bioluminescence and Chemiluminescence. Fundamentals and Applied Aspects.
- Firefly luciferase is a 61 kDa monomeric protein that does not require post-translational processing for enzymatic activity. Thus, it functions as a genetic reporter immediately upon translation. Photon emission is achieved through oxidation of beetle luciferin in a reaction that requires ATP, Mg 2+ and O 2 ( Figure 1).
- Renilla luciferase is a 36 kDa monomeric protein that is composed of 3% carbohydrate when purified from its natural source, Renilla reniformis. Like firefly luciferase, post-translational modification o ⁇ Renilla luciferase is not required for its activity, and it functions as a genetic reporter immediately following translation.
- the luminescent reaction catalyzed by Renilla luciferase utilizes O 2 and coelenterate-luciferin, also called coelenterazine ( Figure 2). Luminescent reactions can be used to detect very small quantities of a particular analyte, the substance being identified and measured in an analysis.
- luminescent reactions can be used to detect and quantify proteases, lipases, phosphatases, peroxidases, glycosidases, and various metabolites such as ATP or NADH. Luminescent reactions can also be used to detect and quantify analytes through binding interactions, such as those mediated by antibodies and nucleotide probes. Typically, luminescent reactions can be used to detect less than lxl 0 "16 moles of analyte in a sample, often less than lxlO "19 moles. In luminescence, commonly detected analytes are the luciferases, especially firefly luciferase and Renilla luciferase.
- luminescent enzymes used as analytes include, but are not limited to, aequorin, Vargula luciferase, and other marine luciferases.
- Luminescence When using luminescence to measure an analyte, it is preferred that little or no light is produced by reactions that are not dependent on the presence of the analyte. This is the case with firefly luciferase. Under typical firefly luciferase assay conditions, luminescence cannot be detected when the firefly luciferase is not present. In contrast to assays employing firefly luciferase, light can generally be detected in Renilla luciferase assay systems when the Renilla luciferase is not present. Luminescence that is not dependent on the catalytic activity of a luminescent enzyme is termed autoluminescence. For example, auto luminescence can be caused by spontaneous oxidation of the lumino genie substrate coelenterazine.
- Luminescence that is not dependent on the on the presence of an analyte can limit the usefulness of an analytical assay by reducing the ability to accurately measure the quantity of light resulting from the activity of the analyte.
- the sensitivity of luminescent assays containing coelenterazine or its structural analogs is reduced due to autoluminescence.
- the addition of various components to the assay system such as lipids (especially above the critical micelle concentration or CMC), hydrophobic proteins (especially those with a defined three-dimensional structure), and cells or other biological materials containing hydrophobic microenvironments, can greatly increase autoluminescence.
- Assay sensitivity may also be reduced by luminescence from an unrelated luminogenic molecule.
- the unrelated luminogenic molecule may be present due to contamination of the analytical assay, or due to a separate analytical luminescence assay performed in the same reaction mixture. In either case, the sensitivity of an analytical luminescence assay could be improved by reducing the luminescence that is not dependent on the presence of the analyte.
- the sensitivity of luminescence assays can be improved by carrying out the assay in the presence of one or more organic compounds that reduce analyte-independent luminescence.
- Applicant has unexpectedly discovered that the analyte-independent luminescence can be reduced without similarly reducing analyte-dependent luminescence.
- the analyte-dependent luminescence is reduced by a lower fold than the analyte-independent luminescence, or the analyte dependent luminescence remains about the same or increases.
- the invention provides a method for increasing the sensitivity of a luminescent assay comprising carrying out the assay in the presence of an organic compound that reduces luminescence that is not dependent on the presence of an analyte by a factor of at least about 10 fold, and that reduces luminescence that is dependent on the presence of an analyte by less than about 7 fold.
- the invention also provides a method for increasing the sensitivity of a luminescent assay comprising carrying out the assay in the presence of an organic compound that reduces luminescence generated by . luminogenic molecules not bound to an enzyme by at least about 10 fold, and that reduces the luminescence generated by luminogenic molecules bound to an enzyme by less than about 7 fold.
- the invention also provides a method for increasing the sensitivity of a luminescent assay comprising carrying out the assay in the presence of an organic compound that reduces autoluminescence by at least about 10 fold, and that reduces luminescence that is dependent on the presence of an analyte by less than about 7 fold.
- the invention also provides a method for increasing the sensitivity of a luminescent assay comprising carrying out the assay in the presence of an organic compound that comprises a selenium atom.
- the invention also provides a method for increasing the sensitivity of a luminescent assay comprising carrying out the assay in the presence of an organic compound that comprises a carbon-selenium bond.
- the invention also provides a method for increasing the sensitivity of a luminescent assay comprising carrying out the assay in the presence of an organic compound that comprises a carbon-selenium single bond (C-Se),
- the invention also provides a method for increasing the sensitivity of a luminescent assay comprising carrying out the assay in the presence of an organic compound that comprises a carbon-sulfur double bond (OS).
- OS carbon-sulfur double bond
- the invention also provides a method for increasing the sensitivity of a luminescent assay comprising carrying out the assay in the presence of an organic compound that comprises a carbon atom bound to both a selenium atom and a nitrogen atom.
- the invention also provides a method for increasing the sensitivity of a luminescent assay comprising carrying out the assay in the presence of an organic compound that comprises a carbon atom bound to both a sulfur atom and a nitrogen atom.
- the invention also provides a method for increasing the sensitivity of a luminescent assay comprising carrying out the assay in the presence of an organic compound that comprises a sulfur atom bound to two carbon atoms, wherein the analyte-independent luminescence is reduced by at least about 10 fold.
- the analyte-dependent luminescence is reduced by less than 7 fold.
- the invention also provides an assay kit comprising packaging material containing 1) a luminogenic substrate of a luminescent enzyme, or a luminogenic enzyme; and 2) an organic compound for increasing the sensitivity of a luminescent assay.
- the organic molecule is capable of 1) reducing the luminescence that is not dependent on the presence of an analyte by a factor of at least about 10 fold, and reducing the luminescence that is dependent on the presence of an analyte by less than about 7 fold; 2) reducing the luminescence generated by luminogenic molecules not bound to an enzyme by at least about 10 fold, and reducing the luminescence generated by luminogenic molecules bound to an enzyme by less than about 7 fold; or 3) reducing autoluminescence by at least about 10 fold, and reducing luminescence that is dependent on the presence of an analyte by less than about 7 fold.
- the invention also provides novel compounds disclosed herein that are useful to increase the sensitivity of a luminescent assay.
- FIG. 1 illustrates chemiluminescent reaction catalyzed by firefly luciferase.
- FIG. 2 illustrates chemiluminescent reaction catalyzed by Renilla luciferase.
- FIG. 3 illustrates a dioxetane intermediate in the colenterazine autoluminescence pathway.
- FIG. 4 shows representative compounds (1-11) that reduce autoluminescence.
- halo denotes fluoro, chloro, bromo, or iodo.
- Alkyl alkoxy, “alkenyl”, “alkynyl”, etc. as used herein denote both branched and unbranched groups; but reference to an individual radical such as “propyl” embraces only the straight, unbranched chain radical, a branched chain isomer such as “isopropyl” being specifically referred to.
- Aryl denotes a monocyclic or polycyclic hydrocarbon radical comprising 6 to 30 atoms wherein at least one ring is aromatic.
- aryl denotes a phenyl radical or an ortho-fused bicyclic carbocyclic radical having about nine to ten ring atoms in which at least one ring is aromatic.
- Heteroaryl encompasses a radical of a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(X) wherein X is absent or is H, O, (C,- C 4 )alkyl, phenyl or benzyl, as well as a radical of a polycyclic ring comprising 8 to 30 atoms derived therefrom.
- heteroaryl encompasses a radical attached via a ring carbon of a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(X) wherein X is absent or is H, O, (C r C 4 )alkyl, phenyl or benzyl, as well as a radical of an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benz-derivative or one derived by fusing a propylene, trimethylene, or tetramethylene diradical thereto.
- analyte is a substance to be detected in a test sample.
- commonly detected analytes include the luciferases, especially firefly luciferase and Renilla luciferase.
- Other luminescent enzymes used as analytes include, but are not limited to, aequorin, Vargula luciferase, and other marine luciferases.
- luminescent reactions can be used to detect and quantify analytes such as proteases, lipases, phosphatases, peroxidases, glycosidases, and various metabolites such as ATP or NADH.
- Luminescent reactions can also be used to detect and quantify analytes through binding interactions, such as those mediated by antibodies and nucleotide probes.
- analyte-dependent luminescence can be coupled to the activity of a luminescent enzyme.
- alkaline phosphatase AP
- AP alkaline phosphatase
- luciferin is generated by the action of AP, which then yields light by reaction with luciferase.
- the instant invention would allow the AP assay to be run after a separate horseradish peroxidase/luminol reaction.
- analyte AP a compound as described herein, could be added to reduce the analyte- independent luminescence caused by horseradish peroxidase.
- autoluminesence refers to the release of light from a luminogenic molecule that does not result from enzymatic action on the luminogenic molecule.
- the term "increase the sensitivity of a luminescent assay” as used herein means increasing the precision of the assay or improving the ability to measure the presence of a small amount of an analyte with the assay.
- the sensitivity of a luminescent assay can be increased by reducing analyte-independent luminescence.
- analyte- independent luminescence is reduced, and a minimal reduction, no reduction, or an increase in analyte-dependent luminescence results.
- analyte-independent luminesence is preferably reduced by a greater fold than analyte-dependent luminescence.
- the term "luminescent,” as used herein, includes bioluminescence (i.e light produced by a living organism), chemi-luminescence (light produced when a chemical reaction proceeds), and electrochemical- luminescence.
- bioluminescence i.e light produced by a living organism
- chemi-luminescence light produced when a chemical reaction proceeds
- electrochemical- luminescence When the enzyme involved has evolved in an organism by natural selection for the purpose of generating light, or the enzyme involved is a mutated derivative of such an enzyme, the luminescent reactions are also called “bioluminescent reactions” and the enzyme involved is also called a “bioluminescent enzyme.” Examples are firefly luciferase, Renilla luciferase, Cypridina luciferase, Aequorin photoprotein, Obelin photoprotein, and the like.
- luminescent assay or “luminescence assay” includes any assay that generates light based on the presence of an analyte. Such assays include assays that employ one or more luciferase enzymes (e.g. firefly luciferase, Renilla luciferase, Cypridina luciferase, and the like).
- luciferase enzymes e.g. firefly luciferase, Renilla luciferase, Cypridina luciferase, and the like.
- luminogenic enzyme as used herein includes enzymes that catalyze a reaction that produces light, or that lead to the production of light. For example, the term includes firefly luciferase, Renilla luciferase, Cypridina luciferase, Aequorin photoprotein, Obelin photoprotein, and the like.
- a luminogenic molecule refers to a molecule capable of creating light via a chemical reaction (e.g. luciferin, coelenterazine, or a functional analog thereof).
- a luminogenic molecule is either a high energy molecular species (e.g. a stabilized dioxetane), or it is transformed into a high energy molecular species by a chemical reaction.
- the chemical reaction is usually oxidation by oxygen, superoxide, or peroxide.
- the energy within the luminogenic molecule is released by the chemical reaction. Although at least some of this energy is released as photons of light, the energy can also be released in other forms, such as heat.
- the luminogenic molecules that do not yield light disperse their energy through alternative modes, often termed "dark pathways".
- luminogenic molecule not bound to an enzyme includes a luminogenic molecule that is not bound to an enzyme (e.g. firefly luciferase, Renilla luciferase, Cypridina luciferase, and the like) that catalyzes a reaction that produces light.
- an enzyme e.g. firefly luciferase, Renilla luciferase, Cypridina luciferase, and the like
- luminogenic molecules bound to an enzyme includes a luminogenic molecule that is bound to an enzyme that catalyzes a reaction that produces light.
- luminescence that is dependent on the presence of an analyte includes luminescence that results from a chemical reaction that involves an analyte, as well as luminescence that correlates with the presence of an analyte either directly or indirectly.
- luminescence that is not dependent on the presence of an analyte includes luminescence resulting from autoluminescence of a luminogenic substrate as well as luminescence resulting from an unrelated luminogenic molecule present in an assay mixture.
- quench means to reduce the yield of photons from a luminescent reaction.
- the term includes preventing an analyte from being detected or being detectable, and may occur either directly or indirectly.
- Agents that can be used to quench a reaction are known as "quenching agents.”
- Applicant has discovered that it is possible to increase the sensitivity of a luminescent assay by carrying out the assay in the presence of an organic compound that reduces analyte-independent luminescence. This finding is unexpected. Using procedures similar to those described herein, one skilled in the art can identify compounds that are suitable for increasing the sensitivity of a luminescent assay. The structure of the compound is not critical provided the compound is capable of increasing the sensitivity of a luminescent assay.
- compounds that comprise a sulfur atom or a selenium atom are particularly useful for increasing the sensitivity of a luminescent assay.
- the remaining chemical structure of the compound that comprises a selenium atom or a sulfur atom is not critical, provided the structure does not interfere with the function of the compound.
- Preferred compounds have low toxicity at concentrations used in the invention, and can be stored, transported, and disposed of inexpensively.
- Suitable compounds that comprise a carbon sulfur double bond or a carbon selenium double bond include for example compounds of
- R ! and R 2 are each independently hydrogen, (C r C 20 )alkyl, (C 3 -C 8 )cycloalkyl, (C 1 -C 20 )alkoxy, (C 2 -C 20 )alkenyl, (C 2 -C 20 )alkynyl, aryl, heteroaryl, or NR a R b ; or R x and R 2 together with the carbon to which they are attached form a 5, 6, 7, or 8 membered saturated or unsaturated ring comprising carbon and optionally comprising 1, 2, or 3 heteroatoms selected from oxy (-O-), thio (-S-), or nitrogen (-NR C )-, wherein said ring is optionally substituted with 1, 2, or 3 halo, hydroxy, oxo, thioxo, carboxy, (C 1 -C 20 )alkyl, (C 3 -C 3 )cycloalkyl, (C C 20 )alk
- Suitable compounds that comprise a mercapto group include for example compounds of the formula R 3 SH wherein: R 3 is (C 1 -C 20 )alkyl, (C 3 -C 3 )cycloalkyl,(C 2 -C 20 )alkenyl, (C 2 -C 20 )alkynyl, aryl, or heteroaryl; wherein any (C r C 20 )alkyl, (C 3 -C 8 )cycloalkyl, (C 2 -C 20 )alkenyl, or (C 2 - C 20 )alkynyl of R 3 is optionally substituted with one or more (e.g 1, 2, 3, or 4) halo, hydroxy, mercapto oxo, thioxo, carboxy, (C 1 -C 20 )alkanoyl, (C C 20 )alkoxycarbonyl, aryl, heteroaryl, or NR d R e ; wherein R d and R
- R 4 is (C 1 -C 20 )alkyl, (C 3 -C 8 )cycloalkyl,(C 2 - C 20 )alkenyl, (C 2 -C 20 )alkynyl, aryl, or heteroaryl; wherein any (C I -C 20 )alkyl, (C 3 -C 8 )cycloalkyl, (C 2 -C 20 )alkenyl, or (C 2 -C 20 )alkynyl of R 3 is optionally substituted with one or more (e.g 1 , 2, 3, or 4) halo, hydroxy, mercapto oxo, thioxo, carboxy, (C ⁇ -C 2o )alkanoyl, (C 1 -C 20 )alkoxycarbonyl, aryl, heteroaryl, or NR f R g ; wherein R f and R g are each
- Suitable compounds that comprise a carbon-selenium single bond or a carbon sulfur single bond include compounds of formula R 5 - X-R 6 wherein:
- X is -S- or -Se-
- R 5 is (C r C 20 )alkyl, (C 3 -C 8 )cycloalkyl,(C 2 -C 20 )alkenyl, (C 2 - C 20 )alkynyl, aryl, or heteroaryl; and R 6 is hydrogen, (C 1 -C 20 )alkyl, (C 3 - C 8 )cycloalkyl,(C 2 -C 20 )alkenyl, (C 2 -C 20 )alkynyl, aryl, or heteroaryl; or R 5 and R 6 together with X form a heteroaryl; wherein any (C 1 -C 20 )alkyl, (C 3 -C 8 )cycloalkyl, (C 2 -C 20 )alkenyl, or (C 2 -C 20 )alkynyl of R 5 or R 6 is optionally substituted with one or more (e.g 1, 2, 3, or 4) halo, hydroxy, mercapto
- (C 1 -C 20 )alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl;
- (C 3 - C g )cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl;
- (C r C 20 )alkoxy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy;
- (C 2 -C 2 o) lkenyl can be vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,-pentenyl, 2- pentenyl, 3-pentenyl,
- C 20 )alkanoyl, (C r C 20 )alkoxycarbonyl, or (C 2 -C 20 )alkynyl of R j , R 2 , R ⁇ , and R b is optionally substituted with 1 or 2 halo, hydroxy, mercapto, oxo, thioxo, carboxy, (C r C 20 )alkanoyl, (C 1 -C 20 )alkoxycarbonyl, aryl, or heteroaryl; and wherein any aryl or heteroaryl is optionally substituted with one or more halo, hydroxy, mercapto, carboxy, cyano, nitro, trifluoromethyl, trifluoromethoxy, (C 1 -C 20 )alkanoyl, (C r C 20 )alkanoyloxy, sulfo or (C r C 20 )alkoxycarbonyl.
- R, and R 2 can each independently be hydrogen, (C 1 -C 10 )alkyl,(C 2 -C 10 )alkenyl, (C 2 -C 10 )alkynyl, aryl, orNR a R b .
- Rj and R 2 together with the carbon to which they are attached can form a 5 or 6 membered saturated or unsaturated ring comprising carbon and optionally comprising 1 or 2 heteroatoms selected from oxy (-O-), thio (-S-), or nitrogen (-NR 0 )-, wherein said ring is optionally substituted with 1, 2, or 3 halo, hydroxy, oxo, thioxo, carboxy, (C 1 -C 20 )alkyl, (C 3 -C 8 )cycloalkyl, (C C 20 )alkoxy, (C r C 20 )alkanoyl, (C r C 20 )alkoxycarbonyl, (C 2 -C 20 )alkenyl, (C 2 -C 20 )alkynyl, aryl, or heteroaryl; wherein R c is hydrogen, (C r C 20 )alkyl, (C 3 -C 8 )cycloalkyl,(C 2 r
- R, and R 2 can each independently be NR ⁇ R,,; wherein R, and R b are each independently hydrogen, (C 1 -C 20 )alkyl, (C 3 - C 8 )cycloalkyl, (C 2 -C 20 )alkenyl, (C ⁇ -C 20 )alkanoyl, (C r C 20 )alkoxycarbonyl, (C 2 - C 20 )alkynyl, aryl, heteroaryl; wherein any (C ⁇ -C 20 )alkyl, (C 3 -C 8 )cycloalkyl, (C 2 -C 20 )alkenyl (C 1 -C 20 )alkanoyl, (C 1 -C 20 )alkoxycarbonyl, or (C 2 -C 20 )alkynyl is optionally substituted with one or more halo, hydroxy, mercapto, oxo, thioxo, carboxy, aryl, or
- C 20 alkyl, (C 1 -C 20 )alkylamino, allylamino, 2-hydroxyethylamino, phenylamino, or 4-thiazoylamino.
- R t and R 2 can each independently be amino, methyl, allylamino, 2-hydroxyethylamino, phenylamino, or 4-thiazoylamino.
- a specific value for R 3 is optionally substituted with one or more halo, mercapto oxo, thioxo, carboxy, (C 1 -C 20 )alkanoyl, (C r C 20 )alkoxycarbonyl, aryl, heteroaryl, or NR d R e .
- a specific value for R 3 is 2-aminoethyl, 2-amino-2- carboxyethyl, or 2-acylamino-2-carboxyethyl.
- a specific value for R 4 is aryl, optionally substituted with one or more halo, mercapto, hydroxy, oxo, carboxy, cyano, nitro, trifluoromethyl, trifluoromethoxy, (C 1 -C 20 )alkanoyl, (C I -C 20 )alkanoyloxy, sulfo or (C r C 20 )alkoxycarbonyl.
- R 5 is (C r C 10 )alkyl, (C 3 -C 6 )cycloalkyl,(C 2 - C 10 )alkenyl, (C 2 -C 10 )alkynyl, aryl, or heteroaryl; and R 6 is hydrogen, (C r C 10 )alkyl, (C 3 -C 6 )cycloalkyl,(C 2 -C 10 )alkenyl, (C 2 -C 10 )alkynyl, aryl, or heteroaryl.
- R 5 and R 6 together with X form a heteroaryl.
- Preferred organic compounds exclude polypeptides and proteins comprising one or more mercapto (C-SH) groups. Preferred organic compounds exclude compounds that comprise one or more mercapto (C-SH) groups.
- a preferred organic compound is a compound of formula 1-11 as shown in figure 4.
- a more preferred compound is thiourea.
- salts include organic acid addition salts, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, ⁇ -ketoglutarate, and ⁇ - glycerophosphate salts.
- suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.
- Salts can be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound with a suitable acid.
- Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts can also be used.
- the compounds described herein can be present in a luminescence reaction at any concentration that increases the sensitivity of the assay. The optimum concentration of a given compound will depend on the luminescent reagent(s) employed, and on the specific conditions under which a given assay is carried out. However, suitable concentrations can be determined using standard techniques that are available in the art.
- the compound that can increase the sensitivity of the assay can be present in a luminescence reaction at a concentration of at least about 0.1 ⁇ M, or at a concentration of at least about 0.1 mM. More specifically, the compound can be present in the luminescence reaction at a concentration in the range from about 0.1 ⁇ M to about 500 mM (inclusive), or in the range from about l ⁇ M to about 250 mM (inclusive). Preferably, the compound is present at a concentration in the range from about 10 ⁇ M to about 100 mM (inclusive).
- the assay can be performed in the presence of whole cells.
- the assay can be carried out in a solvent comprising at least about 10% water. More specifically, the invention can be carried out in a solvent comprising at least about 25% water, or at least about 40% water.
- the analyte-independent luminescence is reduced by at least about 10 fold, or more preferably by at least about 20 fold, at least about 50 fold, or at least about 100 fold in the present of a compound, while the analyte-dependent luminescence is reduced by less than about 7 fold, about 5 fold, about 3 fold, or about 2 fold.
- a relative light unit value of 5 produced in the presence of the compound while a relative light unit value of 100 produced in the absence of the compound reflects a decrease in luminescence in the presence of the compound by 20 fold.
- the luminescence generated by luminogenic molecules not bound to an enzyme is reduced by at least about 10 fold, or more preferably by at least about 20 fold, at least about 50 fold, or at least about 100 fold, while the luminescence generated by luminogenic molecules bound to an enzyme is reduced by less than about 7 fold, about 5 fold, about 3 fold, or about 2 fold.
- the luminescence generated by luminonogenic molecules bound to an enzyme is preferably reduced by a lower fold than the fold decrease in luminescence generated by luminogenic molecules not bound to an enzyme.
- autoluminescence is reduced by at least about 10 fold, or more preferably by at least about 20 fold, at least about 50 fold, or at least about 100 fold, while luminescence that is dependent on the presence of an analyte is reduced by less than about 7 fold, about 5 fold, about 3 fold, or about 2 fold.
- the luminescence that is dependent on the presence of an analyte is preferably reduced by a lower fold than the fold decrease in autoluminescence.
- analyte-independent luminescence is reduced by at least about 10 fold, or more preferably by at least about 20 fold, at least about 50 fold, or at least about 100 fold.
- an assay when an assay is carried out in the presence of a compound that comprises a sulfur atom or a selenium atom, analyte-dependent luminescence is reduced by less than about 7 fold, about 5 fold, about 3 fold, or about 2 fold.
- the enzyme substrate, enzyme, and the compound can each be contained in a separate container, or they can be contained in a single container.
- the kit can optionally comprise a buffer solution suitable for use in a luminescent assay, and the enzyme substrate or enzyme, and the buffer solution can optionally be contained in a single container. Additionally, the compound and the buffer solution can optionally be contained in a single container.
- the kits can also optionally comprise a second substrate (e.g. a substrate for firefly luciferase or Renilla luciferase), or a quenching agent for a luminescent enzyme reaction.
- the kits can also optionally comprise ATP, or can optionally comprise both a luminogenic substrate of a luminescent enzyme, and a luminogenic enzyme.
- the ability of a compound to increase the sensitivity of a luminescent assay can be determined using assays that are well known to the art, or using the assays described in the Examples herein below.
- Compounds identified herein have been shown to be useful for increasing the sensitivity of luminescent assays.
- the compounds are particularly useful for reducing luminescence that results from the decomposition of intermediate dioxetane rings.
- the compounds are also useful for reducing luminescence in other systems that involve intermediate dioxetane rings and the like.
- Renilla luciferase enzyme was added to F-12 cell culture media containing 1% gelatin, to a concentration of approximately 2.5ng/50 ⁇ l media. Reactions in the absence of Renilla luciferase reveal the effect of the compound on autoluminescence while reactions in the presence of enzyme reveal the effect of the compound on Renilla luciferase- catalyzed luminescence.
- the S+G reagent was prepared as per manufacturer's instructions, with the exception that for these experiments a S+G solvent three times more concentrated than normal was used to resuspend the S+G substrate. Under these conditions, a higher concentration of coelenterazine in the S+G was needed for substrate to reach saturation conditions.
- the compound to be tested was re-suspended in either SG or
- a mixture containing all of the components in sufficient amounts for four reactions i.e. 200 ⁇ l media, 200 ⁇ l SG, 200 ⁇ l S+G
- 150 ⁇ l was dispensed into triplicate wells on a 96-well plate.
- reactions were sometimes assembled in each well of the plate by adding each of the 50 ⁇ l portions and mixing. The plate was incubated at 22°C and after 5 minutes the luminescence was measured using a Dynex plate luminometer (1 second measurement per well).
- Format B Experiments were performed in a reaction volume of 150 ⁇ l Matthew's Buffer (referred to herein as MB) as either a standard MB composition or a modified MB composition as described below. As with Format A, reactions with and without Renilla luciferase were carried out to observe the effect of the organic compounds on assay sensitivity. In order to be able to add the reaction components such as enzyme, substrate, detergent, and compound to be tested; the reaction was assembled in 3 portions as follows:
- Enzyme was added to IX MB to a concentration of approximately 2.5 ng/50 ⁇ l buffer
- 2X MB was used to malce IX MB without or with detergent at a concentration of l%Tergitol NP-9, 1% antifoaming agent
- coelenterazine was added at a concentration of 180 ⁇ M in the version with detergent and of 60 ⁇ M in the version without detergent. These levels of substrate are needed to reach saturation conditions.
- IX MB 5 2X MB was used to make IX MB with the compound to be tested at various concentrations and water.
- IX MB was made with only water or with water and the addition of the solvent used to solubilize the compound to be tested.
- buffer standard composition consists of: lOOmM potassium phosphate
- bovine serum albumin pH 7.4
- the BSA functions as an enzyme stabilizer and, in the standard MB composition, enhances coelenterazine autoluminescence but not to the extent of the autoluminescence generated when detergent is present.
- BSA was replaced with porcine gelatin as the enzyme stabilizer at a final concentration of 0.15 mg/ml or 0.45 mg/ml. Taking all the variants into account the format can be sub-divided in 4 different versions:
- Reactions were carried out in triplicate by adding each of the 50 ⁇ l portions to microtiter plate wells and mixing.
- the resulting relative light units generated per well was measured immediately using a Dynex MLX Microtiter plate luminometer or a Wallac 1450 MicroBeta Trilux plate luminometer (1 second/well) or alternatively, the plate was incubated at 22°C and read after 5 minutes in the same fashion.
- results in Table 1 herein below are shown as: a) fold-decrease in non-enzymatic autoluminescence measurement in the presence of the compound when compared to the absence of the compound and, b) effect of the compound on enzymatic luminescence measurement in the presence of the compound when compared to control samples lacking only the compound.
- a result of "decreased 7.4 fold” indicates that the luminescence measurement in the presence of the compound was 7.4 times less than the luminescence measurement in the absence of the compound.
- the fold decrease in luminescence not associated with the presence of Renilla luciferase autoluminescence
- the compounds reduce the luminescence not associated with enzymatic activity of the analyte to a greater degree than the luminescence associated with the enzymatic activity of the analyte.
- CDP-St ⁇ r® is a stabilized 1,2 -dioxetane chemiluminescent enzyme substrate, a high energy luminogenic molecule, used in the detection of alkaline phosphatase and alkaline phosphatase conjugates in solution and in membrane-based assays.
- CDP-St ⁇ r® was obtained from Tropix PE Biosystems, Bedford, Massachusetts.
- CDP-Star® substrate produces a light signal when it is activated by alkaline phosphatase. Alkaline phosphatase dephosphorylates the substrate, yielding an anion that accumulates due to its long half-life.
- Luminol (5-Amino-2,3-didydro-l,4-phthalazinedione) was obtained from Sigma, St. Louis, Missouri. Luminol is a widely used chemiluminescent reagent, that luminesces upon oxidation. Experimental conditions are grouped under Format D, sub-divided as follows:
- duplicate plates of cells were prepared using 96 well microtiter plates. One plate was used to examine viability, and the other plate was used to examine the effect on autoluminescence.
- the media was removed from the cells and replaced with media containing the substrate for firefly luciferase, beetle luciferin, and the organic compounds at various concentrations. Passive diffusion of luciferin across the cell membrane together with the ATP oxygen and luciferase enzyme already contained within the cell, results in light production. Whereas, in compromised or damaged cells, intracellular ATP concentration is rapidly depleted, decreasing the firefly luminescence.
- the level of luminescence was compared to controls containing only luciferin to identify the effect of the compounds, if any, on light output as an indicator of cell viability.
- the media was removed from the cells and replaced with media containing the compounds at various concentrations and coelenterazine. Since there is no Renilla luciferase enzyme being expressed in these cells, the only luminescence observed is autoluminescence.
- the level of autoluminescence was compared to controls containing only coelenterazine to identify the effect of the compounds on reducing autoluminescence. Half of the microtiter plate contained no cells.
- the reagents were added as to the cell counterpart to measure cell- independent luminescence (i.e. background luminescence).
- the reagents were prepared as follows: a) Reagent to examine firefly luminescence (cell viability)
- Luciferin substrate available from Promega Corporation, Madison WI, USA was initially prepared in lOmM sodium phosphate buffer, pH 7.4 as a lOOmM stock. This luciferin stock was used to make DMEM solution containing a final concentration of 2mM luciferin.
- 6-aza-thio-thymidine was dissolved in DMSO as a 750mM stock.
- the 6-aza-thio-thymidine was subsequently added to the DMEM/luciferin reagent at a final concentration of 30mM, 10 mM, and 1 mM, while maintaining a final DMSO concentration of 4%.
- a DMEM/luciferin reagent was used as the control with which to compare the effect of the compound and was also made to contain a final DMSO concentration of 4%.
- DMEM/coelenterazine reagents were made in a similar fashion as the DMEM/luciferin reagents described in a).
- the cell culture medium was removed from the cells and replaced with medium containing substrate (+/- compound) and luminescence was measured immediately. All luminescent values obtained from wells containing cells were background subtracted using the corresponding luminescent values from those wells that did not contain cells. Fold reduction in autoluminescence was calculated by dividing the background-subtracted autoluminescence in minus compound controls by the background-subtracted autoluminescence containing the compounds. Results for representative compounds are shown in the following Table.
Abstract
Description
Claims
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AU7532501A AU7532501A (en) | 2000-06-09 | 2001-06-07 | Method for increasing luminescence assay sensitivity |
CA002411179A CA2411179A1 (en) | 2000-06-09 | 2001-06-07 | Method for increasing luminescence assay sensitivity |
AU2001275325A AU2001275325B2 (en) | 2000-06-09 | 2001-06-07 | Method for increasing luminescence assay sensitivity |
AT01942027T ATE496298T1 (en) | 2000-06-09 | 2001-06-07 | METHOD TO INCREASE THE SENSITIVITY OF A LUMINESSENCE ASSAY |
EP01942027A EP1297337B1 (en) | 2000-06-09 | 2001-06-07 | Method for increasing luminescence assay sensitivity |
JP2002510941A JP4685325B2 (en) | 2000-06-09 | 2001-06-07 | Methods to increase luminescence assay sensitivity |
DE60143888T DE60143888D1 (en) | 2000-06-09 | 2001-06-07 | METHOD TO INCREASE THE SENSITIVITY OF LUMINESIS ASSAY |
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0437013A2 (en) * | 1989-04-10 | 1991-07-17 | Ela Technologies Inc. | Luminescent assays |
US5629168A (en) * | 1992-02-10 | 1997-05-13 | British Technology Group Limited | Chemiluminescent enhancers |
US5744307A (en) * | 1993-02-26 | 1998-04-28 | Mochida Pharmaceutical Co., Ltd. | Method for measuring adenyl group-containing susbstances |
Family Cites Families (25)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
SE428379B (en) | 1978-05-31 | 1983-06-27 | Lkb Produkter Ab | DETERMINATION OF ATOL AND REAGENTS OF BIOLUMINISM |
DE2828658C3 (en) | 1978-06-29 | 1981-10-22 | Lkb-Produkter Ab, Stockholm | Method for the photometric determination of subunit B of creatine kinase and reagent therefor |
DE2901919A1 (en) | 1979-01-18 | 1980-07-24 | Berthold Lab Prof Dr | APPLICATION OF A - IN PARTICULAR AUTOMATIC - METHOD FOR LIGHT MEASUREMENT AND DEVELOPMENTS OF THIS METHOD AND DEVICES FOR IMPLEMENTING IT |
SE432112B (en) * | 1979-07-12 | 1984-03-19 | Lkb Produkter Ab | PROCEDURE FOR DETERMINING CREATINKINASES IN SAMPLES CONTAINING ATP |
DE3048884A1 (en) | 1980-12-23 | 1982-07-15 | Boehringer Mannheim Gmbh, 6800 Mannheim | METHOD FOR ENZYME IMMUNE DETERMINATION IN HETEROGENEOUS PHASE |
US4650770A (en) * | 1981-04-27 | 1987-03-17 | Syntex (U.S.A.) Inc. | Energy absorbing particle quenching in light emitting competitive protein binding assays |
AU7399487A (en) | 1986-05-22 | 1987-12-22 | Unilever Plc | Solid phase immunoassay method |
GB8811294D0 (en) | 1988-05-12 | 1988-06-15 | Kabivitrum Peptide Hormones Ab | Luminescent/luminometric assays |
DE69026851T2 (en) * | 1989-02-14 | 1996-10-31 | Wako Pure Chem Ind Ltd | Process for increasing chemiluminescence |
JPH03167288A (en) | 1989-11-27 | 1991-07-19 | Chisso Corp | Method for sensitized luminescence of aequorin by surface active agent |
US5283179A (en) * | 1990-09-10 | 1994-02-01 | Promega Corporation | Luciferase assay method |
AT401526B (en) | 1993-02-10 | 1996-09-25 | Scheirer Winfried | REAGENT SOLUTION TO STABILIZE LUMINESCENCE IN LUCIFERASE MEASUREMENT |
JPH0767696A (en) | 1993-09-06 | 1995-03-14 | Tosoh Corp | Method for reducing back ground luminescence |
US5744320A (en) * | 1995-06-07 | 1998-04-28 | Promega Corporation | Quenching reagents and assays for enzyme-mediated luminescence |
US6602657B1 (en) | 1995-12-28 | 2003-08-05 | Tropix, Inc. | Multiple reporter gene assay |
US5795729A (en) * | 1996-02-05 | 1998-08-18 | Biometric Imaging, Inc. | Reductive, energy-transfer fluorogenic probes |
US6416960B1 (en) * | 1996-08-08 | 2002-07-09 | Prolume, Ltd. | Detection and visualization of neoplastic tissues and other tissues |
US5814504A (en) * | 1996-08-22 | 1998-09-29 | Kikkoman Corporation | Protein involved in regenerating firefly luciferin |
JPH1087621A (en) | 1996-09-13 | 1998-04-07 | Sankyo Co Ltd | Enhancer for lucigenin chemiluminescence |
US6200762B1 (en) | 1997-08-01 | 2001-03-13 | Aurora Biosciences Corporation | Photon reducing agents and compositions for fluorescence assays |
US6221612B1 (en) | 1997-08-01 | 2001-04-24 | Aurora Biosciences Corporation | Photon reducing agents for use in fluorescence assays |
ATE261583T1 (en) | 1999-09-01 | 2004-03-15 | Invitrogen Corp | PHOTON DAMPER FOR FLUORESCENCE ASSAY |
US7118878B1 (en) | 2000-06-09 | 2006-10-10 | Promega Corporation | Method for increasing luminescence assay sensitivity |
US6867005B2 (en) * | 2001-10-24 | 2005-03-15 | Beckman Coulter, Inc. | Method and apparatus for increasing the dynamic range and accuracy of binding assays |
US20040224377A1 (en) | 2003-02-12 | 2004-11-11 | Erika Hawkins | Compositions and methods to quench light from optical reactions |
-
2000
- 2000-06-09 US US09/590,884 patent/US7118878B1/en not_active Expired - Lifetime
-
2001
- 2001-06-07 DE DE60143888T patent/DE60143888D1/en not_active Expired - Lifetime
- 2001-06-07 AU AU2001275325A patent/AU2001275325B2/en not_active Ceased
- 2001-06-07 JP JP2002510941A patent/JP4685325B2/en not_active Expired - Lifetime
- 2001-06-07 AT AT01942027T patent/ATE496298T1/en not_active IP Right Cessation
- 2001-06-07 AU AU7532501A patent/AU7532501A/en active Pending
- 2001-06-07 EP EP01942027A patent/EP1297337B1/en not_active Expired - Lifetime
- 2001-06-07 CA CA002411179A patent/CA2411179A1/en not_active Abandoned
- 2001-06-07 WO PCT/US2001/018363 patent/WO2001096862A2/en active Application Filing
-
2003
- 2003-10-24 US US10/692,587 patent/US7078181B2/en not_active Expired - Lifetime
-
2004
- 2004-11-18 US US10/991,759 patent/US7108996B2/en not_active Expired - Lifetime
-
2010
- 2010-10-18 JP JP2010234065A patent/JP2011064690A/en active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0437013A2 (en) * | 1989-04-10 | 1991-07-17 | Ela Technologies Inc. | Luminescent assays |
US5629168A (en) * | 1992-02-10 | 1997-05-13 | British Technology Group Limited | Chemiluminescent enhancers |
US5744307A (en) * | 1993-02-26 | 1998-04-28 | Mochida Pharmaceutical Co., Ltd. | Method for measuring adenyl group-containing susbstances |
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JP2010046097A (en) * | 2002-12-23 | 2010-03-04 | Promega Corp | Method and kit for protecting luciferase enzyme activity |
US7741067B2 (en) | 2002-12-23 | 2010-06-22 | Promega Corporation | Luciferase-based assays |
JP2012105676A (en) * | 2002-12-23 | 2012-06-07 | Promega Corp | Improved luciferase-based assay |
US8361739B2 (en) | 2002-12-23 | 2013-01-29 | Promega Corporation | Luciferase-based assays |
JP2008206523A (en) * | 2002-12-23 | 2008-09-11 | Promega Corp | Improved luciferase-based assays |
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JP2006517413A (en) * | 2003-02-12 | 2006-07-27 | プロメガ コーポレイション | Method and kit for double enzyme assay to eliminate light from luminescence reaction |
US8512968B2 (en) | 2007-10-29 | 2013-08-20 | Perkinelmer Health Sciences B.V. | Methods, reagents and kits for luciferase assay |
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Also Published As
Publication number | Publication date |
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US7078181B2 (en) | 2006-07-18 |
US7108996B2 (en) | 2006-09-19 |
DE60143888D1 (en) | 2011-03-03 |
ATE496298T1 (en) | 2011-02-15 |
EP1297337A2 (en) | 2003-04-02 |
AU2001275325B2 (en) | 2006-10-05 |
WO2001096862A3 (en) | 2002-07-18 |
CA2411179A1 (en) | 2001-12-20 |
JP4685325B2 (en) | 2011-05-18 |
JP2011064690A (en) | 2011-03-31 |
US7118878B1 (en) | 2006-10-10 |
AU7532501A (en) | 2001-12-24 |
EP1297337B1 (en) | 2011-01-19 |
US20040096924A1 (en) | 2004-05-20 |
US20060051827A1 (en) | 2006-03-09 |
JP2004503777A (en) | 2004-02-05 |
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