WO1996025667A1 - Dosage par transfert d'energie chimioluminescente - Google Patents

Dosage par transfert d'energie chimioluminescente Download PDF

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Publication number
WO1996025667A1
WO1996025667A1 PCT/US1995/001506 US9501506W WO9625667A1 WO 1996025667 A1 WO1996025667 A1 WO 1996025667A1 US 9501506 W US9501506 W US 9501506W WO 9625667 A1 WO9625667 A1 WO 9625667A1
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Prior art keywords
enzyme
dioxetane
hydrophobic
attophos
substrate
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PCT/US1995/001506
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English (en)
Inventor
Irena Bronstein
Brooks Edwards
John Voyta
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Tropix, Inc.
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Publication date
Application filed by Tropix, Inc. filed Critical Tropix, Inc.
Priority to JP8524903A priority Critical patent/JPH11500222A/ja
Priority to AU19118/95A priority patent/AU704940B2/en
Priority to PCT/US1995/001506 priority patent/WO1996025667A1/fr
Priority to EP95911618A priority patent/EP0809804A4/fr
Publication of WO1996025667A1 publication Critical patent/WO1996025667A1/fr

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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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • 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

Definitions

  • This invention relates to the energy transfer
  • chemiluminescent assays for the determination of the presence or amount of a biological substance in surface-bound assays using 1,2-dioxetanes in connection with hydrophobic
  • fluorometric substrates such as AttoPhos TM as chemiluminescent substrates for enzyme-labeled fluorometric substrate targets or probes.
  • the chemiluminescence of the dioxetane AttoPhos TM acceptor substrate pair can be enhanced by the addition of a polymeric enhancer. Further enhancement can be achieved by adding, in sequence, AttoPhos TM and then the 1,2-dioxetane.
  • Chemiluminescent assays for the detection of the presence or concentration of a biological substance have received increasing attention in recent years as a fast, sensitive and easily read method of conducting bioassays.
  • a chemiluminescent compound is used as a reporter molecule, the reporter molecule chemiluminescing in response to the presence or the absence of the suspected biopolymer.
  • 1,2-dioxetanes A wide variety of chemiluminescent compounds have been identified for use as reporter molecules.
  • One class of compounds receiving particular attention is the 1,2-dioxetanes.
  • 1,2-dioxetanes can be stabilized by the addition of a stabilizing group to at least one of the carbon atoms of the dioxetane ring.
  • An exemplary stabilizing group is spirobound adamantane.
  • Such dioxetanes can be further substituted at the other carbon position with an aryl moiety, preferably phenyl or naphthyl, the aryl moiety being substituted by an oxygen which is, in turn, bound to an enzyme-labile group.
  • Such dioxetanes represent an advance over earlier-recognized dioxetanes, such as 3-(4-methoxyspiro [1,2-dioxetane-3,2'-tricyclo]-3.3.1.1 3 ' 7 ]decan]-4-yl) phenylphosphate, and in particular, the disodium salt thereof, generally identified as AMPPD.
  • AMPPD the disodium salt thereof
  • AMPGD AMPGD are also well-known, and can be used as reporter molecules. These dioxetanes, and their preparation, do not constitute an aspect of the invention herein, per se.
  • DNA assays the target biological substance is bound by a DNA probe with an enzyme covalently or indirectly linked thereto, the probe being admixed with the sample immobilized on a membrane, to permit hybridization. Thereafter, excess enzyme complex is removed, and dioxetane added to the hybridized sample. If hybridization has occurred, the dioxetane will be activated by the bound enzyme, leading to decomposition of the dioxetane, and chemiluminescence.
  • the enzyme In solution-phase assays, the enzyme is frequently conjugated to a nucleic acid probe or immune complexed with an antibody responsive to the target biological substance, unbound components being removed, and the dioxetane added, chemiluminescence being produced by the decomposition of the dioxetane activated by the amount of enzyme present. In cases where the enzyme itself is the target, the dioxetane need only be added to the sample.
  • containing or lacking the analyte in question are generally biological samples, these assays generally take place in an aqueous environment.
  • the light-quenching reactions therefor may substantially reduce the chemiluminescence actually observed from the decomposition of the dioxetan.
  • assays involving low-level detections of particular analytes, such as nucleic acids, viral antibodies and other proteins are generally biological samples, these assays generally take place in an aqueous environment.
  • the light-quenching reactions therefor may substantially reduce the chemiluminescence actually observed from the decomposition of the dioxetan.
  • assays involving low-level detections of particular analytes such as nucleic acids, viral antibodies and other proteins
  • the reduced chemiluminescence observed, coupled with unavoidable background signals may reduce the
  • TMQ poly(vinylbenzyltrimethylammonium chloride)
  • TBQ poly(vinylbenzyltributylammonium chloride)
  • BDMQ poly(vinylbenzyldimethylbenzylammonium chloride)
  • This enhancement is achieved, at least in part, through the formation of hydrophobic regions in which the dioxetane oxyanion is sequestered. Decomposition in these hydrophobic regions enhances chemiluminescence, because water-based light quenching reactions are suppressed. Among the recognized water-soluble quaternary polymer salts employed, TBQ provides unexpectedly superior enhancement, through this hydrophobic region-forming mechanism.
  • chemiluminescent enhancement achieved by the addition of water-soluble polymeric substances such as ammonium, phosphonium and sulfonium polymeric salts can be further improved by the inclusion, in the aqueous sample, of an additive, which improves the ability of the quaternary
  • polymeric salt to sequester the dioxetane oxyanion and the resulting excited state emitter reporting molecule in a hydrophobic region.
  • additive which, when a surfactant or water-soluble polymer itself, may enhance chemiluminescence to a limited degree.
  • the synergistic combination of the polymeric quaternary salt and additives gives enhancement effects making low-level, reliable detection possible even in aqueous samples through the use of 1,2-dioxetanes.
  • the polymeric quaternary salts, coupled with the additives, are sufficiently powerful
  • U.S. Patent 5,208,148 describes a class of fluorescent substrates for detection of cells producing the glycosidase enzyme.
  • the substrate is a fluorescein diglycoside which is a non-fluorescent substrate until hydrolyzed by glycosidase enzyme inside a cell to yield a fluorescent detection product excitable between about 460 nm and 550 nm.
  • the fluorescent enzymatic hydrolysis products are specifically formed and adequately retained inside living cells, and are non-toxic to the cells.
  • the substrates can penetrate the cell membrane under physiological conditions. Therefore, the invention permits analysis, sorting and cloning of the cells and
  • the fluorescent cosurfactant which exists in the bulk phase of the buffer solution used.
  • the fluorescent cosurfactant is present in a form capable of energy transfer-based
  • fluorescent moiety tends to remain associated with the micelle in the bulk phase. If any fluorescent co-surfactant is deposited on the solid phase, this occurs indiscriminately, in areas containing the immobilized ligand binding pair, and in areas which do not contain said pair. Thus a problem results in that the fluorescent emitters never are, or do not remain associated with the immobilized enzyme conjugate. Thus the close proximity needed for energy transfer from the dioxetane to the fluorescent emitter is not efficient. Further because the fluorescent emitters can be deposited anywhere on the solid phase matrix, this method does not allow for specificity when used in bound assay. The majority of the examples in the 1182 and 1565 patents are solution phase enzyme assays or chemical triggering experiments not utilizing enzymes.
  • this fluorescent co-surfactant is not a non-fluorescent enzyme substrate such as AttoPhos.
  • the present invention wherein a fluorescent energy acceptor is produced directly, and locally on a surface, by the same enzyme which catalytically decomposes the dioxetane energy donor, is not suggested by these art references.
  • dephosphorylated chromophore has to be performed with a laser or a lamp with a filter or a monochromator. These light sources are not only cumbersome, but increase the expense of the assay. This necessary and key excitation step which is accomplished with UV/blue light results in a second problem which is auto fluorescence of the membrane or surf ace and other solid supports which ordinarily contain fluorescent brighteners and other excitable fluorophores, as well as exciting chromophores contained in the biological sample
  • 1,2-dioxetane compounds have already been developed which show excellent potential as reporter molecules for such chemiluminescent assays. However, it is still necessary to improve upon the sensitivity and specificity of the
  • the present invention provides a method for determining the presence or the amount of a biological substance in a biological sample, wherein the method comprises the steps of: a) forming an enzyme conjugated binder (antibody or DNA probe) with the biological ligand from the sample; b) adding a hydrophobic fluorometric substrate such as AttoPhos TM and a 1, etane to the bound enzyme conjugated binder; c) wherein the enzyme of the enzyme conjugated biopolymer cleaves an enzyme
  • cleavable group such as a phosphate moiety from the AttoPhos TM and from the dioxetane causing the dioxetane to decompose through an excited state emitter form such that energy
  • kits for conducting a bioassay for the presence or concentration of a biological substance which is detected either bound to a surface or in a solution assay comprising: a) an enzyme complex which will stably bind to a surface-bound biological
  • Figure 1 is an illustration of the method of the present invention showing the energy transfer from CS-D to dephosphorylated Atto, thereby releasing energy in the form of fluorescence.
  • Figure 2 (A) - (D) is a CCD image of Western blot
  • Figure 3 is a graph of a Western blot analysis of rabbit IgG on Nitrocellulose Membrane showing chemiluminescent intensity (average and maximum).
  • Figure 4 (A) - (D) is a CCD image of Western blot analysis of rabbit IgG on PVDF membrane.
  • Figure 4 is
  • Figure 5 is a graph of a Western blot analysis of rabbit IgG on PVDF membrane showing chemiluminescent intensity
  • Figure 6 (A) - (B) are graphs of PSA (Prostate Specific Antigen), ng/mL versus RLU, 5 sec of chemiluminescent
  • Figure 7 is a chemiluminescent emission spectrum
  • Figure 8 is a chemiluminescence spectrum (intensity v. wavelength) obtained with 1.0 mM CSPD, 50% AttoPhos TM , and alkaline phosphatase, as described in Example 3.
  • Figure 9 is a chemiluminescence spectrum (intensity v. wavelength) obtained with 0.1 mM CSPD, 50% AttoPhos TM , 20% BDMQ, and alkaline phosphatase, as described in Example 3.
  • Figure 10 is a chemiluminescence spectrum (intensity v. wavelength) obtained with 0.25 mM CSPD, 50% AttoPhos TM , 20% BDMQ, and alkaline phosphatase, as described in Example 3.
  • Figure 11 is a chemiluminescence spectrum (intensity v. wavelength) obtained with 0.5 mM CSPD, 50% AttoPhos TM , 20% BDMQ, and alkaline phosphatase, as described in Example 3.
  • Figure 12 is a chemiluminescence spectrum (intensity v. wavelength) obtained with 1.0 mM CSPD, 50% AttoPhos TM , 20% BDMQ, and alkaline phosphatase, as described in Example 3.
  • Figure 13 is a chemiluminescence spectrum (intensity v. wavelength) obtained with 1.0 mM CSPD, 50% AttoPhos TM , 10% BDMQ, and alkaline phosphatase, as described in Example 3.
  • Figure 14 is a chemiluminescence spectrum (intensity v. wavelength) obtained with 1.0 mM CSPD, 10% AttoPhos TM , 20% BDMQ, and alkaline phosphatase, as described in Example 3.
  • Figure 15 is a chemiluminescent emission spectrum
  • Figure 16 is a chemiluminescent emission spectrum
  • Figure 17 is a chemiluminescent emission spectrum
  • Figure 18 is a graph showing the ratio of emission at 545 nm/465 nm obtained from the data in Figures 7-14 and
  • Figure 19 is a graph showing the sum of emission at 465 nm and 545 nm, obtained from the data in Figures 7-14 and Figure 17.
  • Figure 20 is a graph showing the ratio of emission at 545 nm/465 nm obtained from the data in Figures 15 and 16.
  • Figure 21 is a graph showing the sum of emission at 465 nm and 545 nm, obtained from the data in Figures 15 and 16.
  • Figure 22 is a CCD camera image detecting the presence of biotinylated DNA. Best Mode for Carrying Out the Invention
  • This invention makes use of a hydrophobic fluorometric substrate. By this is intended a compound which upon
  • activation by an enzyme can be induced to emit in respon ⁇ e to energy transfer from an excited state dioxetane decomposition product donor.
  • the substrate when activated, must be sufficiently hydrophobic as to be sequestered in the same hydrophobic regions to which the donor migrates, for energy and transfer to occur.
  • the present invention is described in terms of a method for determining the presence or amount of a substance or determined in a solution-phase assay biological substance using 1,2-dioxetanes using the hydrophobic fluorometric substrate AttoPhos TM .
  • the kit of the present invention also for determining the presence or amount of a substance, is described using a suitable enzyme conjugate, a 1,2-dioxetane and AttoPhos TM .
  • Other fluorometric substrates may be used.
  • these assays using 1,2-dioxetane in connection with AttoPhos TM alleviate the need for light sources necessary for excitation.
  • the present invention uses the high quantum yield of fluorescence, affinity for surfaces possessed by AttoPhos TM , coupled with the enzyme activated chemiluminescence of 1,2-dioxetane as the excitation source for the
  • dephosphorylated AttoPhos TM is produced at the surface and stays in close proximity with the enzyme environment throughout the assay, and the
  • excitation of the acceptor—dephosphorylated AttoPhos TM can be performed without any external instrumentation and without possible excitation of chromophores which are other than the dephosphorylated AttoPhos TM .
  • the method can be used for determining the presence or the amount of a biological substance in a biological sample.
  • the method comprises the steps of: a) forming a enzyme
  • dioxetane to decompose through an excited state form such that an energy transfer occurs from the excited state donor of dioxetane to the dephosphorylated AttoPhos TM acceptor, causing it to luminesce; and d) determining the presence or amount of the biological substance as a function of the amount of luminescence.
  • the kit of the present invention is also for determining the presence or concentration of a biopolymer and comprises: a) an enzyme complex which will bind to a biological substance upon admixture therewith; b) a 1,2-dioxetane which when contacted by the enzyme of the enzyme complex will be caused to decompose into a decomposition product which is in an excited state; and c) AttoPhos TM .
  • the assays and kits of this invention employ watersoluble chemiluminescent 1,2-dioxetanes.
  • these dioxetanes are well established in the art, and their identity and preparation do not constitute a novel aspect of this invention, per se.
  • any chemiluminescent dioxetane which exhibits sufficient solubility and stability in aqueous buffers to conduct the assay, and which may be caused to decompose and chemiluminesce by interaction with an enzyme, and cleavage, by the enzyme, of an enzyme labile group inducing the decomposition, can be used in connection with this invention.
  • 1,2-dioxetanes useful in this invention will have the general formula:
  • Z H, Cl, other halogens, alkyl, carboxy, or alkoxy groups
  • R 1 is C 1 -C 20 alkyl or C 1-l2 aryl or aralkyl
  • Y is phenyl or naphthyl, unsubstituted or substituted with an electron donating or electron withdrawing group
  • R 2 is meta-substituted or non-conjugated on Y with respect to the dioxetane, and is OX, wherein;
  • X is an enzyme cleavable group which, when cleaved, leaves the dioxetane phenoxy or naphthoxy anion.
  • Suitable dioxetanes are those disclosed in U.S. Patent Application 08/057,903, the entire disclosure of which is incorporated herein by reference.
  • Preferred dioxetanes include dioxetanes in which X is a phosphate moiety.
  • Particularly preferred dioxetanes include AMPPD, and in particular, its disodium salt, as well as CSPD, and in particular, its disodium salt.
  • Methods of preparing these dioxetanes are disclosed in the afore-referenced, commonly-assigned patents, as well as, e.g., U.S. Patent 4,857,652, assigned to Wayne State University. The preparation, purification and isolation of the dioxetanes does not constitute a novel aspect of the invention disclosed and claimed herein per se.
  • AttoPhos TM is a highly sensitive fluorometric substrate for the detection of alkaline phosphatase.
  • the chemical structure of AttoPhos TM is not known at the present time.
  • AttoPhos TM the chemical properties of AttoPhos TM are known.
  • AttoPhos TM was developed by JBL Scientific and can be obtained from the JBL-Scientific catalog (1993) at catalog number
  • AttoPhos TM is a pale, yellow crystalline solid having a molecular weight of approximately 580 gram ⁇ /mol.
  • turnover number for AttoPhos TM is 85,400 molecules of AttoPhos TM per minute per molecule of alkaline phosphatase in 2.40 M DEA (diethanolamine) pH 9.0, 0.23 mM MgCl 2 and 0.005% NaN, by weight.
  • the solubility of AttoPhos TM is ⁇ 10 mM in aqueous 2.4 M DEA buffer at a pH of 9.0.
  • AttoPhos TM has a Km value of 0.030 mM and a molar absorptivity of 31.412.
  • AttoPhos TM When contacted with alkaline phosphatase, AttoPhos TM is known to become a fluorescent emitter.
  • the molecular weight of the fluorescent emitter is approximately 290 g/mole.
  • This fluorescent emitter has an excitation maximum in the visible range at 430-450 nm with fluorescence monitored at 550-570 nm, in a DEA buffer. Best conditions are at 440 nm for excitation with 550 nm emission.
  • the fluorescent emitter also has an emission maximum at 560 nm, and a large Stokes Shift of
  • the Water Raman emission occurs at 470 nm with an excitation at 413 nm.
  • the fluorescent emitter has a maximum at 418 nm with an coefficient of 26,484 in 0.392 M Na 2 CO 3 and a pH of li.o and is fully ionized at a pH > 10.0.
  • the dioxetane is added to an enzyme complex which is bound to a biological binder (antibody or nucleic probe) .
  • the enzyme complex is also bound to the target biological substance.
  • the dioxetane is therefore the substrate for the enzyme, the enzyme-catalyzed cleavage of the labile groups of the substrate from the body of the dioxetane resulting in the formation of the unstable oxyanion, and subsequent
  • the enzyme is usually any organic compound having the decomposition of the dioxetane.
  • the enzyme is usually any organic compound having the decomposition of the dioxetane.
  • the enzyme is usually any organic compound having the decomposition of the dioxetane.
  • binder moiety such as a DNA probe in a hybridization step or suitable antibody in an incubation step, so as to help bind to the biological substance.
  • the hybridization step can be carried out using standard, wellknown procedures and using a suitable probe.
  • an incubation step can be carried out in the usual manner using a suitable antibody.
  • the enzyme conjugate can be any enzyme conjugate capable of stably binding to the biological substance.
  • the enzyme conjugate are any ligand-binder pair, probe with a covalently attached enzyme, or antibody labeled directly with alkaline phosphatase.
  • the nucleic acid probes and antibodies may be labelled indirectly with enzymes via a biotin-[strept ⁇ avidin or antigen-antibody (such as
  • alkaline phosphatase such as Streptavidin-alkaline phosphatase alkaline phosphatase labeled antibodies and DNA probes, are the preferred enzyme conjugates useful in the present invention.
  • AttoPhos TM and the 1,2-dioxetane are added to the bound enzyme conjugate complexed with biological substance either simultaneously, or AttoPhos TM is added first, allowed to dephosphorylate, and subsequently, a 1,2-dioxetane is added.
  • AttoPhos TM itself, like other fluorometric enzyme substrates is non-fluorescent in the bulk phase. Thus, any non-enzymatic decomposition of the dioxetane, which would produce a noise signal, is not amplified by energy transfer in the bulk phase. Thus it is an enzyme reaction which produces the hydrophobic, fluorescent form allowing immobilization on the surface used to perform the assay. It will also be apparent that other hydrophobic, fluorimetric enzyme substrates can also be used in the invention.
  • hydrophobic hydroxyfluoresceins shown in this patent as precursors to the diglycosidee may instead by phosphorylated using known art to give hydrophobic fluorescein mono- and diphosphate derivatives which are useful in the present invention.
  • the enzyme cleaves a phosphate moiety from both the 1,2-dioxetane and AttoPhos TM . ⁇ s the 1,2-dioxetane becomes dephosphorylated by the enzyme, the formed oxyanion becomes the excited state donor, and its energy is transferred to the closely positioned acceptor—the dephosphorylated AttoPhos TM emitter, causing it to emit.
  • Figure 1 illustrates the energy transfer from the 1,2dioxetane (CS"D) to the dephosphorylated AttoPhos TM , which in turn, releasing energy in the form of luminescence.
  • the energy transfer efficiency is enhanced as the dephosphorylated product of AttoPhos TM ⁇ acceptor, is hydrophobic and is immobilized in the surface/biological substance sites and therefore is in very close proximity to the chemiluminescent dephosphorylated 1,2-dioxetane's excited state fragment which is the energy donor.
  • the 1,2-dioxetane is added to the bound enzyme conjugate complexed with biological substance in an amount of from 0.01 to 2.5 mM, preferably 0.25 to 1 mM. Most preferably, the 1,2-dioxetane is added in an amount of 0.25 mM.
  • AttoPhos TM in the 2.40 M diethanolamine (DEA) in water buffer is added to the enzyme or enzyme conjugated binder completed with biological substance in an amount of from 1-100%, preferably 25 to 75% by volume. Most preferably, 10 to 50% by volume AttoPhos TM is added.
  • AttoPhos TM is added first, allowed to dephosphorylate, and subsequently, a 1,2-dioxetane is added. The time period between addition of
  • AttoPhos TM and addition of a 1,2-dioxetane is preferably 10 to 60 minutes, more preferably 20 to 40 minutes, and most
  • the signal can be further enhanced by the addition of a water-soluble macromolecule along with AttoPhos TM or other hydropic fluorometric enzyme substrate.
  • Preferred water-soluble polymers useful in practicing the invention are based, in general, on polymeric onium salts, particularly quaternary salts based on phosphonium, sulfonium and,
  • the polymers have the general formula I shown below:
  • each of R 1 , R 2 and R 3 can be a straight or branched chain unsubstituted alkyl group having from 1 to 20 carbon atoms, inclusive, e.g., methyl, ethyl, n-butyl, t-butyl, hexyl, or the like; a straight or branched chain alkyl group having from 1 to 20 carbon atoms, inclusive, substituted with one or more hydroxy, alkoxy, e.g., methoxy, ethoxy, benzyloxy or polyoxethylethoxy, aryloxy, e.g., phenoxy, amino or substituted amino, e.g., methylamino, amido, e.g.,
  • acetamido or ureido e.g., phenyl ureido; or fluoroalkane or fluoroaryl, e.g., heptafluorobutyl, groups, an unsubstituted monocycloalkyl group having from 3 to 12 carbon ring carbon atoms, inclusive, e.g., cyclohexyl or cyclooctyl, a
  • substituted monocycloalkyl group having from 3 to 12 ring carbon atoms, inclusive, substituted with one or more alkyl, alkoxy or fused benzo groups, e-g-,, methoxycyclohexyl or 1,2,3,4-tetrahydronaphthyl, a polycycloalkyl group having 2 or more fused rings, each having from 5 to 12 carbon atoms, inclusive, unsubstituted or substituted with one or more alkyl, alkoxy or aryl groups, e.g., 1-adamantyl or 3-phenyl-1-adamantyl, an aryl, alkaryl or aralkyl group having at least one ring and from 6 to 20 carbon atoms in toto, unsubstituted or substituted with one or more alkyl, aryl, fluorine or hydroxy groups, e.g., phenyl, naphthyl, pentafluorophenyl, ethyl
  • R 1 , R 2 and R 3 together with the quaternary nitrogen atom to which they are bonded, can form a saturated or unsaturated, unsubstituted or substituted nitrogencontaining, phosphorus-containing or sulfur-containing ring having from 3 to 5 carbon atoms, inclusive, and 1 to 3 heteroatoms, inclusive, and which may be benzoannulated, e.g., 1-pyridinium, 1-(3-alkyl or aralkyl) imidazolium, morpholino, alkyl morpholinium, alkylpiperidinium, N-acylpiperidinium, piperidino or acylpiperidino, benzoxazolium, benzthiazolium or benzamidazolium.
  • X represents a counterion which can include, alone or in combination, moieties such as halide, i.e., fluoride, chloride, bromide or iodide, sulfate,
  • alkylsulfonate e.g., methylsulfonate, arylsulfonate, e.g., p-toluenesulfonate, substituted arylsulfonate, e.g.,
  • diphenylanthracenesulfonate Perchlorate
  • alkanoate e.g., acetate
  • arylcarboxylate e.g., fluorescein or fluorescem derivatives
  • benzoheterocyclic arylcarboxylate e.g., 7-diethylamino-4-cyanocoumarin-3-carboxylate
  • organic dianions such as p-terephthalate may also be represented by X-.
  • n represents a number such that the molecular weight of such poly (vinylbenzyl Quaternary salts) will range from about 800 to about 200,000 (weight average), and
  • the symbol M may also represent phosphorous or sulfur whereupon the corresponding sulfonium or phosphonium polymers have been described in the prior art: U.S. Patents 3,236,820 and 3,065,272.
  • Copolymers containing 2 or more different pendant onium groups may also be utilized in the invention described herein:
  • the symbols X, M', R 1 ', R 2 ', R 3 ' are as described above for X, M, R 1 -R 3 .
  • the symbols Y and Z represent the mole fraction of the individual monomers comprising the copolymer. The symbols Y and Z may thus individually vary from .01 to .99, with the sum always equalling one.
  • M is N or P
  • R 1 -R 3 are preferred moieties
  • polycycloalkyl e.g. adamantane
  • aralkyl or aryl having 1 to 20 carbon atoms, unsubstituted or further substituted with hydroxy1, amino, amido, ureido groups, or combine to form via a spiro linkage to the M atom a heterocyclic (aromatic, aliphatic or mixed, optionally including other N, S or O hetero atoms) onium moiety.
  • heterocyclic aromatic,
  • X is preferably selected to improve solubility and to change ionic strength as desired, and is preferably halogen, a sulfate, a sulfonate.
  • each of R 1 -R 3 may be the same as or different from the corresponding R 1 -R 3 '.
  • Examples of preferred polymers include the following:
  • vinylbenzyl quaternary ammonium salt polymers can be prepared by free radical polymerization of the appropriate precursor monomers or by exhaustive alkylation of the
  • polyvinylbenzyl chicride or copolymers containing a pendant benzyl chloride function. This same approach can be taken using other polymeric alkylating agents such as
  • chloromethylated polyphenylene oxide or polyepichlorohydrin chloromethylated polyphenylene oxide or polyepichlorohydrin.
  • the same polymeric alkylating agents can be used as initiators of oxazoline ring-opening polymerization, which, after
  • polyethyleneimine graft copolymers Such copolymers can then be quaternized, preferably with aralkyl groups, to give the final polymer.
  • each R 4 is the same or a different aliphatic radical
  • substituent and X 1 is an anion, as disclosed and claimed in Bronstein-Bonte et al U.S. Patent 4,124,388, can also be used in practicing this invention.
  • poly(vinylbenzyl quaternary ammonium salts) of formula I above can also be copolymerized with other ethylenically unsaturated monomers having no quaternary ammonium functionality, to give polymers such as those disclosed and claimed in Land et al U.S. Patent 4,322,489; Bronstein-Bonte et al U.S. Patent
  • polymers can also be used as enhancer substances in practicing this invention.
  • these quaternized polymers will have molecular weights within the ranges given above for the
  • poly(vinylbenzyl quaternary ammonium salts) of Formula I poly(vinylbenzyl quaternary ammonium salts) of Formula I.
  • cationic microgels or crosslinked latices are more suitable for the direct formation of cast membranes, but can also be used for the overcoating of preformed membranes.
  • crosslinking moiety substituted with two ethylenically
  • Quaternary ammonium or phosphonium salt containing latices can be prepared using methodologies
  • Formula IV generally represents a useful subset of such water-soluble latex copolymers wherein the symbols X-, R 1 , R 2 and R 3 are as described above. The symbols X, Y and Z are mole fractions which must add together to give unity.
  • a polymeric enhancer such as BDMQ is added to the enzyme or enzyme conjugate biological substance sources in an amount of 0.01 to 26% (0.1 to 250 mg/ml), more preferably 0.025 to 15% (25 to 150 mg/ml). Most preferably, BDMQ is added in an amount of 0.1 to 0.2% (i to 2 mg/ml).
  • the emitted signal resulting from the dephosphorylated AttoPhos TM is by way of an energy transfer excitation from the excited state dioxetane dense fragment.
  • the emitted signal can be captured on a green sensitive film or in a luminometer, CCD camera. The amount of emission detected will be
  • the amount of biological substance is a function of the intensity of the emission.
  • kits of the present invention can be used to determine the presence or concentration of any substance
  • RNA Ribonucleic acid
  • DNA Ribonucleic acid
  • proteins Ribonucleic acid
  • inventions can be used for detections performed on membranes such as Western, Southern, Northern blotting and DNA
  • sequencing can also be used for solution-phase assays.
  • solutions-based assay or when enhancing polymers are employed they may require the dephosphorylated products of both AttoPhos TM and 1,2-dioxetane substrates, and thereby increasing the proximity between the donor and acceptor moieties.
  • Dilutions of rabbit IgG were electrophoresed on a 10% polyacrylamide gel using standard, known methods.
  • the IgG samples were 200, 66.7, 22.2, 7.4 and 2.4 ng per lane for nitrocellulose and 100, 33.3, 11.1, 3.7 and 1.2 ng per lane for PVDF.
  • the protein was then transferred to the membrane as follows: the gel was equilibrated in transfer buffer (5 mM MOPS, 2 mM sodium acetate, 20% methanol, pH 7.5) and then electrotransferred to nitrocellulose (Schleicher and Schuell BAS85) or PVDF (Tropix) at 90 volts for 1 hour at 4oC.
  • the membranes were rinsed with phosphate buffered saline (PBS), blocked with 0.2% casein, 0.1% Tween-20 in PBS (blocking buffer), incubated for 30 minutes with a 1-10,000 dilution of alkaline phosphatase conjugated goat anti-rabbit .ntibody (GAR-AP) in blocking buffer, the PVDF
  • membranes were washed twice for 5 minutes in blocking buffer, the nitrocellulose membranes were washed twice in 0.1% Tween- 20 in PBS, all membranes were washed twice for 5 minutes in 0.1 M diethanolamine, 1 mM MgCl 2 , pH 10 (substrate buffer), incubated for 5 minutes in a 1-20 dilution of Nitro-Block (Tropix) in substrate buffer, washed twice for 5 minutes in substrate buffer, incubated for 5 minutes in 0.25 mM CSPD in substrate buffer and AttoPhos TM under various conditions, sealed in a plastic report cover, incubated for approximately 1 hour and imaged for 5 minutes with a Star I CCD camera
  • Chemiluminescent images were obtained by integration of the chemiluminescent signal for 5 minutes with a Star 1 CCD camera interfaced to an Apple Macintosh Ilci computer using IPLab Spectrum software.
  • the CCD images were transferred into the NIH Image software package, and average and maximum pixel intensities were measured for each band.
  • the CCD images are compositee of the Western blot images.
  • Blot A was incubated in 0.25 mM CSPD in substrate buffer.
  • Blot B was incubated in 0.25 mM CSPD and 50% AttoPhos TM (50% AttoPhos TM buffer) simultaneously.
  • Blot C was incubated first in 50% AttoPhos TM (50% substrate buffer) for 30 minutes, the AttoPhos TM was removed, the
  • Substrate was then added to each tube.
  • the following three substrate compositions (200 ⁇ L per tube) were tested: 0.25 mM CSPD, 1 mg/mL BDMQ in substrate buffer added at time zero;
  • CSPD 0.25 mM CSPD, 1 mg/mL BDMQ, 50% AttoPhos TM in substrate buffer added at time zero; 50% AttoPhos TM , 1 mg/mL BDMQ in substrate buffer for 30 minutes followed by the addition of CSPD (final concentration 0.25 mM) .
  • the chemiluminescent signal was measured 25 minutes after the addition of CSPD (or
  • Figures 6(A) and (B) demonstrate that both the signal and signal/noise ratios are greater with CSPD and AttoPhos TM than with CSPD alone. Therefore, increased signal was the result of use of CSPD in connection with AttoPhos TM .
  • Fig. 13 1.0 mM CSPD, 50% AttoPhOs TM , 10% BDMQ
  • Fig. 15 1.0 mM CSPD, 50% AttoPhos TM , 2.0 mg/ml
  • Fig. 16 1.0 mM CSPD, 50% AttoPhos TM , 2.0 mg/ml
  • Fig. 17 a 30 minute preincubation of alkaline phosphatase in 50% AttoPhos TM , 20% BDMQ, followed by the addition of CSPD (0.25 mM final concentration) at time zero
  • alkaline phosphatase was added to each sample (f inal concentration, 1.12 ⁇ 10 -11 M) and the cuvette was inserted into the fluorimeter (Spex Fluorolog). Emission spectra were obtained with the monochrometer slits set at 10 mm and signal was integrated for 0.5 seconds per nm.
  • This set of experiments shows energy transfer from CSPD to AttoPhos TM in a buffer, such solution-based assays are used with immunoassays which are performed in buffers.
  • Figures 7-21 demonstrate that there is an energy transfer between the dephosphorylated emitter of CSPD and the
  • FIG. 14 demonstrates that the green signal originates from Atto', because when the concentration of AttoPhos TM is low, the energy transfer signal is also very low.
  • Figure 12 shows that the relative energy transfer signal when the substrates are added sequentially, i.e., first adding AttoPhos TM which becomes dephosphorylated creating the ground state emitter, followed by CSPD addition which upon dephosphorylanon, fragments and, generates the excited state donor which transfers its energy to the accumulated acceptor from the dephosphorylated
  • AttoPhos TM AttoPhos TM .
  • Biotinylated DNA was detected by binding streptavidin alkaline phosphatase, and then subsequently incubating with either CSPD 1,2-dioxetane substrate for alkaline phosphatase or mixtures of CSPD and the fluorescent alkaline phosphatase substrate AttoPhos TM .
  • biotinylated 35mer was spotted on to Pall Biodyne A nylon membrane, 210 pg in the top spot followed by successive 1:3 dilutions. DNA was detected by performing the Tropix Southern-LightTm procedure up to the substrate incubation step. Each membrane was then
  • AttoPhos TM solution The image was obtained using a Photometries Star 1 CCD Camera in a light-tight box without any external light source.
  • Figure 22 shows an increased light signal from the samples of AttoPhos TM in combination with CSPD.

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Abstract

L'invention présente des dosages par chimioluminescence permettant de déterminer la présence ou la quantité d'un biopolymère, dans des dosages par liaison superficielle utilisant des 1, 2-dioxétanes en association avec AttoPhosTM comme substrats chimiluminescents pour des cibles ou des sondes marquées par une enzyme. Elle présente, en outre, un nécessaire permettant de procéder à un dosage biologique afin de déterminer la présence ou la concentration d'un biopolymère; ce nécessaire comprend a), un complexe enzymatique, b), un 1, 2-dioxétane et, c), AttoPhosTM.
PCT/US1995/001506 1995-02-13 1995-02-13 Dosage par transfert d'energie chimioluminescente WO1996025667A1 (fr)

Priority Applications (4)

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JP8524903A JPH11500222A (ja) 1995-02-13 1995-02-13 化学ルミネセンスエネルギー移動アッセイ
AU19118/95A AU704940B2 (en) 1995-02-13 1995-02-13 Chemiluminescent energy transfer assays
PCT/US1995/001506 WO1996025667A1 (fr) 1995-02-13 1995-02-13 Dosage par transfert d'energie chimioluminescente
EP95911618A EP0809804A4 (fr) 1995-02-13 1995-02-13 Dosage par transfert d'energie chimioluminescente

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Cited By (5)

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US6355441B1 (en) * 1998-07-28 2002-03-12 Tropix, Inc. Benzothiazole dioxetanes
WO2003062790A2 (fr) 2002-01-17 2003-07-31 Applera Corporation Phases solides optimisees pour la detection par chimioluminescence
US6660529B2 (en) 1998-07-28 2003-12-09 Pe Corporation Heteroaryl substituted benzothiazole dioxetanes
US7091051B2 (en) 2003-08-28 2006-08-15 Tosoh Corporation Chemiluminescence method of 1,2-dioxetane and composition for chemiluminescence
WO2007142314A1 (fr) 2006-06-08 2007-12-13 Fujirebio Inc. Activateur de luminescence

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KR101960113B1 (ko) * 2011-05-03 2019-03-19 라이프 테크놀로지스 코포레이션 섬광 및 백열광 １,２-디옥세탄들

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US5112960A (en) * 1989-07-17 1992-05-12 Bronstein Irena Y Chemiluminescent 3-(substituted adamant-2'-ylidene) 1,2-dioxetanes

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EP0736174B1 (fr) * 1993-12-23 2002-04-03 Tropix, Inc. Dosages chimioluminescents par transfert d'energie

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US5004565A (en) * 1986-07-17 1991-04-02 The Board Of Governors Of Wayne State University Method and compositions providing enhanced chemiluminescence from 1,2-dioxetanes
US5112960A (en) * 1989-07-17 1992-05-12 Bronstein Irena Y Chemiluminescent 3-(substituted adamant-2'-ylidene) 1,2-dioxetanes

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ANALYTICAL BIOCHEMISTRY, Volume 203, issued 1992, EVANGELISTA et al., "Alkyl- and Aryl-Substituted Salicyl Phosphates as Detection Reagents in Enzyme-Amplified Fluorescence DNA Hybridization Assays on Solid Support", pages 218-226. *
BIOTECHNIQUES, Volume 12, Number 5, issued May 1992, BRONSTEIN et al., "Improved Chemiluminescent Western Blotting Procedure", pages 748-753. *
JOURNAL OF APPLIED BACTERIOLOGY, Volume 72, issued 1992, CANO et al., "Detection of Salmonellas by DNA Hybridization with a Fluorescent Alkaline Phosphatase Substrate", pages 393-399. *
See also references of EP0809804A4 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6852548B2 (en) 1998-07-28 2005-02-08 Applera Corporation Benzothiazole dioxetanes
US6355441B1 (en) * 1998-07-28 2002-03-12 Tropix, Inc. Benzothiazole dioxetanes
US6660529B2 (en) 1998-07-28 2003-12-09 Pe Corporation Heteroaryl substituted benzothiazole dioxetanes
US7112413B2 (en) 1998-07-28 2006-09-26 Applera Corporation Heteroaryl substituted benzothiazole dioxetanes
US7368296B2 (en) 2002-01-17 2008-05-06 Applied Biosystems Solid phases optimized for chemiluminescent detection
EP1470422A4 (fr) * 2002-01-17 2006-06-07 Applera Corp Phases solides optimisees pour la detection par chimioluminescence
EP1470422A2 (fr) * 2002-01-17 2004-10-27 Applera Corporation Phases solides optimisees pour la detection par chimioluminescence
WO2003062790A2 (fr) 2002-01-17 2003-07-31 Applera Corporation Phases solides optimisees pour la detection par chimioluminescence
EP2163897A1 (fr) * 2002-01-17 2010-03-17 Applied Biosystems, LLC Phases solides optimisées pour détection de chimiluminescence
US7091051B2 (en) 2003-08-28 2006-08-15 Tosoh Corporation Chemiluminescence method of 1,2-dioxetane and composition for chemiluminescence
WO2007142314A1 (fr) 2006-06-08 2007-12-13 Fujirebio Inc. Activateur de luminescence
EP2028250A1 (fr) * 2006-06-08 2009-02-25 Fujirebio Inc. Activateur de luminescence
EP2028250A4 (fr) * 2006-06-08 2010-07-21 Fujirebio Kk Activateur de luminescence

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AU704940B2 (en) 1999-05-06
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JPH11500222A (ja) 1999-01-06
EP0809804A1 (fr) 1997-12-03

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