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  Chemiluminescence enhancerUSPTO Application #: 20070059786Title: Chemiluminescence enhancer Abstract: The present invention provides a chemiluminescence enhancer treated to retain favorable dispersibility of fine solid carriers and stably exert a chemiluminescence enhancing action. The invention provides a chemiluminescence enhancer used for signal detection in a solid phase immunoassay using antigen or/and antibody immobilized onto fine solid carriers dispersible in a liquid medium, consisting of a water soluble macromolecular quaternary ammonium salt, a quaternary sulfonium salt or a quaternary phosphonium salt in order to enhance emission of light caused by an enzymatic reaction of a chemiluminescent substrate having dioxetane, wherein the chemiluminescence enhancer is given an aggregation inhibition treatment of the fine solid carriers by the treatment with an oxidizing agent or a reducing agent, and a chemiluminescence method and a kit using the chemiluminescence enhancer. (end of abstract) Agent: Oblon, Spivak, Mcclelland, Maier & Neustadt, P.C. - Alexandria, VA, US Inventors: Kazushige Moriyama, Tetsuji Tanimoto, Tatsuki Matsuno, Yoshihiro Ashihara USPTO Applicaton #: 20070059786 - Class: 435007920 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Antigen-antibody Binding, Specific Binding Protein Assay Or Specific Ligand-receptor Binding Assay, Assay In Which An Enzyme Present Is A Label, Heterogeneous Or Solid Phase Assay System (e.g., Elisa, Etc.) The Patent Description & Claims data below is from USPTO Patent Application 20070059786. Brief Patent Description - Full Patent Description - Patent Application Claims
TECHNICAL FIELD [0001] The present invention relates to a chemiluminescence enhancer used for enhancing chemiluminescence which is caused by enzymatic action of a chemiluminescent substrate in signal detection in a solid phase immunoassay using antigen or/and antibody immobilized onto fine solid carriers dispersible in a liquid medium. BACKGROUND ART [0002] In the field of measuring trace components, particularly in the field of clinical diagnostics, measurement methods of applying principles of immunology are utilized and immunoassays using fine solid carriers as a solid phase are widely used. The fine solid carriers include erythrocytes, gelatin particles, latex particles and the like, and a quantitative analysis is performed by absorbing antigen or/and antibody on the surface thereof and reacting immunologically the antibody or/and the antigen against antigen or/and antibody in a test sample. It is well-known that the immunoassays using these fine solid carriers are also commonly used even in the fields other than clinical diagnosis. [0003] Chemiluminescence measurement utilizing chemiluminescence reaction in which the chemiluminescence is caused by allowing an enzyme such as alkaline phosphatase to act upon a chemiluminescent substrate such as 1,2-dioxetanes can rapidly and sensitively measure the presence or concentration of a measurement subject in a specimen, and has been widely used to measure viruses such as HIV and HCV, and other trace components in vivo (JP 96-507694). [0004] It is well-known that a quenching reaction occurs in a liquid medium, particularly in an aqueous medium with chemiluminescence by decomposition of chemiluminescent substrate having dioxetane. Many test samples are biological samples in general, and thus the measurement of the samples by such a method is generally performed in an aqueous medium. Therefore, the quenching reaction sometimes reduces substantially the chemiluminescence occurred by the decomposition of the chemiluminescent substrate having dioxetane, which is actually obsereved. In the measurement methods of certain test samples, e.g., nucleic acids, a viral antibody and other proteins, in which a detection at a low level is required, the chemiluminescence reduced by the quenching reaction in combination with unavoidable background signals reduces the sensitivity of the measurement method, and thus in some cases, those at an extremely low level can not be detected. In order to improve these quenching reactions, an addition of a water soluble macromolecule including both naturally occurring and synthetic molecules (see U.S. Pat. No. 5,145,722), an addition of various water soluble enhancers to test samples (see U.S. Pat. No. 4,978,614), or water soluble polymerized quaternary ammonium salts such as poly(vinylbenzyltrimethyl ammonium chloride) (TMQ), poly(vinylbenzyltributyl ammonium chloride) (TBQ) and poly(vinylbenzyldimethylbenzyl ammonium chloride) (BDMQ) as the water soluble polymerized quaternary ammonium salts has been used (see U.S. Pat. No. 5,112,960 and JP 8-507694 T). [0005] In the meanwhile, the chemiluminescence enhancers such as TMQ, TBQ and BDMQ described above are polymers with high molecular weight. In the case of being used for a signal detection in a solid phase immunoassay using antigen or/and antibody immobilized onto a fine solid carrier dispersible in a liquid medium, when the carriers are once physically aggregated, for example, in order to wash after an immune reaction, these polymers prevent the fine solid carriers from dispersing thereafter, inhibit luminescence caused by an enzymatic reaction of the chemiluminescent substrate having dioxetane depending on the concentration of the subject substance to be detected, and have sometimes had a problem in that no precise measurement value can be obtained. DISCLOSURE OF THE INVENTION [0006] The aim of the invention is to provide a chemiluminescence enhancer made by treating a known chemiluminessence enhancer to retain favorable dispersibility of fine solid carriers in a chemiluminescence reaction by a chemiluminescent substrate and stably exert a chemiluminescence enhancing action. [0007] As a result of intensive study, the inventors of the present invention have found that a chemiluminescence enhancer such as water soluble macromolecular quaternary ammonium salt, quaternary sulfonium salt or quaternary phosphonium salt treated with a reagent having an oxidation or reduction property more stably enhances emission of light caused by an enzymatic reaction of a chemiluminescent substrate having dioxetane in signal detection in a solid phase immunoassay and provides more precise measurement results, and have completed the invention. [0008] That is, the invention provides a chemiluminescence enhancer used for signal detection in a solid phase immunoassay using antigen or/and antibody immobilized onto fine solid carriers dispersible in a liquid medium, consisting of a water soluble macromolecular quaternary ammonium salt, a quaternary sulfonium salt or a quaternary phosphonium salt in order to enhance emission of light caused by an enzymatic reaction of a chemiluminescent substrate having dioxetane, wherein the chemiluminescence enhancer is given an aggregation inhibition treatment of the fine solid carriers by the treatment with an oxidizing agent or a reducing agent, and a chemiluminescence method and a kit using the chemiluminescence enhancer. [0009] A preferable aspect of the enhancer is the chemiluminescence enhancer which does not substantially contain a component of a molecular weight more than about 400,000 daltons in the molecular weight separated by an ultrafiltration method. BEST MODE FOR CARRYING OUT THE INVENTION [0010] As described above, the chemiluminescence enhancer of the invention is water soluble macromolecular quaternary ammonium salt, quaternary sulfonium salt or quaternary phosphonium salt, or the like which is treated with a reagent having oxidation or reduction property, and is represented by following general formula (I): [0011] In general formula (I), each of R.sub.5, R.sub.6 and R.sub.7 may be a straight or branched, unsubstituted alkyl group having 1 to 20 carbon atoms (e.g., methyl group, ethyl group, n-butyl group, t-butyl group or hexyl group or the like); a straight or branched alkyl group having 1 to 20 carbon atoms substituted with one or more of a hydroxyl group, an alkoxy group (e.g., methoxy, ethoxy, benzyloxy or polyoxyethylethoxy group), an aryloxy group (e.g., phenoxy group), an amino group, a substituted amino group (e.g., methylamino), an amide group (e.g., acetamide group) or an ureide group (e.g., phenylureide group); a fluoroalkane group; a fluoroaryl group (e.g., heptafluorobutyl group); an unsubstituted monocycloalkyl group having 3 to 12 ring carbon atoms (e.g., cyclohexyl or cyclooctyl group); a substituted monocycloalkyl group having 3 to 12 ring carbon atoms substituted with one or more of an alkyl group, an alkoxy group or a condensed benzo group (e.g., methoxycyclohexyl or 1,2,3,4-tetrahydronaphthyl group); polycycloalkyl group having two or more condensed rings each having 5 to 12 carbon atoms, which is unsubstituted or substituted with one or more of an alkyl group, an alkoxy group or an aryl group (e.g., 1-adamantyl or 3-phenyl-1-adamantyl group); an aryl group, an alkaryl group or an aralkyl group having at least one ring and totally 6 to 20 carbon atoms, which is unsubstituted or substituted with one or more of an alkyl group, an aryl group, fluorine or a hydroxyl group (e.g., phenyl, naphthyl, pentafluorophenyl, ethylphenyl, benzyl, hydroxybenzyl, phenylbenzyl or dehydroabiethyl group). At least two of R.sub.5, R.sub.6 and R.sub.7 mean the groups capable of forming together with a quaternary atom to which they are bound a saturated or unsaturated, unsubstituted or substituted ring having 3 to 5 carbon atoms and 1 to 3 heteroatoms and containing nitrogen, phosphorus or sulfur, to which a benzene ring may be condensed (e.g., 1-pyridinium, 1-(3-alkyl)imidazolium, 1-(3-aralkyl)imidazolium, morpholino, alkyl morpholinium, alkyl piperidinium, N-acyl piperidinium, piperidino, acyl piperidino, benzoxazolium, benzothiazolium or benzamidazolium). [0012] A symbol Y in the general formula [I] alone or in combination represents a counter ion capable of including moieties such as a halogen ion, i.e., fluorine ion, chlorine ion, bromine ion and iodine ion, sulfate ion, alkyl sulfonate ion (e.g., methyl sulfonate ion), aryl sulfonate ion (e.g., p-toluene sulfonate ion), substituted aryl sulfonate ion (e.g., anilinonaphthylene sulfonate ion and various isomers thereof), diphenylantracene sulfonate ion, perchlorate ion, alkanoate ion (e.g., acetate ion), aryl carboxylate ion (e.g., fluorescein or fluorescein derivatives), benzene heterocyclic aryl carboxylate ion (e.g., 7-diethylamino-4-cyanocoumarin-3-carboxylate ion). Organic dianions such as p-terephthalate ion may be represented by Y. [0013] Furthermore, a symbol n represents a number over the range of about 500 to about 500,000 (average molecular weight), preferably about 20,000 to about 70,000 when the molecular weight of such poly(vinylbenzyl quaternary salt) is measured using an intrinsic viscosity or an LALLS method. Methods of preparing these polymers where M is nitrogen, related copolymers and related starting materials are disclosed in Jones, G. D., Journal of Polymer Science, Vol. 25:201, 1958; U.S. Pat No. 2,780,604, U.S. Pat. No. 3,178,396, U.S. Pat. No. 3,770,439, U.S. Pat. No. 4,308,335, U.S. Pat. No. 4,340,522, U.S. Pat. No. 4,424,326 and German published Patent No. 2,447,611. A symbol M may be phosphorus or sulfur, and here, corresponding phosphonium or sulfonium polymers are described in prior art (U.S. Pat. No. 3,236,820 and U.S. Pat. No. 3,065,272). [0014] As the chemiluminescence enhancer having the structure denoted by the general formula [I], those selected from poly[vinylbenzyl(benzylmethyl ammonium chloride)] (BDMQ), poly(vinylbenzyltrimethyl ammonium chloride) (TMQ), poly[vinylbenzyl(tributyl ammonium chloride)] (TBQ), benzylmethylcetyl ammonium chloride (BDMCAC), polymethacrylamidepropylenemethyl ammonium chloride (poly MAPTAC), poly[vinylbenzyl(triethyl ammonium chloride)] (TEQ), poly[vinylbenzyl(2-benzylamino)ethyldimethyl ammonium chloride] (BAEDM), poly[vinylbenzyldimethyl(2-hydroxy)ethyl ammonium chloride] (DME(OH)B), poly[vinylbenzyl(trimethylphosphonium chloride)] (TM), poly[vinylbenzyl(tributylphosphonium chloride)] (TB) and poly[vinylbenzyl(trioctylphosphonium chloride)] (TO) and copolymers thereof are suitable. [0015] The chemiluminescence enhancers of general formula [I] which is particularly preferable in the invention are those selected from poly[vinylbenzyl(benzylmethyl ammonium chloride)] (BDMQ), poly(vinylbenzyltrimethyl ammonium chloride) (TMQ), poly(vinylbenzyl(tributyl ammonium chloride)] (TBQ), poly[vinylbenzyl(triethyl ammonium chloride)] (TEQ), poly[vinylbenzyl(trimethylphosphonium chloride)] (TM), poly[vinylbenzyl(tributylphosphonium chloride)] (TB) and poly[vinylbenzyl(trioctylphosphonium chloride)] (TO) and copolymers thereof, more preferably those selected from poly(vinylbenzyltrimethyl ammonium chloride) (TMQ), poly[vinylbenzyl(tributyl ammonium chloride)] (TBQ) and poly[vinylbenzyl(benzylmethyl ammonium chloride)] (BDMQ), poly[vinylbenzyl(triethyl ammonium chloride)] (TEQ), and copolymers thereof, and most preferably poly[vinylbenzyl(tributyl ammonium chloride)] (TBQ) and poly[vinylbenzyl(benzylmethyl ammonium chloride)] (BDMQ). [0016] As described in the following general formula [II], a copolymer having two or more different onium side chains can be also utilized in the invention set forth here. The symbols Y, M', R.sub.5', R.sub.6' and R.sub.7' are the same as defined above Y, M, R.sub.5, R.sub.6 and R.sub.7. The symbols y and z represent a molar fraction of an individual monomer which constitutes the copolymer. Therefore, a sum of the symbols y and z is always equal to 1, and each of them may vary from 0.01 to 0.99. As a suitable moiety, M is nitrogen or phosphorus, and R.sub.5 to R.sub.7 are each independently an alkyl, cycloalkyl, polycycloalkyl (e.g., adamantane group), aralkyl or aryl group having 1 to 20 carbon atoms unsubstituted or further substituted with a hydroxyl, amino, amide or ureide group, or together form a heterocyclic (in some cases, aromatic or aliphatic or mixed including other heteroatoms such as nitrogen, sulfur or oxygen) onium group via a spiro-bond with an M atom. [0017] The chemiluminescence reaction itself in which the chemiluminescence enhancer of the invention is used is known. Enzymes for performing the chemiluminescence reaction can include acid phosphatase, alkali phosphatase, galactosidase, glucosidase, glucuronidase or esterase, and preferable examples can include acid phosphatase, alkali phosphatase, glucosidase, galactosidase and esterase. The most preferable is alkali phosphatase. These enzymes can be purified from animals, plants, bacteria and the like by methods known publicly, and are also commercially available. Commercially available articles can be also preferably used. These enzymes may be in a free state or in a state bound to the other substance such as an antigen, an antibody and a hapten. [0018] The substrate of the chemiluminescence reaction used here can include a dioxetane derivative represented by the following general formula [III]: wherein R.sub.2 is an aryl group substituted with an X-oxy group, which forms 1,2-dioxetane compound which is an unstable oxide intermediate when X is eliminated by an activator selected from acid, base, salt, enzyme, organic or inorganic catalyst and electron donor to induce a reaction, and the unstable 1,2-dioxetane compound is decomposed with releasing electron energy to produce light and two carbonyl containing compounds, further, X is a chemically easily reactive group which is eliminated by an enzyme; R.sub.1 is selected from the group consisting of an alkyl group, alkoxy group, aryloxy group, dialkylamino group, trialkylsilyloxy group, arylsilyloxy group, aryl group, and an aryl group which forms a polycyclic aryl group of X-oxy substituent spiro-bound to a 1,2-dioxetane ring by binding to an aryl group R.sub.2; R.sub.3 and R.sub.4 are each an alkyl or heteroalkyl group and R.sub.3 and R.sub.4 may be bound one another to form a polycyclic alkylene group spiro-bound to the 1,2-dioxetane ring. [0019] In general formula [III], when R.sub.1 is not bound to R.sub.2, this R.sub.1 is an alkyl, alkoxy, aryloxy, dialkylamino, trialkylsilyloxy, arylsilyloxy or aryl group as described above, and preferably a lower alkyl or alkoxy group having 1 to 8 carbon atoms. R.sub.1 may be also an aryl, aryloxy or arylsilyloxy group having 6 to 20 carbon atoms. When R.sub.1 is bound to R.sub.2 which is an aryl group to form a polycyclic aryl group spiro-bound to the 1,2-dioxetane ring, it is preferable that the polycyclic aryl group has carbon atoms up to 30. The polycyclic aryl group in this case may be one where an oxygen atom is included in place of a carbon atom as a xanthenyl group, and fluorenyl or xanthenyl where the spiro-bound polycyclic aryl group is spiro-bound to the 1,2-dioxetane ring at position C9 of the group is preferable. [0020] R.sub.2 is an aryl group substituted with X-oxy group (OX group), and the group including the aryl group may be a phenyl, biphenyl, bound phenyl group or other aryl group, include 6 to 30 carbon atoms, and include another substituent. X is a group eliminated from dioxetane by the activator in order to decompose a stable dioxetane structure to produce chemiluminescence (signal). It is preferred that the OX group is selected from a hydroxyl, alkylsilyloxy, arylsilyloxy group, an inorganic oxy acid salt (particularly, phosphate salt or sulfate salt), pyranoside oxygen, an arylcarboxylester or alkylcarboxylester group. When the OX group is a hydroxy group, a hydrogen atom of the group is easily reactive with an organic base such as potassium t-butoxide or an inorganic base such as potassium hydroxide, and can be decomposed by the base to produce the chemiluminescence. When the activator is an enzyme typically frequently used as a label for an immunoassay or a detection of a DNA probe, the OX group having X which is easily reactive with the enzyme could be appropriately selected. For example, when the activator is alkali phosphatase, .beta.-galactosidase, aryl or acetylcholine esterase, or the like commonly used as one detected by a colorimetric substrate or a fluorescent substrate in the immunoassay or the detection of the DNA probe, a phosphate salt, pyranoside oxygen or an acetate ester group can be selected as the OX group. Continue reading... Full patent description for Chemiluminescence enhancer Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Chemiluminescence enhancer patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. 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