WO1993015219A1 - Method of quantitative determination of substance with coumarin derivative - Google Patents

Method of quantitative determination of substance with coumarin derivative Download PDF

Info

Publication number
WO1993015219A1
WO1993015219A1 PCT/JP1993/000128 JP9300128W WO9315219A1 WO 1993015219 A1 WO1993015219 A1 WO 1993015219A1 JP 9300128 W JP9300128 W JP 9300128W WO 9315219 A1 WO9315219 A1 WO 9315219A1
Authority
WO
WIPO (PCT)
Prior art keywords
substituted
compound
hydrogen peroxide
formula
peroxidase
Prior art date
Application number
PCT/JP1993/000128
Other languages
French (fr)
Japanese (ja)
Inventor
Norihito Aoyama
Hideki Takenaka
Akira Miike
Original Assignee
Kyowa Medex Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kyowa Medex Co., Ltd. filed Critical Kyowa Medex Co., Ltd.
Priority to JP5513100A priority Critical patent/JP2980681B2/en
Publication of WO1993015219A1 publication Critical patent/WO1993015219A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • 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
    • 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
    • C12Q2326/00—Chromogens for determinations of oxidoreductase enzymes

Definitions

  • the present invention relates to a method for quantifying a peroxide active substance, hydrogen peroxide and a coumarin derivative represented by the following formula (I) or ( ⁇ ) by a chemiluminescence method using a coumarin derivative.
  • the antigen, antibody or DNA is labeled by labeling the antigen, antibody or DNA with peroxidase, an oxidase other than peroxidase or a luminescent compound, and quantifying peroxidase activity or hydrogen peroxide or a luminescent compound generated by the reaction of the oxidase. There is a known method for quantifying the amount.
  • the coumarin derivatives represented by the following formulas (I) and ( ⁇ ) are known as fluorescent compounds and are commercially available [Fluorescence Analytical Chemistry, pp. 159-161 (1987) published by Baifukan; Japan Chemical Society Journal, Volume 3, p. 644 (1972); Heterocycles, Volume 7, p. 933 (1977); Industry Chemical Magazine, Vol. 71, p. 1010 (1968); Eastman Kodak Co., Ltd., Wako Pure Chemical Industries' power tag, etc.].
  • the present invention relates to hydrogen peroxide and a compound of the formula (I)
  • R 1 , R 2 , R :! , R, R s and R 6 are the same or different and each represents a hydrogen atom, substituted or unsubstituted lower alkyl, lower alkoxy, substituted or unsubstituted Aralkyl, substituted or unsubstituted aryl, halogen atom, cyano, nitro, sulfo, carboxy, alkoxycarbonyl, alkyl rubamoyl, substituted or unsubstituted aryl carbamoyl, carbamoyl, hydroxy, substituted Or an unsubstituted amino, lower alkanoyl, lower alkanoyloxy or heterocyclic group, or R 1 and R 2 together form an alkylene or alkenylene, or a R 5 together form connexion - CH 2 CH 2 CH 2 NH - the Kumari down derivative or a salt thereof to form), is reacted in the presence of a peroxidative substance, the reaction solution Meas
  • the principle of the present invention is based on the fact that the above reaction proceeds stoichiometrically, and the luminescence amount or light intensity of the reaction solution is proportional to the amount of the peroxide active substance, hydrogen peroxide or coumarin derivative. I have.
  • the structure of the coumarin derivative has not been changed after the light emission due to the reaction, it was not confirmed by chromatography that the presence of a substance that was distinguished from the coumarin derivative before the reaction was confirmed. It is clear that a compound that emits light is generated by the reaction.
  • the alkyl moiety in lower alkyl, lower alkoxy, lower alkanoyl, lower alkanoyloxy, alkoxyl-rubonyl and alkyl-rubamoyl is preferably a straight-chain or 1-8 carbon atom.
  • Branched alkyl for example, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, etc., aralkyl having 7 to 15 carbon atoms, for example, benzyl , Phenyl, etc .; aryl in aryl, aryl carbamoyl represents phenyl, naphthyl, etc .; halogen atom represents iodine, bromine, chlorine, fluorine, etc., and heterocyclic ring.
  • the group represents pyridyl, benzothiazolinyl and the like.
  • Substituents in the substituted alkyl, substituted aralkyl, substituted aryl and substituted aryl are the same or different and are substituted with 1 to 5 cyano, halogen atoms, substituted or unsubstituted amino.
  • the alkyl moiety and the halogen atom in lower alkyl, lower alkoxy and alkoxycarbonyl are as defined above.
  • the substituents in the substituted amino are the same or different and represent a substituted or unsubstituted lower alkyl, substituted or unsubstituted heterocyclic group.
  • Lower alkyl is as defined above, and the heterocyclic group is, for example, triazinyl, virazinyl Represents pyridyl, pyrimidinyl, etc.
  • Substituents in the substituted heterocyclic group may be the same or different and each represents a substituted or unsubstituted cyano, halogen atom, amino, lower alkoxy, hydroxy, dialkylamino, or the like.
  • Acceptable salts of compound (I) or (II) include acid addition salts, for example, inorganic acid salts such as hydrochloride, sulfate, phosphate and the like, acetate, maleate, fumarate, citrate Organic acid salts.
  • inorganic acid salts such as hydrochloride, sulfate, phosphate and the like, acetate, maleate, fumarate, citrate Organic acid salts.
  • Table 1 shows specific compounds used in the present invention.
  • peroxidase active substance used in the method of the present invention is a general term for compounds having peroxidase activity, and includes, for example, peroxidase derived from plants, animals or microorganisms [EC. 1. 11.1. 7), hemoglobin, heme, iron oxide, iron chloride, sodium iodide, ammonium iodide, molybdate and the like.
  • the reactions generally performed by the following are hydrochloric acid, acetic acid, acetate, succinate, oxalate, borate, phthalate, glycine, valpital salt, In a buffer solution ( ⁇ . ⁇ ) adjusted to pH 2 to 8 by combining a buffering agent such as GOOD.
  • the components other than the component to be measured are added to the buffer solution to make a reagent solution, and the sample containing the component to be measured is added to this solution at 110 to 90 ° C, preferably 20 to 90 ° C. React at 50 ° C.
  • the total luminescence of the reaction solution or the luminescence during a certain period of time is measured with a luminescence photometer or the like in the wavelength range of 190 to 750 nm, and the calibration curve is prepared using a known amount of the sample containing the component to be measured.
  • the components to be quantified in the sample can be quantified.
  • the luminescence amount corresponds to the amount of the component to be measured, it can be quantified from the integrated luminescence amount measured at the end of the reaction, but is generally calculated from the integrated luminescence amount for a certain period of time.
  • the object can be achieved by measuring the amount of change in the amount of luminescence per unit time.
  • a surfactant for example, Triton X-100 (manufactured by Yoneyama Pharmaceutical Co., Ltd.) having a concentration of 0. It can be added so as to be 01 to 5 wt%.
  • a protein, a polyalkyl quaternary amine, a fluorescent agent, dimethyl sulfoxide or the like can be used as necessary. If necessary, an alkaline solution can be added.
  • proteins include human serum albumin (BSA), human serum albumin (HSA), human immunoglobulin, and ovalbumin.
  • polyalkyl quaternary amines include: POLYGEAR LILIM CYLAMMONIUM CHROMIDE, POLY
  • fluorescent agent examples include fluorescein, 4-fluorene 7-nitole benzofurazan, and ⁇ -fluoro-4-nitrobenzenezoxa.
  • a conjugate of diazole and amide, amino acid, peptide or protein, or a derivative thereof can be used.
  • aqueous solution for example, an aqueous solution of sodium hydroxide, aqueous hydroxide or the like can be used.
  • luminescence enhancers are used at a concentration of 0.0001 to 10 wt% of the reaction solution.
  • the hydrogen peroxide that can be quantified according to the present invention can quantify not only hydrogen peroxide dissolved in a sample but also hydrogen peroxide that is quantitatively generated by an enzymatic reaction.
  • any of the substances used in the above-mentioned reaction can be quantified.
  • the present invention can be applied to the measurement of hydrogen peroxide or peroxidase activity conventionally performed in the field of diagnostic agents.
  • stoichiometry is performed using enzymes from a substrate in a biological sample.
  • Substrate quantification is performed by generating hydrogen oxide and quantifying it.
  • the enzyme activity in a biological sample is measured by measuring the rate of hydrogen peroxide generated by performing an enzymatic reaction by adding an appropriate enzyme or substrate in order to measure the enzyme activity in the sample. ing.
  • Specific examples thereof include, for example, specific oxidase (eg, glucose oxidase, galactose oxidase, cholesterol oxidase, pericase, etc.). Measurement of the activities of enzymes such as oxidase and cholinesterase can be mentioned.
  • specific oxidase eg, glucose oxidase, galactose oxidase, cholesterol oxidase, pericase, etc.
  • peroxidase is used as a labeling substance, and the enzyme is produced by the peroxidase activity after the antigen-antibody reaction or by the action of this activity.
  • Hydrogen peroxide has been quantified.
  • each method described in the enzyme immunoassay (Eiji Ishikawa et al., 1987, Medical College), for example, an antigen is reacted with an immobilized antibody, and an enzyme such as peroxidase or glucose oxidase is used as the antigen.
  • a method of reacting a labeled antibody and quantifying the activity of the enzyme itself or hydrogen peroxide generated by the action of the enzyme can be mentioned.
  • drugs contained in serum, urine, etc. Hormones or carcinoembryonic antigens (CEA), ⁇ -proteins used as indicators of clinical tests It is possible to measure trace components in the body, such as protein (AFP) and prostatic acid phosphatase (PAP).
  • CEA carcinoembryonic antigens
  • AFP protein
  • PAP prostatic acid phosphatase
  • trace components can be quantified.
  • the compound (I) or (II) is used as a labeling substance, the reaction is carried out by the method of the present invention using the antigen-antibody reactant, and the amount of the antigen or the luminescence is measured by measuring the amount or intensity of luminescence. Antibody can be quantified.
  • the present invention can be applied to a polynucleotide measurement method.
  • the polynucleotide measurement method is complementary to the tested polynucleotide.
  • Labeling a polynucleotide that binds to an enzyme or a compound (I) or (H) such as peroxidase or dalcosoxidase, and labeling the enzyme or the compound that binds to the test polynucleotide complementarily Examples include a method of measuring the enzymatic activity of a polynucleotide or quantifying a labeled compound to determine the amount of a test polynucleotide. The method is described in “DNA Probe” (disc, 1988 edition), “DNA Probe II” (disc, 1990 edition) and the like.
  • the feature of the present invention using the coumarin derivative represented by the formula (I) or ( ⁇ ) is that the range of pH at which light is emitted is as wide as ⁇ 1 to 10; In the acidic to neutral range, a strong luminescence is obtained. Luminol, isolminol, lucigenin, acridinium ester, etc., which have been used in the past, cannot satisfactorily produce luminescence under strong alkaline conditions, and are optimal for enzymes such as peroxidase. When H was less than neutral, detection was difficult. By using the method of the present invention, even when the optimum pH of an enzyme is neutral or lower, it is possible to perform measurement at the optimum pH of the enzyme.
  • Figure 1 shows the changes in the emission intensity of compounds 1, 59 and luminol when using various concentrations of hydrogen peroxide.
  • X—Nore Figure 2 shows the changes in the emission intensity of compounds 10, 12, 14, 23 and luminol when various concentrations of hydrogen peroxide were used.
  • Figure 3 shows the changes in the emission intensity of Compound 1 and luminol at various pH values.
  • Figure 4 shows the changes in the emission intensity of Compound 1 and luminol when various concentrations of peroxidase were used.
  • Figure 5 shows the change in the emission intensity of Compound 1 when various concentrations of CEA were used.
  • FIG. 6 shows the change in the luminescence intensity of Compound 35 when various concentrations of AFP were used.
  • FIG. 7 shows the change in the luminescence intensity of compound 35 when various concentrations of PAP were used. Explanation of reference numerals
  • Figure 8 shows the results obtained for compounds 31 and 35 and acridinium ester (acridinium I: Dojin Chemical Laboratories) using various concentrations of BSA.
  • Example 1 chemiluminescence using a coumarin derivative by peroxidase reaction
  • the coumarin derivative used in the present example showed an emission intensity at pH 6.0 equal to or higher than that of luminol by reacting with peroxidase in the presence of hydrogen peroxide. .
  • the coumarin derivative used showed an emission intensity equal to or higher than that of luminol.
  • Example 3 Intensity of chemiluminescence using coumarin derivative by peroxidase reaction at various pH
  • FIG. 3 shows the ratio of the amount of luminescence at each pH when the luminescence at pH showing the maximum luminescence was 100%.
  • the optimal luminescence pH of Compound 1 is in the acidic region, It was shown to be extremely wide compared to
  • the G0D labeling for the anti-CEA antibody was performed according to the method of Ishikawa et al. (Oxygen immunoassay, page 82, edited by Eiji Ishikawa et al., 1987, published by The Medical College).
  • C—N-succinimidyl 4 -— (N-Maleimide methyl) was used to introduce a maleimide group into the GOD using hexane-111-carboxylate [manufactured by PIERCE].
  • the F (ab ') z fraction obtained by digesting the anti-CEA antibody with pepsin was reduced to prepare F (ab') from which the SH group was released. Both were mixed and reacted to obtain a GOD-labeled anti-CEA antibody.
  • Magnosphere (Magnosphere: trade name) manufactured by Stelogy Bioseparation Co., Ltd .; same hereafter) was used as the magnetic particles for the magnetic particles immobilized with the anti-CEA antibody.
  • An equal amount of a CEA antibody solution was added to the fine particles, sodium borohydride as a coupling reagent was added to a final concentration of 0.1 mol, and the mixture was stirred and reacted for 2 hours. After collecting the particles with a magnet and removing the supernatant by suction, 500 ⁇ l of a phosphate buffer containing 0.1% BSA and 0.1% NaN was added to obtain anti-CEA antibody-immobilized magnetic particles. And saved.
  • the particles were collected by a magnet, the supernatant was removed by suction, and glucose was dissolved at a concentration of 54 mgZml in 1 O miol citrate buffer (01115.0) as a substrate for G0D bound to CEA. After 100 1 was added and the mixture was reacted at 37 ° C. for 30 minutes, 50 mmol of glycine buffer (pH 11) was added as a stop solution. The particles were separated by a magnet, the supernatant 101 of this solution was added to the reagent solution attached to the luminescence photometer, and the integrated luminescence amount for one minute was measured. Figure 5 shows the results.
  • a labeled compound of compound 35 against the anti-AFP antibody was prepared as follows. That is, the carboxyl group of compound 35 and the amino group of the anti-AFP antibody react with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (hereinafter referred to as EDC) at pH 7.0. Then, compound 35 was recognized as anti-AFP antibody.
  • EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide
  • Magnospher was used as the magnetic particles for the anti-AFP antibody-immobilized magnetic particles.
  • An equal volume of an anti-AFP antibody solution dissolved in 0.1 niol phosphate buffer (pH 7.0) is added to the microparticles, and sodium borohydride, a coupling reagent, is added to a final concentration of 0.1.
  • the reaction mixture was stirred to react for 2 hours.
  • the particles were collected with a magnet, the supernatant stored after aspirated off, the-phosphate buffer containing 0. 1% BSA and 0. 1% N a N 3 as 5 0 0 1
  • anti-AFP antibody-immobilized magnetic particles did.
  • a labeled compound 35 against anti-PAP antibody was prepared as follows. That is, 50 mg of compound 35 was dissolved in 10 ml of dioxane, 75 mg of EDC and 50 mg of N-hydroxysuccinic acid imid were added, and the mixture was stirred at room temperature for 24 hours. Concentrated to dryness. The solid was extracted with ethyl acetate water, and the organic layer was concentrated to dryness. This was redissolved in 10 ml of dioxane, 50 mg of -alanine was added, and the mixture was reacted at room temperature for 4 hours, and concentrated to dryness to obtain a solid.
  • the solid was reacted with goat anti-human PAPP antibody at pH 7.0 using EDC and allowed to bind to obtain a compound 35-labeled anti-PAPP antibody.
  • Magnosphere was used as the magnetic particles for the anti-PAP antibody-immobilized magnetic particles.
  • An equal volume of an anti-PAP antibody solution dissolved in 0.1 mol phosphate buffer (pH 7.0) is added to the microparticles, and the final concentration of sodium borohydride, a coupling reagent, is added. 0.1 mol was added, and the mixture was stirred and reacted for 2 hours. The particles were collected with a magnet, the supernatant was removed by suction, and then the anti-PAP solid immobilized magnetic particles were added with 501 in phosphate buffer containing 0.1% BSA and 0.1% NaN. saved.
  • phosphate buffer pH 6.0
  • acridinium ester acridinium I: Dojindo Chemical
  • a slight amount of hydrogen peroxide, a peroxide active substance or a coumarin derivative, or a peroxide is used.
  • Active substances, oxidases, or substances chemically coupled with coumarin derivatives eg, CEA, AFP, PAP, etc.

Abstract

A method of quantitative determination of a peroxidation-active substance, hydrogen peroxide or a coumarin derivative represented by general formula (I) or (II) by reacting hydrogen peroxide with the derivative or a salt thereof in the presence of the active substance and determining the quantity or intensity of luminescence of the reaction mixture. In formula (I) or (II) R?1, R2, R3, R4 R5 and R6¿ may be the same or different from one another and each represents hydrogen, (un)substituted lower alkyl, (un)substituted aralkyl, (un)substituted aryl, halogen, or the like.

Description

明 細 書 クマリ ン誘導体を用いた物質の定量方法  Description Method for quantification of substances using coumarin derivatives
技 術 分 野 Technical field
本発明はクマリ ン誘導体を用いた化学発光法による過酸化活性物質、 過酸化水素および後記式 （ I ) も しく は （ Π ) で表されるク マ リ ン誘 導体の定量方法に関する。  The present invention relates to a method for quantifying a peroxide active substance, hydrogen peroxide and a coumarin derivative represented by the following formula (I) or (後) by a chemiluminescence method using a coumarin derivative.
従 来 の 技 術 Conventional technology
ごく微量な過酸化水素やペルォキシダーゼ活性を正確に定量するこ とは分析化学および生化学において重要なことであり、 特に臨床検査 の分野では測定対象物質を最終的に過酸化水素に変換して定量する手 法が多用されている。 また抗原、 抗体または D N Aをペルォキシダー ゼ、 ペルォキシダーゼ以外の酸化酵素または発光化合物で標識し、 ぺ ルォキシダーゼ活性または該酸化酵素の反応により生じる過酸化水素 または発光化合物を定量することにより、 抗原、 抗体または D N Aを 定量する方法が知られている。  Accurate quantification of very small amounts of hydrogen peroxide and peroxidase activities is important in analytical chemistry and biochemistry.Especially in the field of clinical testing, the substance to be measured is finally converted to hydrogen peroxide for quantification. Many methods are used. The antigen, antibody or DNA is labeled by labeling the antigen, antibody or DNA with peroxidase, an oxidase other than peroxidase or a luminescent compound, and quantifying peroxidase activity or hydrogen peroxide or a luminescent compound generated by the reaction of the oxidase. There is a known method for quantifying the amount.
過酸化水素やペルォキシダ一ゼ活性の定量法のなかで、 高感度な定 量法と しては、 生物発光やペルォキシダ一ゼの基質となる化合物の化 学発光を利用する方法が知られている。 特に利用できる発光化合物と してル ミ ノ ール、 イ ソル ミ ノ ール、 ノレシゲニン、 ァ ク リ ジニゥムエス テル等の化合物が知られている 〔生物発光と化学発光、 今井一洋編、 P.82- 89, (1989), 廣川書店〕 力^ 反応液中にタンパク質が共存した り反応液の pHが酸性側であると、 発光強度が低下する等の問題点があ る  Among the methods for quantifying the activity of hydrogen peroxide and peroxidase, methods that utilize bioluminescence and chemical luminescence of a compound serving as a substrate for peroxidase are known as highly sensitive methods. . Compounds such as luminol, isolminol, noresigenin, and acridinester are known as particularly useful luminescent compounds (Bioluminescence and chemiluminescence, edited by Kazuhiro Imai, p. 82-89, (1989), Hirokawa Shoten) Power ^ If there is a protein in the reaction solution or if the pH of the reaction solution is on the acidic side, there are problems such as a decrease in luminescence intensity.
後記式 （ I ) および式 （ Π ) で表されるクマリ ン誘導体は蛍光化合 物と して知られており、 また市販されている 〔蛍光分析化学、 159— 161 頁 （ 1987) 培風館発行 ； 日本化学会誌、 3卷、 644 頁 （ 1972) ； ヘテロサイ ク ロルス （Heterocycles) 、 7卷、 933 頁（1977) ； 工業 化学雑誌、 71卷、 1010頁（1968) ； イース トマンコダック社、 和光純 薬工業の力タ口グ等〕 。 The coumarin derivatives represented by the following formulas (I) and (Π) are known as fluorescent compounds and are commercially available [Fluorescence Analytical Chemistry, pp. 159-161 (1987) published by Baifukan; Japan Chemical Society Journal, Volume 3, p. 644 (1972); Heterocycles, Volume 7, p. 933 (1977); Industry Chemical Magazine, Vol. 71, p. 1010 (1968); Eastman Kodak Co., Ltd., Wako Pure Chemical Industries' power tag, etc.].
クマリ ン誘導体が過酸化活性物質の存在下、 過酸化水素によって発 光するという ことは知られていない。  It is not known that coumarin derivatives emit light with hydrogen peroxide in the presence of a peroxide active substance.
発 明 の 開 示 Disclosure of the invention
本発明は過酸化水素と式 （ I )  The present invention relates to hydrogen peroxide and a compound of the formula (I)
Figure imgf000004_0001
Figure imgf000004_0001
または式 （H)  Or formula (H)
Figure imgf000004_0002
Figure imgf000004_0002
(式中、 R 1 、 R 2 、 R :! 、 R 、 R s および R 6 は同一または異な つて、 水素原子、 置換もしく は非置換の低級アルキル、 低級アルコキ シ、 置換も しく は非置換のァラルキル、 置換もしくは非置換のァリー ル、 ハロゲン原子、 シァノ、 ニ トロ、 スルホ、 カルボキシ、 アルコキ シカルボニル、 アルキル力ルバモイル、 置換もしく は非置換のァリ一 ルカルバモイル、 力ルバモイル、 ヒ ドロキシ、 置換もしく は非置換の ァミ ノ、 低級アルカノィル、 低級アルカノィルォキシまたは複素環基 を表すかまたは R 1 と R2 が一緒になってアルキレンもしく はァルケ 二レンを形成するか、 または と R 5 が一緒になつて- CH2CH2CH2NH - を形成する） で表されるクマリ ン誘導体またはその塩を、 過酸化活性 物質の存在下に反応させ、 反応液の発光量または発光強度を測定する こ とを特徴とする過酸化活性物質、 過酸化水素または式 （ I ) も しく は式 （Π ) で表されるクマリ ン誘導体を定量する方法に関する。 (Wherein, R 1 , R 2 , R :! , R, R s and R 6 are the same or different and each represents a hydrogen atom, substituted or unsubstituted lower alkyl, lower alkoxy, substituted or unsubstituted Aralkyl, substituted or unsubstituted aryl, halogen atom, cyano, nitro, sulfo, carboxy, alkoxycarbonyl, alkyl rubamoyl, substituted or unsubstituted aryl carbamoyl, carbamoyl, hydroxy, substituted Or an unsubstituted amino, lower alkanoyl, lower alkanoyloxy or heterocyclic group, or R 1 and R 2 together form an alkylene or alkenylene, or a R 5 together form connexion - CH 2 CH 2 CH 2 NH - the Kumari down derivative or a salt thereof to form), is reacted in the presence of a peroxidative substance, the reaction solution Measuring the light emission amount or emission intensity The present invention relates to a method for quantifying a peroxide active substance, hydrogen peroxide or a coumarin derivative represented by the formula (I) or the formula (II) characterized by the above.
本発明の原理は上記反応が化学量論的に進行し反応液の発光量また は光の強度が過酸化活性物質、 過酸化水素またはクマリ ン誘導体の量 に比例しているこ とに基づいている。  The principle of the present invention is based on the fact that the above reaction proceeds stoichiometrically, and the luminescence amount or light intensity of the reaction solution is proportional to the amount of the peroxide active substance, hydrogen peroxide or coumarin derivative. I have.
反応によって発光した後、 クマリ ン誘導体が如何なる構造に変化し ているか確認されていないが、 反応前のクマ リ ン誘導体と区別される 物質の存在がク ロマ ト グラフ ィ ーによって確認されており、 反応によ つて発光する化合物が生成していることは明らかである。  Although the structure of the coumarin derivative has not been changed after the light emission due to the reaction, it was not confirmed by chromatography that the presence of a substance that was distinguished from the coumarin derivative before the reaction was confirmed. It is clear that a compound that emits light is generated by the reaction.
式 （ I ) または式 （ Π ) における定義中、 低級アルキル、 低級アル コキシ、 低級アルカノ ィル、 低級アルカノ ィルォキシ、 アルコキシ力 ルボニルおよびアルキル力ルバモイルにおけるアルキル部分は、 炭素 数 1 〜 8の直鎖または分岐状のアルキル、 例えばメ チル、 ェチル、 プ 口 ピル、 イ ソプロ ピル、 ブチル、 t e r t—ブチル、 ペンチル、 へキシル、 ヘプチル、 ォクチル等を表し、 ァラルキルは炭素数 7〜 1 5 の、 例え ばベンジル、 フヱネチル等を表わし、 ァ リ ール、 ァ リ 一ルカルバモイ ルにおけるァ リ ール部分はフ ヱニル、 ナフチル等を表し、 ハロゲン原 子はヨウ素、 臭素、 塩素、 フ ッ素等を表し、 複素環基とは、 ピリ ジル、 ベンゾチアゾリ二ル等を表す。  In the definition in formula (I) or formula (（), the alkyl moiety in lower alkyl, lower alkoxy, lower alkanoyl, lower alkanoyloxy, alkoxyl-rubonyl and alkyl-rubamoyl is preferably a straight-chain or 1-8 carbon atom. Branched alkyl, for example, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, etc., aralkyl having 7 to 15 carbon atoms, for example, benzyl , Phenyl, etc .; aryl in aryl, aryl carbamoyl represents phenyl, naphthyl, etc .; halogen atom represents iodine, bromine, chlorine, fluorine, etc., and heterocyclic ring. The group represents pyridyl, benzothiazolinyl and the like.
置換アルキル、 置換ァラルキル、 置換ァ リ ールおよび置換ァ リ ール 力ルバモイルにおける置換基は、 同一または異なって置換数 1 〜 5 の シァノ、 ハロゲン原子、 置換も し く は非置換のァ ミ ノ、 低級アルキル- 低級アルコキシ、 カルボキシ、 アルコキシカルボニル、 ヒ ドロキシ等 を表す。 低級アルキル、 低級アルコキシおよびアルコキシカルボニル におけるアルキル部分およびハロゲン原子は前記と同義である。  Substituents in the substituted alkyl, substituted aralkyl, substituted aryl and substituted aryl are the same or different and are substituted with 1 to 5 cyano, halogen atoms, substituted or unsubstituted amino. , Lower alkyl-lower alkoxy, carboxy, alkoxycarbonyl, hydroxy and the like. The alkyl moiety and the halogen atom in lower alkyl, lower alkoxy and alkoxycarbonyl are as defined above.
置換ァ ミ ノ における置換基は、 同一または異なって置換数 1 〜 2 の 低級アルキル、 置換も し く は非置換の複素環基を表す。 低級アルキル は前記と同義であり、 複素環基は、 例えば ト リ ァジニル、 ビラ ジニル ピリ ジル、 ピリ ミ ジニル等を表す。 置換複素環基における置換基は、 同一または異なって置換数 1〜 2のシァノ、 ハロゲン原子、 ァミ ノ、 低級アルコキシ、 ヒ ドロキシ、 ジアルキルアミ ノ等を表す。 低級アル コキシおよびアルキルァ ミ ノ におけるアルキル部分は前記と同義であ アルキレンとしては炭素数 2〜4の基を包含し、 -（CH2)2-、 -（CH2)3 -. -(CH2)4-等が挙げられ、 アルケニレンと しては炭素数 2〜 4の基を包 含し、 -CH=CH- 、 -CH=CH-CH2- 、 - CH=CH- CH=CH- 等が挙げられる。 The substituents in the substituted amino are the same or different and represent a substituted or unsubstituted lower alkyl, substituted or unsubstituted heterocyclic group. Lower alkyl is as defined above, and the heterocyclic group is, for example, triazinyl, virazinyl Represents pyridyl, pyrimidinyl, etc. Substituents in the substituted heterocyclic group may be the same or different and each represents a substituted or unsubstituted cyano, halogen atom, amino, lower alkoxy, hydroxy, dialkylamino, or the like. The alkyl moiety in lower alkoxy and alkylamino has the same meaning as described above, and alkylene includes a group having 2 to 4 carbon atoms;-(CH 2 ) 2 -,-(CH 2 ) 3 -.-(CH 2 ) 4 -, and the like, in the alkenylene and packaging containing the group number of 2 to 4 carbon atoms, -CH = CH-, -CH = CH -CH 2 -, - CH = CH- CH = CH- , etc. Is mentioned.
化合物 （ I ) または （Π) の許容される塩としては、 酸付加塩、 例 えば塩酸塩、 硫酸塩、 燐酸塩等の無機酸塩および酢酸塩、 マレイン酸 塩、 フマル酸塩、 クェン酸塩の有機酸塩があげられる。  Acceptable salts of compound (I) or (II) include acid addition salts, for example, inorganic acid salts such as hydrochloride, sulfate, phosphate and the like, acetate, maleate, fumarate, citrate Organic acid salts.
本発明で用いられている具体的化合物を第 1表に示す。 Table 1 shows specific compounds used in the present invention.
Figure imgf000007_0001
Figure imgf000007_0001
Figure imgf000008_0001
—1
Figure imgf000008_0001
—1
Figure imgf000009_0001
Figure imgf000009_0001
Figure imgf000010_0001
Figure imgf000010_0001
CI N  CI N
a -C00C2HS β ： VNH-
Figure imgf000010_0002
第 1 表に記載したクマリ ン誘導体は、 公知化合物であり、 例えば日 本化学会誌、 3巻、 644 頁 （ 1972) ； ヘテロサイ クルス、 7巻、 933 頁 （ 1977) ； 工業化学雑誌、 71巻、 1010頁 （ 1968) 等に記載の方法と 同様にして製造することができる。 また該化合物は、 イース トマンコ ダック社、 アルドリ ッチ社、 ダイ トーケミ ックス社等から購入するこ とも可能である。 a -C00C 2 H S β: VNH-
Figure imgf000010_0002
The coumarin derivatives described in Table 1 are known compounds. For example, Journal of the Chemical Society of Japan, vol. 3, p. 644 (1972); Heterocycles, vol. 7, p. 933 (1977); It can be produced in the same manner as described on page 1010 (1968). The compounds can also be purchased from Eastman Kodak, Aldrich, Die-Toymix, and the like.
本発明方法で用いられる過酸化活性物質とはペルォキシダーゼ活性 を有する化合物の総称であって、 例えば、 植物、 動物または微生物由 来のペルォキシダーゼ 〔EC。 1。 11.1. 7) 、 ヘモグロ ビン、 ヘム、 酸 化鉄、 塩化鉄、 ヨウ化ナ ト リ ウム、 ヨウ化アンモニゥム、 モ リ ブデン 酸塩等があげられる。  The term "peroxidase active substance" used in the method of the present invention is a general term for compounds having peroxidase activity, and includes, for example, peroxidase derived from plants, animals or microorganisms [EC. 1. 11.1. 7), hemoglobin, heme, iron oxide, iron chloride, sodium iodide, ammonium iodide, molybdate and the like.
本発明方法によつて定量分析を行う際一般に下記によって行われる 反応は塩酸、 酢酸、 酢酸塩、 コハク酸塩、 シ ョ ウ酸塩、 ホウ酸塩、 フタル酸塩、 グリ シン、 バルピタール塩、 グッ ド （G O O D) 等の緩 衝剤を組合せて pH 2〜8 に調整された緩衝液（Ο. ΟΟδ ΖπιοΐΖΐ)中で行 わ レる 。  When performing quantitative analysis according to the method of the present invention, the reactions generally performed by the following are hydrochloric acid, acetic acid, acetate, succinate, oxalate, borate, phthalate, glycine, valpital salt, In a buffer solution (Ο. ΟΟδΖπιοΐΖΐ) adjusted to pH 2 to 8 by combining a buffering agent such as GOOD.
化合物 （ I ) または （ Π ) は 0.01 molZl〜100molZl 、 過酸化水 素は 10 z mol / 1 〜100mmolZl 、 過酸化活性物質は 10 〜 2 x 102 mgZml好ま しく はペルォキシダーゼが 10―9〜 102UZmlで用いられる。 Compound (I) or ([pi) is 0.01 molZl~100molZl, peroxide Hydrogen is 10 z mol / 1 ~100mmolZl, peroxidative substance is 10 ~ 2 x 10 2 mgZml favored properly Peruokishidaze is 10 9-10 2 Used in UZml.
これらの 3成分の中の測定すべき成分以外の成分を緩衝液に加えて 試薬液と し、 これに測定すべき成分を含有する試料を加えて一 10〜90 °C、 好ま しく は 20〜50°Cで反応させる。 反応液の全発光量あるいは一 定時間の発光量を 190〜750nm の波長領域で発光光度計等により測定 し、 予め既知量の測定すべき成分含有試料を用いて作成した検量線を 利用 して試料中の定量すべき成分を定量することができる。  Of the three components, the components other than the component to be measured are added to the buffer solution to make a reagent solution, and the sample containing the component to be measured is added to this solution at 110 to 90 ° C, preferably 20 to 90 ° C. React at 50 ° C. The total luminescence of the reaction solution or the luminescence during a certain period of time is measured with a luminescence photometer or the like in the wavelength range of 190 to 750 nm, and the calibration curve is prepared using a known amount of the sample containing the component to be measured. The components to be quantified in the sample can be quantified.
発光量は測定すべき成分の量に対応しているので反応の終了に伴い 測定した積算発光量から定量できるが、 一般に一定時間の積算発光量 から計算される。 測定すべき成分が酵素活性の場合には、 発光量の単位時間当りの変 化量を測定することによって目的を達成できる。 Since the luminescence amount corresponds to the amount of the component to be measured, it can be quantified from the integrated luminescence amount measured at the end of the reaction, but is generally calculated from the integrated luminescence amount for a certain period of time. When the component to be measured is an enzyme activity, the object can be achieved by measuring the amount of change in the amount of luminescence per unit time.
本発明方法を実施する際、 反応液の濁りの発生防止等のため、 必要 に応じて界面活性剤 〔例えば、 ト リ ト ン X— 100 (米山薬品工業製） 〕 を、 その濃度が 0. 01〜 5 w t % になるように加えることができる。  In carrying out the method of the present invention, a surfactant (for example, Triton X-100 (manufactured by Yoneyama Pharmaceutical Co., Ltd.)) having a concentration of 0. It can be added so as to be 01 to 5 wt%.
発光化合物の発光強度を増強するため、 必要に応じてタンパク質、 ポリアルキル 4級ァミ ン、 蛍光剤、 ジメチルスルホキシ ド等を用いる ことができる。 さらに必要に応じて、 アルカ リ溶液を添加することが できる。 タンパク質としては、 例えば、 ゥシ血清アルブミ ン（B S A )、 ヒ ト血清ァルブミ ン （ H S A ) 、 ヒ ト免疫グロブリ ン、 卵白アルブミ ン等があげられ、 ポリアルキル 4級ァミ ンとしては、 例えば、 ポリ ジ ァ リールジメ チルアンモニゥムク ロライ ド、 ポリ 〔ビュルべンジル In order to enhance the luminescence intensity of the luminescent compound, a protein, a polyalkyl quaternary amine, a fluorescent agent, dimethyl sulfoxide or the like can be used as necessary. If necessary, an alkaline solution can be added. Examples of proteins include human serum albumin (BSA), human serum albumin (HSA), human immunoglobulin, and ovalbumin. Examples of polyalkyl quaternary amines include: POLYGEAR LILIM CYLAMMONIUM CHROMIDE, POLY
(ベンジルジメチル一ァンモニゥムクロライ ド） 〕 等があげられ、 蛍 光剤としては、 例えば、 フルォレツセイン、 4 —フルォ口一 7—ニト 口ベンゾフラザン、 Ί —フルオロー 4 —二 トロべンゾキサジァゾール とァミ ン、 ア ミ ノ酸、 ペプチ ドまたは夕ンパク質との結合物もしく は それらの誘導体等を使用することができる。 アル力 リ溶液としては、 例えば、 水酸化ナト リゥム、 水酸化力 リゥム等の水溶液を使用するこ とができる。 これら発光増強剤は反応液の 0. 0001〜10w t %の濃度で用 いられる。 (Benzyldimethylammonium chloride)]. Examples of the fluorescent agent include fluorescein, 4-fluorene 7-nitole benzofurazan, and Ί-fluoro-4-nitrobenzenezoxa. A conjugate of diazole and amide, amino acid, peptide or protein, or a derivative thereof can be used. As the aqueous solution, for example, an aqueous solution of sodium hydroxide, aqueous hydroxide or the like can be used. These luminescence enhancers are used at a concentration of 0.0001 to 10 wt% of the reaction solution.
本発明によって定量できる過酸化水素は、 試料中に溶存する過酸化 水素のみならず酵素反応によって定量的に生成する過酸化水素も定量 できる。  The hydrogen peroxide that can be quantified according to the present invention can quantify not only hydrogen peroxide dissolved in a sample but also hydrogen peroxide that is quantitatively generated by an enzymatic reaction.
定量できる過酸化活性物質と しては前述の反応に用いられる物がい ずれも定量できる。  As the peroxidative active substance that can be quantified, any of the substances used in the above-mentioned reaction can be quantified.
本発明は従来診断薬の分野において行われている過酸化水素または ペルォキシダーゼ活性の測定に適用することができる。  The present invention can be applied to the measurement of hydrogen peroxide or peroxidase activity conventionally performed in the field of diagnostic agents.
従来、 例えば生体試料中の基質から酵素を利用して化学量論的に過 酸化水素を生成させ、 これを定量することによる基質の定量が行われ ている。 Conventionally, for example, stoichiometry is performed using enzymes from a substrate in a biological sample. Substrate quantification is performed by generating hydrogen oxide and quantifying it.
また生体試料中の酵素活性を測定するために適当な酵素や基質を加 えて酵素反応を行わせ生成する過酸化水素の生成速度を測定すること によつて試料中の酵素活性の定量が行われている。  In addition, the enzyme activity in a biological sample is measured by measuring the rate of hydrogen peroxide generated by performing an enzymatic reaction by adding an appropriate enzyme or substrate in order to measure the enzyme activity in the sample. ing.
これらの具体例と して、 例えば、 特異的ォキシダ一ゼ （例えば、 グ ルコースォキシダーゼ、 ガラ ク ト一スォキシダーゼ、 コ レステロール ォキシダ一ゼ、 ゥ リ カーゼ等） 反応の基質の定量や、 モノア ミ ンォキ シダーゼ、 コ リ ンエステラーゼ等の酵素の活性測定があげられる。  Specific examples thereof include, for example, specific oxidase (eg, glucose oxidase, galactose oxidase, cholesterol oxidase, pericase, etc.). Measurement of the activities of enzymes such as oxidase and cholinesterase can be mentioned.
さ らに、 抗原抗体反応を酵素免疫測定法 （E IA 法） 用いて定量する 際標識物質と してペルォキシダーゼが用いられ、 抗原抗体反応後のぺ ルォキシダ一ゼ活性またはこの活性の作用によって生成させた過酸化 水素の定量が行われている。 例えば、 酵素免疫測定法 （石川榮治ら編、 1987年、 医学書院） に記載の各方法、 例えば固定化した抗体に抗原を 反応させ、 その抗原に例えば、 ペルォキシダーゼ、 グルコースォキシ ダーゼ等の酵素を標識した抗体を反応させ、 該酵素活性自身または該 酵素の作用により生成する過酸化水素を定量する方法があげられる。 本発明を該 E IA法等に用いることにより、 血清、 尿等に含まれる薬物. ホルモンまたは臨床検査の指標物質と して用いられている癌胎児性抗 原（CEA) 、 α —フ ヱ トプロテイ ン （AFP)、 前立腺酸性フ ォスフ ァタ一 ゼ（PAP) 等生体内微量成分等を測定することが可能である。  In addition, when quantifying an antigen-antibody reaction using an enzyme immunoassay (EIA method), peroxidase is used as a labeling substance, and the enzyme is produced by the peroxidase activity after the antigen-antibody reaction or by the action of this activity. Hydrogen peroxide has been quantified. For example, each method described in the enzyme immunoassay (Eiji Ishikawa et al., 1987, Medical College), for example, an antigen is reacted with an immobilized antibody, and an enzyme such as peroxidase or glucose oxidase is used as the antigen. A method of reacting a labeled antibody and quantifying the activity of the enzyme itself or hydrogen peroxide generated by the action of the enzyme can be mentioned. By using the present invention in the EIA method, etc., drugs contained in serum, urine, etc. Hormones or carcinoembryonic antigens (CEA), α-proteins used as indicators of clinical tests It is possible to measure trace components in the body, such as protein (AFP) and prostatic acid phosphatase (PAP).
上記に例示される過酸化水素またはペルォキシダーゼ活性の測定に 本発明を適用することによつて微量の成分を定量できる。  By applying the present invention to the measurement of hydrogen peroxide or peroxidase activity exemplified above, trace components can be quantified.
さ らに化合物 （ I ) または （ Π ) を標識物質と して用い、 抗原抗体 反応物を用いて本発明方法による反応を行わせ、 発光量または発光強 度を測定することによつて抗原または抗体を定量できる。  Further, the compound (I) or (II) is used as a labeling substance, the reaction is carried out by the method of the present invention using the antigen-antibody reactant, and the amount of the antigen or the luminescence is measured by measuring the amount or intensity of luminescence. Antibody can be quantified.
また、 本発明はポリ ヌ ク レオチ ド測定法に応用することができる。 ポリ ヌ ク レオチ ド測定法と しては、 被検査ポリ ヌ ク レオチ ドと相補的 に結合するポリヌ ク レオチ ドにペルォキシダーゼ、 ダルコースォキシ ダーゼ等の酵素又は化合物 （ I ) 又は （H ) を標識し、 被検ポリ ヌク レオチ ドと相捕的に結合した該酵素標識化又は該化合物標識化ポリ ヌ ク レオチ ドの酵素活性を測定または標識化合物を定量して被検ポリ ヌ ク レオチ ド量を定量する方法があげられる。 当該方法は、 「D N Aプ ローブ」 （ジスク社、 1988年版） 、 「D N AプローブII」 （ジスク社、 1990年版） 等に記載されている。 Further, the present invention can be applied to a polynucleotide measurement method. The polynucleotide measurement method is complementary to the tested polynucleotide. Labeling a polynucleotide that binds to an enzyme or a compound (I) or (H) such as peroxidase or dalcosoxidase, and labeling the enzyme or the compound that binds to the test polynucleotide complementarily Examples include a method of measuring the enzymatic activity of a polynucleotide or quantifying a labeled compound to determine the amount of a test polynucleotide. The method is described in “DNA Probe” (disc, 1988 edition), “DNA Probe II” (disc, 1990 edition) and the like.
式 （ I ) または （ Π ) で表されるクマリ ン誘導体を用いる本発明の 特徴は、 発光を生じる p H範囲が ρ Η 1〜 1 0 と広いことであり、 特 に p H 5〜 7の酸性〜中性域においては強い発光量が得られることで ある。 従来用いられていたルミ ノール、 イ ソルミ ノ一ル、 ルシゲニン 、 ァク リ ジニゥムエステル等は強アル力 リ条件下でなければ満足のい く発光量が得られず、 ペルォキシダーゼ等の酵素の至適 p Hが中性以 下の場合は検出が困難であった。 本発明方法を用いることにより、 酵 素の至適 p Hが中性以下の場合でも、 その酵素の至適 p Hでの測定を 行うことが可能である。 また、 これにより至適 p Hが酸性領域にあり 、 ルミ ノール等公知の発光物質を用いた定量法では使えなかった酸化 酵素の使用が可能になる。 従って本発明方法の場合、 上記の性質を持 つ酵素の活性を測定するとき、 酵素の最も活性の高い領域で行えるの で、 従来より微量の酵素でも検出することが可能である。  The feature of the present invention using the coumarin derivative represented by the formula (I) or (Π) is that the range of pH at which light is emitted is as wide as ρΗ1 to 10; In the acidic to neutral range, a strong luminescence is obtained. Luminol, isolminol, lucigenin, acridinium ester, etc., which have been used in the past, cannot satisfactorily produce luminescence under strong alkaline conditions, and are optimal for enzymes such as peroxidase. When H was less than neutral, detection was difficult. By using the method of the present invention, even when the optimum pH of an enzyme is neutral or lower, it is possible to perform measurement at the optimum pH of the enzyme. In addition, this makes it possible to use an oxidase that has an optimum pH in the acidic region and cannot be used in a quantitative method using a known luminescent substance such as luminol. Therefore, in the case of the method of the present invention, when the activity of the enzyme having the above properties is measured, it can be carried out in the region where the enzyme has the highest activity.
図面の簡単な説明 BRIEF DESCRIPTION OF THE FIGURES
図 1 は、 各種濃度の過酸化水素を用いたときの化合物 1， 5 9お よびルミ ノールの発光強度の変化を示す。  Figure 1 shows the changes in the emission intensity of compounds 1, 59 and luminol when using various concentrations of hydrogen peroxide.
符号の説明  Explanation of reference numerals
—— O—— 化合物 1  —— O—— Compound 1
——秦—— 化合物 5 ——Qin—— Compound 5
一一□一一 化合物 9  11-11 Compound 9
X— ノレ ミ ノ 一ノレ 図 2は各種濃度の過酸化水素を用いたときの化合物 10， 12, 14， 23 およびルミ ノ一ルの発光強度の変化を示す。 X—Nore Figure 2 shows the changes in the emission intensity of compounds 10, 12, 14, 23 and luminol when various concentrations of hydrogen peroxide were used.
符号の説明 Explanation of reference numerals
—— ·—— 化合物 1 0  —— · —— Compound 1 0
- -□- - 化合物 1 2  --□--Compound 1 2
Δ—— 化合物 1 4  Δ—— Compound 1 4
—— O—— 化合物 2 3  —— O—— Compound 2 3
—— X—— ノレ ミ ノ ーノレ —— X——
図 3 は各種 p Hにおける化合物 1 およびルミ ノールの発光強度の変 化を示す。  Figure 3 shows the changes in the emission intensity of Compound 1 and luminol at various pH values.
符号の説明 Explanation of reference numerals
——〇—— 化合物 1  ——〇—— Compound 1
□ ノレ ミ ノ ーノレ  □ ノ レ ミ ノ ノ レ
図 4 は各種濃度のペルォキシダ一ゼを用いたときの化合物 1 および ルミ ノールの発光強度の変化を示す。  Figure 4 shows the changes in the emission intensity of Compound 1 and luminol when various concentrations of peroxidase were used.
符号の説明 Explanation of reference numerals
——鲁—— 化合物 1  —— 鲁 —— Compound 1
□ ノレミ ノーノレ 図 5 は各種濃度の C E Aを用いたときの化合物 1の発光強度の変化 を示す。  Figure 5 shows the change in the emission intensity of Compound 1 when various concentrations of CEA were used.
符号の説明 Explanation of reference numerals
——翁—— 化合物 1  ——Okin—— Compound 1
図 6 は各種濃度の A F Pを用いたときの化合物 3 5の発光強度の変 化を示す。  FIG. 6 shows the change in the luminescence intensity of Compound 35 when various concentrations of AFP were used.
符号の説明  Explanation of reference numerals
——〇—— 化合物 3 5  ——〇—— Compound 3 5
図 7 は各種濃度の P A Pを用いたときの化合物 3 5の発光強度の変 化を示す。 符号の説明 FIG. 7 shows the change in the luminescence intensity of compound 35 when various concentrations of PAP were used. Explanation of reference numerals
—— O—— 化合物 3 5  —— O—— Compound 3 5
図 8 は各種濃度の B S Aを用いたときの化合物 3 1， 3 5およびァ ク リ ジニゥムエステル （ァク リ ジニゥムー I ： 同仁化学研究所） の B Figure 8 shows the results obtained for compounds 31 and 35 and acridinium ester (acridinium I: Dojin Chemical Laboratories) using various concentrations of BSA.
S A無添加時の発光強度に対する比率を示す。 The ratio to the emission intensity when no SA was added is shown.
符号の説明 Explanation of reference numerals
——□—— 化合物 3 1  —— □ —— Compound 3 1
—— 化合物 3 5  —— Compound 3 5
〇 ァク リ ジニゥムエステル  Polyethylene ester
発明を実施するための最良の形態 BEST MODE FOR CARRYING OUT THE INVENTION
以下に本発明の実施例を示す。  Hereinafter, examples of the present invention will be described.
実施例 1 (ペルォキシダーゼ反応によるクマリ ン誘導体を用いる化学 発光） Example 1 (chemiluminescence using a coumarin derivative by peroxidase reaction)
2 O mmo lのリ ン酸緩衝液 （ p H 6. 0 ) 1 0 0 mlに西洋ヮサビ由来の ペルォキシダーゼ （東洋紡績製 ： 以下同じ） 1 0 0 U、 5 0 の ト リ トン X— 1 0 0および第 2表に記載のクマリ ン誘導体または比較化合 物としてのルミ ノール （東京化成製 ： 以下同じ） 各 1 0 mgを溶解し試 薬液とした。  2 Ommol of phosphate buffer (pH 6.0) in 100 ml of horseradish peroxidase (Toyobo: same hereafter) 100 U, 50 Triton X—10 Luminol as a coumarin derivative or a comparative compound shown in Tables 0 and 2 (manufactured by Tokyo Chemical Industry; the same applies hereinafter) was dissolved in 10 mg each to prepare a reagent solution.
試験管に該試薬液を 4 0 0 // 1 とり、 3 7 °Cに 1 0分間放置したの ち、 3 7 °Cの恒温状態に保たれた発光光度計 （ルミカウンター 1000 ： 日音医理科器械製作所製 ： 以下同じ） に装着し 3 mmo l 1 の過酸化水 素を 1 0 1および 1 Nの水酸化ナ ト リ ウム水溶液を 1 0 1添加し. 反応液の 1分間の積算発光量 〔発光強度 （cpm)〕 を測定した。 その結 果を第 2表に示す。 2 表 Take the reagent solution into a test tube at 400 // //, leave it at 37 ° C for 10 minutes, and then keep it at a constant temperature of 37 ° C (Lumicounter 1000: Nisson Medical) 3 mmol 1 of hydrogen peroxide was added to 101 and 1 N of 1 N sodium hydroxide aqueous solution, and 101 was added. The integrated emission of the reaction solution for 1 minute was added. The amount [emission intensity (cpm)] was measured. Table 2 shows the results. 2 Table
リ J ¾ Λ兀ΐ ¾ ス ィ 1卜し 口 物 "Γク J 7t ソし1 ½ ¾Κ 1 I又f J J ¾ Λ ΐ ΐ ΐ ¾ 1 1 1 1 又 Γ 1
( c p m) ( c p m) レ « rJ; R < QJ 丄 1 A: 丄 し ? 0) ^ 2 7 2 3 U (c p m) (c p m) «« rJ; R <QJ 丄 1 A: Yes? 0) ^ 2 7 2 3 U
1 7 fi Q fi 7 C Q O 1 7 fi Q fi 7 C Q O
丄 丄 / わ ΰ D ΰ y 丄 （3 D ζ 9 ^ 0 4 A Π U ft 0 丄 丄 / W ΰ D ΰ y 丄 (3D ζ 9 ^ 0 4 A Π U ft 0
A 1 O Λ C 0 厶 1 4 5 5 8 0 1 4 1 1 U b o o ό 1 8 4 9 6 7 1 D 4 0 b 7 5 4  A 1 O Λ C 0 m 1 4 5 5 8 0 1 4 1 1 U b oo ό 1 8 4 9 6 7 1 D 4 0 b 7 5 4
η ΐ 八 v n  η ΐ eight v n
4 4 1 2 7 0 9 1 6 1 1 6 o 6 b  4 4 1 2 7 0 9 1 6 1 1 6 o 6 b
n  n
0 丄 8 d d 1 Ζ 1 1 ϋ ύ U n 9 0 n 0 c c Ο Q Q Ο o O O Q Q β D 1 o 4 o o 丄 0 Z 9 0 b 0 9 9 q  0 丄 8 d d 1 Ζ 1 1 ϋ ύ Un 9 0 n 0 c c Ο Q Q Ο o O O Q Q β D 1 o 4 o o 丄 0 Z 9 0 b 0 9 9 q
丄 丄 y 丄 1 y Q  丄 丄 y 丄 1 y Q
丄 y ύ q o i  丄 y ύ q o i
4 O Q 丄 1 < Q3 丄 1 Π U li: 4 Λ 1  4 O Q 丄 1 <Q3 丄 1 Π U li: 4 Λ 1
丄 i QD t 0 Q 1  丄 i QD t 0 Q 1
丄 乙 丄 B
Ό 丄 O »J O 丄 1 O O ^ \J 0 7 7 0 3 9 9 2 2 1 5 0 8 0 9 1 5 0 2 9 0 0 2 3 7 6 9 1 9 8 発 光 強 度 化 合 物 発 光 強 度 ( c p m) c p m) 丄 丄 O »JO 丄 1 OO ^ \ J 0 7 7 0 3 9 9 2 2 1 5 0 8 0 9 1 5 0 2 9 0 0 2 3 7 6 9 1 9 8 Luminous intensity Compound Luminous intensity (cpm) cpm)
1 3 2 5 4 0 5 2 5 9 2 5 3 1 9 1 5 2 3 5 3 6 3 5 2 0 2 2 6 3 5 8 1 5 4 8 6 2 9 7 3 1 4 3 0 0 3 5 5 6 6 5 3 4 5 1 1 1 3 1 2 5 6 5 7 2 3 5 7 7 9 4 5 1 2 8 8 0 2 1 6 2 8 0 5 9 0 7 6 2 5 3 8 9 5 8 3 2 1 0 9 1 7 2 0 1 1 3 2 9 5 1 第 2表によれば、 本実施例に用いたクマリ ン誘導体は、 過酸化水素 の存在下ペルォキシダーゼと反応させることにより、 p H6. 0 におい てルミ ノールと同等かそれ以上の発光強度を示した。 1 3 2 5 4 0 5 2 5 9 2 5 3 1 9 1 5 2 3 5 3 6 3 5 2 0 2 2 6 3 5 8 1 5 4 8 6 2 9 7 3 1 4 3 0 0 3 5 5 6 6 5 3 4 5 1 1 1 3 1 2 5 6 5 7 2 3 5 7 7 9 4 5 1 2 8 8 0 2 1 6 2 8 0 5 9 0 7 6 2 5 3 8 9 5 8 3 2 1 0 9 1 7 2 0 1 1 3 2 9 5 1 According to Table 2, the coumarin derivative used in the present example showed an emission intensity at pH 6.0 equal to or higher than that of luminol by reacting with peroxidase in the presence of hydrogen peroxide. .
実施例 2 (過酸化水素の定量） Example 2 (quantification of hydrogen peroxide)
2 0 mmolのリ ン酸緩衝液 （ p H6. 0 ) 1 0 0 mlにペルォキシダーゼ 1 0 0 U、 5 O mgの ト リ ト ン X— 1 0 0、 および化合物 1、 5、 9、 1 0、 1 2、 1 4、 2 3、 比較化合物と してルミ ノ ールをそれぞれ 1 0 mg溶解し試薬液と した。  100 mmol of phosphate buffer (pH 6.0) in 100 ml of peroxidase 100 U, 50 mg of triton X—100, and compounds 1, 5, 9, 10 , 12, 14, 23, and 10 mg of luminol were each dissolved as a comparative compound to prepare a reagent solution.
試験管に該試薬液を 4 0 0 i 1 とり、 3 7 °Cに 1 0分間放置したの ち、 3 7 °Cの恒温状態に保たれた発光光度計に装着し各種濃度 （ 5 X 1 0 1 1 〜 2 X 1 0 ·" gZ反応液） の過酸化水素を 1 0 1 および 1 Nの水酸化ナ ト リウム水溶液を 1 添加し、 反応液の 1分間の積 算発光量を測定した。 その結果を図 1 および図 2 に示す。 Take 400 ml of the reagent solution into a test tube, leave it at 37 ° C for 10 minutes, and attach it to a luminescence photometer maintained at a constant temperature of 37 ° C to attach various concentrations (5 X 1 0 1 1 ~ 2 X 1 0 · "gZ reaction) of hydrogen peroxide was 1 added 1 0 1 and 1 N hydroxide Na preparative potassium aqueous solution of a was measured totalized light emission amount of 1 minute of the reaction solution The results are shown in Figures 1 and 2.
図 1 および図 2 によれば、 用いられたクマリ ン誘導体はルミ ノール と同等かそれ以上の発光強度を示した。  According to FIGS. 1 and 2, the coumarin derivative used showed an emission intensity equal to or higher than that of luminol.
実施例 3 (各種 p Hにおけるペルォキシダーゼ反応によるクマリ ン誘 導体を用いた化学発光の強度） Example 3 (Intensity of chemiluminescence using coumarin derivative by peroxidase reaction at various pH)
2 0 mmolフタル酸緩衝液 （ p H 2. 5〜5. 5 ) 、 2 0 mmolリ ン酸緩衝 液 （ p H5. 5〜8. 0 ) または 2 0 mmolホウ酸緩衝液 （ p H8. 0〜10) 各 1 0 0 mlにペルォキシダ一ゼ 1 0 0 U、 ト リ ト ン X— 1 0 0を 5 0 mgおよび化合物 1 または比較化合物としてルミ ノールをそれぞれ 1 0 mg溶解し試薬液とした。  20 mmol phthalate buffer (pH 2.5-5.5), 20 mmol phosphate buffer (pH 5.5-8.0) or 20 mmol borate buffer (pH 8.0 ~ 10) 100 U of peroxidase and 100 mg of Triton X-100 were dissolved in 100 mL of each, and 10 mg of luminol was dissolved as a compound 1 or a comparative compound to prepare a reagent solution. .
試験管に試薬液を 4 0 0 1 とり、 3 7 °Cに 1 0分間放置したのち. 3 7 °Cの恒温状態に保たれた発光光度計に装着し 3 mmol/1 の過酸化 水素を 1 0 // 1添加し、 反応液の 1 分間の積算発光量を測定した。 最 大発光量を示した p Hにおける発光量を 1 0 0 %と したときの各 p H における発光量の比を図 3 に示す。  Transfer the reagent solution to a test tube and leave it at 37 ° C for 10 minutes.Then, attach it to an emission photometer maintained at a constant temperature of 37 ° C and remove 3 mmol / 1 of hydrogen peroxide. 1 0 // 1 was added, and the integrated luminescence amount of the reaction solution for 1 minute was measured. FIG. 3 shows the ratio of the amount of luminescence at each pH when the luminescence at pH showing the maximum luminescence was 100%.
図 3 によれば、 化合物 1 の至適発光 p Hは酸性領域にあり、 ルミ ノ ールと比較して極めて広いことが示された。 According to FIG. 3, the optimal luminescence pH of Compound 1 is in the acidic region, It was shown to be extremely wide compared to
実施例 4 (ペルォキシダ一ゼ活性の測定） Example 4 (Measurement of peroxidase activity)
2 0 mmolのリ ン酸緩衝液 （ p H6. 0 ) 1 0 0 mlに、 5 0 mgの トリ ト ン X— 1 0 0、 および化合物 1 または比較化合物と してのルミ ノール 各 1 0 mg溶解し試薬液 1 とした。 対照として 1 0 0 mmolのホウ酸緩衝 液 （ P H9. 5 ) 1 0 0 mlに、 5 0 mgの ト リ トン X— 1 0 0およびルミ ノールを 1 0 mg溶解し試薬液 2 とした。 試験管に試薬液 1 または試薬 液 2を 4 0 0 1 とり、 3 7 °Cに 1 0分間放置したのち、 3 7 °Cの恒 温状態に保たれた発光光度計に装着し 3 0 0 mmol/1 の過酸化水素を 1 0 1および各濃度のペルォキシダ一ゼ溶液を 1 0 1添加し、 反 応液の 1分間の積算発光量を測定した。 その結果を図 4に示す。  In 100 ml of 20 mmol phosphate buffer (pH 6.0), 50 mg of triton X—100, and 10 mg each of luminol as compound 1 or comparative compound This was dissolved and used as reagent solution 1. As a control, 100 mg of triton X—100 and 100 mg of luminol were dissolved in 100 ml of 100 mmol of a borate buffer solution (PH9.5) to prepare a reagent solution 2. Take reagent solution 1 or reagent solution 2 in a test tube, leave it at 37 ° C for 10 minutes, and attach it to an emission photometer maintained at a constant temperature of 37 ° C. 101/1 of hydrogen / 1 of hydrogen peroxide and 101 of a peroxidase solution of each concentration were added, and the amount of luminescence of the reaction solution for 1 minute was measured. Fig. 4 shows the results.
図 4 によれば、 化合物 1 を用いた場合、 ルミ ノールを用いた場合よ り低濃度でペルォキシダーゼ活性の検量線が得られた。  According to FIG. 4, a calibration curve of peroxidase activity was obtained at a lower concentration when compound 1 was used than when luminol was used.
実施例 5 〔癌胎児性抗原 （C E A) の定量〕 Example 5 [Quantification of carcinoembryonic antigen (CEA)]
(1) ゥサギ抗ヒ ト C E A抗体のグルコースォキシダーゼ （G O D) に よ 5Bイし  (1) 5G of glucose oxidase (GOD) of heron anti-human CE antibody
抗 C E A抗体に対する G 0 Dの標識は石川らの方法 （酸素免疫測定 法、 8 2頁、 石川榮治ら編、 1987年、 医学書院刊） に従って作成した c すなわち、 N—サク シ二ミ ジル一 4— (N—マレイ ミ ドメチル） シク 口へキサン一 1 一カルボキシレー ト 〔ピアス （P I E R C E) 社製〕 を用いて G O Dにマレイ ミ ド基を導入した。 一方抗 C E A抗体をぺプ シン消化して得られた F ( a b ' ) z 画分を還元し、 S H基を遊離さ せた F ( a b ' ) を調製した。 両者を混合し反応させ、 G O D標識抗 C E A抗体を得た。  The G0D labeling for the anti-CEA antibody was performed according to the method of Ishikawa et al. (Oxygen immunoassay, page 82, edited by Eiji Ishikawa et al., 1987, published by The Medical College). C—N-succinimidyl 4 -— (N-Maleimide methyl) was used to introduce a maleimide group into the GOD using hexane-111-carboxylate [manufactured by PIERCE]. On the other hand, the F (ab ') z fraction obtained by digesting the anti-CEA antibody with pepsin was reduced to prepare F (ab') from which the SH group was released. Both were mixed and reacted to obtain a GOD-labeled anti-CEA antibody.
(2) 抗 C E A抗体固定化磁性粒子の調製  (2) Preparation of anti-CEA antibody-immobilized magnetic particles
磁性粒子としてマグノスフヱァ 〔（Magnosphere ：商品名） ステロジ エンバイォセパレーショ ン社製、 以下同じ〕 を抗 C E A抗体固定化磁 性粒子に使用した。 0. 1 mo 1 リ ン酸緩衝液 （ p H7. 0 ) に溶解した抗 C E A抗体溶液を該微粒子に等量添加し、 カ ップリ ング試薬である水 素化ホウ素ナ ト リ ウムを終濃度 0. 1 mol になるように添加し、 2時間 攪拌し反応させた。 磁石で粒子を集め、 上清を吸引除去後、 0. 1 % B S Aおよび 0. 1 % N a N , を含むリ ン酸緩衝液を 5 0 0 u 1 加え抗 C E A抗体固定化磁性粒子と して保存した。 Magnosphere [(Magnosphere: trade name) manufactured by Stelogy Bioseparation Co., Ltd .; same hereafter) was used as the magnetic particles for the magnetic particles immobilized with the anti-CEA antibody. 0.1 mo 1 Phosphate buffer dissolved in phosphate buffer (pH 7.0) An equal amount of a CEA antibody solution was added to the fine particles, sodium borohydride as a coupling reagent was added to a final concentration of 0.1 mol, and the mixture was stirred and reacted for 2 hours. After collecting the particles with a magnet and removing the supernatant by suction, 500 μl of a phosphate buffer containing 0.1% BSA and 0.1% NaN was added to obtain anti-CEA antibody-immobilized magnetic particles. And saved.
(3) C E Aの定量 ' (3) Quantitation of CEA ''
2 0 mmo 1のリ ン酸緩衝液 （ p H 6. 0 ) 1 0 0 mlにペルォキシダーゼ 1 0 0 U、 5 0 mgの ト リ ト ン X— 1 0 0および化合物 1 を 1 0 mg溶解 し試薬液と した。 試験管に試薬液を 4 0 0 〃 1 とり、 3 7 °Cに 1 0分 間放置したのち、 3 7 °Cの恒温状態に保たれた発光光度計に装着した c 別に、 各濃度の標準 C E Aを 5 0 1 、 抗 C E A抗体固定化磁性粒子 を 1 0 0 1 、 G O D標識抗 C E A抗体を 1 0 0 / 1 混合し、 3 7 °C にて 1 時間反応させ、 C E Aを G O D標識抗 C E A抗体および抗 C E A抗体固体化磁性粒子に結合させた。 磁石で粒子を集め、 上清を吸引 除去後、 C E Aと結合した G 0 Dの基質と して 1 O miolのクェン酸緩 衝液 （ 0115. 0 ) に 5 4 mgZmlの濃度でグルコースを溶解した液 100 1 を加え 3 7 °Cにて 3 0分反応させた後、 停止液と して 5 0 mmolグ リ シン緩衝液 （ p H l l. 0 ) を 1 0 1 添加した。 磁石で粒子を分 離し、 この液の上清 1 0 1 を発光光度計に装着しておいた試薬液に 添加し 1分間の積算発光量を測定した。 その結果を図 5 に示す。  Dissolve 10 mg of peroxidase 100 U, 50 mg of Triton X—100 and Compound 1 in 100 ml of phosphate buffer (pH 6.0) at 20 mmo 1. The reagent solution was used. Transfer the reagent solution to a test tube at 400 ° C, leave it at 37 ° C for 10 minutes, and attach it to an emission photometer maintained at a constant temperature of 37 ° C. Mix CEA with 501, anti-CEA antibody-immobilized magnetic particles with 1001, and GOD-labeled anti-CEA antibody with 100/1, and react at 37 ° C for 1 hour. Antibodies and anti-CEA antibodies were bound to solidified magnetic particles. The particles were collected by a magnet, the supernatant was removed by suction, and glucose was dissolved at a concentration of 54 mgZml in 1 O miol citrate buffer (01115.0) as a substrate for G0D bound to CEA. After 100 1 was added and the mixture was reacted at 37 ° C. for 30 minutes, 50 mmol of glycine buffer (pH 11) was added as a stop solution. The particles were separated by a magnet, the supernatant 101 of this solution was added to the reagent solution attached to the luminescence photometer, and the integrated luminescence amount for one minute was measured. Figure 5 shows the results.
実施例 6 (ペルォキシダーゼ反応によるクマリ ン誘導体を用いる化学 発光） Example 6 (Chemiluminescence using coumarin derivative by peroxidase reaction)
2 0 mmolの酢酸緩衝液 （ p Η4· 0 ) 1 0 0 mlにペルォキシダーゼ 1 0 0 U、 5 0 mgの ト リ ト ン X— 1 0 0および第 3表に記載のクマリ ン誘導体または比較化合物と してのルミ ノ ール各 1 O mgを溶解し試薬 液と した。  Peroxidase 100 U, 100 mg of Triton X—100 in 100 ml of 20 mmol acetate buffer (pΗ4.0) and 100 mg of the coumarin derivative or comparative compound shown in Table 3 Then, 1 O mg of each of the luminols was dissolved and used as a reagent solution.
試験管に該試薬液を 4 0 0 〃 1 とり、 3 7 °Cに 1 0分間放置したの ち、 3 7 °Cの恒温状態に保たれた発光光度計に装着し 3 mmolZl の過 酸化水素を 1 0 ^ 1および 1 Nの水酸化ナ トリゥム水溶液を 1 0 1 添加し、 反応液の 1分間の積算発光量を測定した。 その結果を第 3表 に示す。 Take 400 ml of the reagent solution into a test tube, leave it at 37 ° C for 10 minutes, attach it to a luminescence photometer maintained at a constant temperature of 37 ° C, and add 3 mmolZl Hydrogen oxide was added to an aqueous solution of 10 ^ 1 and 1N aqueous sodium hydroxide in an amount of 101, and the integrated luminescence of the reaction solution for 1 minute was measured. Table 3 shows the results.
第 3 表 化 合 物 発 光 強 度 化 合 物 発 光 強 度  Table 3 Compound emission intensity Compound emission intensity
c m ρ ) c m p ) ルミ ノ ール 4 7 2 1 3 2 7 8 1 5 2 0  cm ρ) cm p) Luminor 4 7 2 1 3 2 7 8 1 5 2 0
1 1 7 3 4 5 3 0 3 3 1 3 4 9 2 1 5 1 1 7 3 4 5 3 0 3 3 1 3 4 9 2 1 5
2 6 2 3 0 5 3 4 1 2 6 1 8 2 6 2 3 0 5 3 4 1 2 6 1 8
7 3 3 4 1 4 3 5 3 6 2 5 8 7 7 3 3 4 1 4 3 5 3 6 2 5 8 7
8 2 8 2 3 1 3 8 1 1 3 5 3 8 2 8 2 3 1 3 8 1 1 3 5 3
2 3 3 8 0 6 7 1 3 4 0 2 4 8 3 1 2 3 3 8 0 6 7 1 3 4 0 2 4 8 3 1
2 4 9 2 5 1 8 4 1 3 1 5 2 2 2 4 9 2 5 1 8 4 1 3 1 5 2 2
2 6 5 8 0 5 7 4 2 1 3 5 0 9 0 2 6 5 8 0 5 7 4 2 1 3 5 0 9 0
2 9 3 4 0 0 4 4 3 2 4 4 4 2 2 9 3 4 0 0 4 4 3 2 4 4 4 2
3 0 3 4 9 5 6 6 4 4 1 2 4 1 8 3 0 3 4 9 5 6 6 4 4 1 2 4 1 8
3 1 1 5 5 2 1 9 9 5 6 9 7 5 1 6 第 3表によれば、 本実施例に用いたクマリ ン誘導体は、 ペルォキシ ダーゼの存在下、 過酸化水素と反応させることにより、 p H4. 0の酸 性条件においてもルミ ノールをはかるに上回る発光強度を示した。 実施例 7 (クマリ ン誘導体を用いる化学発光によるクマリ ン誘導体の 定量） 3 1 1 5 5 2 1 9 9 5 6 9 7 5 1 6 According to Table 3, the coumarin derivative used in the present example, when reacted with hydrogen peroxide in the presence of peroxidase, emits light that far exceeds luminol even under acidic conditions of pH 4.0. The strength was indicated. Example 7 (Quantification of coumarin derivative by chemiluminescence using coumarin derivative)
2 0 mmolの酢酸緩衝液 （ p H4. 0 ) または 2 0 mmolのリ ン酸緩衝液 ( H 6. 0 ) 1 0 0 mlにペルォキシダーゼ 1 0 0 U、 5 0 mgの ト リ ト ン X— 1 0 0および第 4表に記載のクマリ ン誘導体を所定の濃度に溶 解し試薬液と した。  In 100 ml of 20 mmol acetate buffer (pH 4.0) or 20 mmol phosphate buffer (H 6.0), 100 uU of peroxidase, 50 mg of triton X— The coumarin derivatives described in 100 and Table 4 were dissolved at a predetermined concentration to obtain a reagent solution.
試験管に該試薬液を 4 0 0 β 1 とり、 3 7 °Cに 1 0分間放置したの ち、 3 7 °Cの恒温状態に保たれた発光光度計に装着し、 3 mmolZl の 過酸化水素を 1 0 1 添加し、 反応液の 1 分間の積算発光量を測定し た。 その結果を第 4表に示す。 Take the reagent solution at 400 β1 in a test tube, leave it at 37 ° C for 10 minutes, attach it to a luminescence photometer maintained at a constant temperature of 37 ° C, and add 3 mmolZl of peroxide. Hydrogen was added in 101, and the integrated luminescence of the reaction solution for 1 minute was measured. Table 4 shows the results.
Figure imgf000024_0001
化 難夜中のクマリン^ ίΦ»離 (mg/ml) 口 H
Figure imgf000024_0001
Coumarin in difficult nights ^ ίΦ »release (mg / ml) Mouth H
物 102 ιυ 丄 υ 丄 U 100 107 108 Object 10 2 ιυ 丄 υ 丄 U 10 0 10 7 10 8
31 4 1.13X10" 1.10X10" 9.00X10 3.80X10 37X10:' 1.02X10:' 8.20X102 31 4 1.13X10 "1.10X10" 9.00X10 3.80X10 37X10: '1.02X10:' 8.20X10 2
32 4 6.93Χ10Γ' 5.11X10" 462X10'' 1.90X10 ― 32 4 6.93 Χ10 Γ '5.11X10 "462X10''1.90X10 ―
33 4 1.10X10" 9.00Χ10' 8.32X10^ 1.20X10 ―  33 4 1.10X10 "9.00Χ10 '8.32X10 ^ 1.20X10 ―
34 4 1.21 104 8.30Χ10:ι 418X10:' 34 4 1.21 10 4 8.30Χ10 : ι 418X10: '
35 4 3.60X105 Ζ50Χ10Γ' 9.00X101 9.30X103 2.30X103 1.11X103 7.00X102 35 4 3.60X10 5 Ζ50Χ10 Γ '9.00X10 1 9.30X10 3 2.30X10 3 1.11X10 3 7.00X10 2
37 6 8.05X10' Z43X10 8.87X10:' 37 6 8.05X10 'Z43X10 8.87X10:'
38 4 1.36X10' 7.28X10'1 1.00Χ10:1 38 4 1.36X10 '7.28X10' 1 1.00Χ10 : 1
40 4 a ooxio' 6.71X10:' 440X10:'  40 4 a ooxio '6.71X10:' 440X10: '
41 4 3.59ΧΚΤ 5.00 10:ι 1.92Χ10;ι 41 4 3.59ΧΚΤ 5.00 10 : ι 1.92Χ10 ; ι
42 4 1.51X10r' 8.11X10' 6.50X10:' 1.00Χ10:' 一 42 4 1.51X10 r '8.11X10' 6.50X10: '1.00Χ10 : '
43 4 2.10X104 6.18X103 2.00X103 43 4 2.10X10 4 6.18X10 3 2.00X10 3
44 4 1.06X104 7.60X10'' 1.03X10'' 44 4 1.06X10 4 7.60X10 '' 1.03X10 ''
46 6 2.74 X 10s 9.18X10" 1.27X101 6.59X10:' ― 46 6 2.74 X 10 s 9.18X10 "1.27X10 1 6.59X10: '―
47 6 6.25X104 1.68X10" 7.36X10:' 2.50X103 一 47 6 6.25X10 4 1.68X10 "7.36X10: '2.50X10 3
49 6 5.11XKT 2.41X10^ 1.81X10' 5.63Χ10η 49 6 5.11XKT 2.41X10 ^ 1.81X10 '5.63Χ10 η
50 6 1.18X10r' 7.33X101 5.40X10:' 1.00X10:' 50 6 1.18X10 r '7.33X10 1 5.40X10:' 1.00X10: '
l. yxiハu「》 6.10X101 418X10:' l. yxi ha u ">> 6.10X10 1 418X10: '
52 6 6.11 101 1.32X10' 5.91X10:' 52 6 6.11 10 1 1.32X10 '5.91X10:'
53 6 7.00X101 δ,θδχιο11 a 16X10:' 53 6 7.00X10 1 δ, θδχιο 11 a 16X10: '
54 6 a i5xio4 了.00X10:' 1.13X103 54 6 a i5xio 4 ending.00X10: '1.13X10 3
55 6 5.90X104 3.21X10" 1.00X103 55 6 5.90X10 4 3.21X10 "1.00X10 3
56 4 1.21X10r' 6.48X104 469X10:' 1) - ：舰していない 56 4 1.21X10 r '6.48X10 4 469X10:' 1)-: not
2 )
Figure imgf000025_0001
( c p m)を^ 。 第 4表によれば、 化合物 3 1および化合物 3 5を筆頭に、 化合物 4， 5， 8， 9， 1 2， 2 3， 3 2， 3 3， 4 2 , 4 6， 4 7， 4 9およ び 5 0 自体は極めて低い濃度まで化学発光法によつて定量することが できることが判る。 2)
Figure imgf000025_0001
(cpm) ^. According to Table 4, Compounds 3, 5, 8, 9, 12, 23, 32, 33, 42, 46, 47, 49, with Compound 31 and Compound 35 at the top. Also, it can be seen that 50 itself can be quantified by chemiluminescence to very low concentrations.
実施例 8 〔 —フ ヱ トプロテイ ン （A F P) の定量〕 Example 8 [Quantification of photoprotein (AFP)]
(1) ャギ抗ヒ ト A F P抗体の化合物 3 5 による標識化  (1) Labeling of goat anti-human AFP antibody with compound 35
抗 A F P抗体に対する化合物 3 5の標識体は次のようにして作製し た。 すなわち、 化合物 3 5の有するカルボキシル基と抗 A F P抗体の 有するァ ミ ノ基とを 1 —ェチルー 3 — ( 3 —ジメチルァ ミノプロピル） カルボジイ ミ ド （以下 E D C) を用いて p H 7. 0 にて反応、 結合させ、 化合物 3 5檫識抗 A F P抗体を得た。  A labeled compound of compound 35 against the anti-AFP antibody was prepared as follows. That is, the carboxyl group of compound 35 and the amino group of the anti-AFP antibody react with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (hereinafter referred to as EDC) at pH 7.0. Then, compound 35 was recognized as anti-AFP antibody.
(2) 抗 A F P抗体固定化磁性粒子の調製  (2) Preparation of anti-AFP antibody-immobilized magnetic particles
磁性粒子としてマグノスフ 二ァを抗 A F P抗体固定化磁性粒子に使 用した。 0. 1 niol リ ン酸緩衝液 （ p H7. 0 ) に溶解した抗 A F P抗体 溶液を該微粒子に等量添加し、 カ ップリ ング試薬である水素化ホウ素 ナ ト リ ウムを終濃度 0. 1 mol になるように添加し、 2時間攪拌し反応 させた。 磁石で粒子を集め、 上清を吸引除去後、 0. 1 %B S Aおよび 0. 1 %N a N3 を含むリ ン酸緩衝液を 5 0 0 1加え抗 A F P抗体固 定化磁性粒子として保存した。 Magnospher was used as the magnetic particles for the anti-AFP antibody-immobilized magnetic particles. An equal volume of an anti-AFP antibody solution dissolved in 0.1 niol phosphate buffer (pH 7.0) is added to the microparticles, and sodium borohydride, a coupling reagent, is added to a final concentration of 0.1. The reaction mixture was stirred to react for 2 hours. The particles were collected with a magnet, the supernatant stored after aspirated off, the-phosphate buffer containing 0. 1% BSA and 0. 1% N a N 3 as 5 0 0 1 In addition anti-AFP antibody-immobilized magnetic particles did.
(3) A F Pの定量  (3) Quantification of AFP
2 0 mmoiの酢酸緩衝液 （ p H 4. 0 ) 1 0 0 mlにペルォキシダーゼ 1 0 0 Uおよび 5 0 mgの ト リ トン X— 1 0 0を溶解し試薬液とした。 別に、 各濃度の標準 A F Pを 5 0 1、 抗 A F P抗体固定化磁性粒子 を 1 0 0 1、 化合物 3 5標識抗 A F P抗体を 1 0 0 1混合し、 3 7 °Cにて 1時間反応させ、 A F Pを化合物 3 5標識抗 A F P抗体お よび抗 A F P抗体固定化磁性粒子に結合させた。 磁石で粒子を集め、 上清を吸引除去後、 3 7 °Cの恒温状態に保たれた発光光度計に装着し、 予め 3 7 °Cに保温した試薬液 4 0 0 1 および 3 mmol/1 の過酸化水 素を 2 0 1添加し、 反応液の 1 分間の積算発光量を測定した。 その 結果を図 6 に示す。 100 U of peroxidase and 100 mg of Triton X—100 were dissolved in 100 ml of 20 mmoi acetate buffer (pH 4.0) to prepare a reagent solution. Separately, mix 501 of standard AFP at each concentration, 1001 of anti-AFP antibody-immobilized magnetic particles, and 1001 of compound 35-labeled anti-AFP antibody, and react at 37 ° C for 1 hour. AFP was bound to compound 35-labeled anti-AFP antibody and anti-AFP antibody-immobilized magnetic particles. The particles were collected with a magnet, the supernatant was removed by suction, and then attached to an emission photometer maintained at a constant temperature of 37 ° C. Peroxide water Then, 201 was added, and the integrated luminescence amount of the reaction solution for 1 minute was measured. Figure 6 shows the results.
実施例 9 〔前立腺酸性フ ォスフ ァ タ一ゼ （ P A P ) の定量〕  Example 9 [Quantification of prostatic acid phosphatase (PAP)]
(1) ャギ抗ヒ ト P A P抗体の化合物 3 5 による標識化  (1) Labeling of goat anti-human PAP antibody with compound 35
抗 P A P抗体に対する化合物 3 5の標識体は次のようにして作製し た。 すなわち、 5 0 mgの化合物 3 5 をジォキサン 1 0 mlに溶解し、 E D C 7 5 mgおよび N —ヒ ドロキシコハク酸ィ ミ ド 5 0 mgを添加後、 室温にて 2 4時間攪拌し、 反応液を濃縮乾固した。 固形分を酢酸ェチ ル 水にて抽出し、 有機層を濃縮乾固した。 これをジォキサン 1 0 ml に再溶解し、 —ァラニン 5 0 mgを添加して室温にて 4時間反応後、 濃縮乾固し、 固形分を得た。  A labeled compound 35 against anti-PAP antibody was prepared as follows. That is, 50 mg of compound 35 was dissolved in 10 ml of dioxane, 75 mg of EDC and 50 mg of N-hydroxysuccinic acid imid were added, and the mixture was stirred at room temperature for 24 hours. Concentrated to dryness. The solid was extracted with ethyl acetate water, and the organic layer was concentrated to dryness. This was redissolved in 10 ml of dioxane, 50 mg of -alanine was added, and the mixture was reacted at room temperature for 4 hours, and concentrated to dryness to obtain a solid.
上記固形分とャギ抗ヒ ト P A P抗体とを E D Cを用いて p H 7. 0 に て反応、 結合させ、 化合物 3 5標識抗 P A P抗体を得た。  The solid was reacted with goat anti-human PAPP antibody at pH 7.0 using EDC and allowed to bind to obtain a compound 35-labeled anti-PAPP antibody.
(2) 抗 P A P抗体固定化磁性粒子の調製  (2) Preparation of anti-PAP antibody-immobilized magnetic particles
磁性粒子としてマグノ スフエアを抗 P A P抗体固定化磁性粒子に使 用した。 0. 1 mo l リ ン酸緩衝液 （ p H 7. 0 ) に溶解した抗 P A P抗体 溶液を該微粒子に等量添加し、 カ ップリ ング試薬である水素化ホウ素 ナ ト リ ゥムを終濃度 0. 1 mo l になるように添加し、 2時間攪拌し反応 させた。 磁石で粒子を集め、 上清を吸引除去後、 0. 1 % B S Aおよび 0. 1 % N a N を含むリ ン酸緩衝液を 5 0 0 1加えた抗 P A P固体 固定化磁性粒子と して保存した。  Magnosphere was used as the magnetic particles for the anti-PAP antibody-immobilized magnetic particles. An equal volume of an anti-PAP antibody solution dissolved in 0.1 mol phosphate buffer (pH 7.0) is added to the microparticles, and the final concentration of sodium borohydride, a coupling reagent, is added. 0.1 mol was added, and the mixture was stirred and reacted for 2 hours. The particles were collected with a magnet, the supernatant was removed by suction, and then the anti-PAP solid immobilized magnetic particles were added with 501 in phosphate buffer containing 0.1% BSA and 0.1% NaN. saved.
(3) P A Pの定量  (3) Quantification of PAP
2 0 mmo lの酢酸緩衝液 （ p H 4. 0 ) 1 0 0 mlにペルォキシダーゼ 1 0 0 Uおよび 5 0 mgの ト リ ト ン X— 1 0 0を溶解し試薬液と した。 別に、 各濃度の標準 P A Pを 5 0 μ. 1 、 抗 P A Ρ抗体固定化磁性粒子 を 1 0 0 /Z 1 、 化合物 3 5標識抗 P A P抗体を 1 0 0 〃 1混合し、 3 7 °Cにて 1 時間反応させ、 P A Pを化合物 3 5標識抗？八？抗体ぉ よび抗 P A P抗体固定化磁性粒子に結合させた。 磁石で粒子を集め、 上清を吸引除去後 3 7 °Cの恒温状態に保たれた発光光度計に装着し、 予め 3 7 °Cに保温した試薬液 4 0 0 1および 3 mmol/1 の過酸化水 素を 2 0 / 1添加し、 反応液の 1分間の積算発光量を測定した。 その 結果を図 7 に示す。 100 U of peroxidase and 100 mg of Triton X—100 were dissolved in 100 ml of 20 mM acetate buffer (pH 4.0) to prepare a reagent solution. Separately, mix 50 μl of standard PAP at each concentration, 100 / Z 1 anti-PA Ρ antibody-immobilized magnetic particles, and 100 〃 1 of compound 35-labeled anti-PAP antibody at 37 ° C. And react for 1 hour with PAP. Eight? The antibody was bound to the magnetic particles immobilized on the antibody and anti-PAP antibody. Collect particles with a magnet, After removing the supernatant by suction, it was attached to an emission photometer maintained at a constant temperature of 37 ° C, and the reagent solution 4001 and 3 mmol / 1 hydrogen peroxide, which had been pre-incubated at 37 ° C, were added for 2 hours. 0/1 was added, and the integrated luminescence amount of the reaction solution for 1 minute was measured. Figure 7 shows the results.
実施例 1 0 (タンパク質の影響） Example 10 (Effect of protein)
2 0 mmolの酢酸緩衝液 （ p H 4. 0 ) 1 0 0 mlにペルォキシダーゼ 1 0 0 U、 5 0 mgの ト リ ト ン X— 1 0 0および化合物 3 1 または化合 物 3 5を 1 0 mg溶解し、 それぞれ試薬液と した。  Add 100 U of peroxidase, 100 U of peroxidase, 50 mg of Triton X—100 and Compound 31 or Compound 35 to 100 mL of 20 mmol acetate buffer (pH 4.0). mg were dissolved and used as reagent solutions.
対照化合物として、 2 0 mmolのリ ン酸緩衝液 （ p H6. 0 ) 1 0 0 ml に 5 0 mgの ト リ トン X _ 1 0 0およびァク リ ジニゥムエステル （ァク リ ジニゥムー I ： 同仁化学研究所） 0. l mgを溶解し試薬液とした。 各試薬液に所定の濃度の B S Aを添加し、 この試薬液を試験管に 4 0 0 1 とり、 3 7 °Cに 1 0分間放置したのち、 3 7 °Cの恒温状態 に保たれた発光光度計に装着した。 化合物 3 1 または化合物 3 5の場 合は、 3 mmol/1 の過酸化水素を 1 0 1 、 ァク リ ジニゥムエステル の場合は 3 mmol/1 の過酸化水素を 1 0 z 1 および 1 Nの水酸化ナ ト リゥム水溶液を 1 0 1添加し、 反応液の 1分間の積算発光量を測定 した。 その結果を図 8 に示す。  As a control compound, 20 mg of phosphate buffer (pH 6.0) in 100 ml of 50 mg of triton X — 100 and acridinium ester (acridinium I: Dojindo Chemical) (Laboratory) 0.1 mg was dissolved and used as a reagent solution. A predetermined concentration of BSA was added to each reagent solution, 4001 of this reagent solution was placed in a test tube, left at 37 ° C for 10 minutes, and then luminescence kept at a constant temperature of 37 ° C Attached to the photometer. In the case of compound 31 or compound 35, 3 mmol / 1 of hydrogen peroxide is 101, and in the case of acridium ester, 3 mmol / 1 of hydrogen peroxide is 10z1 and 1N water. An aqueous solution of sodium oxide was added in a quantity of 101, and the integrated luminescence of the reaction solution for one minute was measured. Figure 8 shows the results.
図 8 によりば、 ァク リ ジニゥムエステルに比較して、 化合物 3 1お よび化合物 3 5 は共存する B S Aの影響を極めてうけにくいことが判 る  According to FIG. 8, it is found that Compound 31 and Compound 35 are much less susceptible to the coexisting BSA than the acrylic ester.
発 明 の 効 果  The invention's effect
本発明のクマリ ン誘導体を用いる化学発光方法によれば、 反応液の •P Hが中性から酸性側であっても微量の過酸化水素、 過酸化活性物質 もしく はクマリ ン誘導体、 または過酸化活性物質、 酸化酵素も しく は クマリ ン誘導体を化学結合させた物質 （例えば、 C E A， A F P, P A P等） をタンパク質の影響をほとんど受けることなく定量できる  According to the chemiluminescence method using the coumarin derivative of the present invention, even when the pH of the reaction solution is from neutral to acidic, a slight amount of hydrogen peroxide, a peroxide active substance or a coumarin derivative, or a peroxide is used. Active substances, oxidases, or substances chemically coupled with coumarin derivatives (eg, CEA, AFP, PAP, etc.) can be quantified with little effect from proteins

Claims

1. 過酸化水素と式 （ I ) 1. Hydrogen peroxide and formula (I)
請Contract
Figure imgf000029_0001
Figure imgf000029_0001
または式 （ Π ) Or the formula (Π)
の 範  Range of
( Π囲 )
Figure imgf000029_0002
(Surrounding)
Figure imgf000029_0002
(式中、 R 1 、 R2 、 R3 、 R4 、 R 5 および は同一または異な つて、 水素原子、 置換も しく は非置換の低級アルキル、 低級アルコキ シ、 置換も し く は非置換のァラルキル、 置換も しく は非置換のァ リ ー ル、 ハロゲン原子、 シァノ、 ニ トロ、 スルホ、 カルボキシ、 アルコキ シカルボニル、 アルキル力ルバモイル、 置換も しく は非置換のァ リ ー ルカルバモイル、 力ルバモイル、 ヒ ドロキシ、 置換も しく は非置換の ァ ミ ノ、 低級アルカノ ィル、 低級アルカ ノ ィルォキシまたは複素環基 を表すかまたは R ' と R 2 が一緒になってアルキレンも し く はァルケ 二レ ンを形成する力、、 または R4 と R5 がー緒になって- CH2CH2CH2NH- を形成する） で表されるクマリ ン誘導体またはその塩を、 過酸化活性 物質の存在下に反応させ、 反応液の発光量または発光強度を測定する ことを特徴とする過酸化活性物質、 過酸化水素または式 （I ) も し く は式 （Π) で表される クマリ ン誘導体の定量方法。 (Wherein, R 1 , R 2 , R 3 , R 4 , R 5 and R are the same or different and each represents a hydrogen atom, a substituted or unsubstituted lower alkyl, a lower alkoxy, a substituted or unsubstituted Aralkyl, substituted or unsubstituted aryl, halogen atom, cyano, nitro, sulfo, carboxy, alkoxycarbonyl, alkyl rubamoyl, substituted or unsubstituted aryl carbamoyl, carbamoyl, phenyl Represents droxy, substituted or unsubstituted amino, lower alkanol, lower alkanoyloxy or heterocyclic group, or R 'and R 2 taken together form alkylene or alkenylene. Or a salt thereof, or R 4 and R 5 are linked together to form —CH 2 CH 2 CH 2 NH—) in the presence of a peroxide active substance. reaction A method for quantifying a peroxide active substance, hydrogen peroxide or a coumarin derivative represented by the formula (I) or the formula (II), wherein the luminescence amount or the luminescence intensity of the reaction solution is measured.
2. 式 （ I ) において、 R 1 が置換もしく は非置換のァリールまたは シァノであり、 R 2 、 R 3 、 R 4 及び が水素原子または置換も し く は非置換の低級アルキルであり、 R 5 が置換もく しは非置換のア ミ ノ、 二 トロまたは低級アルコキシである請求項 1記載の定量方法。 2. In the formula (I), R 1 is a substituted or unsubstituted aryl or cyano, R 2 , R 3 , R 4 and are a hydrogen atom or a substituted or unsubstituted lower alkyl, R 5 is a substituted heather Mr unsubstituted a Mi Roh, quantitative method of claim 1, wherein a two-Toro or lower alkoxy.
3. 式 （Π ) において、 R ' がシァノ、 アルコキシカルボニルまたは カルボキシであり、 R 2 及び R 3 が水素原子である請求項 1記載の定 量方法。 3. The method according to claim 1, wherein in formula (Π), R ′ is cyano, alkoxycarbonyl or carboxy, and R 2 and R 3 are hydrogen atoms.
4. 定量される成分が過酸化活性物質である請求項 1記載の定量方法 < 4. The method according to claim 1, wherein the component to be determined is a peroxide active substance.
5. 過酸化活性物質がペルォキシダーゼである請求項 4記載の定量方 5. The method according to claim 4, wherein the peroxide active substance is peroxidase.
6. ペルォキシダーゼが抗原または抗体に標識されている請求項 5記 載の定量方法。 6. The method according to claim 5, wherein peroxidase is labeled on the antigen or the antibody.
7. 定量される成分が過酸化水素である請求項 1記載の定量方法。 7. The method according to claim 1, wherein the component to be determined is hydrogen peroxide.
8. 過酸化水素が酵素反応に由来する請求項 7記載の定量方法。 8. The method according to claim 7, wherein the hydrogen peroxide is derived from an enzymatic reaction.
PCT/JP1993/000128 1992-02-04 1993-02-03 Method of quantitative determination of substance with coumarin derivative WO1993015219A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP5513100A JP2980681B2 (en) 1992-02-04 1993-02-03 Determination of substances using coumarin derivatives

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP1904392 1992-02-04
JP4/19043 1992-02-04

Publications (1)

Publication Number Publication Date
WO1993015219A1 true WO1993015219A1 (en) 1993-08-05

Family

ID=11988399

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP1993/000128 WO1993015219A1 (en) 1992-02-04 1993-02-03 Method of quantitative determination of substance with coumarin derivative

Country Status (1)

Country Link
WO (1) WO1993015219A1 (en)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000004008A1 (en) * 1998-07-16 2000-01-27 Gentest Corporation Novel cyp2d fluorescent assay reagents
US6207404B1 (en) 1999-09-30 2001-03-27 Gentest Corporation Coumarin-based CYP3A fluorescent assay reagents
DE102008049675A1 (en) 2008-09-30 2010-04-01 Markus Dr. Heinrich Process for the preparation of 3-aminobiphenyls
US8475776B2 (en) 2005-04-28 2013-07-02 Paloma Pharmaceuticals, Inc. Compositions and methods to treat diseases characterized by cellular proliferation and angiogenesis
US20130171664A1 (en) * 2010-01-29 2013-07-04 Dalibor Sames Ph-responsive fluorescent false neurotransmitters and their use
US9381187B2 (en) 2011-02-16 2016-07-05 Paloma Pharmaceuticals, Inc. Radiation countermeasure agents
USRE46558E1 (en) 2005-04-28 2017-09-26 Paloma Pharmaceuticals, Inc. Compositions and methods to treat diseases characterized by cellular proliferation and angiogenesis
CN111039910A (en) * 2019-12-31 2020-04-21 云南大学 Photoinitiated method for synthesizing 3-aryl flavone or coumarin compound and application thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FENXI HUAXUE, Vol. 19, No. 7, (1991), G. XIE et al., "Optimization of Determining Hydrogen Peroxide by Chemiluminescence Detection", pages 823-825. *
J. BIOLUMIN CHEMILUMIN, Vol. 4, No. 1, (1989), D. SLAWINSKA et al., "Hydroxycoumarins as Sensitizers and Reactants of Chemiluminescent Oxidative Reactions", pages 226-230. *
J. CHROMATOGR., Vol. 542, No. 2, (1991), M. TOD et al., "Luminarin 4 as a Labelling Reagent for Carboxylic Acids in Liquid Chromatography With Peroxyoxalate Chemiluminescence Detection", pages 295-306. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000004008A1 (en) * 1998-07-16 2000-01-27 Gentest Corporation Novel cyp2d fluorescent assay reagents
US6130342A (en) * 1998-07-16 2000-10-10 Gentest Corporation CYP2D fluorescent assay reagents
US6207404B1 (en) 1999-09-30 2001-03-27 Gentest Corporation Coumarin-based CYP3A fluorescent assay reagents
US8475776B2 (en) 2005-04-28 2013-07-02 Paloma Pharmaceuticals, Inc. Compositions and methods to treat diseases characterized by cellular proliferation and angiogenesis
USRE46558E1 (en) 2005-04-28 2017-09-26 Paloma Pharmaceuticals, Inc. Compositions and methods to treat diseases characterized by cellular proliferation and angiogenesis
DE102008049675A1 (en) 2008-09-30 2010-04-01 Markus Dr. Heinrich Process for the preparation of 3-aminobiphenyls
US20130171664A1 (en) * 2010-01-29 2013-07-04 Dalibor Sames Ph-responsive fluorescent false neurotransmitters and their use
US9075014B2 (en) * 2010-01-29 2015-07-07 The Trustees Of Columbia University In The City Of New York pH-responsive fluorescent false neurotransmitters and their use
US9381187B2 (en) 2011-02-16 2016-07-05 Paloma Pharmaceuticals, Inc. Radiation countermeasure agents
CN111039910A (en) * 2019-12-31 2020-04-21 云南大学 Photoinitiated method for synthesizing 3-aryl flavone or coumarin compound and application thereof

Similar Documents

Publication Publication Date Title
USRE49466E1 (en) Solution phase homogeneous assays
FI76380B (en) LYSINOMETRISK LESANDE ELLER LUMINOMETRISK BESTAEMNING.
JP3117459B2 (en) Method for amplifying luminescence signal from fluorescent compound
EP0087959B1 (en) Enhanced luminescent and luminometric assay
JPH0553478B2 (en)
US5424194A (en) 4-(cyanomethylthio)phenol enhanced peroxidase assays
US20100273189A1 (en) Non separation assays with selective signal inhibitors
JP4068137B2 (en) Biotinylated chemiluminescent label, conjugate, assay and assay kit
WO1993015219A1 (en) Method of quantitative determination of substance with coumarin derivative
JPH0648996B2 (en) Amplified emission analysis
US5851785A (en) Method of quantitative determination of substances using coumarin derivatives
JP2980681B2 (en) Determination of substances using coumarin derivatives
JP3792899B2 (en) Chemiluminescent enzyme immunoassay method
JP4028645B2 (en) Chemiluminescent enzyme immunoassay method
JP3746381B2 (en) Chemiluminescent enzyme immunoassay method
JP3815905B2 (en) Enzyme immunoassay
JP4286357B2 (en) Chemiluminescent enzyme immunoassay method
JP3194446B2 (en) Immunological detection method using electron carrier as labeling substance
JP3792886B2 (en) Chemiluminescent enzyme immunoassay method
JP2002221521A (en) New fluorescent source
JP2000180448A (en) Method for measuring enzyme immunity
WO2001035103A1 (en) Stabilizing a luminescent acridinium compound

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): JP US

ENP Entry into the national phase

Ref country code: US

Ref document number: 1993 122582

Date of ref document: 19931001

Kind code of ref document: A

Format of ref document f/p: F