JP2980681B2 - Determination of substances using coumarin derivatives - Google Patents

Description

Description: TECHNICAL FIELD 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 (II) by a chemiluminescence method using a coumarin derivative.

2. Description of the Related Art Accurate quantification of minute amounts of hydrogen peroxide and peroxidase activities is important in analytical chemistry and biochemistry, especially in the field of clinical testing, where the substance to be measured is finally converted to hydrogen peroxide. Quantitative methods are often used. In addition, antigen, antibody or DNA can be used for peroxidase,
There is known a method for quantifying an antigen, an antibody or DNA by labeling with an oxidase or a luminescent compound other than peroxidase and quantifying peroxidase activity or hydrogen peroxide or a luminescent compound generated by the reaction of the oxidase.

Among the methods for quantifying hydrogen peroxide and peroxidase activities, methods using bioluminescence and chemiluminescence of a compound serving as a substrate for peroxidase are known as highly sensitive quantification methods. Compounds such as luminol, isoluminol, lucigenin, and acridinium ester are known as particularly usable luminescent compounds (bioluminescence and chemiluminescence,
Kazuhiro Imai, eds. P.82-89, (1989), Hirokawa Shoten] has a problem that if the protein coexists in the reaction solution or the pH of the reaction solution is acidic, the luminescence intensity decreases. is there.

The coumarin derivatives represented by the formulas (I) and (II) described below are known as fluorescent compounds and are commercially available [Fluorescence Analytical Chemistry, pp. 159 to 161 (1987), published by Baifukan; Volume, 644 (1972); Heterocycles, 7, 933 (1977); Industrial Chemistry, 71, 1010 (1968); Catalogs of Eastman Kodak Company, Wako Pure Chemical Industries, etc.] .

It is not known that a coumarin derivative emits light by hydrogen peroxide in the presence of a peroxide active substance.

DISCLOSURE OF THE INVENTION The present invention relates to hydrogen peroxide and a compound of formula (I) Or formula (II) (Wherein, R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are the same or different and each represents a hydrogen atom, a substituted or unsubstituted lower alkyl,
Lower alkoxy, substituted or unsubstituted aralkyl, substituted or unsubstituted aryl, halogen atom, cyano,
Nitro, sulfo, carboxy, alkoxycarbonyl,
Represents an alkylcarbamoyl, substituted or unsubstituted arylcarbamoyl, carbamoyl, hydroxy, substituted or unsubstituted amino, lower alkanoyl, lower alkanoyloxy or heterocyclic group, or R ¹ and R ² together represent alkylene or alkenylene. Form or
Or a combination of R ⁴ and R ⁵ to form —CH ₂ CH ₂ CH ₂ NH—) or a salt thereof in the presence of a peroxide active substance; 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 (II), characterized by measuring the amount or luminescence intensity.

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.

After the reaction emits light, it is not confirmed what structure the coumarin derivative has changed to.However, the presence of a substance that is distinguished from the coumarin derivative before the reaction has been confirmed by chromatography, and the reaction produces a compound that emits light. It is clear that you are doing.

In the definition in formula (I) or formula (II), the alkyl moiety in lower alkyl, lower alkoxy, lower alkanoyl, lower alkanoyloxy, alkoxycarbonyl and alkylcarbamoyl is a straight-chain or branched alkyl having 1 to 8 carbon atoms, For example, represents methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, etc., aralkyl represents a group having 7 to 15 carbon atoms, for example, benzyl, phenethyl and the like, aryl, aryl part in aryl carbamoyl Represents phenyl, naphthyl and the like, a halogen atom represents iodine, bromine, chlorine, fluorine and the like, and a heterocyclic group represents pyridyl, benzothiazolinyl and the like.

Substituents in the substituted alkyl, substituted aralkyl, substituted aryl and substituted aryl carbamoyl may be the same or different and each has 1 to 5 cyano, halogen atom, substituted or unsubstituted amino, lower alkyl, lower alkoxy, carboxy, alkoxycarbonyl, Represents hydroxy and the like. The alkyl moiety and the halogen atom in lower alkyl, lower alkoxy and alkoxycarbonyl are as defined above.

The substituents on the substituted amino are the same or different and represent a lower alkyl having 1 or 2 substituents, a substituted or unsubstituted heterocyclic group. Lower alkyl has the same meaning as described above, and the heterocyclic group represents, for example, triazinyl, pyrazinyl, pyridyl, pyrimidinyl and the like. The substituent in the substituted heterocyclic group is
The alkyl moiety in lower alkoxy and alkylamino, which is the same or different and represents a cyano, halogen atom, amino, lower alkoxy, hydroxy, dialkylamino or the like having 1 or 2 substituents, is as defined above.

The alkylene includes a group having 2 to 4 carbon atoms,
(CH ₂ ) ₂ —, — (CH ₂ ) ₃ —, — (CH ₂ ) ₄ — and the like. Alkenylene includes a group having 2 to 4 carbon atoms, and —CH ＝ CH—, —CH ＝ _{CH-CH 2 -, - CH} = CH-CH = CH
-And the like.

As the acceptable salt of the compound (I) or (II),
Acid addition salts include, for example, inorganic acid salts such as hydrochloride, sulfate and phosphate, and organic acid salts of acetate, maleate, fumarate and citrate.

Table 1 shows specific compounds used in the present invention.

The coumarin derivatives described in Table 1 are known compounds, for example, The Chemical Society of Japan, vol. 3, p. 644 (1972); Heterocycles, vol. 7, p. 933 (1977);
Volume 1, p. 1010 (1968) and the like. The compound can also be purchased from Eastman Kodak Co., Aldrich Co., Ltd., Daito Mixmix Co., or the like.

The peroxidation active substance used in the method of the present invention is a general term for compounds having peroxidase activity, for example,
Peroxidases 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.

When performing a quantitative analysis by the method of the present invention, it is generally performed as follows.

The reaction is performed in a buffer adjusted to pH 2 to 8 by combining a buffer such as hydrochloric acid, acetic acid, acetate, succinate, succinate, borate, phthalate, glycine, barbital salt, and good (GOOD). (0.005 to 2 mol / l).

Compound (I) or (II) is 0.01 μmol / l to 100 mol /
l, hydrogen peroxide is 10 μmol / l to 100 mmol / l, and the peroxide active substance is 10 ⁻⁹ to 2 × 10 ² mg / ml, preferably peroxidase.
Used at 10 ^-9 to 10 ² U / ml.

Components other than the component to be measured among these three components are added to a buffer solution to prepare a reagent solution, and a sample containing the component to be measured is added to the reagent solution at -10 to 90 ° C, preferably 20 to 50 ° C. Let react. The total light emission amount of the reaction solution or the light emission amount for a certain period of time is measured by a light emission photometer or the like in a wavelength region of 190 to 750 nm,
The component to be quantified in the sample can be quantified using a calibration curve prepared using a known amount of the sample containing the component to be measured.

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.

When performing the method of the present invention, a surfactant [for example, Triton X
-100 (manufactured by Yoneyama Pharmaceutical Co., Ltd.)] with a concentration of 0.01 to 5 wt%
Can be added.

In order to enhance the emission intensity of the light emitting compound, a protein, a polyalkyl quaternary amine, a fluorescent agent, dimethyl sulfoxide, or the like can be used as necessary. Further, if necessary, an alkaline solution can be added. Examples of proteins include bovine serum albumin (BSA),
Examples include human serum albumin (HSA), human immunoglobulin, ovalbumin, and the like, and examples of polyalkyl quaternary amines include polydiaryldimethylammonium chloride and poly [vinylbenzyl (benzyldimethyl-ammonium chloride)]. Examples of the fluorescent agent include fluorescein and 4-fluoro-7.
A conjugate of -nitrobenzofurazan, 7-fluoro-4-nitrobenzoxadiazole with an amine, amino acid, peptide or protein or a derivative thereof can be used. As the alkaline solution, for example,
An aqueous solution such as sodium hydroxide or potassium hydroxide can be used. These luminescence enhancers are used in an amount of 0.0001 to
Used at a concentration of 10 wt%.

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.

As the peroxidative active substance that can be quantified, any substance 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.

2. Description of the Related Art Conventionally, for example, hydrogen peroxide is stoichiometrically generated from a substrate in a biological sample using an enzyme, and the amount of the hydrogen peroxide is quantified to quantify the substrate.

In addition, in order to measure the enzyme activity in a biological sample, the enzyme activity in the sample is quantified by measuring the rate of hydrogen peroxide generated by performing an enzyme reaction by adding an appropriate enzyme or substrate. .

Specific examples thereof include quantification of a substrate for a specific oxidase (eg, glucose oxidase, galactose oxidase, cholesterol oxidase, uricase, etc.) reaction, and measurement of the activity of an enzyme such as monoamine oxidase, cholinesterase.

In addition, when quantifying the antigen-antibody reaction using the enzyme immunoassay (EIA method), peroxidase is used as a labeling substance, and the peroxidase activity after the antigen-antibody reaction or hydrogen peroxide generated by the action of this activity is quantified. Have been done. For example, enzyme immunoassay (Eiji Ishikawa et al.,
1987, Medical Shoin), for example, by reacting an antigen with an immobilized antibody, and reacting the antigen with, for example, an antibody labeled with an enzyme such as peroxidase or glucose oxidase. Of hydrogen peroxide produced by the action of By using the present invention for the EIA method or the like, a carcinoembryonic antigen (CEA), α-fetoprotein (AFP), It is possible to measure in vivo trace components such as acid phosphatase (PAP).

By applying the present invention to the measurement of hydrogen peroxide or peroxidase activity exemplified above, trace components can be quantified.

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 an antigen-antibody reactant, and the antigen or antibody can be quantified by measuring the amount or intensity of luminescence.

Further, the present invention can be applied to a polynucleotide measuring method. As a polynucleotide measuring method, a polynucleotide that complementarily binds to a test polynucleotide is labeled with an enzyme such as peroxidase or glucose oxidase, or a compound (I) or (II), and then complementarily binds to the test polynucleotide. Examples of the method include measuring the enzyme activity of the enzyme-labeled or compound-labeled polynucleotide or quantifying the labeled compound to determine the amount of the test polynucleotide. The method is described in "DNA Probe" (disc, 1988 edition) and "DNA Probe II" (disc, 1990).
Year version).

The feature of the present invention using the coumarin derivative represented by the formula (I) or (II) is that the pH range at which light emission occurs is as wide as pH 1 to 10, especially in the acidic to neutral range of pH 5 to 7. That is, a light emission amount can be obtained. Conventionally used luminol, isoluminol, lucigenin, acridinium ester, etc. cannot obtain a satisfactory luminescence amount unless under strong alkaline conditions, and when the optimal pH of an enzyme such as peroxidase is 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 which has an optimum pH in an 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, so that it is possible to detect even a trace amount of the enzyme compared to the conventional method.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows compounds using various concentrations of hydrogen peroxide.
The change of the luminescence intensity of 1,5,9 and luminol is shown.

Explanation of symbols − −− Compound 1 − ● − Compound 5 −− □ −− Compound 9 −− × −− Luminol Figure 2 shows Compound 1 when various concentrations of hydrogen peroxide were used.
5 shows changes in the emission intensity of 0, 12, 14, 23 and luminol.

Explanation of symbols − ● − Compound 10 −− □ −− Compound 12 − △ − Compound 14 − ○ − Compound 23 −−−−− Luminol FIG. 3 shows changes in the luminescence intensity of Compound 1 and Luminol at various pHs.

Explanation of reference symbols -O- Compound 1-----------------------------Luminol FIG.

Explanation of Symbols-●-Compound 1 ---- □-Luminol FIG. 5 shows changes in the emission intensity of Compound 1 when various concentrations of CEA were used.

Explanation of Symbols-●-Compound 1 FIG. 6 shows the change in the emission intensity of Compound 35 when various concentrations of AFP were used.

Explanation of Symbols -−- Compound 35 FIG. 7 shows the change in the luminescence intensity of Compound 35 when various concentrations of PAP were used.

Explanation of Symbols -−- Compound 35 FIG. 8 shows the ratio of the compound 31, 35 and the acridinium ester (acridinium-I: Dojindo Laboratories) to the luminescence intensity without BSA when various concentrations of BSA were used. Show.

DESCRIPTION OF SYMBOLS-□-Compound 31-◇-Compound 35-○-Acridinium ester BEST MODE FOR CARRYING OUT THE INVENTION Examples of the present invention are shown below.

Example 1 (chemiluminescence using coumarin derivative by peroxidase reaction) 100 U of a horseradish peroxidase (manufactured by Toyobo: same hereafter) in 100 ml of 20 mmol phosphate buffer (pH 6.0),
50 mg of Triton X-100 and 10 mg of each of the coumarin derivatives listed in Table 2 or 10 mg of luminol as a comparative compound (manufactured by Tokyo Chemical Industry; the same applies hereinafter) were dissolved to prepare reagent solutions.

400 μl of the reagent solution is placed in a test tube, left at 37 ° C. for 10 minutes, and then kept at a constant temperature of 37 ° C. by a luminescence photometer (Lumicounter 1000: manufactured by Nichion Medical Science Instrument Co., Ltd .: the same applies hereinafter).
Then, 10 μl of 3 mmol / l hydrogen peroxide and 10 μl of a 1N aqueous solution of sodium hydroxide were added, and the integrated luminescence amount [luminescence intensity (cpm)] of the reaction solution for 1 minute was measured. Table 2 shows the results.

According to Table 2, the coumarin derivative used in the present example showed a luminescence intensity equal to or higher than that of luminol at pH 6.0 by reacting with oxidase in the presence of hydrogen peroxide.

Example 2 (Quantification of hydrogen peroxide) 100 U of peroxidase, 50 mg of Triton X-100, and compound 1 in 100 ml of 20 mmol phosphate buffer (pH 6.0)
5, 9, 10, 12, 14, 23 and 10 mg of luminol as a comparative compound were each dissolved to prepare a reagent solution.

Take 400 μl of the reagent solution 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 attach it to various concentrations (5 × 10 ¹¹ to 2 × 10 ^-8 g / reaction). Liquid) and 10 μl of 1N aqueous sodium hydroxide solution
The reaction mixture was added, and the integrated luminescence of the reaction solution for one minute was measured. The results are shown in FIGS.

According to FIGS. 1 and 2, 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) 20 mmol phthalate buffer (pH 2.5 to 5.5), 20 mmol phosphate buffer (pH 5.5 to 8.0) or 20 mmol borate buffer Liquid (pH 8.
0-10) Peroxidase 100U, Triton X-
50 mg of 100 and 10 mg of luminol as the compound 1 or the comparative compound were each dissolved to prepare a reagent solution.

Take 400 μl of the reagent solution in 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.
10 μl of 3 mmol / l hydrogen peroxide was added, and the integrated luminescence amount of the reaction solution for one minute was measured. FIG. 3 shows the ratio of the luminescence amount at each pH when the luminescence amount at the pH showing the maximum luminescence amount is 100%.

FIG. 3 shows that the optimum luminescence pH of Compound 1 is in an acidic region, which is much wider than that of Luminol.

Example 4 (Measurement of peroxidase activity) 50 mg of Triton X-100 and 10 mg of each of compound 1 or luminol as a comparative compound were dissolved in 100 ml of a 20 mmol phosphate buffer (pH 6.0) to obtain a reagent solution 1. As a control
Reagent solution 2 was obtained by dissolving 50 mg of Triton X-100 and 10 mg of luminol in 100 ml of 100 mmol of borate buffer (pH 9.5). Take 400 μl of reagent solution 1 or reagent solution 2 in a test tube, leave it at 37 ° C. for 10 minutes, and attach it to a luminescence photometer kept at a constant temperature of 37 ° C., and add 300 μm / l of hydrogen peroxide at 10 μl.
and 10 μl of a peroxidase solution of each concentration were added, and the integrated luminescence amount of the reaction solution for one minute was measured. FIG. 4 shows the results.

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.

Example 5 [Quantification of carcinoembryonic antigen (CEA)] (1) Labeling of rabbit anti-human CEA antibody with glucose oxidase (GOD) GOD labeling of anti-CEA antibody was carried out according to the method of Ishikawa et al. Page, edited by Eiji Ishikawa et al., 1987, published by the Medical School.)
Created according to. That is, N-succinimidyl-4
A maleimide group was introduced into the GOD using-(N-maleimidomethyl) cyclohexane-1-carboxylate (manufactured by PIERCE). On the other hand, the F (ab ') ₂ fraction obtained by digesting the anti-CEA antibody with pepsin was reduced to prepare F (ab') from which the SH group was released. Mix and react both,
GOD-labeled anti-CEA antibody was obtained.

(2) Preparation of anti-CEA antibody-immobilized magnetic particles Magnosphere ((Magnosphere: trade name, manufactured by Sterogen Bioseparation Co., Ltd .; the same applies hereinafter)) was used as the anti-CEA antibody-immobilized magnetic particles. An equal amount of an anti-CEA antibody solution dissolved in 0.1 mol phosphate buffer (pH 7.0) is added to the fine particles, and sodium borohydride as a coupling reagent is added to a final concentration of 0.1 mol, and the mixture is added for 2 hours. The mixture was stirred and reacted. After collecting the particles with a magnet and removing the supernatant by suction, 500 μl of phosphate buffer containing 0.1% BSA and 0.1% NaN ₃ was added.
μl was added and stored as anti-CEA antibody-immobilized magnetic particles.

(3) Quantification of CEA 100 U of peroxidase, 50 mg of Triton X-100 and compound 1 were added to 100 ml of a 20 mmol phosphate buffer (pH 6.0) in 100 ml.
g was dissolved to give a reagent solution. Take 400 μl of the reagent solution into a test tube,
After being left at 37 ° C. for 10 minutes, it was attached to a luminescence photometer maintained at a constant temperature of 37 ° C. Separately, add 50 μl of standard CEA at each concentration.
l, 100 μl of anti-CEA antibody-immobilized magnetic particles, GOD-labeled anti-CEA
100 μl of the antibody was mixed and reacted at 37 ° C. for 1 hour.
GOD-labeled anti-CEA antibody and anti-CEA antibody were bound to solidified magnetic particles. After collecting the particles with a magnet and removing the supernatant by suction, CEA
10 mmol citrate buffer (p
H5.0) in which glucose was dissolved at a concentration of 54 mg / ml, 100μ
After the reaction at 37 ° C for 30 minutes, 50 mm
100 μl of ol glycine buffer (pH 11.0) was added. The particles were separated with a magnet, and 10 μl of the supernatant of this solution was added to the reagent solution mounted on the luminometer, and the integrated luminescence amount for one minute was measured. The result is shown in FIG.

Example 6 (chemiluminescence using coumarin derivative by peroxidase reaction) 100 U of peroxidase, 50 mg of triton X-100 in 100 ml of 20 mmol acetate buffer (pH 4.0) and coumarin derivative shown in Table 3 or luminol as a comparative compound each
10 mg was dissolved to prepare a reagent solution.

Take 400 μl of the reagent solution in a test tube, leave it at 37 ° C. for 10 minutes, attach it to an emission photometer kept at a constant temperature of 37 ° C., attach 10 μl of 3 mmol / l hydrogen peroxide and 1N aqueous sodium hydroxide solution. Was added, and the integrated luminescence amount of the reaction solution for one minute was measured. Table 3 shows the results.

According to Table 3, the coumarin derivative used in this example showed a luminescence intensity far higher than that of luminol even under acidic conditions of pH 4.0 by reacting with hydrogen peroxide in the presence of peroxidase.

Example 7 (Quantification of coumarin derivative by chemiluminescence using coumarin derivative) In 100 ml of 20 mmol acetate buffer (pH 4.0) or 20 mmol phosphate buffer (pH 6.0), 100 U of peroxidase, 50 mg of Triton X-100 and The coumarin derivatives shown in Table 4 were dissolved at a predetermined concentration to prepare a reagent solution.

Take 400 μl of the reagent solution in a test tube, leave it at 37 ° C. for 10 minutes, attach it to an emission photometer kept at a constant temperature of 37 ° C., add 10 μl of 3 mmol / l hydrogen peroxide, and add the reaction solution. 1
The integrated light emission amount per minute was measured. Table 4 shows the results.

According to Table 4, compound 31 and compound 35 are the leading,
It can be seen that compounds 4, 5, 8, 9, 12, 23, 32, 33, 42, 46, 47, 49 and 50 themselves can be quantified by chemiluminescence to very low concentrations.

Example 8 [Quantification of α-fetoprotein (AFP)] (1) Labeling of goat anti-human AFP antibody with compound 35 A labeled compound of compound 35 for anti-AFP antibody was prepared as follows. That is, the carboxyl group of the compound 35 and the amino group of the anti-AFP antibody were combined with 1-ethyl-3-
(3-dimethylaminopropyl) carbodiimide (hereinafter referred to as
The mixture was reacted and bound at pH 7.0 using EDC) to obtain a compound 35-labeled anti-AFP antibody.

(2) Preparation of anti-AFP antibody-immobilized magnetic particles Magnosphere was used as anti-AFP antibody-immobilized magnetic particles as magnetic particles. An equal amount of an anti-AFP antibody solution dissolved in 0.1 mol phosphate buffer (pH 7.0) was added to the fine particles, and sodium borohydride as a coupling reagent was added to a final concentration of 0.1 m
ol, and the mixture was stirred and reacted for 2 hours. After collecting the particles with a magnet and removing the supernatant by suction, 0.1% BSA and 0.1%
500 μl of a phosphate buffer solution containing% NaN ₃ was added and stored as anti-AFP antibody-immobilized magnetic particles.

(3) Determination of AFP A reagent solution was prepared by dissolving 100 U of peroxidase and 50 mg of Triton X-100 in 100 ml of 20 mmol acetate buffer (pH 4.0). Separately, 50 μl of standard AFP at each concentration, 100 μl of anti-AFP antibody-immobilized magnetic particles, and 100 μl of compound 35-labeled anti-AFP antibody
After mixing and reacting 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 by a magnet, and the supernatant was removed by suction.Then, the sample was attached to an emission photometer maintained at a constant temperature of 37 ° C., and 400 μl of a reagent solution preliminarily maintained at 37 ° C. and 20 μl of 3 mmol / l hydrogen peroxide were added. The integrated luminescence amount of the reaction solution for one minute was measured. FIG. 6 shows the result.

Example 9 [Quantification of prostatic acid phosphatase (PAP)] (1) Labeling of goat anti-human PAP antibody with compound 35 A labeled compound of anti-PAP antibody with compound 35 was prepared as follows. That is, 50 mg of compound 35 was added to dioxane 10
The mixture was dissolved in 50 ml, and added with 75 mg of EDC and 50 mg of N-hydroxysuccinimide, stirred at room temperature for 24 hours, and 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 β-alanine was added, and the mixture was reacted at room temperature for 4 hours, and concentrated to dryness to obtain a solid.

The solid content and the goat anti-human PAP antibody were adjusted to pH 7.
Reaction and binding were performed at 0 to obtain a compound 35-labeled anti-PAP antibody.

(2) Preparation of anti-PAP antibody-immobilized magnetic particles Magnosphere was used as anti-PAP antibody-immobilized magnetic particles as magnetic particles. An equivalent amount of an anti-PAP antibody solution dissolved in 0.1 mol phosphate buffer (pH 7.0) is added to the fine particles, and sodium borohydride as a coupling reagent is added to a final concentration of 0.1 m
ol, and the mixture was stirred and reacted for 2 hours. After collecting the particles with a magnet and removing the supernatant by suction, 0.1% BSA and 0.1%
It was stored as anti-PAP solid immobilized magnetic particles to which 500 μl of a phosphate buffer containing% NaN ₃ was added.

(3) Determination of PAP 100 U of peroxidase and 50 mg of Triton X-100 were dissolved in 100 ml of 20 mM acetate buffer (pH 4.0) to prepare a reagent solution. Separately, 50 μl of standard PAP at each concentration, 100 μl of anti-PAP antibody-immobilized magnetic particles, and 100 μl of compound 35-labeled anti-PAP antibody
After mixing and reacting at 37 ° C. for 1 hour, PAP was bound to the compound 35-labeled anti-PAP antibody and the magnetic particles immobilized with the anti-PAP antibody. The particles were collected by a magnet, the supernatant was removed by suction, attached to an emission photometer maintained at a constant temperature of 37 ° C., and 400 μl of a reagent solution previously heated to 37 ° C. and 20 μl of 3 mmol / l hydrogen peroxide were added. The integrated luminescence amount of the reaction solution for one minute was measured. FIG. 7 shows the result.

Example 10 (Effect of Protein) 100 U of peroxidase, 50 mg of Triton X-100, and 10 mg of Compound 31 or Compound 35 were dissolved in 100 mL of 20 mmol acetate buffer (pH 4.0) to prepare reagent solutions.

As a control compound, 20 mmol phosphate buffer (pH 6.0) 1
50 mg of Triton X-100 and 0.1 mg of acridinium ester (acridinium-I: Dojindo Laboratories) were dissolved in 00 ml to prepare a reagent solution.

A predetermined concentration of BSA is added to each reagent solution, 400 μl of this reagent solution is placed in a test tube, and left at 37 ° C. for 10 minutes.
It was mounted on a luminescence photometer maintained at a constant temperature of ° C. Compound
In the case of 31 or compound 35, 3 mmol / l hydrogen peroxide is added to 10
μl, in the case of acridinium ester, 10 μl of 3 mmol / l hydrogen peroxide and 10 μl of 1N aqueous sodium hydroxide solution.
μl was added, and the integrated luminescence amount of the reaction solution for one minute was measured.
FIG. 8 shows the result.

According to FIG. 8, compared to the acridinium ester,
It can be seen that Compound 31 and Compound 35 are extremely unlikely to be affected by coexisting BSA.

Effects of the Invention 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 trace amount of hydrogen peroxide, a peroxide active substance or a coumarin derivative, or a peroxide active substance In addition, a substance (for example, CEA, AFP, PAP, etc.) to which an oxidase or a coumarin derivative is chemically bonded can be quantified almost without being affected by proteins.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int.Cl. ⁶ , DB name) C12Q 1/28 CA (STN) REGISTRY (STN)

Claims (15)

(57) [Claims] 1. Hydrogen peroxide and a compound of the formula (I) Or formula (II) (Wherein, when R ⁵ is lower alkoxy, nitro, or substituted or unsubstituted amino, R ¹ , R ² , R ³ , R ⁴ and R ⁶ are the same or different and each represents a hydrogen atom, a substituted or unsubstituted Represents lower alkyl, lower alkoxy, substituted or unsubstituted aryl, halogen atom, cyano, carboxy, alkoxycarbonyl, substituted or unsubstituted arylcarbamoyl, hydroxy, substituted or unsubstituted amino, lower alkanoyl or heterocyclic group, or or R ¹ and R ² together form an alkylene or alkenylene, or R ⁴ and R ⁵ together form a -CH ₂ CH ₂ CH ₂ NH-, when R ⁵ is hydroxy, R ¹ is a substituted or unsubstituted lower alkyl, substituted or unsubstituted aryl, halogen atom, carboxy, or a heterocyclic group, R ² is a substituted or unsubstituted Lower alkyl, R ³ is a hydrogen atom, R ⁴ is a hydrogen atom or an alkoxy, and R ⁶ represents a hydrogen atom, a substituted or unsubstituted alkyl or substituted or unsubstituted amino)
A coumarin derivative or a salt thereof is reacted in the presence of a peroxide active substance, and the luminescence amount or luminescence intensity of the reaction solution is measured. ) Or a method for quantifying a coumarin derivative represented by the formula (II) or a salt thereof. 2. In the formula (I), R ¹ is a hydrogen atom, substituted or unsubstituted aryl, cyano, lower alkoxy, carboxy, alkoxycarbonyl, lower alkanoyl or heterocyclic group, and R ² , R ³ , R ⁴ and ⁶ are the same or different and are a hydrogen atom or a substituted or unsubstituted lower alkyl, R ⁵ is a substituted or unsubstituted amino, nitro, lower alkoxy, or R ¹ and R ² together or form an alkylene or alkenylene, or R ⁴ and quantification method of claim 1, wherein R ⁵ together and forms a -CH ₂ CH ₂ CH ₂ NH-. 3. In the formula (II), R ¹ is cyano, lower alkoxycarbonyl, carboxy, hydrogen atom, lower alkoxy, substituted or unsubstituted aryl, lower alkanoyl, heterocyclic group, and R ² and R ³ are The same or different, hydrogen, substituted or unsubstituted substituted lower alkyl,
Or the method of claim ¹ , wherein R ¹ and R ² together form alkylene or alkenylene. 4. In the formula (I), R ¹ is a substituted or unsubstituted aryl or cyano, and R ² , R ³ , R ⁴ and R ⁶
Is the same or different and is a hydrogen atom or a substituted or unsubstituted lower alkyl, and R ⁵ is a substituted or unsubstituted amino, nitro or lower alkoxy. 5. The method according to claim 1, wherein in the formula (II), R ¹ is cyano, alkoxycarbonyl or carboxy, and R ² and R ³ are hydrogen atoms. 6. The method according to claim 1, wherein the component to be determined is a peroxide active substance. 7. The method according to claim 6, wherein the peroxidation active substance is peroxidase. 8. The method according to claim 7, wherein the peroxidase is labeled on an antigen or an antibody. 9. The method according to claim 1, wherein the component to be determined is hydrogen peroxide. 10. The method according to claim 9, wherein the hydrogen peroxide is derived from an enzymatic reaction. 11. The formula (I) Or formula (II) (Wherein, when R ⁵ is lower alkoxy, nitro, or substituted or unsubstituted amino, R ¹ , R ² , R ³ , R ⁴ and R ⁶ are the same or different and each represents a hydrogen atom, a substituted or unsubstituted Represents lower alkyl, lower alkoxy, substituted or unsubstituted aryl, halogen atom, cyano, carboxy, alkoxycarbonyl, substituted or unsubstituted arylcarbamoyl, hydroxy, substituted or unsubstituted amino, lower alkanoyl or heterocyclic group, or or R ¹ and R ² together form an alkylene or alkenylene, or R ⁴ and R ⁵ together, CH ₂ CH ₂ CH ₂ NH- to form, when R ⁵ is hydroxy, R ¹ is a substituted or unsubstituted lower alkyl, substituted or unsubstituted aryl, halogen atom, carboxy, or a heterocyclic group, R ² is a substituted or unsubstituted Lower alkyl, R ³ is a hydrogen atom, R ⁴ is a hydrogen atom or an alkoxy, and R ⁶ represents a hydrogen atom, a substituted or unsubstituted alkyl or substituted or unsubstituted amino)
A reagent for quantitatively determining hydrogen peroxide by a luminescence amount or luminescence intensity measurement method, comprising a coumarin derivative or a salt thereof represented by the formula: 12. The formula (I) Or formula (II) (Wherein, when R ⁵ is lower alkoxy, nitro, or substituted or unsubstituted amino, R ¹ , R ² , R ³ , R ⁴ and R ⁶ are the same or different and each represents a hydrogen atom, a substituted or unsubstituted Represents lower alkyl, lower alkoxy, substituted or unsubstituted aryl, halogen atom, cyano, carboxy, alkoxycarbonyl, substituted or unsubstituted arylcarbamoyl, hydroxy, substituted or unsubstituted amino, lower alkanoyl or heterocyclic group, or or R ¹ and R ² together form an alkylene or alkenylene, or R ⁴ and R ⁵ together form a -CH ₂ CH ₂ CH ₂ NH-, when R ⁵ is hydroxy, R ¹ is a substituted or unsubstituted lower alkyl, substituted or unsubstituted aryl, halogen atom, carboxy, or a heterocyclic group, R ² is a substituted or unsubstituted Lower alkyl, R ³ is a hydrogen atom, R ⁴ is a hydrogen atom or an alkoxy, and R ⁶ represents a hydrogen atom, a substituted or unsubstituted alkyl or substituted or unsubstituted amino)
A reagent for quantifying a peroxide active substance by a luminescence amount or luminescence intensity measurement method, comprising a coumarin derivative represented by the formula (1) or a salt thereof and hydrogen peroxide. 13. A compound of formula (I) comprising a peroxide active substance and hydrogen peroxide, which is determined by a method for measuring the amount or intensity of luminescence. Or formula (II) (Wherein, when R ⁵ is lower alkoxy, nitro, or substituted or unsubstituted amino, R ¹ , R ² , R ³ , R ⁴ and R ⁶ are the same or different and each represents a hydrogen atom, a substituted or unsubstituted Represents lower alkyl, lower alkoxy, substituted or unsubstituted aryl, halogen atom, cyano, carboxy, alkoxycarbonyl, substituted or unsubstituted arylcarbamoyl, hydroxy, substituted or unsubstituted amino, lower alkanoyl or heterocyclic group, or or R ¹ and R ² together form an alkylene or alkenylene, or R ⁴ and R ⁵ together form a -CH ₂ CH ₂ CH ₂ NH-, when R ⁵ is hydroxy, R ¹ is a substituted or unsubstituted lower alkyl, substituted or unsubstituted aryl, halogen atom, carboxy, or a heterocyclic group, R ² is a substituted or unsubstituted Lower alkyl, R ³ is a hydrogen atom, R ⁴ is a hydrogen atom or an alkoxy, and R ⁶ represents a hydrogen atom, a substituted or unsubstituted alkyl or substituted or unsubstituted amino)
A reagent for quantifying a coumarin derivative or a salt thereof represented by the formula: 14. In the formula (I), R ¹ is a hydrogen atom, substituted or unsubstituted aryl, cyano, lower alkoxy,
A carboxy, alkoxycarbonyl, lower alkanoyl or heterocyclic group, R ² , R ³ , R ⁴ and ⁶ are the same or different and are a hydrogen atom or a substituted or unsubstituted lower alkyl, and R ⁵ is substituted or unsubstituted amino, nitro, lower alkoxy, or R ¹ and one R ² form an alkylene or alkenylene together, or R ⁴ and R ⁵ together with -CH ₂ CH ₂ CH ₂ NH- 14. The quantitative reagent according to claim 11, 12 or 13, which is formed. 15. In the formula (II), R ¹ is cyano, lower alkoxycarbonyl, carboxy, hydrogen atom, lower alkoxy, substituted or unsubstituted aryl, lower alkanoyl, heterocyclic group, and R ² and R ³ are The same or different, hydrogen, substituted or unsubstituted substituted lower alkyl,
14. The quantification reagent according to claim 11, 12 or 13, wherein R ¹ and R ² are combined to form an alkylene or an alkenylene.