JPH07167862A - Chemiluminescent compound and assaying method using the same - Google Patents

Abstract

PURPOSE:To obtain a chemiluminescent compound having high stability by acridinium compound or its isomer which is represented by a specific formula and in which a group which does not disturb its chemiluminescence may have at an arbitrary position of its acridine ring or phenyl ring. CONSTITUTION:This acridinium compound is represented by a formula and contains isomer (R1: alkyl, alkenyl, alkynyl, etc., R2: alkyl fluoride, R3: alkyl, alkyl fluoride or alkoxy, X and Y are O or S, and A<-> is anion which does not disturb chemiluminescence). A group which does not disturb the chemiluminescence may have at an arbitrary position of its acridine ring or phenyl ring. As an assaying method using this compound, a composite material is formed by mixing a substance to be inspected, labeled at a sample with this compound with bonding substance to be specifically bonded to the substance to be inspected. The labeled bonding substance liberated therefrom is separated, chemiluminescence of this compound bonded to the composite material is detected, and a quantity of the substance to be inspected in the sample is decided from its luminescent amount.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel chemiluminescent compound useful as a labeling substance in a chemiluminescent analysis method, a luminescent complex to which the chemiluminescent compound is bound, and an assay using the chemiluminescent compound. Concerning the law.

[0002]

2. Description of the Related Art The application of chemiluminescent compounds to analytical techniques has been expanding more and more in recent years due to their high sensitivity. Luminol, acridinium, stabilized dioxetane, peroxalate, and the like have high emission yields and are being put to practical use in various fields as reagents for high-sensitivity analysis. Among them, an acridinium compound has a detection sensitivity of 10 ⁻¹⁸ mol level and can be easily bound to various ligand molecules, and thus has attracted attention as a safe labeling substance instead of a radioisotope.

Particularly in the field of clinical examinations, there is an increasing demand for a ligand assay utilizing an antigen / antibody reaction or complementary binding of nucleic acids, and high precision and speedy measurement by an automatic analyzer is desired. On the other hand, since the acridinium compound emits flash-type light emission in seconds, it is a reagent that can sufficiently meet such requirements in the field of clinical examination.

N-methylacridinium-9-carboxylic acid aryl ester chemically changes into excited N-methylacridone by hydrogen peroxide under alkaline conditions and emits light. (Prog. Org. Chem., 8, 231-277, 1973). afterwards,
It was first applied by Woodhead et al. As a labeled substance for immunoassay (Clin. Chem., 29, 1474-1479, 1983). The acridinium ester synthesized by Woodhead et al. For labeling was a compound represented by the following formula, and was unstable in a solvent system (pH neutral aqueous solution) usually used for immunoassay.

[0005]

[Chemical 2]

As a means for solving the instability problem of this acridinium-9-carboxylic acid aryl ester,
The introduction of a methyl group at two ortho positions of a phenoxy ring which constitutes a part of an ester component was disclosed by Seiyon et al. (JP-A-63-101368). Similar effects are also shown by map couplers etc.
Issue). The stability was improved by using the labeling compound in which an orthodimethyl group was introduced into the phenoxy ring.

[0007]

However, with the improvement in accuracy or speed of the analytical method including the apparatus, it is desired to develop a more stable and high-performance labeled compound.

An object of the present invention is to provide a novel chemiluminescent compound having high stability. Another object of the present invention is to provide a highly stable luminescent complex prepared from a novel chemiluminescent compound. A further object of the present invention is to provide an assay method using a novel chemiluminescent compound.

[0009]

Means for Solving the Problems As a result of intensive studies to achieve the above object, the present inventors have found that acridinium-
The present invention has been completed by finding that the introduction of a fluorinated alkyl group at the ortho position of the phenoxy ring of 9-carboxylic acid phenyl ester significantly improves the stability without impairing the light emitting property.

The present invention has the general formula (I)

[0011]

[Chemical 3]

(Wherein R ₁ is a group that does not interfere with chemiluminescence, R ₁ is
₂ is fluorinated alkyl, R ₃ is alkyl, fluorinated alkyl or alkoxy, X and Y each independently represent O or S, and A ⁻ represents an anion that does not interfere with chemiluminescence. ), And an acridinium compound which may have a group that does not interfere with chemiluminescence at any position of its acridine ring or phenyl ring or an isomer thereof (hereinafter referred to as chemiluminescent compound (I)). In the present specification, the “phenyl ring” refers to a phenyl ring bonded to Y unless otherwise specified.

The present invention also relates to a luminescent complex composed of a biologically active substance and a chemiluminescent compound (I) bound thereto.

In the present invention, (a) a sample is mixed with a binding substance that is labeled with a chemiluminescent compound (I) and that specifically binds to the test substance in the sample. A complex with a binding substance is formed, (b) the complex and the free labeled binding substance are separated, and (c) the chemiluminescence of the chemiluminescent compound (I) bound to the complex is detected. And (d) an assay method for the test substance in the sample (hereinafter referred to as assay method 1), which comprises determining the amount of the test substance in the sample from the detected luminescence amount.

The present invention further provides (a) a binding substance which specifically binds a sample to (i) a test substance in the sample, and
(ii) mixing with a test substance labeled with a chemiluminescent compound (I) to form a complex of the test substance and a binding substance,
(B) separating the complex from the free labeled analyte,
(C) Chemiluminescent compound (I) bound to the complex
(D) determining the amount of the test substance in the sample from the detected luminescence amount (d), and assaying the test substance in the sample (hereinafter referred to as assay method 2).

As used herein, the term "biologically active substance" means an antigen (including hapten), an antibody, a protein, a peptide,
It is meant to include nucleic acids, hormones, drugs, drug metabolites and the like.

As used herein, the term "group that does not interfere with chemiluminescence" means a group that does not interfere with the generation of effective chemiluminescence in chemiluminescence analysis. Specifically, the group that does not interfere with chemiluminescence represented by R ₁ includes alkyl, alkenyl, alkynyl, aryl or aralkyl, or a reactive functional group for labeling. One or more hydrogen atoms in the alkyl, alkenyl, alkynyl, aryl and aralkyl may be optionally substituted by a suitable substituent. Suitable substituents here are those selected from nitro, halogen, amino, protected amino, alkoxy, aryloxy, hydroxy, protected hydroxy and the like. Further, one or more carbon atoms in the alkyl, alkenyl, alkynyl, aryl and aralkyl may be optionally substituted with a hetero atom or carbonyl. Here, the hetero atom is one selected from nitrogen, phosphorus, sulfur and oxygen.

The alkyl, alkenyl and alkynyl have 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 4 carbon atoms, and the aryl and aralkyl have 6 to 20 carbon atoms, preferably 6 to carbon atoms. Twelve.
Examples of the aryl include phenyl and naphthyl, and examples of the aralkyl include benzyl. Preferably R ₁ is alkyl having 1 to 4 carbons.

The above-mentioned "reactive functional group for labeling" means a reactive group capable of binding to an amino group, a carboxyl group, a mercapto group, or another functional group usually contained in biologically active substances. As a specific example, -Q-RW, -RW or -W [In the formula, Q represents O or S, R represents alkylene or arylene, and W represents

[0020]

[Chemical 4]

(Where R_FourIs alkyl, aryl or
Aralkyl, R_FiveIs alkylene or arylene, X₁
Is halogen and Z is bromine or iodine)
A group represented by Archi here
Le, aryl and aralkyl are as defined above for R ₁Definition
Is the same meaning as. Also, alkylene and arylene are charcoal.
Prime number 1-20, preferably carbon number 1-10, more preferably
It preferably has 1 to 6 carbon atoms. Preferably R is methylene,
Ethylene, propylene, or ortho-, meta- or
It is para-phenylene. Halogen is chlorine, bromine, iodine
Elemental or fluorine, preferably chlorine or bromine
It

R ₂ is fluorinated alkyl, preferably C 1-4 fluorinated alkyl. The fluorinated alkyl may be perfluoroalkyl in which all hydrogen atoms of the alkyl are fluorine-substituted, or may be one in which a part of the hydrogen atoms is fluorine-substituted, and preferably perfluoroalkyl. Preferably R ₂ is trifluoromethyl.

R₃Is alkyl, fluorinated alkyl or
Alkoxy, preferably alkyl having 1 to 4 carbon atoms,
C1-C4 fluorinated alkyl or C1-C4
It is an alkoxy. The fluorinated alkyl is alkyl
In which all the hydrogen atoms of
It may be alkyl, or some of the hydrogen atoms may be fluorine-substituted.
May be used, preferably perfluoroalkyl
is there. Preferably R ₃Is methyl, trifluoromethy
It is methoxy.

X and Y are each independently O or S, and preferably both X and Y are O. The chemiluminescent compound (I) of the present invention may have a group that does not interfere with chemiluminescence at any position of its acridine ring or phenyl ring. Here, the “group that does not interfere with chemiluminescence” is
In addition to alkyl, alkenyl, alkynyl, aryl or aralkyl defined as the group represented by R ₁ or a reactive functional group for labeling, nitro, halogen (eg chlorine, bromine, iodine, fluorine), alkoxy Substituents selected from, carboxy, protected carboxy, alkoxycarbonyl, amino, protected amino, hydroxy and protected hydroxy are included. Preferably, an alkyl or haloalkyl having 1 to 4 carbon atoms, nitro,
Halogen or alkoxy having 1 to 4 carbon atoms, more preferably methyl, trifluoromethyl, nitro,
Halogen and methoxy.

[0025] A ^- the "anion which does not interfere with the chemiluminescence" represented by the sulfate, halosulfonates (e.g., fluorosulfonate), alkyl sulfonates,
Included are haloalkyl sulfonates (eg, fluoroalkyl sulfonates, etc.), aryl sulfonates, haloborates, haloacetates, halophosphates, phosphates, halides and the like.

In the chemiluminescent compound (I), the "reactive functional group for labeling" is introduced at any position of R ₁ or acridine ring or phenyl ring. Preferably the reactive functional group is introduced in the meta or para position relative to R ₁ or Y on the phenyl ring.

In the present invention, the isomers of general formula (I) are those of general formula (II) in which the acridine ring moiety is a phenanthridine ring.

[0028]

[Chemical 5]

The compounds represented by the formulas (wherein each symbol has the same meaning as defined above) are included.

Suitable as the chemiluminescent compound of the present invention is the above formula (I) in which R ₁ is alkyl having 1 to 4 carbon atoms, R ₂ is fluorinated alkyl having 1 to 4 carbon atoms, and R ₃ is carbon. Alkyl having 1 to 4 carbons, fluorinated alkyl having 1 to 4 carbons or alkoxy having 1 to 4 carbons, both X and Y are O, and the meta position or para position to Y of the phenyl ring is a reactive functional group. A compound substituted with a group, more preferably, R ₁ is methyl, R ₂ is trifluoromethyl, R ₃ is methyl, trifluoromethyl or methoxy, and is meta or para to Y of the phenyl ring. A compound in which the position is substituted with an N-succinimidyloxycarbonylalkyleneoxy group.

The luminescent complex of the present invention comprises an antigen (including a hapten), an antibody, a protein, a peptide, a nucleic acid, a hormone, a drug via a reactive functional group for labeling the chemiluminescent compound (I). It is formed by covalently bonding to various biologically active substances such as drug metabolites.

The acridinium compound of the present invention can be produced according to the following synthetic route.

[0033]

[Chemical 6]

(Wherein R ₁ , R ₂ , R ₃ and A ⁻ have the same meanings as described above) Acridine-9-carboxylic acid (III) or its reactive derivative is treated with 2,6-disubstituted phenol (IV). ) And an acridine-9-carboxylic acid phenyl ester compound (V) are obtained by esterification by a known ester synthesis method. By subjecting the ester compound (V) to quaternary salt according to a conventional method,
An acridinium compound (I) is produced.

The acridinium compound for labeling is prepared by reacting acridine-9-carboxylic acid (III) or its reactive derivative with the following formula:

[0036]

[Chemical 7]

(Wherein R ₂ and R ₃ have the same meanings as described above, and R ₆ represents a reactive functional group for labeling) and are ester-bonded with a substituted phenol.

Alternatively, acridine-9-carboxylic acid (II
I) or its reactive derivative is represented by the following formula

[0039]

[Chemical 8]

[In the formula, R ₂ and R ₃ have the same meanings as described above, and R ₇ is -Q-R-COOR ₈ or -R-COOR ₈
Or -COOR ₈ (wherein Q and R have the same meanings as described above, and R ₈ represents a carboxy-protecting group)], and then an ester bond is formed with the substituted phenol, and then the carboxyl-protecting group is removed. A reactive functional group may be introduced onto the phenyl ring using a compound such as N-hydroxysuccinimide. To introduce a reactive functional group at the position of R ₁ , the method disclosed in JP-A-63-112564 may be used.

The chemiluminescent compound (I) may be bound to a biologically active substance to prepare a luminescent complex according to a conventional method appropriately selected depending on the kind of the reactive functional group. For example, a luminescent compound of the present invention having an N-succinimidyloxycarbonyl group is mixed with a biologically active substance to be labeled in a buffer solution, and the mixture is allowed to stand at room temperature for several minutes to several tens of minutes (usually 15 minutes). It is prepared by leaving it for about 30 minutes.

The assay method of the present invention is characterized by using a luminescent complex labeled with chemiluminescent compound (I) as a tracer. The assay method of the present invention can be applied to any known assay method as long as the assay method uses a biological specific binding reaction. Here, the biological specific binding reaction means an antigen / antibody reaction, complementary binding of nucleic acid, hormone and its binding protein, drug and its binding protein,
It includes a binding reaction between an enzyme and its substrate.

Examples of test substances that can be measured by the assay method of the present invention include various biologically active substances such as antigens (including haptens), antibodies, proteins, peptides, nucleic acids, hormones, drugs, and drug metabolites. To be Examples of combinations of the test substance and the binding substance include an antigen and an antibody, a nucleic acid and a complementary polynucleotide, an enzyme and its substrate, a hormone and its binding protein, a drug and its binding protein, and a tumor marker and its antibody. Examples of such test substances include TSH (thyroid stimulating hormone), hCG (placental gonadotropin), CR
P (C-reactive protein), ASO (anti-streptolysin O), AFP (α-fetoprotein), IgG, Ig
A, IgM, IgD, IgE, CEA (carcinoembryonic antigen), transferrin, ferritin, fibrin, fibrinogen degradation product, haptoglobin, α ₁ -antitrypsin, α ₁ -side glycoprotein, α ₂ −
Examples include macroglobulin and β ₂ -microglobulin.

In the assay method of the present invention, the separation of the complex from the free form (B / F separation) may be carried out by a method usually used in the field of immunoassay. The solid phase method of immobilizing a binding substance on an insoluble carrier such as a polystyrene test tube or well, glass beads, polystyrene latex beads, sepharose, and polyacrylic is advantageous in terms of simplicity.

The chemiluminescence may be detected by a known means. Light emission occurs under alkaline conditions, for example by adding an oxidizing agent such as hydrogen peroxide. The amount of emitted light may be measured with an appropriate measuring device such as a luminometer. Needless to say, instead of measuring the chemiluminescence of the labeled compound bound to the complex, the labeled compound bound to the separated free form may be measured.

A preferred embodiment of the present invention will be described below by taking an immunoassay method utilizing an antigen-antibody reaction as an example. The test substance may be either an antigen or an antibody, but the method for measuring the antigen will be described for convenience.

A preferred embodiment of Assay Method 1 (non-competitive method) is the sandwich method. That is, it is an assay method for an antigen in a sample, which comprises the following steps. (A) a sample is incubated with an immobilized antibody against the antigen in the sample (hereinafter referred to as an immobilized antibody) to form a complex between the immobilized antibody and the antigen, and (b) then a chemiluminescent compound An antibody against an antigen (hereinafter referred to as a labeled antibody) labeled with (I) is added and incubated to form a sandwich consisting of an immobilized antibody, an antigen and a labeled antibody,
(C) separating the sandwich from the free labeled antibody,
(D) The chemiluminescence of the chemiluminescent compound (I) bound to the sandwich is detected, and (e) the amount of the antigen in the sample is determined from the detected luminescence amount. This method can be applied to the measurement of an antigen having two or more antibody binding sites.

A preferred embodiment of Assay Method 2 (competitive method) is
It is an assay method for an antigen in a sample, which comprises the following steps. (A) the sample is incubated with (i) the immobilized antibody and (ii) the chemiluminescent compound (I) -labeled antigen (hereinafter referred to as labeled antigen) to form a complex of the antigen and the antibody, (B) the complex and the free labeled antigen are separated, and (c) the chemiluminescent compound (I) bound to the complex.
(D) The amount of the antigen in the sample is determined from the detected luminescence amount.

[0049]

The present invention will be described in more detail with reference to the following examples and test examples. Example 1 (1) Synthesis of benzyl-4-bromobutyric acid (Compound 1) 4-Bromochlorobutyric acid (13.8 g) was added to ethyl acetate (50
ml), and N-methylmorpholine (7.6 g) was added dropwise with stirring at -20 ° C. After further dropping benzyl alcohol (7 g), stirring was continued at room temperature for 2 hours. Ethyl acetate (50 ml) and 10% -sodium carbonate (400 ml) were added, and the organic layer was separated. The organic layer was washed with purified water (400 ml) and then dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to obtain a crude product (oil). Purification by distillation under reduced pressure (140 ° C./1 mmHg) gave the title compound. (Oil 13 g, yield 77%)

(2) Synthesis of 3,5-bis (trifluoromethyl) -4-nitrophenol (Compound 2) 3,5-bis (trifluoromethyl) -phenol (4
0 g) was dissolved in acetic acid (500 ml), and 13N-nitric acid (500 ml) was added dropwise while stirring under ice cooling.
After heating at 80 ° C. for 50 minutes, the reaction mixture was added to cold water (6 liters), extracted with ethyl acetate (400 ml, 4 times), washed with saturated brine, and the organic layer was dried over anhydrous sodium sulfate. After evaporating the solvent under reduced pressure, the residue was purified by silica gel chromatography (eluent, dichloromethane / ethyl acetate, 30: 1) to obtain the title compound. (1
7 g, yield 35%) Melting point: 154-155 ° C. ¹ H-NMR (DMSO-d ₆ ): δ 11.6 (broad, 1H), 7.50 (s, 2H)

(3) Synthesis of 2,6-bis (trifluoromethyl) -4- (3-benzyloxycarbonylpropyloxy) -nitrobenzene (Compound 3) 3,5-bis (trifluoromethyl) -4-nitro Phenol (4.6 g) and potassium carbonate (4.6 g) were added to acetone (80 ml), and benzyl-4- was added while stirring.
Bromobutyric acid (14.7 g) was added dropwise. After refluxing for 24 hours, the solvent was distilled off under reduced pressure. The product was purified by silica gel chromatography (eluent, n-hexane / ethyl acetate, 6: 1) to obtain the title compound. (Oil 6.
0 g, 80% yield)

(4) Synthesis of 2,6-bis (trifluoromethyl) -4- (3-benzyloxycarbonylpropyloxy) -aniline (Compound 4) 2,6-bis (trifluoromethyl) -4- ( 3-Benzyloxycarbonylpropyloxy) -nitrobenzene (6.0 g) and 10% -palladium / carbon (2.5
g) was added to methanol (200 ml), and hydrogen gas was bubbled through for 2 hours while stirring. After the catalyst was filtered off, the solvent was distilled off under reduced pressure to obtain the title compound as crystals. (4.8
g, 88% yield) Melting point: 95-99 ° C

(5) Synthesis of 2,6-bis (trifluoromethyl) -4- (3-benzyloxycarbonylpropyloxy) -phenol (Compound 5) 2,6-bis (trifluoromethyl) -4- ( 3-benzyloxycarbonylpropyloxy) -aniline (4.3 g) was dissolved in acetic acid (150 ml), and then 0.1N was added.
-Sulfuric acid (740 ml) was added, and an aqueous sodium nitrite solution (1.5 g / 80 ml of water) was added dropwise over 3 hours while stirring under ice cooling. Copper (II) nitrate (300 g) and copper (I) oxide (15 g) were added, and the mixture was stirred at room temperature for 1 hour (gas generation). After filtration, the product in the filtrate was extracted with ethyl acetate (300 ml, 3 times). The organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The obtained crude crystals are subjected to silica gel chromatography (eluate, n-
Purify with hexane / ethyl acetate, 1: 1) to give the title compound. (4.1 g, yield 95%) Melting point: 81-82 ° C

(6) 2,6-bis (trifluoromethyi)
) -4- (3-benzyloxycarbonylpropyl)
Xy) -phenyl acridine-9-carboxylate
Synthesis of (Compound 6) Acridine-9-carboxylic acid chloride hydrochloride (2.8
g), N, N-dimethylaminopyridine (0.2 g),
2,6-bis (trifluoromethyl) -4- (3-ben
Zyloxycarbonylpropyloxy) -phenol
(2.1 g) was added to anhydrous pyridine (60 ml) and added to 20
Reflux for hours. After evaporating the solvent under reduced pressure, the residue was
Kagel chromatography (eluate, n-hexane / vinegar
Purification with ethyl acid, 3: 1) gave the title compound.
(190 mg, 6% yield) IR (KBr): 3399, 2934, 1786, 1709, 1613, 1489, 137
5, 1281, 1177, 1132 cm ^{-1 1} H-NMR (CDCl₃): δ 7.60 (m, 15H), 5.39 (s, 2H), 3.9
1 (m, 4H), 2.55 (t, 2H)

(7) Synthesis of 2,6-bis (trifluoromethyl) -4- (3-N-succinimidyloxycarbonylpropyloxy) -phenylacridine-9-carboxylate (Compound 7) 2,6 10% of -bis (trifluoromethyl) -4- (3-benzyloxycarbonylpropyloxy) -phenylacridine-9-carboxylate (70 mg)
-Crystals obtained by treatment with hydrogen bromide / acetic acid (1 ml) at 100 ° C. for 5 hours, anhydrous dimethylformamide (3 m
l), dicyclohexylcarbodiimide (60 mg),
N, N-Dimethylaminopyridine (10 mg) and N-hydroxysuccinimide (30 mg) were added, and the mixture was stirred at room temperature for 6 hours.
Stir for 9 hours. After the solvent was distilled off under reduced pressure, the residue was dissolved in methylene chloride and the precipitated dicyclohexylurea was removed by filtration. The product was chromatographed on silica gel (eluent, chloroform / ethyl acetate, 6:
Purification according to 1) gave the title compound. (24 mg, yield 34%)

(8) 2,6-bis (trifluoromethyl) -4- (3-N-succinimidyloxycarbonylpropyloxy) -phenyl N-methyl-acridinium-9-carboxylate fluorosulfonate (Compound 8 ) 2,6-bis (trifluoromethyl) -4- (3-N-)
Succinimidyloxycarbonylpropyloxy)-
Phenyl acridine-9-carboxylate (10m
g) was dissolved in anhydrous methylene chloride (1 ml), methylfluorosulfonate (0.03 ml) was added, and the mixture was stirred at room temperature for 1 hour.
Stir for 7 hours. Anhydrous ether (2 ml) is added to obtain crude crystals. The crude crystals were recrystallized from a mixture of acetonitrile and ether to give the title compound. (5 mg, yield 4
2%) Melting point: 169-175 ° C (decomposition) FAB-MS: M ⁺ 649

Comparative Example 4- (2-succinimidyloxycarbonylethyl)
Synthesis of -Phenyl N-methyl-acridinium-9-carboxylate fluorosulfonate (Compound 9) This compound was synthesized according to the method of Woodhead et al. (Methods in Enzymology, Vol. 133, pages 372-374).

Example 2 Labeling of antibody protein with acridinium ester 0.1 M-containing anti-alpha-fetoprotein (AFP) mouse monoclonal antibody (IgG, 200 μg)
Phosphate buffer (pH 8.0), labeling acridinium ester (compound 8) in dimethylformamide 5 mM
5 μl of the thus-dissolved product was added. 3 at room temperature
After standing for 0 minutes, 1M-glycine buffer solution (pH 8.
50 μl of 0) was added and the mixture was left for another 30 minutes. PBS (0.15M-pH containing sodium chloride)
It was passed through a Sephadex G-25 column that had been equilibrated with 7.0 mM of 20 mM phosphate buffer) to obtain a labeled body 8. It was revealed from the spectroscopic method that about 3 molecules of acridinium were labeled per 1 molecule of IgG.

The labeling with compound 9 was carried out according to the method of Woodhead et al. (Methods in Enzymology, Vol. 133, pp. 374-378). In this case, IgG
The number of acridinium bound to was about 3 molecules per IgG molecule (Labeled substance 9).

Test Example 1 Stability of Labeled Body Labeled Body 8 (present invention) and Labeled Body 9 (Comparative Example) contained 0.1% bovine serum albumin at the same concentration.
The daily change in the luminescence amount when diluted with BS and stored at 37 ° C. was examined. Chemiluminescence is 0.3% for the sample (20 μl)
-0.1N-nitric acid (150 µl) containing hydrogen peroxide was added, and 1 minute later, 0.25N-sodium hydroxide (150 µl) was added.
l) was measured by shot (manufactured by Nichine Co., Ltd., Lumi Counter 2500, integration time 5 seconds). The results are shown in Table 1.

[0061]

[Table 1]

The labeled substance 9 (comparative example) shows a remarkable decrease in the amount of luminescence during storage. On the other hand, it is clear that the labeled body 8 (invention) has excellent stability.

Example 3 Chemiluminescence Immunoassay (1) Preparation of Sensitization Test Tube Anti-AFP goat IgG was added to 0.1 M-phosphate buffer solution (pH).
It was diluted to 7.0 μg / ml with 7.0), 300 μl was added to a polystyrene test tube (12 × 75 mm, Nunc Maxisorp tube), and the mixture was allowed to stand at 5 ° C. overnight. Wash twice with PBS containing 0.1% -Tween 20, and 1 ml of PBS containing 0.5% -bovine serum albumin
In addition, it was left to stand at 5 ° C. overnight. (2) Dilution of Labeled Body 8 and Labeled Body 9 Labeled bodies 8 and 9 were diluted with PBS containing 20% -bovine serum so that the luminescence amount per 20 μl was 2 × 10 ⁶ counts. (3) Measurement After removing the liquid in the sensitization test tube by suction, 0.3 ml of PBS containing 1% -bovine serum albumin and 20 μl of the AFP standard solution dilution series were added and left at room temperature for 1 hour. 0.1%-
After washing twice with PBS containing Tween 20, 0.3 ml of the diluted labeling substance 8 or 9 was added, and the mixture was left at room temperature for 1 hour. After washing 4 times with PBS containing 0.1% -Tween 20, 0.1% containing 0.3% -hydrogen peroxide-nitric acid 3
00 μl was added, and 1 minute later, 300 μl of 0.25 N sodium hydroxide was shot to measure the chemiluminescence amount. A
A calibration curve when each dilution series of the FP standard solution is used is shown in FIG.

[0064]

INDUSTRIAL APPLICABILITY The chemiluminescent compound of the present invention is stable, particularly stable in a neutral aqueous solution, as compared with the conventional luminescent acridinium compound because the fluorinated alkyl is introduced into the ortho position of the phenyl ring. It has extremely high properties, and its chemiluminescence property is comparable to or higher than that of conventional compounds. It is useful as a labeling compound in chemiluminescence analysis.

[Brief description of drawings]

FIG. 1 is a graph showing a calibration curve when each dilution series of AFP standard solution in Example 3 is used.

Claims (4)

[Claims] 1. A compound represented by the general formula (I): (In the formula, R ₁ is a group that does not interfere with chemiluminescence, R ₂ is fluorinated alkyl, R ₃ is alkyl, fluorinated alkyl or alkoxy, X and Y each independently represent O or S, and A ⁻ is chemical. Which represents an anion that does not interfere with light emission) and may have a group that does not interfere with chemiluminescence at any position of its acridine ring or phenyl ring, or an isomer thereof. 2. A luminescent complex comprising a biologically active substance and the compound according to claim 1 bound thereto. 3. A sample is mixed with a binding substance which is labeled with the compound according to claim 1 and which specifically binds to the test substance in the sample. Forming a complex, (b) separating the complex and a free labeled binding substance, (c) detecting chemiluminescence of the compound bound to the complex, (d) An assay method for a test substance in a sample, which comprises determining the amount of the test substance in the sample from the amount of luminescence detected. 4. A binding substance which specifically binds (a) a sample to (i) a test substance in the sample, and (ii)
By mixing with the test substance labeled with the compound described above to form a complex of the test substance and the binding substance, (b) separating the complex from the free labeled test substance, and (c) the Detecting chemiluminescence of the compound of claim 1 bound to a complex,
(D) An assay method for a test substance in a sample, which comprises determining the amount of the test substance in the sample from the detected luminescence amount.