JP2002277468A - Method of measuring organic chlorine compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method by which wide organic chlorine compounds, such as the PCB, dioxin, etc., can be measured by an enzyme immunoassay instead of the conventional complicated measuring method. SOLUTION: In this method, the organic chlorine compounds can be measured by an immunoassay by using the carboxylic acid expressed by the general formula (1) of A-S-(CH2 )m -COOH (wherein, A and m respectively denote a substituted aromatic hydrocarbonic group or substituted heterocyclic group and an integer of 5-7) as a solid-phase binding antigen or labeled antigen.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a compound represented by the general formula

Embedded image (Where A is

Embedded image Wherein R ¹ and R ² are a lower alkyl group, X is a carbon atom or a nitrogen atom, or a substituted aryl group represented by the formula:

Embedded image Wherein R ³ and R ⁴ are a halogen atom, and R ⁵ is an alkoxyl group.
Is an integer of 5 to 7. ), Wherein the carboxylic acid is an antigen of a solid phase-bound antigen or an antigen of a labeled antigen when measuring an organic chlorine compound such as PCB or dioxins. Can be used as

[0002]

2. Description of the Related Art Conventionally, PCBs and dioxins have been measured by using an apparatus that integrates gas chromatography and mass spectrometry. However, these measuring apparatuses are very expensive, and the sample is expensive. In the case of soil or water, the concentration operation must be performed before measuring these devices, which is complicated. These problems could be solved by employing an immunoassay. This method is quick and simple,
Although high-sensitivity measurement can be performed at low cost, substances to be measured are limited, and improvement has been desired.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a simple method for measuring an organic chlorine compound. Another object of the present invention is to provide a method for measuring a wide range of organic chlorine compounds.

[0004]

Means for Solving the Problems In order to solve such problems, the present inventors have found a novel carboxylic acid represented by the above general formula (I),
The present inventors have found that organochlorine compounds such as CB and dioxins can be extensively measured by immunoassay, and completed the present invention.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The carboxylic acid represented by the general formula (I) of the present invention can be produced according to the following formula.

[0006]

Embedded image

(Where A is

Embedded image Wherein R ¹ and R ² are a lower alkyl group, X is a carbon atom or a nitrogen atom, or a substituted aryl group represented by the formula:

Embedded image An in represented by a substituted phenyl group (wherein, R ³ and R ⁴ is a halogen atom, R ⁵ is an alkoxyl group.), R ⁶
Is an alkyl group or an aryl group, and m is an integer of 5 to 7. X is a halogen atom. )

In describing the present invention, the term "alkyl group" may be any of a linear, branched or cyclic alkyl group having 1 to 12 carbon atoms, for example, a methyl group, an ethyl group, n-propyl group, 1-methylethyl group,
Cyclopropyl group, n-butyl group, 2-methylpropyl group, 1-methylpropyl group, 1,1-dimethylethyl group, cyclobutyl group, n-pentyl group, 3-methylbutyl group, cyclopentyl group, 2,2-dimethyl Propyl, 1-methylcyclobutyl, cyclobutylmethyl, n-hexyl, 4-methylpentyl, cyclohexyl, 1-methylcyclopentyl, cyclopentylmethyl, (1-methylcyclobutyl) methyl, n
-Heptyl group, 5-methylhexyl group, 4,4-dimethylpentyl group, cycloheptyl group, cyclohexylmethyl group, (1-methylcyclopentyl) methyl group, n-octyl group, 6-methylheptyl group, 5,5- Dimethylhexyl group, (1-methylcyclohexyl) methyl group, n
-Nonyl group, 7-methyloctyl group, 6,6-dimethylheptyl group, n-decyl group, 8-methylnonyl group, 7,
Examples include a 7-dimethyloctyl group, an n-indecacyl group, a 9-methyldecyl group, an 8,8-dimethylnonyl group, an n-dodecasyl group, a 10-methylundecacil group, and a 9,9-dimethyldecacil group. . The “alkyl group” may have a substituent, and examples of the substituent include an aromatic hydrocarbon group such as a phenyl group.

As the "lower alkyl group", among the above-mentioned alkyl groups, a linear, branched or cyclic alkyl group having 1 to 6 carbon atoms can be mentioned.

The "alkoxyl group" is a group in which an oxygen atom is bonded to the above "lower alkyl group", for example, methoxy, ethoxy, n-propoxy, 1-methylethyloxy, n-butoxy, 2-methylpropyloxy group, 1-methylpropyloxy group, 2-methyl-2-
Propyloxy group, 2,2-dimethylethyloxy group,
n-pentyloxy group, 3-methylbutyloxy group, n
-Hexyloxy group, 4-methylpentyloxy group, cyclohexyloxy group and the like.

The term "aryl group" refers to an aromatic hydrocarbon group which is monocyclic or polycyclic and may further have one or more various substituents on a ring. , Methylphenyl, dimethylphenyl, methoxyphenyl, dimethoxyphenyl, nitrophenyl, dinitrophenyl, chlorophenyl, dichlorophenyl, bromophenyl, dibromophenyl, iodophenyl, fluorophenyl, trifluoromethylphenyl, aminophenyl, hydroxyphenyl, mercaptophenyl, α −
Examples include naphthyl and β-naphthyl groups.

As the "halogen atom", chlorine, bromine,
Examples thereof include iodine and fluorine.

(First Step) This step is carried out by the above-mentioned general formula (IV)
Is a process of reacting a substituted thiol represented by the general formula (II) with a halo fatty acid ester represented by the general formula (III) in the presence of a base.

Examples of the thiol derivative represented by the general formula (II) include dimethylthiophenol, diethylthiophenol, ethylmethylthiophenol, dimethylmercaptopyrimidine, diethylmercaptopyrimidine, ethylmethylmercaptopyrimidine, dichloromethoxythiophenol, Examples thereof include dichloroethoxythiophenol, dibromomethoxythiophenol, dibromoethoxythiophenol, difluoromethoxythiophenol, and difluoroethoxythiophenol. Examples of the halofatty acid ester represented by the general formula (III) include, for example, ethyl 6-bromohexanoate, methyl 6-bromohexanoate, t-butyl 6-bromohexanoate, ethyl 7-bromoheptanoate, -Methyl bromoheptanoate, t-butyl 7-bromoheptanoate, ethyl 8-bromooctanoate, methyl 8-bromooctanoate, t-butyl 8-bromooctanoate, ethyl 6-chlorohexanoate, 6-chlorohexanoic acid Methyl, 6
T-butyl chlorohexanoate, ethyl 7-chloroheptanoate, methyl 7-chloroheptanoate, t-butyl 7-chloroheptanoate, ethyl 8-chlorooctanoate, 8-
Methyl chlorooctanoate, t-butyl 8-chlorooctanoate and the like can be used.

(Second Step) This step is a step for producing a carboxylic acid represented by the general formula (I) by hydrolyzing a compound represented by the general formula (IV).

In the present invention, an organic chlorine compound is measured by an immunoassay using the carboxylic acid represented by the above general formula (I). In the competitive reaction method of the present invention, an antibody that recognizes an organochlorine compound as a substance to be measured, an organochlorine compound in a sample, and a carboxylic acid represented by the general formula (I) used as a reagent are competed with each other. And a method using a solid-phase-bound antibody, and a method using a solid-phase-bound antibody. That is, the carboxylic acid bound to the solid phase, the labeled antibody and the sample are subjected to a competitive reaction, and the response based on the amount of the labeled antibody bound to the solid phase is measured. And the like, and a competitive reaction is performed, and a response based on the amount of the labeled antigen (the labeled carboxylic acid) bound to the solid phase is measured.

For bonding the carboxylic acid represented by the general formula (I) to a carrier protein or a labeling substance, a covalent bond is preferable, and a known technique can be appropriately used for the bond. As the joining method, for example,
Examples include an active ester method, a mixed acid anhydride method, and a method using a condensing agent. Here, examples of the ester used in the active ester method include N-hydroxyamine-based active esters such as hydroxysuccinimide ester, N-hydroxyphthalimide ester, and N-hydroxy-5-norbornene-2,3-dicarboximide ester;
phenylester-based active esters having an electron-withdrawing substituent at the o- and p-positions, such as p-nitrophenyl ester, pentachlorophenyl ester, and 2,4,5-trichlorophenyl ester; 8-hydroxyquinolyl ester; Examples include difunctional active esters such as 5-chloro-8-hydroxyquinolyl ester.
Hydroxysuccinimide esters are preferred in terms of operability and reactivity. DCC (N, N-
Dicyclohexyl carbodiimide), CMC (1-Cyclohexyl
-3- (2- morpholinoethyl) carbodiimide), DIC (Di
isopropyl carbodiimide), WSC (1-Ethyl-3- (3-di
methylaminopropyl) carbodiimide hydrochloride),
Woodward's Reagent K (N-ethyl
-3-phenylisoxazolium-3'-sulfonate), CDI (N, N-C
arbonyldiimidazole) and the like.

The labeling substance used in the immunoassay of the present invention is a substance which gives a response for detecting the labeling substance, and includes, for example, an enzyme, a radioisotope, a fluorescent substance and a luminescent substance. Is preferred. As the enzyme of the enzyme-labeled antibody, an enzyme that is not affected by the measurement system, for example,
Alkaline phosphatase or the like can be used.

Examples of the carrier protein used in the present invention include KLH and BSA. The antibody that recognizes an organic chlorine compound may be any antibody as long as it can carry out the present invention. JP 2000
The monoclonal antibody described in 191699 is also preferable from the viewpoint of improving sensitivity. The organic chlorine compounds that can be measured according to the present invention are PCBs, dioxins, and the like.

[0020]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Reference Examples and Examples, but the present invention is not limited thereto.

Reference Example 1 6- (4,6-dimethyl-2-)
Synthesis of ethyl pyrimidylthio) hexanoate

[0022]

Embedded image

Under an argon gas atmosphere, the suspension was cooled on ice and a suspension of 240 mg (6.00 mmol) of sodium hydride in 10 ml of anhydrous dimethylformamide was added to 715 mg (5.10 mm) of 4,6-dimethyl-2-mercaptopyrimidine.
ol) in anhydrous dimethylformamide (5 ml) was added dropwise and stirred for 30 minutes. Further, 0.89 ml (5.00 mmol) of ethyl bromohexanoate was added, and the mixture was added at room temperature for 3 hours.
The mixture was stirred for 5 minutes. After completion of the reaction, the reaction solution was poured into a saturated aqueous solution of ammonium chloride and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (hexane-ethyl acetate = 5: 1) to give 1.34 g of ethyl 6- (4,6-dimethyl-2-pyrimidylthio) hexanoate (yield 93.2).
%).

NMR (400 MHz, CDCl ₃ ):
1.25 (t, J = 7.1 Hz, 3H), 1.44-
1.53 (m, 2H), 1.64 to 1.80 (m, 4
H), 2.31 (t, J = 7.6 Hz, 2H), 2.3
9 (s, 6H), 3.15 (t, J = 7.3 Hz, 2
H), 4.14 (q, J = 7.1 Hz, 2H), 6.6.
7 (s, 1H) ppm. IR (liquid film): 2940,173
8, 1580, 1538, 1268, 1190 cm ^-1 Mass (m / z,%): 282 (M ⁺ , 55), 23
7 (45), 167 (76), 154 (100), 14
0 (94), 108 (36).

Reference Example 2 6- (4,6-dimethyl-2-)
Synthesis of pyrimidylthio) hexanoic acid

[0026]

Embedded image

Under an argon gas atmosphere, ethyl 6- (4,6-dimethyl-2-pyrimidylthio) hexanoate 500
mg (1.77 mmol) of ethanol solution (10 m
To 1), 1.0 ml of a 4N aqueous lithium hydroxide solution was added, and the mixture was stirred at room temperature for 15 hours and 10 minutes. After completion of the reaction, the reaction solvent was distilled off under reduced pressure, and the residue was dissolved in chloroform to give 10%
The extract was washed sequentially with a citric acid aqueous solution and a saturated saline solution, and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure.
(4,6-Dimethyl-2-pyrimidylthio) hexanoic acid was obtained quantitatively.

NMR (400 MHz, DMSO-
_{d 6): 1.37~1.44 (m,} 2H), 1.49~
1.58 (m, 2H), 1.60 to 1.69 (m, 2
H), 2.21 (t, J = 7.3 Hz, 2H), 2.3
4 (s, 6H), 3.07 (t, J = 7.3 Hz, 2
H), 6.93 (s, 1H), 11.7 to 12.4 (b
r, 1H) ppm. IR (liquid film): 3050, 293
6,1712,1582,1538,1438,126
6 cm ^-1 Mass (m / z,%): 254 (M ⁺ , 36), 16
7 (65), 154 (100), 140 (92), 10
8 (35).

Reference Example 3 N-succinimidyl-6
Synthesis of (4,6-dimethyl-2-pyrimidylthio) hexanoate

[0030]

Embedded image

To a solution of 132 mg (0.52 mmol) of 6- (4,6-dimethyl-2-pyrimidylthio) hexanoic acid in 10 ml of anhydrous dichloromethane, 62 mg (0.54 mmol) of N-hydroxysuccinimide, 1 ml of hydrochloric acid
104 mg (0.54 mmol) of -ethyl-3- (3-dimethylaminopropyl) carbodiimide was added, and the mixture was stirred at room temperature for 4 days. After completion of the reaction, the reaction solvent was distilled off under reduced pressure, and the residue was purified by silica gel chromatography (chloroform-methanol = 20: 1) to give
N-succinimidyl-6- (4,6-dimethyl-2-
150 mg of pyrimidylthio) hexanoate (yield 8
2.0%).

NMR (400 MHz, DMSO-
_{d 6): 1.43~1.52 (m,} 2H), 1.63~
1.72 (m, 4H), 2.34 (s, 6H), 2.6
9 (t, J = 7.3 Hz, 2H), 2.81 (s, 4
H), 3.08 (t, J = 7.3 Hz, 2H), 6.9
4 (s, 1H) ppm. IR (liquid film): 2940,181
6,1786,1740,1580,1538,143
6, 1268, 1208, 1068 cm ^-1 Mass (m / z,%): 351 (M ⁺ , 36), 23
7 (100), 167 (46), 154 (74), 14
0 (72).

Reference Example 4 6- (4,6-dimethyl-2-)
Synthesis of pyramidylthio) hexanoic acid-bound BSA 5.0 mg of bovine serum albumin (BSA) in 0.1 M
Dissolved in 900 μl of phosphate buffer (pH 7.5)
-Succinimidyl-6- (4,6-dimethyl-2-pyrimidylthio) hexanoate (1.0 mg) in anhydrous dimethylformamide (100 µl) was added, and the mixture was stirred at room temperature for 5 hours. Thereafter, the reaction solution was dialyzed in PBS and desalted to obtain BSA bound to 6- (4,6-dimethyl-2-pyrimidylthio) hexanoic acid.

Reference Example 5 6- (4,6-dimethyl-2-)
Preparation of BSA-sensitized particles containing pyrimidylthio) hexanoic acid Carboxylated particles (manufactured by Nippon Paint Co., Ltd.) at 0.1 M
After washing three times with a phosphate buffer (pH 5.0), suspending with 1 ml of the same buffer, and adjusting to 50 to 400 μg / ml, the 6- (4,6-dimethyl-2) prepared in Reference Example 4 was used. −
1 ml of a BSA solution containing pyrimidylthio) hexanoic acid was added, and the mixture was rotated at 25 ° C. for 2 hours using a rotator. After washing the particles, 0.05 M female buffer (pH 5.5)
Suspended in 1 ml, and 80 mg / ml of 1-ethyl-3-
50 μl of an aqueous solution of (3-dimethylaminopropyl) carbodiimide hydrochloride (manufactured by Nakarai Task Co., Ltd.) was added, and
Rotation reaction was performed at 25 ° C. for 30 minutes using a rotator. After washing the particles, 2 ml of a post-coat buffer was added, and the mixture was reacted with a rotator at 37 ° C overnight. After washing the particles, the particle concentration was adjusted to 1.5% to obtain 6- (4,6-dimethyl-2-pyrimidylthio) hexanoic acid-bound BSA-sensitized particles.

Reference Example 6 Alkaline phosphatase (A
LP) labeled anti-PCB # 169 (3, 3 ', 4, 4',
Preparation of 5,5′-Hexachlorobiphenyl) Antibody Anti-PCB # 169 Monoclonal Antibody (PCB169E)
Antibody; manufactured by KRI) and coupled with alkaline phosphatase (manufactured by Oriental) by maleimide method
A P-labeled anti-PCB # 169 antibody was obtained.

Example 1 Measurement of PCB # 169 PCB # 169 was measured using a fully automatic chemiluminescence immunoassay system (Lumipulse f; manufactured by Fujirebio).
layer One step competition method was used. PCB # 169
130 μl of standard antigen solution and ALP-labeled anti-PCB # 16
The antibody mixture was immunoreacted with 10 μl of the 9 antibody solution at 37 ° C. for 10 minutes. At 37 ° C
The immune reaction was performed for 10 minutes. After washing, substrate (AMPPD)
200 μl of the solution was added, and an enzyme reaction was carried out at 37 ° C. for 5 minutes, and then the luminescence was measured.

The standard antigen solution of the PCB # 169 is PC
B # 169 (manufactured by GL Sciences) was dissolved in a 10% dimethyl sulfoxide solution, and the solution was adjusted to a concentration of 0 to 5 ng / ml. Response of each standard antigen solution when the count value of standard antigen 0 concentration is 100% (B / B
0 (%)). The result is shown in FIG. Also, B / B0 = 90% estimated from the standard curve,
Table 1 shows the values of 85% and 50%.

[0038]

[Table 1]

In addition, P-sensitized BSA-sensitized particles using 6- (4,6-dimethyl-2-pyrimidylthio) hexanoic acid were used.
Cross-reactivity of the PCB congener with the CB # 169 assay and the relatively high toxicity (toxic equivalent coefficient; TEF 0.1
Table 2 and Table 3 show the measurement results of cross-reactivity with 11 types of dioxins. As a result, the PCB # 169 measurement system showed a large number of cross-reactivity with PCBs and homologues of dioxin.

[0040]

[Table 2]

[0041]

[Table 3]

[0042]

The present invention provides a method for measuring an organic chlorine compound using the carboxylic acid represented by the general formula (I),
Organochlorine compounds such as PCBs and dioxins can be measured by an enzyme immunoassay. Further, according to the present invention, a wide range of organic chlorine compounds can be easily measured.

[Brief description of the drawings]

FIG. 1 shows a standard curve when PCB # 169 was measured.

Claims (10)

[Claims] 1. A compound of the general formula A method for immunoassay of an organochlorine compound using a carboxylic acid represented by Wherein R ¹ and R ² are a lower alkyl group, X is a carbon atom or a nitrogen atom, or a substituted aryl group represented by the formula: Wherein R ³ and R ⁴ are a halogen atom, and R ⁵ is an alkoxyl group.
Is an integer of 5 to 7. ). 2. The immunoassay according to claim 1, wherein m is 5. (3) A is The immunoassay according to claim 1 or 2, which is a group represented by the formula: (4) A is The immunoassay according to claim 1 or 2, which is a group represented by the formula: (5) A is The immunoassay according to claim 1 or 2, which is a group represented by the formula: 6. The immunoassay according to claim 1, wherein a competitive reaction between the compound according to any one of claims 1 to 5 bound to a solid phase and an organic chlorine compound in a sample is used. 7. The immunoassay according to claim 6, wherein the compound according to any one of claims 1 to 5 uses an antigen-binding solid phase bound via a carrier protein. 8. The immunoassay according to claim 1, wherein a competitive reaction between the labeled compound according to any one of claims 1 to 5 and an organochlorine compound in a sample is used. 9. The immunoassay according to claim 1, wherein the organochlorine compound is one or more compounds selected from the group consisting of PCBs and dioxins. 10. An immunoassay kit for performing the immunoassay method according to any one of claims 1 to 9.