JP2002195999A - Immunoassay technique for determining organic halogen compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a simple method for determining a variety of organic halogen compounds. SOLUTION: Organic halogen compounds are determined by this immunoassay technique using a carboxylic acid represented by the general formula (wherein R1 and R2 are a fluorine atom, a boron atom or a trifluoromethyl group; R3 is either a hydrogen atom or a group of all the three expressed by R1; m is 5 or 7 as an integer) as a solid phase binding antigen or as an antigen of the labelled one.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

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

Embedded image (Wherein R ¹ and R ² are a fluorine atom, a bromine atom or a trifluoromethyl group, R ³ is a hydrogen atom or any group of R ¹ , and m is an integer of 5 to 7). An immunoassay method for an organic halogen compound using a carboxylic acid to be used, wherein the carboxylic acid is used as an antigen of a solid-phase-bound antigen or an antigen of a labeled antigen when measuring an organic halogen compound such as PCB or dioxin. Can be.

[0002]

2. Description of the Related Art Conventionally, PCBs and dioxins have been measured using instruments that integrate gas chromatography and mass spectrometry. However, these measuring instruments are very expensive, and samples are soiled. Or in the case of water,
A concentration operation had to be performed before measuring these instruments, which was complicated. These problems could be solved by employing an immunoassay. This method enables quick, simple, and low-cost high-sensitivity measurement, but the number of substances to be measured is limited, and improvement has been desired.

[0003]

An object of the present invention is to provide a simple method for measuring an organic halogen compound. Another object of the present invention is to provide a method for measuring a wide range of organic halogen 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 organic halogen compounds such as CB and dioxin 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 R¹And R^TwoIs a fluorine atom, bromine
An atom or a trifluoromethyl group, R ^ThreeIs a hydrogen atom or
Is R¹Any group of R^FourIs an alkyl group or aryl
The group, m, is an integer from 5 to 7. X is a halogen atom
is there. )

(First step) This step is carried out according to the general formula (IV)
Is a process of reacting a substituted phenol represented by the general formula (II) with a halo fatty acid ester represented by the general formula (III) in the presence of a base.

The substituted phenol represented by the general formula (II) includes, for example, difluorophenol, dibromophenol, bistrifluoromethylphenol, bromo-fluorophenol, fluoro-trifluoromethylphenol, bromo-trifluoromethylphenol, Trifluorophenol, tribromophenol,
Tristrifluoromethylphenol, bromo-difluorophenol, dibromo-fluorophenol and the like can be mentioned. 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, t-butyl 6-chlorohexanoate, ethyl 7-chloroheptanoate, methyl 7-chloroheptanoate, 7
T-butyl chloroheptanoate, ethyl 8-chlorooctanoate, methyl 8-chlorooctanoate, t-butyl 8-chlorooctanoate and the like can be used.

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

In the present invention, an organic halogen compound is measured by an immunoassay using the carboxylic acid represented by the general formula (I). In the competitive reaction method of the present invention, an antibody that recognizes an organic halogen compound as a substance to be measured, an organic halogen 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, a carboxylic acid bound to the solid phase, a labeled antibody and a sample are subjected to a competitive reaction, and a response based on the amount of the labeled antibody bound to the solid phase is measured. It means a method such as measuring the response based on the amount of labeled antigen (labeled carboxylic acid) bound to the solid phase after a competitive reaction.

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-hydroxysuccinimide ester, N-hydroxyphthalimide ester, and N-hydroxy-5-norbornene-2,3-dicarboximide ester.
Hydroxyamine-based active ester, p-nitrophenyl ester, pentachlorophenyl ester, 2,4,5
Phenylester-based active esters having an electron-withdrawing substituent at the o- and p-positions such as -trichlorophenylester, 8-hydroxyquinolyl ester, 5-chloro-8
Examples thereof include bifunctional active esters such as -hydroxyquinolyl ester, and hydroxysuccinimide esters are preferred from the viewpoint of operability. As a condensing agent, DCC (N, N-Dicyclohexyl ca.
rbodiimide), CMC (1-Cyclohexyl-3- (2-morphol
inoethyl) carbodiimide), DIC (Diisopropyl carbo)
diimide), WSC (1-Ethyl-3- (3-dimethylaminoprop)
yl) carbodiimide hydrochloride), Woodwar
d's Reagent K (N-ethyl-3-phenylisox
azolium-3'-sulfonate), CDI (N, N-Carbonyldiimida)
zole).

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, enzymes, radioisotopes, fluorescent substances and luminescent substances. Because it is a compound,
Preferably, an enzyme is employed. As the enzyme of the enzyme-labeled antibody, an enzyme that is not affected by the measurement system, such as alkaline phosphatase, can be used.

Examples of the carrier protein used in the present invention include KLH and BSA. The antibody that recognizes an organic halogen compound may be any antibody as long as it can carry out the present invention. WO99
/ 43799 is also preferable from the viewpoint of improving sensitivity. The organic halogen compound that can be measured according to the present invention is PCB, dioxin, or the like.

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 Synthesis of ethyl 6- (3,4-difluorophenoxy) hexanoate

[0017]

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Under a stream of argon, 200 mg (5.00 mmol) of 60% oily sodium hydride was added to a solution of 650 mg (5.00 mmol) of 3,4-difluorophenol in 15 ml of anhydrous dimethylformamide, and the mixture was stirred at room temperature for 15 minutes. Subsequently, 445 μl (2.50 mmol) of ethyl 6-bromohexanoate was added, and the mixture was added at room temperature for 24 hours.
Stirred for hours. After completion of the reaction, the mixture was made weakly acidic with a 10% aqueous citric acid solution, extracted with ethyl acetate, washed sequentially with a saturated aqueous sodium hydrogen carbonate solution and saturated brine, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. . The residue was chromatographed on silica gel (hexane-ethyl acetate = 2
0: 1) to give 537 mg (yield 78.8%) of ethyl 6- (3,4-difluorophenoxy) hexanoate.

¹ H-NMR (400 MHz, CDC
l ₃ ): 1.26 (t, J = 7.1 Hz, 3H);
45 to 1.53 (m, 2H), 1.60 to 1.74
(M, 2H), 1.74 to 1.82 (m, 2H), 2.
33 (t, J = 7.4 Hz, 2H), 3.89 (t, J
= 6.3 Hz, 2H), 4.13 (q, J = 7.1H)
z, 2H), 6.54-6.59 (m, 1H), 6.6.
6 to 6.72 (m, 1H) 7.00 to 7.08 (m, 1
H) ppm. IR (liquid film): 2948, 173
6,1608,1518,1256,1214,116
2 cm ^-1 Mass (m / z,%): 272 (M ⁺ , 24), 22
7 (22), 143 (100), 130 (47), 11
5 (32), 97 (67), 69 (63).

Reference Example 2 Synthesis of 6- (3,4-difluorophenoxy) hexanoic acid

[0021]

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336 mg (1.23 mmol) of ethyl 6- (3,4-difluorophenoxy) hexanoate was dissolved in 3 ml of acetic acid, 2 ml of concentrated hydrochloric acid was added, and the mixture was heated under reflux for 3 hours. After completion of the reaction, the mixture was neutralized with a saturated aqueous solution of sodium hydrogen carbonate, and the precipitated crystals were collected by filtration. Recrystallization from ether-hexane gave 193 mg of 6- (3,4-difluorophenoxy) hexanoic acid (yield 63.9%).

Mp: 65.0-66.0 ° C. ¹ H-NMR (400 MHz, CDCl ₃ ): 1.48-
1.57 (m, 2H), 1.67 to 1.76 (m, 2
H), 1.76 to 1.83 (m, 2H), 2.40.
(T, J = 7.4 Hz, 2H), 3.90 (t, J =
6.3 Hz, 2H), 6.55-6.59 (m, 1
H), 6.66 to 6.72 (m, 1H), 7.00 to
7.08 (m, 1H) ppm. IR (KBr): 3064, 2960, 1714, 1
614, 1520, 1436, 1306, 1206, 1
164, 1124 cm ^-1 Mass (m / z,%): 244 (M ⁺ , 60), 13
0 (100), 115 (86), 97 (56), 69
(70).

Reference Example 3 N-succinimidyl-6
Synthesis of (3,4-difluorophenoxy) hexanoate

[0025]

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To a solution of 899 mg (3.68 mmol) of 6- (3,4-difluorophenoxy) hexanoic acid in 20 ml of anhydrous dichloromethane, 508 mg (4.42 mmol) of N-hydroxysuccinimide and 1-ethyl-3- (3 -Dimethylaminopropyl) carbodiimide (847 mg, 4.42 mmol) was added at room temperature.
Stirred for hours. After completion of the reaction, the mixture was made weakly acidic with a 10% aqueous citric acid solution, extracted with ethyl acetate, 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 = 1: 1) to obtain 1.11 g (yield: 88.1%) of N-succinimidyl-6- (3,4-difluorophenoxy) hexanoate.

¹ H-NMR (400 MHz, CDC
_{l 3): 1.56~1.64 (m,} 2H), 1.77~
1.87 (m, 4H), 2.65 (t, J = 7.4H
z, 2H), 2.84 (bs, 4H), 3.91 (t,
J = 6.2 Hz, 2H), 6.55 to 6.60 (m, 1
H), 6.67-6.73 (m, 1H), 7.00-
7.08 (m, 1H) ppm. IR (liquid film): 2948, 181
6,1788,1742,1608,1518,121
2, 1162, 1070 cm ^-1 Mass (m / z,%): 341 (M ⁺ , 85), 22
7 (61), 130 (56), 97 (100), 69
(94).

Reference Example 4 Bovine Serum Albumin (BSA)
Synthesis of 3,4-difluorophenoxy derivative 5.0 mg of bovine serum albumin was dissolved in 900 μl of 0.1 M phosphate buffer (pH 7.5), and N-succinimidyl-6- (3,4-difluorophenoxy) hexanoate was dissolved. 100 mg of an anhydrous dimethylformamide solution (1.0 mg) 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 the BSA-bound 3,4-difluorophenoxy derivative.

Reference Example 5 Bovine serum albumin binding 3,
Preparation of Sensitized Particles of 4-Difluorophenoxy Derivative Carboxylated particles (manufactured by Nippon Paint Co., Ltd.) at 0.1 M
The BSA-bound 3,4-difluorophenoxy derivative solution prepared in Reference Example 4 was washed three times with a phosphate buffer (pH 5.0), suspended in 1 ml of the same buffer, and adjusted to 5 to 40 μg / ml. Add 1 ml, 25 ° C for 2 hours,
Rotation reaction was performed with a rotator. After washing the particles, 0.0
Suspended in 1 ml of 5M female buffer (pH 5.5), 80m
g / ml of an aqueous solution of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (manufactured by Nacalai Task Co.) was added in an amount of 50 µl, and the mixture was rotated at 25 ° C with a rotator.
A rotation reaction was performed for 0 minutes. 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 bovine serum albumin-bound 3,4-difluorophenoxy derivative-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 by a one-step competition method using a fully automatic chemiluminescence immunoassay system (Lumipulse f, manufactured by Fujirebio). Bovine serum albumin-conjugated 3,4-difluorophenoxy derivative-sensitized particles prepared in Reference Example 5 150 μl of standard antigen solution of PCB # 169
90 μl and 50 μl of ALP-labeled anti-PCB # 169 antibody solution
After washing, 200 µl of a substrate (AMPPD) solution was added, and an enzymatic reaction was carried out at 37 ° C for 5 minutes, and then the amount of luminescence was measured.

The standard antigen solution of PCB # 169 is PC
B # 169 (manufactured by GL Sciences) was dissolved in a 10% dimethyl sulfoxide solution and adjusted to a concentration of 0 to 10 ng / ml. Set the count value of standard antigen 0 concentration to 100
% Response of each standard antigen solution (B / B0 (%))
The standard curve was determined by. The result is shown in FIG.

Further, bovine serum albumin-bound 3,4-
PCB # using difluorophenoxy derivative-sensitized particles
Cross-reactivity of PCB congeners to 169 measurement system and relatively high toxicity (toxicity equivalent coefficient; TEF 0.1 or more)
Tables 1 and 2 show the measurement results of the cross-reactivity with 11 kinds of dioxins. As a result, the PCB # 169 measurement system showed a large number of cross-reactivity with PCBs and homologues of dioxin.

[0034]

[Table 1]

[0035]

[Table 2]

[0036]

The substituted phenoxycarboxylic acid represented by the general formula (I) of the present invention is useful as a reagent for measuring an organic halogen compound such as PCB. The substituted phenoxycarboxylic acid represented by the general formula (I) of the present invention can be combined with, for example, BSA and used for measurement of organic halogen compounds such as PCB and dioxin by an enzyme immunoassay.

[Brief description of the drawings]

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

[Procedure amendment]

[Submission date] February 20, 2001 (2001.2.2)
0)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0014

[Correction method] Change

[Correction contents]

Examples of the carrier protein used in the present invention include KLH and BSA. The antibody that recognizes the organic halogen compound may be any antibody as long as it can carry out the present invention. JP 20
The monoclonal antibody described in 00-191699 is also preferable from the viewpoint of improving sensitivity. The organic halogen compound that can be measured according to the present invention is PCB, dioxin, or the like.

Claims (12)

[Claims] 1. A compound of the general formula Wherein the carboxylic acid represented by the formula is used as an antigen for a solid-phase-bound antigen or a labeled antigen, wherein R ¹ and R ² are a fluorine atom, a bromine atom or a trifluoromethyl group, and R ³ is A hydrogen atom or a group represented by R ¹ , and m is an integer of 5 to 7). 2. The immunoassay according to claim 1, wherein m is 5. 3. The immunoassay according to claim 1, wherein R ¹ and R ² are a fluorine atom and R ³ is a hydrogen atom. 4. The immunoassay according to claim ¹ , wherein R ¹ , R ² and R ³ are a fluorine atom. 5. The immunoassay according to claim 1, wherein R ¹ and R ² are a bromine atom and R ³ is a hydrogen atom. 6. The immunoassay according to claim ¹ , wherein R ¹ , R ² and R ³ are bromine atoms. 7. R ¹ and R ² are trifluoromethyl groups,
Immunoassay method according to claim 1 or 2 R ³ is a hydrogen atom. 8. The immunoassay according to claim 1, wherein a competitive reaction between the compound according to any one of claims 1 to 7 bound to a solid phase and an organic halogen compound in a sample is used. 9. The immunoassay according to claim 5, wherein the compound according to any one of claims 1 to 7 uses an antigen-bound solid phase bound via a carrier protein. 10. The immunoassay according to claim 1, wherein a competitive reaction between the labeled compound according to any one of claims 1 to 7 and an organic halogen compound in a sample is used. 11. The immunoassay according to claim 1, wherein the organic halogen compound is PCB or dioxin. 12. An immunoassay kit for performing the immunoassay method according to any one of claims 1 to 11.