JP2002243738A - Method of measuring organic chloro compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for measuring wide-ranging organic chloro compounds such as PCB and dioxin by an enzyme immunoassay, instead of a conventional complicated method. SOLUTION: The organic chloro compounds are measured by the immunoassay, by using a carboxylic acid expressed by a general formula [1] (where R1, R2 represent lower alkyl groups, R3 represents hydrogen atom or a lower alkyl group, m is an integar of 5-7) as an antigen for a solid phase- bonded antigen or a labeled antigen.

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 lower alkyl groups, R ³ is a hydrogen atom or a lower alkyl group, and m is an integer of 5 to 7). This is a measurement method, and the carboxylic acid can be used as 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.

[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]

An object of the present invention is to provide a simple method for immunoassay of an organic chlorine compound. It is another object of the present invention to provide a safer immunoassay method by using a relatively low-toxicity compound as a solid phase antigen or a labeled antigen, instead of PCBs or dioxins themselves. Further, the present invention provides an immunoassay method for measuring a wide range of organic chlorine compounds at one time.

[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

Wherein R ¹ and R ² are a lower alkyl group,
R ³ is a hydrogen atom or a lower alkyl group, R ⁴ 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 “alkyl group” may be any of a straight-chain, branched-chain or cyclic alkyl group having 1 to 12 carbon atoms, such as a methyl group, an ethyl group, or an n-alkyl group. Propyl, 1-methylethyl, cyclopropyl, n-butyl, 2-methylpropyl, 1-methylpropyl, 1,1-dimethylethyl, cyclobutyl, n-pentyl, 3-methylbutyl A cyclopentyl group, a 2,2-dimethylpropyl group, a 1-methylcyclobutyl group, a cyclobutylmethyl group, an n-hexyl group, a 4-methylpentyl group, a cyclohexyl group, a 1-methylcyclopentyl group, a cyclopentylmethyl group, (1
-Methylcyclobutyl) methyl group, 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, 7,7-dimethyloctyl group, n-indecacyl group, 9-methyldecyl group,
8,8-dimethylnonyl group, n-dodecasyl group, 10-
Examples thereof include a 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 alkyl groups, one having 1 to carbon atoms
And 6 linear, branched or cyclic alkyl groups. The “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, for example, phenyl, methylphenyl , Dimethylphenyl, methoxyphenyl, dimethoxyphenyl, nitrophenyl, dinitrophenyl, chlorophenyl, dichlorophenyl, bromophenyl, dibromophenyl, iodophenyl, fluorophenyl, trifluoromethylphenyl, aminophenyl, hydroxyphenyl, mercaptophenyl, α
-Naphthyl, β-naphthyl group and the like.
Examples of the “halogen atom” include chlorine, bromine, iodine, fluorine and the like.

(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.

Examples of the substituted phenol represented by the general formula (II) include dimethylphenol, trimethylphenol, diethylphenol, triethylphenol, ethylmethylphenol, ethyldimethylphenol, diethylmethylphenol and the like. Examples of the halo fatty acid ester represented by the general formula (III) include, for example, ethyl 6-bromohexanoate,
Methyl 6-bromohexanoate, 6-bromohexanoic acid t
-Butyl, ethyl 7-bromoheptanoate, methyl 7-bromoheptanoate, t-butyl 7-bromoheptanoate, 8
-Ethyl bromooctanoate, methyl 8-bromooctanoate, t-butyl 8-bromooctanoate, ethyl 6-chlorohexanoate, methyl 6-chlorohexanoate, t-butyl 6-chlorohexanoate, 7-chloroheptanoic acid Ethyl, methyl 7-chloroheptanoate, t-butyl 7-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 (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 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.

In order to bond the carboxylic acid represented by the general formula (I) with a carrier protein or a labeling substance, a covalent bond is preferable, and a known technique can be appropriately used for the bond. Examples of the bonding method include an active ester method, a mixed acid anhydride method, and a method using a condensing agent. Here, as the ester used in the active ester method, for example, hydroxysuccinimide ester, N-hydroxyphthalimide ester,
N-hydroxyamine-based active esters such as -hydroxy-5-norbornene-2,3-dicarboximide ester, o-, p such as p-nitrophenyl ester, pentachlorophenyl ester and 2,4,5-trichlorophenyl ester Examples thereof include phenylester-based active esters having an electron-withdrawing substituent at the -position, dihydroxyfunctional active esters such as 8-hydroxyquinolylester, and 5-chloro-8-hydroxyquinolylester. Hydroxysuccinimide esters are preferred in view of reactivity and operability. As a condensing agent, DCC
(N, N-Dicyclohexyl carbodiimide), CMC (1-Cycl
ohexyl -3- (2- morpholinoethyl) carbodiimide), D
IC (Diisopropyl carbodiimide), WSC (1-Ethyl
-3- (3-dimethylaminopropyl) carbodiimide hydrochlori
de) Woodward's Reagent K (N- et
hyl-3-phenylisoxazolium-3'-sulfonate), CDI (N,
N-Carbonyldiimidazole) 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.

[0015]

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

[0017]

Embedded image

366 mg of 3,4-dimethylphenol
455 mg (3.3 mmol) of potassium carbonate was added to a solution (3.0 mmol) of anhydrous dimethylformamide (10 ml).
l), 669 mg of ethyl 6-bromohexanoate (3.0
mmol) and stirred at room temperature for 24 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 50% aqueous potassium carbonate solution and saturated 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 = 50: 1),
Ethyl (3,4-dimethylphenoxy) hexanoate 47
5 mg (60.0% yield) was obtained.

¹ H-NMR (400 MHz, CDC
l ₃ ): δ 1.26 (t, J = 7.1 Hz, 3H),
1.44 to 1.54 (m, 2H), 1.65 to 1.74
(M, 2H), 1.74 to 1.82 (m, 2H), 2.
18 (s, 3H), 2.23 (s, 3H), 2.33
(T, J = 7.5 Hz, 2H), 3.92 (t, J =
6.5 Hz, 2H), 4.13 (q, J = 7.1 Hz,
2H), 6.63 (dd, J = 8.2 and 2.7H)
z, 1H), 6.70 (d, J = 2.7 Hz, 1H),
7.01 (d, J = 8.2 Hz, 1H) ppm. IR (liquid film): 2944, 174
0, 1612, 1306, 1256, 1166 cm ^-1 Mass (m / z,%): 264 (M ⁺ , 44), 14
3 (83), 122 (100), 97 (34), 69
(36).

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

[0021]

Embedded image

313 mg (1.18 mmol) of ethyl 6- (3,4-dimethylphenoxy) hexanoate is dissolved in 5 ml of ethanol, and 1 ml of a 4N lithium hydroxide aqueous solution is dissolved.
(4.0 mmol) and stirred at room temperature for 14 hours.
After completion of the reaction, the reaction solution was concentrated under reduced pressure and acidified with 10% citric acid. The precipitated crystals were collected by filtration, washed with purified water and hexane, and dried under reduced pressure to obtain 274 mg of 6- (3,4-dimethylphenoxy) hexanoic acid (98.8% yield).

Mp: 105.0-106.5 ° C. ¹ H-NMR (400 MHz, CDCl ₃ ): δ 1.4
8 to 1.57 (m, 2H), 1.67 to 1.83 (m,
4H), 2.19 (s, 3H), 2.23 (s, 3
H), 2.39 (t, J = 7.4 Hz, 2H), 3.9
3 (t, J = 6.4 Hz, 2H), 6.63 (dd, J
= 8.2 and 2.7 Hz, 1H), 6.70 (d, J
= 2.7 Hz, 1H), 7.01 (d, J = 8.2H)
z, 1H) ppm. IR (KBr): 3050, 2948, 1716, 16
16, 1506, 1254, 1206, 1124, 10
20 cm ^-1 Mass (m / z,%): 236 (M ⁺ , 72), 12
2 (100), 107 (51).

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

[0025]

Embedded image

To a solution of 1.04 g (4.42 mmol) of 6- (3,4-dimethylphenoxy) hexanoic acid in 20 ml of anhydrous dichloromethane, 560 mg (4.86 mmol) of N-hydroxysuccinimide and 1-ethyl hydrochloride were added.
3- (3-dimethylaminopropyl) carbodiimide 9
32 mg (4.86 mmol) was added, and the mixture was stirred at room temperature for 3 days. After completion of the reaction, a 10% aqueous solution of citric acid was added, extracted with ethyl acetate, washed with saturated saline, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was chromatographed on silica gel (hexane-ethyl acetate =
1: 1) and recrystallized from ether-hexane-ethyl acetate to give 1.28 g of N-succinimidyl-6- (3,4-dimethylphenoxy) hexanoate (yield 8).
7.0%).

Mp: 84.0-85.0 ° C. ¹ H-NMR (400 MHz, CDCl ₃ ): δ 1.5
5 to 1.64 (m, 2H), 1.77 to 1.87 (m,
4H), 2.19 (s, 3H), 2.23 (s, 3
H), 2.65 (t, J = 7.5 Hz, 2H), 2.8
4 (bs, 4H), 3.94 (t, J = 6.3 Hz, 2
H), 6.64 (dd, J = 8.2 and 2.7 Hz,
1H), 6.71 (d, J = 2.7 Hz, 1H), 7.
01 (d, J = 8.2 Hz, 1H) ppm. IR (KBr): 2952, 1812, 1784, 17
40,1622,1506,1202,1066 cm
^-1 Mass (m / z,%): 333 (M ⁺ , 73), 21
9 (48), 122 (100), 97 (36), 69
(28).

Reference Example 4 Synthesis of ethyl 6- (3,4,5-trimethylphenoxy) hexanoate

[0029]

Embedded image

Under a stream of argon, 200 mg (5.0 mmol) of 60% oily sodium hydride was added to a solution (681 ml) of 681 mg (5.0 mmol) of 3,4,5-trimethylphenol in anhydrous dimethylformamide, and the mixture was stirred at room temperature for 15 minutes. did. Subsequently, 1.34 g (5.1 mmol) of ethyl 6-bromohexanoate was added, and the mixture was stirred at room temperature for 18 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 subjected to silica gel chromatography (hexane-ethyl acetate = 15:
Purification in 1) gave 1.23 g of ethyl 6- (3,4,5-trimethylphenoxy) hexanoate (88.2% yield).

¹ H-NMR (400 MHz, CDC
l ₃ ): δ 1.25 (t, J = 7.1 Hz, 3H),
1.44 to 1.53 (m, 2H), 1.65 to 1.73
(M, 2H), 1.74-1.81 (m, 2H), 2.
09 (s, 3H), 2.25 (s, 6H), 2.32
(T, J = 7.5 Hz, 2H), 3.91 (t, J =
6.4 Hz, 2H), 4.13 (q, J = 7.1 Hz,
2H), 6.57 (s, 2H) ppm. IR (liquid film): 2952, 174
0, 1608, 1490, 1318, 1202, 114
8,1060 cm ^-1 Mass (m / z,%): 278 (M ⁺ , 46), 14
3 (96), 136 (100), 121 (43), 97
(35), 69 (36).

Reference Example 5 Synthesis of 6- (3,4,5-trimethylphenoxy) hexanoic acid

[0033]

Embedded image

792 mg (2.84 mmol) of ethyl 6- (3,4,5-trimethylphenoxy) hexanoate
Was dissolved in 10 ml of ethanol, and 2.13 ml (8.53 mmol) of a 4N lithium hydroxide aqueous solution was added.
For 2.5 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure and acidified with 10% citric acid. The precipitated crystals were collected by filtration and recrystallized from ether-hexane to give 6- (3,4,5).
Thus, 561 mg (yield: 78.7%) of (trimethylphenoxy) hexanoic acid was obtained.

Mp: 77.0-78.0 ° C. ¹ H-NMR (400 MHz, CDCl ₃ ): δ 1.4
8 to 1.57 (m, 2H), 1.67 to 1.75 (m,
2H), 1.75 to 1.82 (m, 2H), 2.09
(S, 3H), 2.25 (s, 6H), 2.39 (t,
J = 7.5 Hz, 2H), 3.92 (t, J = 6.4H)
z, 2H), 6.57 (s, 2H) ppm. IR (KBr): 3044, 2932, 1712, 16
06,1490,1322,1230,1150,10
60 cm ^-1 Mass (m / z,%): 250 (M ⁺ , 41), 13
6 (100), 121 (28).

Reference Example 6 N-succinimidyl-6
Synthesis of (3,4,5-trimethylphenoxy) hexanoate

[0037]

Embedded image

To a solution of 375 mg (1.5 mmol) of 6- (3,4,5-trimethylphenoxy) hexanoic acid in 10 ml of anhydrous dichloromethane (207 ml) of N-hydroxysuccinimide 207 mg (1.8 mmol) of hydrochloric acid 1-ethyl-3- (3 -Dimethylaminopropyl) carbodiimide (345 mg, 1.8 mmol) was added, and the mixture was stirred at room temperature for 18 hours. After completion of the reaction, a 10% aqueous citric acid solution was added,
The mixture was extracted with ethyl acetate, washed with saturated saline, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. Ether-
Recrystallization from ethyl acetate-hexane gave 402 mg of N-succinimidyl-6- (3,4,5-trimethylphenoxy) hexanoate (77.2% yield).

Mp: 91.5-92.0 ° C. ¹ H-NMR (400 MHz, CDCl ₃ ): δ 1.5
0 to 1.63 (m, 2H), 1.76 to 1.87 (m,
4H), 2.09 (s, 3H), 2.25 (s, 6
H), 2.64 (t, J = 7.5 Hz, 2H), 2.8
4 (bs, 4H), 3.93 (t, J = 6.4 Hz, 2
H), 6.57 (s, 2H) ppm. IR (KBr): 2944, 1822, 1784, 17
56, 1604, 1318, 1218, 1148, 10
80 cm ^-1 Mass (m / z,%): 347 (M ⁺ , 77), 23
3 (45), 136 (100), 121 (26), 97
(26), 69 (21).

REFERENCE EXAMPLE 7 Synthesis of 3,4-dimethylphenoxyhexanoic acid-bound BSA 5.0 mg of bovine serum albumin (BSA) was added to 0.1 M
Dissolved in 900 μl of phosphate buffer (pH 7.5)
-Succinimidyl-6- (3,4-dimethylphenoxy) 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 conjugated with 3,4-dimethylphenoxyhexanoic acid.

Reference Example 8 Preparation of 3,4-dimethylphenoxyhexanoic acid-bound BSA-sensitized particles Carboxylated particles (manufactured by Nippon Paint Co., Ltd.) were used at 0.1M.
The plate was washed three times with a phosphate buffer (pH 5.0), suspended in 1 ml of the same buffer, and adjusted to 50 to 400 μg / ml. The 3,4-dimethylphenoxyhexanoic acid-bound BSA prepared in Reference Example 7 was prepared. 1 ml of the solution was added, and the mixture was rotated by a rotator at 25 ° C. for 2 hours. After particle washing,
Suspended in 1 ml of 0.05 M female buffer (pH 5.5),
A 50 μl aqueous solution of 80 mg / ml of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (manufactured by Nacalai Task Co., Ltd.) was added, and 2
The reaction was allowed to rotate at 5 ° C. for 30 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 3,4-dimethylphenoxyhexanoic acid-bound BSA-sensitized particles.

Reference Example 9 Alkaline phosphatase (A
LP) labeled anti-PCB # 126 (3, 3 ', 4, 4', 5
Preparation of -Pentachlorobiphenyl) Antibody Using an anti-PCB # 126 monoclonal antibody (PCB77A antibody; manufactured by KRI), alkaline phosphatase (manufactured by Oriental) was bound by a maleimide method to obtain an ALP-labeled anti-PCB # 126 antibody.

Example 1 Measurement of PCB # 126 Measurement of PCB # 126 was performed by a one-step competition method using a fully automatic chemiluminescence immunoassay system (Lumipulse f, manufactured by Fujirebio). 3,4- created in Reference Example 8
Dimethylphenoxyhexanoic acid-bound BSA sensitized particles 1
In 50 μl, 90 μl of PCB # 126 standard antigen solution
Add 50 μl of ALP-labeled anti-PCB # 126 antibody solution,
After performing an immunoreaction at 37 ° C. for 20 minutes, washing the substrate (AMP)
200 μl of the PD) 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 PCB # 126 is PC
B # 126 (manufactured by GL Sciences Inc.) was dissolved in a 10% dimethyl sulfoxide solution and adjusted to a concentration of 0 to 10 ng / ml. Response of each standard antigen solution when the count value of standard antigen 0 concentration is 100% (B /
B0 (%)) to obtain a standard curve. The result is shown in FIG. Also, B / B0 = 90% estimated from the standard curve,
Table 1 shows the values of 85% and 50%.

Further, the cross-reactivity of the PCB homolog with the PCB # 126 assay system using 3,4-dimethylphenoxyhexanoic acid-conjugated BSA-sensitized particles showed relatively high toxicity (toxic equivalent coefficient; TEF 0.1 Table 1 and Table 2 show the measurement results of cross-reactivity with 11 types of dioxins. As a result, in the PCB # 126 measurement system, PCB # 7
7 and 14.3%, PCB # 81 and 5.2%, PCB # 1
56 and 7.7%, 1,2,3,4,7,8-HeCDD
And a cross-reactivity of 20.9%.

Reference Example 10 Alkaline phosphatase (ALP) -labeled anti-PCB # 169 (3, 3 ', 4,
Preparation of 4 ′, 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 2 Measurement of PCB # 169 Measurement of PCB # 169 was performed by a one-step competition method using a fully automatic chemiluminescence immunoassay system (Lumipulse f, manufactured by Fujirebio). 3,4- created in Reference Example 8
Dimethylphenoxyhexanoic acid-bound BSA sensitized particles 1
90 μl of the standard antigen solution of PCB # 169 in 50 μl
Add 50 μl of ALP-labeled anti-PCB # 169 antibody solution,
After performing an immunoreaction at 37 ° C. for 20 minutes, washing the substrate (AMP)
200 μl of the PD) 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 10 ng / ml. Response of each standard antigen solution when the count value of standard antigen 0 concentration is 100% (B /
B0 (%)) to obtain a standard curve. The result is shown in FIG. Also, B / B0 = 90% estimated from the standard curve,
Table 1 shows the values of 85% and 50%.

Further, the cross-reactivity of the PCB homolog with the PCB # 169 assay system using BSA-sensitized particles conjugated with 3,4-dimethylphenoxyhexanoic acid and the relatively high toxicity (toxic equivalent coefficient; TEF 0.1 Table 1 and Table 2 show the measurement results of cross-reactivity with 11 types of dioxins. As a result, in the PCB # 169 measurement system, PCB # 1
It showed a very high cross-reactivity of 91.2% with 26 and 91.2%, and 91.9% with 2,3,4,7,8-PeCDD, and also showed multiple cross-reactivity with other homologues.

Reference Example 11 Synthesis of 3,4,5-trimethylphenoxyhexanoic acid-bound BSA 5.0 mg of bovine serum albumin (BSA) was added to 0.1 M
Dissolved in 900 μl of phosphate buffer (pH 7.5)
-Succinimidyl-6- (3,4,5-trimethylphenoxy) hexanoate (100 mg) in anhydrous dimethylformamide (1.0 mg) was added, and the mixture was stirred at room temperature for 5 hours. The reaction was then dialysed in PBS and desalted.
The title 3,4,5-trimethylphenoxyhexanoic acid-bound BSA was obtained.

Reference Example 12 Preparation of 3,4,5-trimethylphenoxyhexanoic acid-bonded BSA-sensitized particles Carboxylated particles (manufactured by Nippon Paint Co., Ltd.) at 0.1 M
It was washed three times with a phosphate buffer (pH 5.0), suspended in 1 ml of the same buffer, and adjusted to 50 to 400 μg / ml to prepare 3,4,5-trimethylphenoxyhexanoic acid prepared in Reference Example 11. Add 1 ml of bound BSA solution and add 25
Rotation reaction was carried out with a rotator at 2 ° C. for 2 hours. After the particles were washed, the suspension was suspended in 1 ml of a 0.05 M female buffer (pH 5.5), and 50 μl of an 80 mg / ml aqueous solution of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (manufactured by Nacalai Task Co.) was added. Then, a rotation reaction was performed at 25 ° C. for 30 minutes using a rotator. After washing the particles, 2 ml of post-coat buffer was added, and the mixture was rotated with a rotator.
C. The reaction was allowed to rotate overnight. After washing the particles, reduce the particle concentration
3,4,5-Trimethylphenoxyhexanoic acid-bound BSA-sensitized particles were obtained by adjusting the amount to 1.5%.

Example 3 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
9 antibody solution (10 μl) was immunoreacted for 10 minutes at 37 ° C. Then, 120 μl of the reaction mixture and 150 μl of the 3,4,5-trimethylphenoxyhexanoic acid-conjugated BSA sensitized particles prepared in Reference Example 12 were added to The immune reaction was performed for minutes. After washing, substrate (AMPPD) solution 200
The enzyme reaction was performed at 37 ° C. for 5 minutes after adding μl, and then the amount of luminescence was measured.

The standard antigen solution of PCB # 169 was 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%.

Further, PCB # 169 using 3,4,5-trimethylphenoxyhexanoic acid-bound BSA-sensitized particles
Tables 2 and 3 show the measurement results of the cross-reactivity of the PCB homolog with the measurement system and the cross-reactivity with 11 kinds of dioxins having relatively high toxicity (toxic equivalent coefficient; TEF 0.1 or more). As a result, in the PCB # 169 measurement system, PC
It showed multiple cross-reactivity with homologues of B and dioxin.

[0055]

[Table 1]

[0056]

[Table 2]

[0057]

[Table 3]

According to the present invention, there is provided a method for immunoassay of an organic chlorine compound using the carboxylic acid represented by the above general formula (I) as a solid phase antigen or a labeled antigen.
Since it is not CB or dioxin itself, a safer measurement method can be provided. Further, the present invention exhibits a large number of cross-reactivity with PCB and dioxins, so that a wide range of organic chlorine compounds can be easily measured at once.

[Brief description of the drawings]

FIG. 1: 3,4-dimethylphenoxyhexanoic acid bond B
2 shows a standard curve when measuring SA # and PCB # 126 using ALP # 126 antibody.

FIG. 2: 3,4-dimethylphenoxyhexanoic acid bond B
2 shows a standard curve obtained when SA # and PCB # 169 were measured using ALP # 169 antibody.

FIG. 3: PCB # 1 using 3,4,5-trimethylphenoxyhexanoic acid-conjugated BSA and ALP # 169 antibody
The standard curve when 69 is measured is shown.

Claims (9)

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