JPH07143899A - Enhancing of chemiluminescence - Google Patents

Abstract

PURPOSE:To reduce background luminescence and increase the intensity of sample-response luminescence by adding an oxidizing agent, a peroxidase, a chemiluminescence sensitizer and a specified compound to a chemiluminescent substance. CONSTITUTION:A chemiluminescent substance is allowed to emit light by an oxidizing agent, a peroxidase, a chemiluminescent sensitizer and a compound of the formula (R) is hydroxyl Group or its alkali salt, etc.; R2 to R5 are each independently H or a halogen. R2 and R3 or R4 and R5 may respectively bonded to each other to form a benzene ring; R5 and R7 are independently H, a lower alkyl, etc.).

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a chemiluminescence enhancing method. More specifically, the present invention relates to a method for detecting a chemiluminescent substance, and more particularly to a method for enhancing chemiluminescence, which is suitable for improving the sensitivity of immunoassay and enzyme quantification, based on various measurements based on the measurement of chemiluminescence. .

[0002]

2. Description of the Related Art Analytical methods based on chemiluminescent reactions have a possibility of becoming extremely sensitive measuring methods, and thus have been actively studied so far. This analysis method has been particularly attracting attention in the field of immunological measurement, and chemiluminescent immunoassay (CLIA), EI, in which a chemiluminescent substance such as luminol or acridinium ester is directly labeled on an antibody, is used.
Various measurement methods such as chemiluminescent enzyme immunoassay (CLEIA), which utilizes chemiluminescence to measure the activity of the labeling enzyme of A, have been put to practical use [Toshio Imai, Medical Technology, 21 (1), 6-13 (1993)].

The background to which the chemiluminescence method has been applied to the above-described measurement is the discovery of a sensitizer (enhancer). That is, coexistence of a sensitizer such as p-iodophenol in a chemiluminescence reaction system which originally ends in an extremely short time makes it possible to delay the luminescence reaction and increase the amount of luminescence. Further, more excellent sensitizers have been searched for in order to improve the measurement sensitivity, and for example, JP-A-59-171.
839, JP-A-2-174694, JP-A-2-29
No. 1299 and the like. These sensitizers include those that amplify the activity of peroxidase, those that stabilize radicals, and those that sensitize by emitting fluorescence by themselves.

[0004]

However, when the chemiluminescence reaction is enhanced by using a sensitizer, nonspecific luminescence (background) also increases, and as a result, the lower limit of detection is still high, and the objective is to be achieved. In some cases, the components cannot be detected sufficiently. The present invention provides a chemiluminescent system in which an oxidant, a peroxidase and a sensitizer coexist in a chemiluminescent substance, and a fourth compound is added to the chemiluminescent system in addition to the oxidant, the peroxidase and the chemiluminescent sensitizer. It is an object of the present invention to provide a chemiluminescence enhancing method capable of reducing the ground emission and enhancing the emission intensity of sample response.

[0005]

In order to solve the above-mentioned problems, the present inventors have studied various chemiluminescent reaction systems, and as a result, have chemically synthesized a compound represented by the general formula (I) as a fourth compound. It was found that coexistence in a luminescence reaction system reduces background luminescence and enhances luminescence intensity of sample response, and completed the present invention.

That is, according to the present invention, a compound represented by the following general formula (I) coexists in a system in which a chemiluminescent substance emits light using an oxidizing agent, a peroxidase and a chemiluminescent sensitizer (enhancer). A method for enhancing chemiluminescence is provided.

[Chemical 2] (In the formula, R ₁ represents a hydroxyl group, an alkali salt of a hydroxyl group, a substituted or unsubstituted amino group, or an acyloxy group,
R ₂ , R ₃ , R ₄ and R ₅ each independently represent a hydrogen atom or a halogen atom, or R ₂ and R ₃ or R ₄ and R ₅ respectively bond to form a benzene ring, and R ₆
And R ₇ each independently represent a hydrogen atom, a substituted or unsubstituted lower alkyl group or a lower alkoxy group. )

The compound of the general formula (I) used in the chemiluminescence enhancing method of the present invention can be easily obtained as a commercial product. A highly pure reagent purified by a column chromatography method, recrystallization or the like is preferably used.

As the chemiluminescent substance (hereinafter often referred to as a luminescent substrate) used in the present invention, various kinds of luminol, isoluminol or their derivatives, lophine, lucigenin and the like can be used, but preferably luminol, isoluminol and their derivatives. And as a derivative, N-aminobutyl-N-ethylisoluminol, N
-Aminohexyl-N-ethylisoluminol and the like can be used. Further, a system in which these luminescent substrates are indirectly produced may be used.

The oxidizing agent used in the present invention may be an oxidizing agent capable of producing light in a chemiluminescent reaction, and for example, hydrogen peroxide, perborate and the like can be used. Further, a system in which an oxidant is indirectly produced may be used, and a system in which hydrogen peroxide is produced from glucose by using an enzyme such as glucose oxidase may be used. Hydrogen peroxide is preferred.

Specific examples of the compound represented by the general formula (I) include phenol indophenol, 2,6
-Dichlorophenol indophenol sodium, indophenol blue, indophenol sodium,
Indophenol acetate, 2,6-dichloroindo-O-
Examples include, but are not limited to, cresol and N, N-dimethylindoaniline. The use concentration of the compound represented by the general formula (I) is 0.05 to 500 μM, preferably 0.4 to
40 μM. If it is less than 0.05 μM, the chemiluminescence enhancing effect is small, and if it exceeds 500 μM, the chemiluminescent reaction is disturbed.

The chemiluminescent sensitizer which can be used in the present invention is not particularly limited, but an example which can be preferably used is 4- [2 '-(4'-methyl) thiazolyl].
Phenol, 4- (2'-thienyl) phenol, p-
Iodophenol and 4- [4 '-(2'-methyl)
At least one selected from the group consisting of thiazolyl] phenol can be mentioned. A system in which these sensitizers are indirectly produced may also be used.

When these sensitizers are used, the compound represented by the general formula (I) is phenol indophenol (R ₁ ═OH; R ₂ , R ₃ , R ₄ , R ₅ , R ₆ ，
R ₇ = H), 2,6- dichlorophenol indophenol sodium _{(R 1 = ONa; R 2} , R 4, R 6, R
_{7 = H; R 3, R} 5 = Cl), the indophenol blue _{(R 1 = N (CH 3} ) 2; R 2, R 3, R 6, R 7 = H;
R ₄ and R ₅ = benzene ring), sodium indophenol (R ₁ = ONa; R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R)
₇ = H) and indophenol acetate (R ₁ = OCOC)
_{H 3; R 2, R 3} , R 4, R 5, R 6, at least one member selected from the group consisting of R ₇ = H) (hereinafter sometimes referred to as indophenol compound) is preferably.

Peroxidase is used as a free (unbound) type in the assay system, but it can be used as a labeled type for immunochemical measurement by binding with an antigen, a hapten, an antibody and the like.

INDUSTRIAL APPLICABILITY The present invention can be used to detect a measurement target selected from peroxidase or an immunochemically active substance labeled with peroxidase, an oxidizing agent, a luminescent substrate, and a sensitizer. When carrying out the present invention, it is needless to say that the measurement conditions must be optimized according to the measurement system, but the procedure is preferably as follows.

The reaction temperature for chemiluminescence according to the present invention is preferably 0 to 60 ° C., particularly around room temperature. The reaction is preferably carried out using a buffer solution as a solvent, and the pH of the buffer solution is preferably in the vicinity of neutral to alkaline. As the buffer solution, borate buffer solution, phosphate buffer solution, carbonate buffer solution, Tris-HCl buffer solution,
Glycine buffer and the like can be effectively used, but borate buffer, Tris-HCl buffer and glycine buffer are preferable. As reagents, free or labeled peroxidase, an oxidizing agent, a sensitizer, and an indophenol compound solution are prepared at respective predetermined concentrations, and each solution is allowed to coexist to cause a chemiluminescent reaction. The light generated from the reaction solution can be detected by, for example, a self-made or commercially available measuring device equipped with a photomultiplier tube. It is desirable to use a luminometer, chemiluminometer, or fluorometer depending on the shape of the container in which the reaction is performed.

The field of application of the present invention is not limited to immunological measurement, and can be applied to, for example, quantification of hydrogen peroxide in environmental analysis.

[0017]

The present invention will be described in detail with reference to examples.
The following reagents and equipment were used in the examples below. In addition, Table 1
Abbreviations in 3 mean the following. PIP: phenol indophenol DCIP: 2,6-dichlorophenol indophenol
Sodium IPB: Indophenol Blu-IPNa: Indophenol sodium IPA: Indophenol acetate reagent Luminol was a commercially available product dissolved in a 23 mM NaOH aqueous solution. Peroxidase is a conjugate with an antibody, and is an anti-endothelin 1HRP labeled antibody [IBL (IBL)].
16155] manufactured by the same company was used. 30% commercially available as oxidizer
A hydrogen peroxide solution (manufactured by Wako Pure Chemical Industries, Ltd.) was appropriately diluted and used. As the sensitizer, 4- [2 '-(4'-methyl) thiazolyl] phenol (manufactured by Mast Co.), 4- (2'-phenyl) phenol (manufactured by Mast Co.), p-iodophenol (Wako Pure Chemical Industries, Ltd.) Each) and 4- [4 '-(2'-methyl) thiazolyl] phenol (Mast) DMS
O solution was used. Phenol indophenol, 2,6
-Sodium dichloroindophenol, sodium indophenol and indophenol acetate were commercially available products (indophenol acetate manufactured by Kodak Co., Ltd. and others manufactured by Kanto Chemical Co., Inc.) were used after being appropriately dissolved in water. As for indophenol blue, a commercially available product (manufactured by Kanto Chemical Co., Inc.) was dissolved in acetonitrile and used. Commercially available reagent grades were used as the other additives.

The apparatus chemiluminescence reaction was carried out in each well of a 96-well microtiter plate [manufactured by Nunc]. Luminescence was measured using a luminometer (MLR-100 manufactured by Corona Electric Co., Ltd.).

Example 1 (Preparation of solution for chemiluminescence reaction) As a hydrogen peroxide solution,
0.1% milk in 50 mM glycine buffer (pH 9),
0.05% egg albumin, 0.1% Tween20
A 2 mM hydrogen peroxide solution containing was prepared. Anti-endothelin 1HRP labeled antibody (60 μg / ml) was 0.1
It was used after diluting to a predetermined concentration with a PBS solution containing% milk. As a sensitizer solution, 3.2 mM 4- [2'-
A 50 mM glycine buffer solution (pH 9) containing (4′-methyl) thiazolyl] phenol and each indophenol compound at a predetermined concentration was prepared and used. Luminol solution is 2
It was adjusted to 20 mM with a 3 mM NaOH solution.

(Chemiluminescence measurement) In a well of a 96-well microtiter plate, 10 μl of 10 ³ -fold diluted anti-endothelin 1HRP labeled antibody, 100 μl of hydrogen peroxide solution, 50 μl of luminol solution, and 50 μl of sensitizer solution were added. Add and mix, and after 10 minutes, add 4 by luminometer.
The amount of light emitted at 35 nm (Lumicount) was measured. At the same time, a blank measurement was performed without adding the labeled antibody. Table 1
, A blank value (background), a sample emission amount and a signal / blank ratio (S / N) with and without an indophenol compound as an additive.
The ratio) is shown in comparison.

[0021]

[Table 1] Chemiluminescence enhancing effect of additives ───────────────────────────────────── Additive concentration (ΜM) Blank value Sample emission S / N ratio ───────────────────────────────────── None ( Conventional method) 0 386 227,990 591 PIP 1.6 321 229,354 715 DCIP 3.2 305 219,948 720 IPB 1.6 123 155,840 1267 IPNa 3.2 230 191,130 831 IPA 16 207 204,142 986 ────────────────── ──────────────────

As shown in the results of Table 1, coexistence of additives of each indophenol compound suppresses background luminescence and may increase sample luminescence, resulting in improvement of S / N ratio. It was

Example 2 (Preparation of solution for chemiluminescence reaction) As a hydrogen peroxide solution,
0.1% milk, 0.05% egg albumin, 0.1% Tween in 50 mM borate buffer (pH 9.5)
A 2 mM hydrogen peroxide solution containing 20 was prepared. Anti-endothelin 1HRP labeled antibody (60 μg / ml)
It was diluted to a predetermined concentration with a PBS solution containing 0.1% milk and used. As a sensitizer solution, 0.16 mM 4-
50 mM borate buffer solution (pH) of (2'-thienyl) phenol and each indophenol compound at a predetermined concentration
9.5) was prepared and used. Luminol solution is 23 mM
It was adjusted to 20 mM with a NaOH solution.

(Chemiluminescence measurement) In a well of a 96-well microtiter plate, 10 μl of 10 ⁴ -fold diluted anti-endothelin 1HRP-labeled antibody, 100 μl of hydrogen peroxide solution, 50 μl of luminol solution, and 50 μl of sensitizer solution were added. Add and mix, and after 6 minutes 43
The amount of light emission (Lumicount) at 5 nm was measured. At the same time, a blank measurement was performed without adding the labeled antibody. In Table 2, the blank value (background), the sample luminescence amount and the signal / blank ratio (S / N ratio) with and without coexistence of the indophenol compound as an additive
Are shown in comparison.

[0025]

[Table 2] Chemiluminescence enhancing effect of additives ───────────────────────────────────── Additive concentration (ΜM) Blank value Sample emission S / N ratio ───────────────────────────────────── None ( Conventional method) 0 305 10,570 34 PIP 1.6 277 11,016 40 DCIP 3.2 275 10,779 39 IPNa 3.2 300 11.300 38 IPA 32 268 11,150 42 ───────────────────────── ─────────────

As shown in the results of Table 2, the coexistence of the additives of each indophenol compound suppressed the background luminescence, increased the luminescence amount of the sample, and improved the S / N ratio.

Example 3 (Preparation of solution for chemiluminescence reaction) As a hydrogen peroxide solution,
50% Tris-HCl buffer (pH 8.5) with 0.1% milk, 0.05% egg albumin, 0.1% Tween
A 2 mM hydrogen peroxide solution containing n20 was prepared. Anti-endothelin 1HRP labeled antibody (60 μg / ml)
It was diluted to a predetermined concentration with a PBS solution containing 0.1% milk and used. As a sensitizer solution, a 50 mM glycine buffer solution (pH 9) containing 3.2 mM p-iodophenol and each indophenol compound at a predetermined concentration was prepared and used. Luminol solution is 20m with 23mM NaOH solution
Prepared to M.

(Chemiluminescence measurement) In a well of a 96-well microtiter plate, 10 μl of 10 ³ -fold diluted anti-endothelin 1HRP-labeled antibody, 100 μl of hydrogen peroxide solution, 50 μl of luminol solution, and 50 μl of sensitizer solution were added. Add and mix, and after 12 minutes, add 4 by luminometer.
The amount of light emitted at 35 nm (Lumicount) was measured. At the same time, a blank measurement was performed without adding the labeled antibody. Table 3
, A blank value (background), a sample emission amount and a signal / blank ratio (S / N) with and without an indophenol compound as an additive.
The ratio) is shown in comparison.

[0029]

[Table 3] Chemiluminescence enhancing effect of additives ───────────────────────────────────── Additive concentration (ΜM) Blank value Sample emission S / N ratio ───────────────────────────────────── None ( Conventional method) 0 34 54,911 1615 PIP 8 28 68,639 2451 DCIP 16 25 56,751 2270 IPB 1.6 34 55,778 1640 IPNa 16 32 58,820 1838 IPA 32 18 57,174 3176 ────────────────── ──────────────────

As shown in the results of Table 3, the coexistence of each indophenol additive suppressed the background luminescence, increased the luminescence amount of the sample, and improved the S / N ratio.

Example 4 (Preparation of solution for chemiluminescence reaction) As a hydrogen peroxide solution,
0.1% milk in 50 mM borate buffer (pH 10),
0.05% egg albumin, 0.1% Tween20
A 2 mM hydrogen peroxide solution containing was prepared. Anti-endothelin 1HRP labeled antibody (60 μg / ml) was 0.1
It was used after diluting to a predetermined concentration with a PBS solution containing% milk. As a sensitizer solution, 0.32 mM 4- [4'-
(2'-methyl) thiazolyl] phenol and 1.6
A 50 mM borate buffer solution (pH 10) of μM-8 μM phenol indophenol was prepared and used. The luminol solution was adjusted to 20 mM with a 23 mM NaOH solution.

(Chemical luminescence measurement using phenol indophenol) 10 μl of 10 ⁴ -fold diluted anti-endothelin 1HRP was added to the well of a 96-well microtiter plate.
Labeled antibody, then 100 μl hydrogen peroxide solution, 50 μl
1 l of the luminol solution and 50 μl of the sensitizer solution were added and mixed, and after 10 minutes, the amount of light emission (luminous count) at 435 nm was measured by a luminometer. At the same time, a blank measurement was performed without adding the labeled antibody. Table 4 shows a comparison of the blank value (background), the luminescence amount of the sample, and the signal / blank ratio (S / N ratio) with and without the addition of phenol indophenol.

[0033]

[Table 4] Phenol Indophenol A chemiluminescence enhancing effect when coexisting with ────────────────────────────────── ─── Concentration (μM) Blank value Sample emission amount S / N ratio ──────────────────────────────────── ── 0 (conventional method) 79 16,978 216 1.6 43 22,22 717 528 8.0 47 21,185 446 ──────────────────────── ──────────────

As shown in the results of Table 4, addition of phenol indophenol suppresses background luminescence,
Further, the light emission amount of the sample increased and the S / N ratio was improved.

Example 5 (Measurement of change in chemiluminescence with time) In order to measure the chemiluminescence amount with high accuracy, the chemiluminescence reaction used must be stable for a long time. Therefore, a chemiluminescent solution was prepared in the same manner as in Example 4, and measurement was started immediately after mixing to examine the change with time. Phenol indophenol was prepared at 1.6 μM. As a result, as shown in FIG. 1, the amount of light emission reached a plateau in about 10 minutes, and the light emission was stably maintained thereafter. In particular, when phenol indophenol coexisted, the stability was shown at a higher luminescence amount than when coexisted. Further, as shown in FIG. 2, a similar tendency was observed in the S / N ratio.

Example 6 (Preparation of solution for chemiluminescence reaction) As a hydrogen peroxide solution,
0.1% milk in 50 mM borate buffer (pH 10),
0.05% egg albumin, 0.1% Tween20
A 2 mM hydrogen peroxide solution containing was prepared. Anti-endothelin 1HRP labeled antibody (60 μg / ml) was 0.1
Dilution series were made using PBS solution containing% milk.
As a sensitizer solution, 0.32 mM 4- [4 '-(2'-
Methyl) thiazolyl] phenol and 0.16 mM phenol indophenol in 50 mM borate buffer (p
H10) was prepared and used. Luminol solution is 23 mM
It was adjusted to 20 mM with a NaOH solution.

(Measurement of peroxidase activity) The chemiluminescence was measured in the same manner as in Example 4 using the above reaction solution for each dilution series of the anti-endothelin 1HRP-labeled antibody to prepare a dose action curve. The results are shown in FIG.

Example 7 (Preparation of chemiluminescent reaction solution) As a hydrogen peroxide solution,
0.1% milk in 50 mM borate buffer (pH 10),
0.05% egg albumin, 0.1% Tween20
A concentration series of hydrogen peroxide solution containing was prepared. Anti-endothelin 1HRP labeled antibody (60 μg / ml)
It was diluted 1/10 ³ with a PBS solution containing 0.1% milk. As a sensitizer solution, 0.32 mM 4- [4 '
-(2'-methyl) thiazolyl] phenol and 1.
A 50 mM borate buffer solution (pH 10) of 6 μM phenol indophenol was prepared and used. The luminol solution was adjusted to 20 mM with a 23 mM NaOH solution.

(Measurement of hydrogen peroxide) Using the above reaction solution with respect to the hydrogen peroxide concentration series, chemiluminescence was measured in the same manner as in Example 4 to prepare a dose action curve. Figure 4
The results are shown in.

Example 8 (Preparation of solution for chemiluminescence reaction) As a hydrogen peroxide solution,
0.1% milk in 50 mM borate buffer (pH 10),
0.05% egg albumin, 0.1% Tween20
A 2 mM hydrogen peroxide solution containing was prepared. Anti-endothelin 1HRP labeled antibody (60 μg / ml) was 0.1
It diluted 1/10 ³ times using the PBS solution containing% milk. As a sensitizer solution, 0.32 mM 4- [4'-
(2'-methyl) thiazolyl] phenol and 1.6
A 50 mM borate buffer solution (pH 10) of μM phenol indophenol was prepared and used. Luminol solution is 2
A concentration series was prepared with a 3 mM NaOH solution.

(Measurement of Luminol) Using the above reaction solution with respect to the luminol concentration series, chemiluminescence was measured in the same manner as in Example 4 to prepare a dose action curve. Figure 5
The results are shown in.

Example 9 (Preparation of solution for chemiluminescence reaction) As a hydrogen peroxide solution,
0.1% milk in 50 mM borate buffer (pH 10),
0.05% egg albumin, 0.1% Tween20
A 2 mM hydrogen peroxide solution containing was prepared. Anti-endothelin 1HRP labeled antibody (60 μg / ml) was 0.1
It was used after diluting to a predetermined concentration with a PBS solution containing% milk. As a sensitizer solution, 0.32 mM 4- [4'-
(2'-methyl) thiazolyl] phenol and 3.2
A 50 mM borate buffer solution (pH 10) of sodium μM-32 μM 2,6-dichlorophenolindophenol was prepared and used. The luminol solution was adjusted to 20 mM with a 23 mM NaOH solution.

(Chemical Luminescence Measurement Using 2,6-Dichlorophenolindophenol Sodium) In a well of a 96-well microtiter plate, 10 μl of 10 ⁴ -fold diluted anti-endothelin 1HRP-labeled antibody, and then 100 μl of hydrogen peroxide solution, 50 μl luminol solution, 50 μl
The sensitizer solution of was added and mixed, and after 10 minutes, the amount of light emission (lumine count) at 435 nm was measured by a luminometer. At the same time, a blank measurement was performed without adding the labeled antibody. In Table 5, the blank value (background), the luminescence amount of the sample and the signal / blank ratio (S / N ratio) with and without the addition of 2,6-dichlorophenolindophenol sodium are shown.

[0044]

[Table 5] Chemiluminescence enhancing effect when 2,6-dichlorophenolindophenol sodium is allowed to coexist ─────────────────────────── ────────── Additive concentration (μM) Blank value Sample emission S / N ratio ──────────────────────────── ────────── 0 (conventional method) 79 16, 978 216 3.2 69 20,047 291 16 57 19, 349 342 32 61 61,714 323 ────────── ───────────────────────────

As shown in the results of Table 5, the addition of 2,6-dichlorophenolindophenol sodium suppresses background luminescence and increases the amount of luminescence of the sample.
The result is that the / N ratio is improved.

Example 10 (Measurement of Change in Chemiluminescence over Time) In order to accurately measure the chemiluminescence amount, the chemiluminescence reaction to be used must be stable for a long time. Therefore, a chemiluminescence solution was prepared in the same manner as in Example 9, and measurement was started immediately after mixing to examine the change with time. In addition, 2,6-dichlorophenol indophenol sodium was adjusted to 32 μM. As a result, as shown in FIG. 6, the amount of luminescence reached a plateau in about 10 minutes, and thereafter luminescence continued stably. In particular, when 2,6-dichlorophenolindophenol sodium was made to coexist, stability was shown with a higher luminescence amount than when not coexisted. Further, as shown in FIG. 7, a similar tendency was observed in the S / N ratio.

Example 11 (Preparation of solution for chemiluminescence reaction) As a hydrogen peroxide solution,
0.1% milk in 50 mM borate buffer (pH 10),
0.05% egg albumin, 0.1% Tween20
A 2 mM hydrogen peroxide solution containing was prepared. Anti-endothelin 1HRP labeled antibody (60 μg / ml) was 0.1
Dilution series were made using PBS solution containing% milk.
As a sensitizer solution, 0.32 mM 4- [4 '-(2'-
Methyl) thiazolyl] phenol and 32 μM 2,6
-Dichlorophenol sodium indophenol 50
A mM borate buffer solution (pH 10) was prepared and used. The luminol solution was adjusted to 20 mM with a 23 mM NaOH solution.

(Measurement of peroxidase activity) Chemiluminescence was measured in the same manner as in Example 9 by using the above reaction solution for each dilution series of the anti-endothelin 1HRP-labeled antibody to prepare a dose action curve. The result is shown in FIG.

Example 12 (Preparation of solution for chemiluminescence reaction) As a hydrogen peroxide solution,
0.1% milk in 50 mM borate buffer (pH 10),
0.05% egg albumin, 0.1% Tween20
A concentration series of hydrogen peroxide solution containing was prepared. The anti-endothelin 1HRP-labeled antibody (60 μg / m) was added at 0.
It was diluted 1/10 ³ with a PBS solution containing 1% milk. As a sensitizer solution, 0.32 mM 4- [4'-
(2'-methyl) thiazolyl] phenol and 16μ
A 50 mM boric acid buffer solution (pH 10) of M2,6-dichlorophenolindophenol sodium was prepared and used. Luminol solution is 20mM with 23mM NaOH solution
Was prepared.

(Measurement of hydrogen peroxide) Using the above reaction solution for the concentration series of hydrogen peroxide, chemiluminescence was measured in the same manner as in Example 9 to prepare a dose action curve. Figure 9
The results are shown in.

Example 13 (Preparation of solution for chemiluminescence reaction) As a hydrogen peroxide solution,
0.1% milk in 50 mM borate buffer (pH 10),
0.05% egg albumin, 0.1% Tween20
A 2 mM hydrogen peroxide solution containing was prepared. Anti-endothelin 1HRP labeled antibody (60 μg / ml) was 0.1
It diluted 1/10 ³ times with the PBS solution containing% milk. As a sensitizer solution, 0.32 mM 4- [4'-
(2'-methyl) thiazolyl] phenol and 16μ
A 50 mM boric acid buffer solution (pH 10) of M2,6-dichlorophenolindophenol sodium was prepared and used. A concentration series of luminol solution was prepared with a 23 mM NaOH solution.

(Measurement of Luminol) Using the above reaction solution for the concentration series of luminol, chemiluminescence was measured in the same manner as in Example 9 to prepare a dose action curve. Figure 1
The result is shown in 0.

Example 14 (Preparation of solution for chemiluminescence reaction) As a hydrogen peroxide solution,
0.1% milk in 50 mM borate buffer (pH 10),
0.05% egg albumin, 0.1% Tween20
A 2 mM hydrogen peroxide solution containing was prepared. Anti-endothelin 1HRP labeled antibody (60 μg / ml) was 0.1
It was used after diluting to a predetermined concentration with a PBS solution containing% milk. As a sensitizer solution, 0.32 mM 4- [4'-
(2'-methyl) thiazolyl] phenol and 3.2
A 50 mM borate buffer solution (pH 10) of μM indophenol blue was prepared and used. Luminol solution is 23
It was adjusted to 20 mM with a mM NaOH solution.

(Chemiluminescence measurement using indophenol blue) 1 in a well of a 96-well microtiter plate
0 μl of 10 ⁴ -fold diluted anti-endothelin 1HRP labeled antibody, then 100 μl of hydrogen peroxide solution, 50 μl of luminol solution, 50 μl of sensitizer solution were added and mixed,
After 10 minutes, the amount of luminescence at 435 nm (Lumicount) was measured with a luminometer. At the same time, a blank measurement was performed without adding the labeled antibody. Table 6 shows a comparison of the blank value (background), the sample luminescence amount, and the signal / blank ratio (S / N ratio) with and without addition of indophenol blue.

[0055]

[Table 6] Chemiluminescence enhancement effect when indophenol blue coexists ────────────────────────────────── ─── Additive concentration (μM) Blank value Light emission of sample S / N ratio ────────────────────────────────── ─── 0 (conventional method) 79 16,978 216 3.2 62 19,157 309 ─────────────────────────────── ───────

As shown in the results of Table 6, the addition of indophenol blue suppressed the background luminescence, increased the luminescence amount of the sample, and improved the S / N ratio.

Example 15 (Measurement of Change in Chemiluminescence over Time) In order to measure the chemiluminescence amount with high accuracy, the chemiluminescence reaction used must be stable for a long time. Therefore, a chemiluminescence solution was prepared in the same manner as in Example 14, and measurement was started immediately after mixing to examine the change with time. Indophenol blue was prepared at 1.6 μM. As a result, as shown in FIG. 11, the light emission amount reached a plateau in about 10 minutes, and thereafter, the light emission was stably maintained. In addition, in the case of coexisting with indophenol blue, the stability was shown at a higher luminescence amount than in the case of not coexisting. Further, as shown in FIG. 12, a similar tendency was observed in the S / N ratio.

Example 16 (Preparation of solution for chemiluminescence reaction) As a hydrogen peroxide solution,
0.1% milk in 50 mM borate buffer (pH 10),
0.05% egg albumin, 0.1% Tween20
A 2 mM hydrogen peroxide solution containing was prepared. Anti-endothelin 1HRP labeled antibody (60 μg / ml) was 0.1
Dilution series were made using PBS solution containing% milk.
As a sensitizer solution, 0.32 mM 4- [4 '-(2'-
Methyl) thiazolyl] phenol and 3.2 μM indophenol blue in 50 mM borate buffer (pH 1
0) was prepared and used. Luminol solution is 23 mM Na
It was adjusted to 20 mM with an OH solution.

(Measurement of peroxidase activity) The chemiluminescence was measured in the same manner as in Example 14 using the above reaction solution for each dilution series of the anti-endothelin 1HRP-labeled antibody to prepare a dose action curve. The result is shown in FIG.

Example 17 (Preparation of solution for chemiluminescence reaction) As a hydrogen peroxide solution,
0.1% milk in 50 mM borate buffer (pH 10),
0.05% egg albumin, 0.1% Tween20
A concentration series of hydrogen peroxide solution containing was prepared. Anti-endothelin 1HRP labeled antibody (60 μg / ml)
It was diluted 1/10 ³ with a PBS solution containing 0.1% milk. As a sensitizer solution, 0.32 mM 4- [4 '
-(2'-methyl) thiazolyl] phenol and 1.
6 μM indophenol blue 50 mM borate buffer (pH 10) was prepared and used. Luminol solution is 23
It was adjusted to 20 mM with a mM NaOH solution.

(Measurement of Hydrogen Peroxide) Using the above reaction solution for the concentration series of hydrogen peroxide, chemiluminescence was measured in the same manner as in Example 14 to prepare a dose action curve. The result is shown in FIG.

Example 18 (Preparation of solution for chemiluminescence reaction) As a hydrogen peroxide solution,
0.1% milk in 50 mM borate buffer (pH 10),
0.05% egg albumin, 0.1% Tween20
A 2 mM hydrogen peroxide solution containing was prepared. Anti-endothelin 1HRP labeled antibody (60 μg / ml) was 0.1
It diluted 1/10 ³ times with the PBS solution containing% milk. As a sensitizer solution, 0.32 mM 4- [4'-
(2'-methyl) thiazolyl] phenol and 1.6
A 50 mM borate buffer solution (pH 10) of μM indophenol blue was prepared and used. Luminol solution is 23
A concentration series was prepared with mM NaOH solution.

(Measurement of Luminol) Using the above reaction solution for the concentration series of luminol, chemiluminescence was measured in the same manner as in Example 14 to prepare a dose action curve. The results are shown in FIG.

Example 19 (Preparation of solution for chemiluminescence reaction) As a hydrogen peroxide solution,
0.1% milk in 50 mM borate buffer (pH 10),
0.05% egg albumin, 0.1% Tween20
A 2 mM hydrogen peroxide solution containing was prepared. Anti-endothelin 1HRP labeled antibody (60 μg / ml) was 0.1
It was used after diluting to a predetermined concentration with a PBS solution containing% milk. As a sensitizer solution, 0.32 mM 4- [4'-
(2'-methyl) thiazolyl] phenol and 3.2
μM-32 μM Sodium indophenol 50 mM
A borate buffer solution (pH 10) was prepared and used. The luminol solution was adjusted to 20 mM with a 23 mM NaOH solution.

(Chemiluminescence measurement using sodium indophenol) 10 μl of 10 ⁴ -fold diluted anti-endothelin 1HRP was added to the well of a 96-well microtiter plate.
Labeled antibody, then 100 μl hydrogen peroxide solution, 50 μl
1 l of the luminol solution and 50 μl of the sensitizer solution were added and mixed, and after 10 minutes, the amount of light emission (luminous count) at 435 nm was measured by a luminometer. At the same time, a blank measurement was performed without adding the labeled antibody. In Table 7, the blank value (background), the sample luminescence amount, and the signal / blank ratio (S / N ratio) with and without addition of sodium indophenol are shown in comparison.

As shown in the results of Table 7, addition of indophenol sodium suppresses background luminescence,
Further, the light emission amount of the sample increased and the S / N ratio was improved.

[0067]

[Table 7] Chemiluminescence enhancement effect when coexisting with indophenol sodium ────────────────────────────────── ─── Additive concentration (μM) Blank value Light emission of sample S / N ratio ────────────────────────────────── ─── 0 (conventional method) 79 16,978 216 3.2 71 22,22 98 314 16 53 53 19,177 362 32 52 52 17,360 337 ───────────────── ────────────────────

Example 20 (Measurement of Change in Chemiluminescence over Time) In order to accurately measure the chemiluminescence amount, the chemiluminescence reaction to be used must be stable for a long time. Therefore, a chemiluminescent solution was prepared in the same manner as in Example 19, and measurement was started immediately after mixing to examine the change with time. The sodium indophenol was adjusted to 3.2 μM. As a result, as shown in FIG. 16, the amount of luminescence reached a plateau in about 10 minutes, and luminescence continued steadily thereafter. In addition, in particular, in the case of coexisting indophenol sodium, the stability was shown with a higher luminescence amount than in the case of not coexisting. Further, as shown in FIG. 17, a similar tendency was observed in the S / N ratio.

Example 21 (Preparation of solution for chemiluminescence reaction) As a hydrogen peroxide solution,
0.1% milk in 50 mM borate buffer (pH 10),
0.05% egg albumin, 0.1% Tween20
A 2 mM hydrogen peroxide solution containing was prepared. Anti-endothelin 1HRP labeled antibody (60 μg / ml) was 0.1
Dilution series were made using PBS solution containing% milk.
As a sensitizer solution, 0.32 mM 4- [4 '-(2'-
Methyl) thiazolyl] phenol and 16 μM sodium indophenol in 50 mM borate buffer (pH 1
0) was prepared and used. Luminol solution is 23 mM Na
It was adjusted to 20 mM with an OH solution.

(Measurement of peroxidase activity) The chemiluminescence was measured in the same manner as in Example 19 using the above reaction solution for each dilution series of the anti-endothelin 1HRP-labeled antibody to prepare a dose action curve. The results are shown in FIG.

Example 22 (Preparation of solution for chemiluminescence reaction) As a hydrogen peroxide solution,
0.1% milk in 50 mM borate buffer (pH 10),
0.05% egg albumin, 0.1% Tween20
A concentration series of hydrogen peroxide solution containing was prepared. Anti-endothelin 1HRP labeled antibody (60 μg / ml)
It was diluted 1/10 ³ with a PBS solution containing 0.1% milk. As a sensitizer solution, 0.32 mM 4- [4 '
-(2'-methyl) thiazolyl] phenol and 3.
A 50 mM borate buffer solution (pH 10) of 2 μM sodium indophenol was prepared and used. The luminol solution was adjusted to 20 mM with a 23 mM NaOH solution.

(Measurement of hydrogen peroxide) Using the above reaction solution with respect to the hydrogen peroxide concentration series, chemiluminescence was measured in the same manner as in Example 19 to prepare a dose action curve. The results are shown in FIG.

Example 23 (Preparation of solution for chemiluminescence reaction) As a hydrogen peroxide solution,
0.1% milk in 50 mM borate buffer (pH 10),
0.05% egg albumin, 0.1% Tween20
A 2 mM hydrogen peroxide solution containing was prepared. Anti-endothelin 1HRP labeled antibody (60 μg / ml) was 0.1
It diluted 1/10 ³ times with the PBS solution containing% milk. As a sensitizer solution, 0.32 mM 4- [4'-
(2'-methyl) thiazolyl] phenol and 3.2
A 50 mM borate buffer solution (pH 10) of sodium μM indophenol was prepared and used. Luminol solution is 2
A concentration series was prepared with a 3 mM NaOH solution.

(Measurement of Luminol) Using the above reaction solution for luminol concentration series, chemiluminescence was measured in the same manner as in Example 19 to prepare a dose action curve. The result is shown in FIG.

Example 24 (Preparation of solution for chemiluminescence reaction) As a hydrogen peroxide solution,
0.1% milk in 50 mM borate buffer (pH 10),
0.05% egg albumin, 0.1% Tween20
A 2 mM hydrogen peroxide solution containing was prepared. Anti-endothelin 1HRP labeled antibody (60 μg / ml) was 0.1
It was used after diluting to a predetermined concentration with a PBS solution containing% milk. As a sensitizer solution, 0.32 mM 4- [4'-
(2'-methyl) thiazolyl] phenol and 16μ
A 50 mM borate buffer solution (pH 10) of m-160 μM indophenol acetate was prepared and used. The luminol solution was adjusted to 20 mM with a 23 mM NaOH solution.

(Chemical luminescence measurement using indophenol acetate) Into a well of a 96-well microtiter plate, 10
μl of 10 ⁴ -fold diluted anti-endothelin 1HRP-labeled antibody, then 100 μl of hydrogen peroxide solution, 50 μl of luminol solution, 50 μl of sensitizer solution were added and mixed,
After 10 minutes, the amount of luminescence at 435 nm (Lumicount) was measured with a luminometer. At the same time, a blank measurement was performed without adding the labeled antibody. In Table 8, the blank value (background), the sample luminescence amount, and the signal / blank ratio (S / N ratio) with and without the addition of indophenol acetate are shown in comparison.

[0077]

[Table 8] Chemiluminescence-enhancing effect when coexisting with indophenol acetate ────────────────────────────────── ─── Additive concentration (μM) Blank value Light emission of sample S / N ratio ────────────────────────────────── ─── 0 (conventional method) 79 16,978 216 16 65 20 20,941 325 32 62 20,20 348 328 160 160 52 18,890 367 ─────────────────── ─────────────────

As shown in the results of Table 8, when indophenol acetate was added, the background luminescence was suppressed, the luminescence amount of the sample was increased, and the S / N ratio was improved.

Example 25 (Measurement of Change in Chemiluminescence over Time) In order to accurately measure the chemiluminescence amount, the chemiluminescence reaction to be used must be stable for a long time. Therefore, a chemiluminescence solution was prepared in the same manner as in Example 24, and measurement was started immediately after mixing to examine the change with time. Indophenol acetate is 16
μM. As a result, as shown in FIG.
The luminescence amount reached a plateau in minutes, and thereafter, the luminescence continued stably. In particular, when indophenol acetate was allowed to coexist, stability was shown at a higher luminescence amount than when not coexisted. Further, as shown in FIG. 22, a similar tendency was observed in the S / N ratio.

Example 26 (Preparation of solution for chemiluminescence reaction) As a hydrogen peroxide solution,
0.1% milk in 50 mM borate buffer (pH 10),
0.05% egg albumin, 0.1% Tween20
A 2 mM hydrogen peroxide solution containing was prepared. Anti-endothelin 1HRP labeled antibody (60 μg / ml) was 0.1
Dilution series were made using PBS solution containing% milk.
As a sensitizer solution, 0.32 mM 4- [4 '-(2'-
A 50 mM borate buffer (pH 10) of methyl) thiazolyl] phenol and 32 μM indophenol acetate was prepared and used. The luminol solution was adjusted to 20 mM with a 23 mM NaOH solution.

(Measurement of peroxidase activity) Chemiluminescence was measured in the same manner as in Example 24 using the above reaction solution for each dilution series of the anti-endothelin 1HRP-labeled antibody to prepare a dose action curve. The result is shown in FIG.

Example 27 (Preparation of solution for chemiluminescence reaction) As a hydrogen peroxide solution,
0.1% milk in 50 mM borate buffer (pH 10),
0.05% egg albumin, 0.1% Tween20
A concentration series of hydrogen peroxide solution containing was prepared. Anti-endothelin 1HRP labeled antibody (60 μg / ml)
It was diluted 1/10 ³ with a PBS solution containing 0.1% milk. As a sensitizer solution, 0.32 mM 4- [4 '
-(2'-methyl) thiazolyl] phenol and 16
μM Indophenol acetate 50 mM borate buffer (p
H10) was prepared and used. Luminol solution is 23 mM
It was adjusted to 20 mM with a NaOH solution.

(Measurement of Hydrogen Peroxide) Using the above reaction solution for hydrogen peroxide concentration series, chemiluminescence was measured in the same manner as in Example 24 to prepare a dose action curve. The result is shown in FIG.

Example 28 (Preparation of solution for chemiluminescence reaction) As a hydrogen peroxide solution,
0.1% milk in 50 mM borate buffer (pH 10),
0.05% egg albumin, 0.1% Tween20
A 2 mM hydrogen peroxide solution containing was prepared. Anti-endothelin 1HRP labeled antibody (60 μg / ml) was 0.1
It diluted 1/10 ³ times with the PBS solution containing% milk. As a sensitizer solution, 0.32 mM 4- [4'-
(2'-methyl) thiazolyl] phenol and 16μ
50 mM boric acid buffer solution of M indophenol acetate (pH
10) was prepared and used. Luminol solution is 23 mMN
A concentration series was prepared with aOH solution.

(Measurement of Luminol) Using the above reaction solution for luminol concentration series, chemiluminescence was measured in the same manner as in Example 24 to prepare a dose action curve. The result is shown in FIG.

[0086]

INDUSTRIAL APPLICABILITY The present invention can provide various measuring methods based on the measurement of chemiluminescence, particularly methods for enhancing chemiluminescence suitable for improving the sensitivity of immunoassay and enzyme quantification.

[Brief description of drawings]

FIG. 1 is a graph showing a change with time in chemiluminescence amount in a chemiluminescence reaction of Example 5.

FIG. 2 is a graph showing changes with time of S / N ratio in the chemiluminescence reaction of Example 5.

FIG. 3 is a graph showing a dose action curve in the peroxidase activity measurement of Example 6.

FIG. 4 is a graph showing a dose action curve in hydrogen peroxide concentration measurement of Example 7.

FIG. 5 is a graph showing a dose-effect curve in measuring luminol concentration in Example 8.

6 is a graph showing a change with time in chemiluminescence amount in a chemiluminescence reaction of Example 10. FIG.

FIG. 7 is a graph showing changes with time of S / N ratio in the chemiluminescence reaction of Example 10.

FIG. 8 is a graph showing a dose-effect curve in the peroxidase activity measurement of Example 11.

FIG. 9 is a graph showing a dose action curve in hydrogen peroxide concentration measurement of Example 12.

FIG. 10 is a graph showing a dose-effect curve in measurement of luminol concentration in Example 13.

FIG. 11 is a graph showing the change over time in the chemiluminescence amount in the chemiluminescence reaction of Example 15.

FIG. 12 is a graph showing changes with time in S / N ratio in the chemiluminescence reaction of Example 15.

FIG. 13 is a graph showing a dose-effect curve in the peroxidase activity measurement of Example 16.

FIG. 14 is a graph showing a dose action curve in hydrogen peroxide concentration measurement of Example 17.

FIG. 15 is a graph showing a dose-effect curve in measurement of luminol concentration in Example 18.

16 is a graph showing a change with time in chemiluminescence amount in a chemiluminescence reaction of Example 20. FIG.

FIG. 17 is a graph showing the change with time of the S / N ratio in the chemiluminescent reaction of Example 20.

FIG. 18 is a graph showing a dose-effect curve in the peroxidase activity measurement of Example 21.

FIG. 19 is a graph showing a dose action curve in hydrogen peroxide concentration measurement of Example 22.

FIG. 20 is a graph showing a dose-effect curve in measurement of luminol concentration in Example 23.

FIG. 21 is a graph showing a change with time in chemiluminescence amount in a chemiluminescence reaction of Example 25.

22 is a graph showing changes over time in the S / N ratio in the chemiluminescent reaction of Example 25. FIG.

FIG. 23 is a graph showing a dose-effect curve in the peroxidase activity measurement of Example 26.

FIG. 24 is a graph showing a dose action curve in hydrogen peroxide concentration measurement of Example 27.

FIG. 25 is a graph showing a dose-effect curve in measurement of luminol concentration in Example 28.

[Explanation of symbols]

 ○ When indophenol compound does not coexist ● When indophenol compound coexists Anti-ET-HRP: Anti-endothelin 1HRP labeled antibody

Claims (4)

[Claims] 1. A system for causing a chemiluminescent substance to emit light using an oxidizing agent, a peroxidase and a chemiluminescent sensitizer (enhancer), wherein a compound represented by the following general formula (I) is allowed to coexist. Chemiluminescence enhancement method. [Chemical 1] (In the formula, R ₁ represents a hydroxyl group, an alkali salt of a hydroxyl group, a substituted or unsubstituted amino group, or an acyloxy group,
R ₂ , R ₃ , R ₄ and R ₅ each independently represent a hydrogen atom or a halogen atom, or R ₂ and R ₃ or R ₄ and R ₅ respectively bond to form a benzene ring, and R ₆
And R ₇ each independently represent a hydrogen atom, a substituted or unsubstituted lower alkyl group or a lower alkoxy group. ) 2. The chemiluminescence enhancing method according to claim 1, wherein luminol or isoluminol or a derivative thereof is used as the chemiluminescent substance, and hydrogen peroxide is used as the oxidizing agent. 3. As a chemiluminescent sensitizer, 4- [2'-
(4'-Methyl) thiazolyl] phenol, 4- (2 '
-Thienyl) phenol, p-iodophenol and 4- [4 '-(2'-methyl) thiazolyl] phenol are used, at least one selected from the group consisting of: -the chemiluminescence according to claim 1 or 2. Augmentation method. 4. The compound represented by the general formula (I) is
The chemiluminescence enhancing method according to claim 3, wherein the chemiluminescence is at least one selected from the group consisting of phenol indophenol, 2,6-dichlorophenol indophenol sodium, indophenol blue, indophenol sodium and indophenol acetate. .