JPH06242111A - Chemiluminescent analysis method using peroxidase as marker - Google Patents

Abstract

PURPOSE:To improve the S/N ratio (signal/noise ratio by lowering the non- specific luminous reaction, that is, reagent blank (noise) and increasing the specific luminous reation, that is, the signal quantity so as to improve reliability of a measured value. CONSTITUTION:In the case of causing chemical luminous reaction of luminol/ hydrogen peroxide in the presence of a sensitizer to measure a biological component using peroxidase as a marker, at the time of detecting and measuring the luminous quantity in a reaction liquid, secondary alcohol polyethoxylate or defatted milk and/or albumen therewith is forced to exist together with the reaction liquid to detect and measure the luminous quantity.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a chemiluminescence analysis method using peroxidase as a labeling substance, which can be used for analysis of trace constituents of a living body, diagnosis of diseases and the like.

[0002]

2. Description of the Related Art As a method for sensitively detecting and measuring a substance to be analyzed, a specific binding reagent such as an antibody that specifically binds to the substance (often labeled with an enzyme such as peroxidase in advance) There is known a method (so-called enzyme immunoassay method, etc.) of reacting a sample with a sample and subsequently detecting and measuring a signal generated by a catalytic reaction of an enzyme such as peroxidase which is a labeled substance. As the enzyme immunoassay, various improved methods or modified methods such as a one-antibody method, a two-antibody method, a sandwich method, a homogeneous method, and a heterogeneous method are known.

As a method of efficiently measuring the signal generated by the catalytic reaction of an enzyme such as peroxidase, there is a method of measuring the chemiluminescence of luminol / hydrogen peroxide catalyzed by peroxidase. Radical stabilizers that aid one-electron oxidation are used as sensitizers (Methods in Enzymology, Vol.133,
p.331-353,1986; JP-A-2-291299).

On the other hand, it is also known that the activity of peroxidase is increased by adding polyoxyethylene ethers (Clinica Chimica Acta, 109 , 177-181, 198).
1; J. Clin. Chem. Clin. Biochem., 19 , 435-439, 198
1) In addition, in the enzyme immunoassay method, a method has also been proposed in which polyoxyethylene ethers are contained in a buffer solution to enhance the S / N ratio or the measurement sensitivity (Tokuhyohei 2-503).
No. 029 publication). Further, conventionally, for the purpose of blocking nonspecific reaction of a carrier or an antibody (or antigen) -immobilized carrier,
A carrier or an antibody (or antigen) -immobilized carrier is albumin,
It is known to treat with proteins such as milk protein and ovalbumin (Japanese Patent Laid-Open Nos. 1-217266 and 1-224665).

[0005]

When the substance to be analyzed is an ultratrace substance in the living body, etc., it is desired to develop a method having higher sensitivity than anything else. The present invention aims to develop a method that is even more sensitive than conventional methods.

[0006]

In order to develop a highly sensitive method, the present inventors have used peroxidase in the presence of a sensitizer that facilitates the one-electron oxidation of luminol to produce luminol / hydrogen peroxide. When various chemiluminescence analyzes for conducting the reaction in a system were examined, the presence of secondary alcohol polyethoxylates in the reaction solution increased the activity (signal) of peroxidase and decreased the reagent blank (noise), resulting in S / N ratio is improved, and when skimmed milk and / or ovalbumin coexist with the secondary linear alcohol polyethoxylate in the reaction solution, the effects of both act synergistically or additively, and S / The present invention has been completed by finding that the N ratio is significantly improved.

That is, the present invention relates to the following (1) and (2). (1) In a chemiluminescence analysis method in which a substance to be analyzed labeled with peroxidase is allowed to react with luminol and hydrogen peroxide in the presence of a sensitizer, and the generated luminescence amount is detected and measured, luminescence in a reaction solution When detecting and measuring the amount, a secondary alcohol polyethoxylate is present in the reaction solution,
Chemiluminescence analysis method. (2) In a chemiluminescence analysis method in which a substance to be analyzed labeled with peroxidase is reacted with luminol and hydrogen peroxide in the presence of a sensitizer, and the generated luminescence amount is detected and measured, luminescence in the reaction solution A chemiluminescence analysis method, wherein skimmed milk and / or ovalbumin is present together with a secondary alcohol polyethoxylate in the reaction solution when detecting / measuring the amount.

The secondary alcohol polyethoxylate used in the present invention is

[Chemical 1] (In Chemical Formula 1, R ¹ and R ² are the same or different saturated aliphatic alkyl groups, and n is an integer of 1 or more), and is a branched alkyl polyoxyethylene ether.

As such a secondary alcohol polyethoxylate, "Adecatol" manufactured by Asahi Denka Kogyo Co., Ltd.
SO series ”, and ADECATOL SO-80, ADECATOL SO-105, ADECATOL SO-12
0, ADECATOL SO-135, ADECATOL SO-
145, ADECATOL SO-160, etc. can be used.

These secondary alcohol polyethoxylates are contained in the buffer solution or reaction solution used in the reaction. The amount used is an amount that improves at least the S / N ratio of chemiluminescence, and is usually used in the range of 0.01 to 10% by weight, preferably 0.02 to 2% by weight. Usage is 0.0
If it is less than 1% by weight, the degree of improving the S / N ratio of chemiluminescence is low, and if it exceeds 10% by weight, the viscosity of the liquid becomes large and it becomes difficult to uniformly mix the reagents. In the following description, “%” simply means “% by weight” unless otherwise specified.

The chemiluminescence analysis in the present invention is not particularly limited as long as it is an analytical method in which peroxidase, which is a labeling substance, is reacted with luminol and hydrogen peroxide in the presence of a sensitizer, and the generated luminescence is detected and measured. However, it can be used for various analysis methods such as a one-antibody immunoassay method, a two-antibody immunoassay method, a competitive analysis method, a sandwich method, a homogeneous method, a heterogeneous method, a Western analysis method, and a DNA probe method. In particular, it is preferably used in the following solid phase sandwich method. The procedure of the solid-phase sandwich method will be described below.

Procedure: (1) A first binding reagent (for example, a first antibody) capable of binding to a specific site of a substance to be analyzed is immobilized on a solid phase carrier to obtain a first binding reagent-immobilized carrier. (2) In order to prevent non-specific binding, a sample is added directly or with an aqueous solvent to a first binding reagent-immobilized carrier that has been previously treated with protein (blocking treatment), and incubated, Form a complex of the analyte. (3) Wash with the washing solution. (4) In addition to this, it can bind to other specific sites of the substance
A second binding reagent labeled with peroxidase (eg, a second antibody) is added together with an aqueous solvent to form a complex of the first binding reagent-immobilized carrier / analyte / peroxidase-labeled second binding reagent. Let (5) Wash with the washing solution. (6) Luminol, hydrogen peroxide and a sensitizer in an aqueous solvent,
And the secondary alcohol polyethoxylates are added. (7) Measure the amount of emitted light;

As the first binding reagent to be immobilized on the solid-phase carrier, an antibody (polyclonal antibody, monoclonal antibody, antibody fragment or the like) is preferably used when the substance to be analyzed can be an antigen. When is a nucleic acid, a (single-stranded) nucleic acid or nucleic acid fragment complementary thereto is preferably used.

The second binding reagent labeled with peroxidase is attached to the substance to be analyzed at a site different from the site to which the first binding reagent binds, depending on whether the substance to be analyzed can be an antigen or nucleic acid. An antibody or nucleic acid that can bind is used.

To label the second binding reagent with peroxidase, the peroxidase is directly labeled by the maleimide / hinge method, the succinimide method or the like, or the second binding reagent has a chemical group such as a biotinyl group. After that, peroxidase having a chemical group such as avidin that reacts with this may be reacted to label indirectly. As the peroxidase for labeling the second binding reagent, a horseradish-derived basic isozyme is suitable. B, C, D and E types of basic isozymes are known, and of these, the C type is most preferable.

As the solid phase carrier to be used, a microtiter plate is usually suitable. A black module plate (Nu
nc), Micro Fluoroplate Black (manufactured by Dynatech), Micro Fluorimovawell Black (manufactured by Dynatech) and the like. In some cases, white microtiter plates that prevent stray light from adjacent wells can be used. Examples of such a microtiter plate include a white pigment (Microfluoroplate white (manufactured by Dynatech), Microfluororemovawell white (manufactured by Dynatech), a white plate (manufactured by Sumitomo Bakelite), and the like.

As the luminol used for the luminescence reaction, hydrazine and sulfide ions, which are the starting materials for production, are often mixed in the commonly available reagent grade products, so that the product is purified after repeated recrystallization.

The sensitizer used in the present invention has a sensitizing effect by assisting the one-electron oxidation of luminol, and is composed of 4-iodophenol, 4-bromophenol, 4-chlorophenol, 4-phenylphenol and 2 -Chloro-4-
Phenylphenol, 4- (2'-thienyl) phenol, 6-hydroxybenzothiazole, 4- [4'-
(2'-methyl) thiazolyl] phenol, 4- [2 '
-(4'-methyl) thiazolyl] phenol, 4-
(2'-benzothiazolyl) phenol, 3- (10-
Phenothiazyl) -n-propyl-sulfonate and the like.

When the luminescence reaction of luminol / hydrogen peroxide is carried out by the catalytic action of peroxidase in the presence of these sensitizers, the sensitizer becomes a sensitizing radical, which reacts with luminol to emit light.

The solvent of the reaction solution is usually an aqueous solvent.
Examples of the aqueous solvent include water or a buffer solution such as a phosphate buffer solution, a borate buffer solution, and a Good's buffer solution. The pH of these buffers is usually 5 to 12, preferably 7 to 11. pH is 5
If it is less than the above, the amount of luminescence deviates from the optimum pH of peroxidase, and the amount of luminescence becomes weak. If the pH exceeds 12, luminescence (noise) not depending on the peroxidase activity becomes strong.

The skim milk used in the present invention is a milk from which fat is removed, and is also called as skimmed milk powder or skim milk. This is a commercial product, for example, "MILK DILUENT
/ BLOCKING SOLUTION "(KPL, code number: 50-82
-01), "Block Ace" (Dainippon Pharmaceutical, code number: UK-B25), "Block Ace Powder" (Dainippon Pharmaceutical, code number: UK-B80), etc. are easily available.

The ovalbumin used in the present invention has a molecular weight of 45,000 separated from egg white and an isoelectric point of 4.6.
, Which is also a commercially available product, such as "albumin, egg white, 5 times crystal" (Seikagaku Corporation, code number: 250440), "ovalbumin" (Taiyo Kagaku Corporation,
Code number: 300-00711), "Albumin, egg" (Wako Pure Chemical Industries, Ltd., code number: 018-09882), "Albumin
Chicken egg ”(manufactured by Sigma, code number: A7641) and the like are easily available.

The concentrations of these skim milk or ovalbumin used are such that at least the S / N ratio is improved. Usually, the respective concentrations are in the range of 0.01 to 0.5%, preferably 0.02 to 0.2% in the final concentration of the reaction solution. If the concentration is less than 0.01%, the effect of improving the S / N ratio is small, and if it exceeds 0.5%, the specific luminescent reaction is suppressed, which is not preferable.

Other additives, for example, sugar alcohols such as mannitol and sorbitol, may be added to the reaction solution in order to improve performance such as operability and stability.

Experimental examples are shown below. Experimental Example 1 Effect of Addition of Secondary Alcohol Polyethoxylate in Chemiluminescent Reaction in the Presence of 4- [4 ′-(2′-methyl) thiazolyl] phenol The chemiluminescent reaction (no addition control) was performed as follows. It was That is, the black module plate (Nunc
475515), peroxidase (POD)
Labeled anti-endothelin antibody (Yamasa Shoyu Co., MCA
ET-02) 1/10 ⁵ diluted solution [using 50 mM sodium borate buffer (pH 10.0) as a diluting buffer] 100 μl was injected, and then 2 mM 4- [4′-
(2'-Methyl) thiazolyl] phenol dimethylsulfoxide solution 8 μl, 4 mM hydrogen peroxide-containing 50
50 μl of mM sodium borate buffer (pH 10) and 50 μl of 20 mM luminol were added, and the amount of luminescence after 10 minutes was measured with a luminometer (Corner Electric Co., Ltd., MLR-100 type) to obtain a signal (S). : Response 6
4, Sense Auto). Further, instead of the 1/10 ⁵ dilution of the POD-labeled anti-endothelin antibody, 50 mM sodium borate buffer (pH 10.0) was used in the same manner to measure the luminescence amount, which was designated as noise (N). did.

The secondary alcohol polyethoxylate addition test was carried out by using a 50 mM sodium borate buffer (pH 10.0) for diluting the POD-labeled anti-endothelin antibody used.
Adecatol SO-135 (manufactured by Asahi Denka Kogyo Co., Ltd.) was contained in advance at 0.1% by weight, and the same operation as above was performed. In addition, as a comparative addition experiment, ADECATOL SO-135
Instead of polyoxyethylene sorbitan monolaurate (manufactured by Wako Pure Chemical Industries, Ltd., "Tween 20") was used in the same manner. Table 1 shows the measurement results.

When ADECATOL SO-135 was added, the emission noise was about 2/3 and the signal was about 1.
It was 5 times and the S / N ratio was slightly more than 2 times. Meanwhile, Tween 20
In addition, the addition did not show a reduction in luminescence noise as compared to the case without addition, but only a slight increase in the signal was seen.

[Table 1] ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Additive (*) Noise (N) Signal (S) S / N ratio ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ No additives 1,581 1, 968 1.24 ADECATOL SO-135 1,105 3,043 2.75 Tween 20 (Comparison) 1,537 2,800 1.82 ━━━━━━━━━━━━━━━━━━━ ━━━━━━━━━━━━━━━━━ *: As for the concentration of the additives, the concentration of each component is 0.1% except for the non-addition (hereinafter, also in Experimental Examples 2 to 9. the same).

Experimental Example 2 Effect of addition of various nonionic surfactants on chemiluminescence reaction in the presence of 4- [4 '-(2'-methyl) thiazolyl] phenol Secondary alcohols as nonionic surfactants In addition to polyethoxylate (Adecatol SO-135), various compounds were used and tested in the same manner as in Experimental Example 1. The results are shown in Table 2.

[Table 2] ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Additive Noise (N) Signal (S) S / N ratio ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Additive-free 2,442 2,713 1. 11 Adecatol SO-135 1,105 3,043 2.75 Adecatol LO-9 2,091 3,454 1.65 Adecatol NP-695 1,700 3,417 2.01 Adecatol NP-700 2,060 3,991 1.94 Adecatol PC-10 2,598 4,153 1.60 Tween 20 2,674 4,039 1.51 ━━━━━━━━━━━━━━━━━━━━━━━ ━━━━━━━━━━━━━

The order in which the effect of improving S / N is high is ADECATOL SO-135> ADECATOL NP-695> ADECATOL NP-700> ADECATOL LO-9> ADECATOL P.
C-10> Tween 20 (Table 2).

Experimental Example 3 Effect of Addition of Secondary Alcohol Polyethoxylate on Chemiluminescence Reaction in the Presence of 4- [2 '-(4'-methyl) thiazolyl] phenol 2 mM 4- [4' as a sensitizer 20 mM 4- [2 'instead of-(2'-methyl) thiazolyl] phenol
The same procedure as in Experimental Example 1 was performed except that-(4'-methyl) thiazolyl] phenol was used. The results are shown in Table 4.

[Table 3] ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Additive Noise (N) Signal (S) S / N ratio ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ No additive 1,164 1,478 1. 27 Adecatol SO-135 948 3,580 3.77 Tween 20 (Comparison) 1,260 3,371 2.68 ━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━━━━━━━━━━

Experimental Example 4 Effect of addition of secondary alcohol polyethoxylate in chemiluminescence reaction in the presence of 4- (2'-thienyl) phenol 2 mM 4- [4 '-(2'-methyl) as a sensitizer ) Thiazolyl] phenol instead of 1 mM 4- (2'-
The same procedure as in Experimental Example 1 was performed except that thienyl) phenol was used. The results are shown in Table 4.

[0032]

[Table 4] ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Additive Noise (N) Signal (S) S / N ratio ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ No additive 732 827 1.13 ADECATOL SO- 135 570 1,922 3.37 Tween 20 (Comparison) 723 1,226 1.75 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━━━━

Experimental Example 5 3- (10-phenothiazyl)
Addition Effect of Secondary Alcohol Polyethoxylate on Chemiluminescence Reaction in the Presence of -n-Propyl-Sulfonate Instead of 2 mM 4- [4 '-(2'-methyl) thiazolyl] phenol as a sensitizer 20 mM 3- (10
-Phenothiazyl) -n-propyl-sulfonate was used, and the same procedure as in Experimental Example 1 was performed. The results are shown in Table 5.

[Table 5] ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Additive Noise (N) Signal (S) S / N ratio ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Additive-free 1,371 1,493 1. 09 Adecator SO-135 942 3,162 3.36 Tween 20 (comparative) 1,370 1,662 1.21 ━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━━━━━━━━━━

Experimental Example 6 In a chemiluminescence reaction in the presence of 4- [4 '-(2'-methyl) thiazolyl] phenol,
Coexistence Effect of Secondary Alcohol Polyethoxylate and Skim Milk A test without additives (control without addition) was carried out as follows. To a black module plate, 100 μl of a 1/10 ⁵ diluted solution of peroxidase (POD) -labeled anti-endothelin antibody [using 50 mM sodium borate buffer (pH 10.0) as a buffer for dilution] was injected, and then 2 mM 4- [4 '-(2'-methyl) thiazolyl]
Phenol dimethyl sulfoxide solution 8μl, 4m
50 µl of 50 mM sodium borate buffer (pH 10) containing M hydrogen peroxide and 50 µl of 20 mM luminol were added, and the amount of luminescence after 10 minutes was measured with a luminometer to obtain a signal (S). Noise (N) was measured by using 50 mM sodium borate buffer (pH 10.0) instead of the 1/10 ⁵ dilution of POD-labeled anti-endothelin antibody.
It was used.

The secondary alcohol polyethoxylate (Adecatol SO-135), skim milk, and both of them were added to the test solution in a 50 mM sodium borate buffer (pH 10.0) for diluting the POD-labeled anti-endothelin antibody used. , Except that each of them was previously contained in an amount of 0.1% by weight. The results are shown in Table 6. The skim milk is "MILK DILUENT / BLOCKING SOLUTION" (made by KPL,
Code number: 50-82-01) was used (Experimental example 7-
9 and the skim milk used in Examples 1 to 3).

[Table 6] ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Additive Noise (N) Signal (S) S / N ratio ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ No additive (no additive control) 1,581 1,968 1.24 ADECATOL SO-135 1,105 3,043 2.75 Skim milk 135 1,655 12.2 ADECATOL SO-135 ┐ 131 2,082 15.9 + skim milk ┘ ━━━━━━━ ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━

With the addition of both ADECATOL SO-135 and skim milk, the S / N ratio was 15.9.
It was further improved as compared with the case of adding O-135 alone (S / N ratio = 2.75) or the case of adding skim milk alone (S / N ratio = 12.2) (Table 6).

Experimental Example 7 In a chemiluminescence reaction in the presence of 4- [2 '-(4'-methyl) thiazolyl] phenol,
Coexistence effect of secondary alcohol polyethoxylate and skim milk Instead of 2 mM 4- [4 '-(2'-methyl) thiazolyl] phenol as a sensitizer, 20 mM 4- [2'
The same procedure as in Experimental Example 6 was performed using-(4'-methyl) thiazolyl] phenol. The results are shown in Table 7.

[Table 7] ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Additive Noise (N) Signal (S) S / N ratio ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ No addition (no addition control) 1,164 1,478 1.27 ADECATOL SO-135 948 3,580 3.77 nonfat milk 85 493 580 ADECATOL SO-135 ┐ 94 1,114 11.9 + nonfat dry powder ┘ ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━━━━━━━━━━━━━━━━━━━━━━━━

Experimental Example 8 3- (10-phenothiazyl)
-Coexistence effect of secondary alcohol polyethoxylate and skim milk in chemiluminescence reaction in the presence of n-propyl-sulfonate 2 mM 4- [4 '-(2'-methyl) thiazolyl] as sensitizer 20 mM 3- (10 instead of phenol
-Phenothiazyl) -n-propyl-sulfonate was used, and the same procedure as in Experimental Example 6 was performed. The results are shown in Table 8
Shown in.

[0039]

[Table 8] ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Additive Noise (N) Signal (S) S / N ratio ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ No additives (no additives control) 1,371 1,493 1.09 ADECATOL SO-135 942 3,162 3.36 Skim milk 164 1,510 9.2 1 ADECATOL SO-135 ┐ 130 1,569 12.1 + skim milk ┘ ━━━━━━━━━━ ━━━━━━━━━━━━━━━━━━━━━━━━━━━━

Experimental Example 9 Effect of coexistence of secondary alcohol polyethoxylate and skim milk in chemiluminescent reaction in the presence of p-iodophenol Same as Experimental Example 6 except that p-iodophenol was used as a sensitizer. I went to. The results are shown in Table 9.

[Table 9] ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Additive Noise (N) Signal (S) S / N ratio ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ No additive (no additive control) 4,154 4,117 0.99 Adecatol SO-135 1,015 1,997 1.97 Skim milk powder 149 501 3.36 Adecatol SO-135 ┐ 119 420 3.52 + skim milk ┘ ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━━━━━━━━━━━━━━━━━━━━━━━━

[0041]

【Example】

Example 1 Standard curve of endothelin-1 (No. 1) Endothelin-1 (abbreviated as ET-1. Human origin, manufactured by Peptide Institute, Code No. 4198-s) was used as 0.1% Tween 20 and 0. Diluted with Dulbecco's phosphate buffered saline containing 1% skim milk (hereinafter, referred to as a diluting buffer), and diluted with 2, 5, 10, 20, and 40, respectively.
An endothelin-1 standard solution with a concentration of pg / ml was prepared.

In advance, anti-endothelin antibody (manufactured by Immune Biology Institute, anti-endothelin ^15-21 specific antibody, code number:
16155) to 50 mM sodium phosphate (pH 7.0)
200 μl was dispensed into each well of a black module plate (Nunc Co., code No. 475515), and the mixture was allowed to stand overnight at 4 ° C. to adsorb the antibody to the wells of the plate. After washing twice with ion-exchange distilled water, 400 μl of 0.5% skim milk was added to the well, and the mixture was kept at 37 ° C. for 6 hours (blocking treatment), and then 0.1% Tween 20-containing calcium-free Dulbecco's phosphate buffered saline. The plate was washed 3 times with to obtain an antibody-immobilized plate.

200 μl of each standard solution of endothelin-1 prepared above was placed in a well of the plate and left overnight at room temperature. Then, it was washed 10 times with 0.1% Tween-20-containing Dulbecco's phosphate buffered saline (hereinafter referred to as a washing buffer), and diluted with a diluting buffer at 2 μg / m 2.
Peroxidase (POD) -labeled anti-endothelin-1 antibody adjusted to 1 (manufactured by Immune Biology Institute, code number 1)
6165) 200 μl each was added to each well and incubated at 37 ° C. for 2 hours. After washing 10 times with a washing buffer, ADECATOL SO-135 was added to each well in order of 0.1.
50 mM (pH 10) sodium borate buffer containing 2 mM hydrogen peroxide containing 1% (hereinafter referred to as luminescent substrate solution A).
100 μl, then 20 mM Luminol and 0.32 mM
4- [4 '-(2'-methyl) thiazolyl] phenol-containing 50 mM (pH 10) sodium borate buffer 1: 1 (volume ratio) mixed solution (hereinafter referred to as luminescent substrate solution B) 100 μl was added. . Ten minutes later, the amount of luminescence was measured with a luminometer. The reagent blank had the same volume of the buffer for dilution instead of the standard solution of endothelin-1 and was operated in the same manner. The concentration of endothelin-1 was plotted on the horizontal axis, and the amount of luminescence (lumi count) was plotted on the vertical axis, which was plotted on a graph to give a calibration curve for endothelin-1 (1 in FIG. 1).

As the luminescent substrate solution A, a 50 mM sodium borate buffer solution containing 2 mM hydrogen peroxide (pH 10) (neither ADECATOL SO-135 nor skim milk) was used. It was used as a comparative control.

Example 2 Calibration curve of endothelin-1 (No. 2) As a luminescent substrate solution A, 0.1% ADECATOL SO-13
5 and 0.1% skim milk containing 2 mM hydrogen peroxide 50
A calibration curve for endothelin-1 was prepared in the same manner as in Example 1 except that a mM (pH 10) sodium borate buffer was used (2 in FIG. 1). From the results of Example 1 and Example 2 (FIG. 1), ADECATOL SO-135 was observed during the luminescence reaction.
It can be seen that in the presence of the above, particularly when both ADECATOL SO-135 and skim milk coexist, the slope of the calibration curve for endothelin-1 is large and the measurement sensitivity is high.

Example 3 Measurement of Endothelin-1 in Plasma of Healthy Individuals Endothelin-1 (Peptide Institute, Code No. 4198-s), which serves as a standard, was diluted with endothelin-1-free plasma to give 2, 5 respectively. 10, 20 and 40 pg / ml
An endothelin-1 standard solution having a concentration of was prepared. Endothelin-1 standard solution and specimen (plasma of healthy subjects: 6
(Body) was operated in the same manner as in Example 2 to determine the amount of light emission. Luminescence intensity of endothelin-1 standard solution (calibration curve)
Then, the concentration of endothelin-1 in the sample was obtained.

[Table 10] ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Specimen number ET-1 (pg / ml) Specimen number ET-1 (pg / ml) ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ 1 1.47 4 3.00 2 2.12 5 2.79 3 1.87 6 1.55 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━

[0047]

INDUSTRIAL APPLICABILITY The present invention reduces non-specific luminescence reaction, that is, reagent blank (noise) in chemiluminescence analysis using peroxidase as a label, and enhances specific luminescence reaction, that is, signal amount, S / N. Ratio (signal /
Noise ratio). Therefore, the reliability of the measured value is improved. Further, when the present invention is used for the measurement of endothelin-1, it can be directly quantified without pretreatment such as concentration of plasma (or serum).

[Brief description of drawings]

FIG. 1 is a graph showing a calibration curve for endothelin-1.

[Explanation of symbols]

1 ... Calibration curve when the luminescent substrate solution A containing 0.1% ADECATOL SO-135 was used (Example 1). 2 ... Calibration curve using luminescent substrate solution A containing 0.1% ADECATOL SO-135 and 0.1% skim milk (Example 2).

Claims (2)

[Claims] 1. A chemiluminescence analysis method in which a substance to be analyzed labeled with peroxidase is allowed to react with luminol and hydrogen peroxide in the presence of a sensitizer to detect and measure the amount of luminescence produced in the reaction solution. Chemiluminescence analysis method in which a secondary alcohol polyethoxylate is present in the reaction solution when detecting and measuring the luminescence amount of. 2. A chemiluminescence analysis method in which a substance to be analyzed labeled with peroxidase is allowed to react with luminol and hydrogen peroxide in the presence of a sensitizer to detect and measure the amount of luminescence produced in the reaction solution. Chemiluminescence analysis method, wherein skimmed milk and / or ovalbumin are present together with the secondary alcohol polyethoxylate in the reaction solution when detecting / measuring the luminescence amount of.