JP3315775B2 - Chemiluminescence analysis using peroxidase as label - Google Patents

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 label, and can be used for analysis of trace components of a living body, diagnosis of a disease, and the like.

[0002]

2. Description of the Related Art As a method for detecting and measuring a substance to be analyzed with high sensitivity, a specific binding reagent such as an antibody specifically binding to the substance (often labeled in advance with an enzyme such as peroxidase). Is known to react with a sample, and subsequently detect and measure a signal generated by a catalytic reaction of an enzyme such as peroxidase as a label (so-called enzyme immunoassay). Further, as this 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 a signal generated by a catalytic reaction of an enzyme such as peroxidase, there is a method of measuring chemiluminescence of luminol / hydrogen peroxide catalyzed by peroxidase. Radical stabilizers that help 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 known that the activity of peroxidase is increased by the addition of polyoxyethylene ethers (Clinica Chimica Acta, 109 , 177-181, 198).
1; J. Clin. Chem. Clin. Biochem., 19 , 435-439, 198
1) In addition, a method of increasing the S / N ratio or the measurement sensitivity by adding a polyoxyethylene ether to a buffer in an enzyme immunoassay has also been proposed (Tokuhei 2-503).
No. 029). Conventionally, for the purpose of blocking non-specific reactions of carriers and carriers on which antibodies (or antigens) are immobilized,
Albumin, carrier or antibody (or antigen) immobilized carrier
It is known to treat with proteins such as milk protein and ovalbumin (Japanese Patent Application Laid-Open Nos. 1-217266 and 1-222465).

[0005]

When the substance to be analyzed is an ultra-trace substance in a living body, it is desired to develop a method having a higher sensitivity than anything. An object of the present invention is to develop a method with higher sensitivity than the conventional method.

[0006]

SUMMARY OF THE INVENTION To develop a sensitive method, the present inventors have used peroxidase in the presence of a sensitizer that facilitates one-electron oxidation of luminol, using luminol / hydrogen peroxide. Various studies were performed on chemiluminescence analysis in which a reaction was carried out in a system. When a secondary alcohol polyethoxylate was present in the reaction solution, the activity (signal) of peroxidase increased, and the reagent blank (noise) decreased, resulting in a decrease in S. When the / N ratio is improved and skim milk and / or ovalbumin coexist with the secondary linear alcohol polyethoxylate in the reaction solution, the effects of both act synergistically or additively, The inventors have found that the N ratio is greatly improved, and have completed the present invention.

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 reacted with luminol and hydrogen peroxide in the presence of a sensitizer, and the amount of generated luminescence is detected and measured, the luminescence in the reaction solution is measured. 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 amount of generated luminescence is detected and measured, the luminescence in the reaction solution is determined. A chemiluminescence analysis method comprising allowing skim milk and / or ovalbumin to coexist with a secondary alcohol polyethoxylate in a reaction solution when detecting and measuring the amount.

The secondary alcohol polyethoxylate used in the present invention is

Embedded image (Wherein R ¹ and R ² are the same or different saturated aliphatic alkyl groups, and n is an integer of 1 or more).

As such a secondary alcohol polyethoxylate, "ADEKATOL" manufactured by Asahi Denka Kogyo KK
SO Series ”, Adekatrol SO-80, Adekatrol SO-105, Adekatrol SO-12
0, ADEKATOL SO-135, ADEKATOL SO-
145, ADEKATOL SO-160 and the like can be used.

[0010] These secondary alcohol polyethoxylates are contained in a buffer or reaction solution used for the reaction. The amount used is at least an amount that improves 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. Used amount 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 is more than 10% by weight, the viscosity of the liquid becomes large, making it difficult to mix the reagents uniformly. In the following description, the term “%” simply means “% by weight” unless otherwise specified.

The chemiluminescence analysis in the present invention is not particularly limited as long as it is a method of reacting labeled peroxidase with luminol and hydrogen peroxide in the presence of a sensitizer, and detecting and measuring the generated luminescence. It can be used for various analysis methods such as one antibody immunoassay, two antibody immunoassay, competition analysis, sandwich method, homogenous method, heterogeneous method, western analysis method, DNA probe method, etc. In particular, it is suitably used for the following solid phase sandwich method. Hereinafter, the procedure of the solid phase sandwich method will be described.

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 form a first binding reagent-immobilized carrier. (2) In order to prevent non-specific binding, the sample is added directly or together with an aqueous solvent to the first binding reagent-immobilized carrier previously treated with a protein (blocking treatment) and incubated, and the first binding reagent-immobilized carrier / A complex of the analyte is formed. (3) Wash with washing solution. (4) In addition, it can bind to other specific site of the analyte,
A second binding reagent (for example, a second antibody) labeled with peroxidase 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 it. (5) Wash with a washing solution. (6) in an aqueous solvent, luminol, hydrogen peroxide and a sensitizer,
Also add the secondary alcohol polyethoxylate. (7) measuring the amount of emitted light;

As the first binding reagent immobilized on the solid phase carrier, an antibody (polyclonal antibody, monoclonal antibody, antibody fragment, or the like) is suitably used when the substance to be analyzed can be an antigen. 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 may be attached to the analyte at a site different from the site to which the first binding reagent binds, depending on whether the analyte can be an antigen or a nucleic acid. An antibody or nucleic acid capable of binding is used.

To label the second binding reagent with peroxidase, the peroxidase is directly labeled by a maleimide hinge method, a succinimide method, or the like, or the second binding reagent is provided with a chemical group such as a biotinyl group. Thereafter, a peroxidase having a chemical group such as avidin, which reacts with this, may be reacted and indirectly labeled. As the peroxidase that labels the second binding reagent, a basic isozyme derived from horseradish is preferable. B, C, D and E types are known as basic isozymes, and among these, type C is most preferred.

As the solid phase carrier to be used, a microtiter plate is usually suitable. As such a microtiter plate, a black module plate (Nu
nc), microfluoroplate black (manufactured by Dynatech), microfluororemover well black (manufactured by Dynatech) and the like. In some cases, a white microtiter plate that prevents stray light from adjacent wells can be used. Examples of such a microtiter plate include a microfluoroplate white (manufactured by Dynatech), a microfluororemover well white (manufactured by Dynatech), and a white plate (manufactured by Sumitomo Bakelite) containing a white pigment.

The luminol used for the luminescence reaction is usually of a reagent grade which is generally available and contains hydrazine and sulfide ions, which are raw materials for production, in many cases.

The sensitizer used in the present invention has a sensitizing effect by assisting one-electron oxidation of luminol, and comprises 4-iodophenol, 4-bromophenol, 4-chlorophenol, 4-phenylphenol, -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.

When a luminous 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.

An aqueous solvent is usually used as a solvent for the reaction solution.
Examples of the aqueous solvent include buffers such as water or phosphate buffer, borate buffer, and good buffer. The pH of these buffers is usually 5 to 12, preferably 7 to 11. pH 5
When the pH is less than the optimum pH of peroxidase, the luminescence becomes weak, and when the pH exceeds 12, the luminescence (noise) independent of the peroxidase activity becomes strong.

The skim milk used in the present invention refers to milk from which fat has been removed, and is also referred to as skim milk or skim milk. This is a commercial product, for example, "MILK DILUENT
/ BLOCKING SOLUTION "(KPL, code number: 50-82
-01), "Block Ace" (Dainippon Pharmaceutical Co., Ltd., code No. UK-B25), "Block Ace Powder" (Dainippon Pharmaceutical Co., Ltd., code No. UK-B80) and the like can be easily obtained.

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

The use concentration of the skim milk or ovalbumin is a concentration at which the S / N ratio is improved at least. Usually, their concentrations are each 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 luminescence reaction is suppressed, which is not preferable.

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

An experimental example will be described below. Experimental Example 1 Effect of addition of secondary alcohol polyethoxylate on chemiluminescence reaction in the presence of 4- [4 '-(2'-methyl) thiazolyl] phenol Chemiluminescence reaction (no addition control) was performed as follows. Was. That is, a black module plate (Nunc
Peroxidase (POD)
Labeled anti-endothelin antibody (MCA, manufactured by Yamasa Shoyu Co., Ltd.)
100 μl of a 1/10 ⁵ dilution of ET-02) [using 50 mM sodium borate buffer (pH 10.0) as a diluting buffer], then injecting 2 mM 4- [4′-
(2′-methyl) thiazolyl] phenol in dimethyl sulfoxide (8 μl, 50 mM containing 4 mM hydrogen peroxide)
50 μl of a 50 mM sodium borate buffer (pH 10) and 50 μl of 20 mM luminol were added, and the luminescence amount after 10 minutes was measured with a luminometer (manufactured by Corona Electric Co., Ltd., type MLR-100) to obtain a signal (S). : Response 6
4, Sense Auto). Also, in place of the 1/10 ⁵ dilution of the POD-labeled anti-endothelin antibody, the same operation was performed using a 50 mM sodium borate buffer (pH 10.0) to measure the amount of luminescence, which was referred to as noise (N). did.

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

Addition of ADEKATOL SO-135 resulted in a light emission noise of about 2/3 and a signal of about 1.
5 times, the S / N ratio was slightly more than 2 times. On the other hand, Tween 20
In addition, no decrease in emission noise was observed, and only a slight increase in signal was observed, as compared with no addition.

[Table 1] 添加 Additive (*) Noise (N) Signal (S) S / N ratio ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ No additive 1,581 1, 968 1.24 Adecitol SO-135 1,105 3,043 2.75 Tween 20 (comparison) 1,537 2,800 1.82 ━━━━━━━━━━━━━━━━━ *: Concentrations of additives are 0.1% for each component except no additive (hereinafter, Experimental Examples 2 to 9 also apply). 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 alcohol as nonionic surfactant A test was conducted in the same manner as in Experimental Example 1 using various materials in addition to polyethoxylate (Adecitol SO-135). Table 2 shows the results.

[Table 2] ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Additive Noise (N) Signal (S) S / N ratio ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ No additive 2,442 2,713 11 ADEKATOL SO-135 1,105 3,043 2.75 ADEKATOL LO-9 2,091 3,454 1.65 ADEKATOL NP-695 1,700 3,417 2.01 ADEKATOL NP-700 2,060 3,991 1.94 Adecitol 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 as follows: ADEKATOL SO-135> ADEKATOL NP-695> ADEKATOL NP-700> ADEKATOL LO-9> ADEKATOL P
C-10> Tween 20 (Table 2).

Experimental Example 3 Effect of adding secondary alcohol polyethoxylate on chemiluminescence reaction in the presence of 4- [2 '-(4'-methyl) thiazolyl] phenol 2 mM 4- [4' as sensitizer -(2'-Methyl) thiazolyl] phenol instead of 20 mM 4- [2 '
-(4'-Methyl) thiazolyl] phenol was used in the same manner as in Experimental Example 1. The results are shown in Table 4.

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

Experimental Example 4 Effect of adding secondary alcohol polyethoxylate on chemiluminescence reaction in the presence of 4- (2'-thienyl) phenol 2 mM 4- [4 '-(2'-methyl) as sensitizer ) Thiazolyl] phenol instead of 1 mM 4- (2'-
The procedure was performed in the same manner as in Experimental Example 1 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 addition 732 827 1.13 Adecitol SO- 135 570 1,922 3.37 Tween 20 (comparison) 723 1,226 1.75 ━━━━━━

Experimental Example 5 3- (10-phenothiazyl)
Effect of Addition of Secondary Alcohol Polyethoxylate in 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, except that Experimental Example 1 was used. Table 5 shows the results.

[Table 5] ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Additive Noise (N) Signal (S) S / N ratio ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Not added 1,371 1,493 1. 09 Adecitol SO-135 942 3,162 3.36 Tween 20 (comparison) 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 (no control) was operated as follows. 100 μl of a 1/10 ⁵ dilution of peroxidase (POD) -labeled anti-endothelin antibody [using 50 mM sodium borate buffer (pH 10.0) as a dilution buffer) is injected into the black module plate, 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). The measurement of the noise (N) was performed using a 50 mM sodium borate buffer (pH 10.0) instead of the 1/10 ⁵ dilution of the POD-labeled anti-endothelin antibody.
It was used.

The addition test of the secondary alcohol polyethoxylate (Adecitol SO-135), skim milk and both was carried out in a 50 mM sodium borate buffer (pH 10.0) for diluting the POD-labeled anti-endothelin antibody used. , Respectively, except that each was previously contained in an amount of 0.1% by weight. Table 6 shows the results. In addition, skim milk is “MILK DILUENT / BLOCKING SOLUTION” (KPL,
(Code number: 50-82-01) (Experimental Examples 7 to below)
9 and the skim milk used in Examples 1 to 3).

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

With the addition of both ADEKATOL SO-135 and skim milk, the S / N ratio became 15.9,
The results were further improved (Table 6) 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).

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 20 mM 4- [2 'instead of 2 mM 4- [4'-(2'-methyl) thiazolyl] phenol as sensitizer
-(4'-Methyl) thiazolyl] phenol was used in the same manner as in Experimental Example 6. Table 7 shows the results.

[Table 7] ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Additive Noise (N) Signal (S) S / N ratio ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ No additive (No additive control) 1,164 1,478 1.27 ADEKATOL SO-135 948 3,580 3.77 Skim milk 85 493 5.80 ADEKATOL SO-135 ９４ 94 1,114 11.9 + skim milk powder ┘ ━━━━━━━━━━ ━━━━━━━━━━━━━━━━━━━━━━━━━━

Experimental Example 8 3- (10-phenothiazyl)
-Coexistence effect of secondary alcohol polyethoxylate and skim milk on chemiluminescence reaction in the presence of -n-propyl-sulfonate 2 mM 4- [4 '-(2'-methyl) thiazolyl] as sensitizer Instead of phenol, 20 mM 3- (10
-Phenothiazyl) -n-propyl-sulfonate was used, except that Experimental Example 6 was used. Table 8 shows the results.
Shown in

[0039]

[Table 8] ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Additive Noise (N) Signal (S) S / N ratio ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ No additive (No additive control) 1,371 1,493 1.09 ADEKATOL SO-135 942 3,162 3.36 Skim milk 164 1,510 9.21 ADEKATOL SO-135 {130 1,569 12.1 + skim milk} ━━━━━━━━━━━━━━━━━━━━━━━━━━━━

EXPERIMENTAL EXAMPLE 9 Coexistence Effect of Secondary Alcohol Polyethoxylate and Skim Milk in Chemiluminescence Reaction in the Presence of p-Iodophenol Same as Experimental Example 6, except that p-iodophenol was used as a sensitizer. I went. Table 9 shows the results.

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

[0041]

【Example】

Example 1 Calibration Curve of Endothelin-1 (Part 1) A standard endothelin-1 (abbreviated as ET-1; human-derived, peptide research laboratory, code No. 4198-s) was 0.1% Tween 20 and 0.1%. It is diluted with Dulbecco's phosphate buffered saline containing 1% skim milk (hereinafter, referred to as dilution buffer), and 2, 5, 10, 20, and 40, respectively.
An endothelin-1 standard solution having a concentration of pg / ml was prepared.

An anti-endothelin antibody (manufactured by Immune Biological Laboratories, anti-endothelin ^15-21 specific antibody, code number:
16155) with 50 mM sodium phosphate (pH 7.0)
To 200 μl / well in a black module plate (manufactured by Nunc, code number 475515), and allowed to stand at 4 ° C. overnight to allow the antibody to adsorb to the wells of the plate. After washing twice with ion-exchanged distilled water, 400 μl of 0.5% non-fat milk was added to the wells, and the mixture was incubated at 37 ° C. for 6 hours (blocking treatment), and then calcium-free Dulbecco's phosphate buffered saline containing 0.1% Tween 20 was added. And washed three times to obtain an antibody-immobilized plate.

Each 200 μl of the standard solution of endothelin-1 prepared above was placed in a well of a plate, and allowed to stand at room temperature overnight. Thereafter, the plate is washed 10 times with a Dulbecco's phosphate buffered saline containing 0.1% Tween 20 (hereinafter referred to as a washing buffer), and then 2 μg / m 2 with a dilution buffer.
l of peroxidase (POD) -labeled anti-endothelin-1 antibody (manufactured by Immune Biological Laboratory, code number 1)
6165) 200 μl was added to each well and incubated at 37 ° C. for 2 hours. After washing 10 times with the washing buffer, ADECATOL SO-135 was sequentially added to each well in 0.1 ml.
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
100 μl of a 1: 1 (volume ratio) mixed solution of 50 mM (pH 10) sodium borate buffer containing 4- [4 ′-(2′-methyl) thiazolyl] phenol (hereinafter referred to as luminescent substrate solution B) was added. . Ten minutes later, the luminescence was measured with a luminometer. The reagent blank was prepared in the same manner by using the same volume of the dilution buffer in place of the standard solution of endothelin-1. The concentration of endothelin-1 was plotted on the horizontal axis, the luminescence (lumi count) was plotted on the vertical axis, and plotted on a graph to obtain a calibration curve of endothelin-1 (1 in FIG. 1).

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

Example 2 Calibration Curve of Endothelin-1 (Part 2) As a luminescent substrate solution A, 0.1% adecatol SO-13 was used.
50 containing 2 mM hydrogen peroxide with 5 and 0.1% skim milk
A calibration curve of endothelin-1 was prepared in the same manner as in Example 1 except that an mM (pH 10) sodium borate buffer was used (2 in FIG. 1). From the results of Example 1 and Example 2 (FIG. 1), it was found that ADEKATOL SO-135
It can be seen that the slope of the calibration curve of endothelin-1 is large and the measurement sensitivity is high, especially when both ADEKATOL SO-135 and skim milk coexist.

Example 3 Measurement of Endothelin-1 in Healthy Human Plasma Endothelin-1 (manufactured by Peptide Research Institute, code No. 4198-s), which was a standard, was diluted with endothelin-1-free plasma, , 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 person: 6)
) Was operated in the same manner as in Example 2 to determine the amount of luminescence. Emission of Endothelin-1 standard solution (calibration curve)
Was used to determine the concentration of endothelin-1 in the sample.

[Table 10] 検 体 Sample number ET-1 (pg / ml) Sample 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 nonspecific luminescence reaction, ie, reagent blank (noise), and enhances specific luminescence reaction, ie, signal amount, in chemiluminescence analysis using peroxidase as a label. Ratio (signal /
Noise ratio). Therefore, the reliability of the measured value is improved. In addition, 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 the drawings]

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

[Explanation of symbols]

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

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-59-162454 (JP, A) JP-A-4-194664 (JP, A) JP-A-4-235349 (JP, A) JP-A-1- 217266 (JP, A) JP-A-54-17899 (JP, A) JP-A-3-502248 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 33/531 G01N 33 / 536

Claims (4)

(57) [Claims] 1. A chemiluminescence analysis method comprising reacting a substance to be analyzed labeled with peroxidase, luminol and hydrogen peroxide in the presence of a sensitizer, and detecting and measuring the amount of generated light. A chemiluminescence analysis method in which a secondary alcohol polyethoxylate is present in a reaction solution when detecting and measuring the amount of luminescence of the compound. 2. A chemiluminescence analysis method comprising reacting a substance to be analyzed labeled with peroxidase, luminol and hydrogen peroxide in the presence of a sensitizer, and detecting and measuring the amount of luminescence produced. A chemiluminescence analysis method in which skim milk and / or ovalbumin are present together with a secondary alcohol polyethoxylate in a reaction solution when detecting and measuring the amount of luminescence of the compound. 3. A sensitizer, luminol, hydrogen peroxide,
A reagent for chemiluminescence analysis for measuring an analyte to be labeled with peroxidase, comprising a secondary alcohol polyethoxylate. 4. A sensitizer, luminol, hydrogen peroxide,
Secondary alcohol polyethoxylate, skim milk and / or
Or a reagent for chemiluminescence analysis for measuring a substance to be analyzed labeled with peroxidase, comprising ovalbumin or ovalbumin.