JPH08261943A - Stabilized chemiluminescence method, chemiluminescent reagent, and enzyme immunoassay using them - Google Patents

Abstract

PURPOSE: To provide a stable high-sensitivity chemiluminescence method and a chemiluminescent reagent adaptable to a clinical examination medicine. CONSTITUTION: While peroxidase is determined by the chemiluminescence generated when a 2,3-dihydro-1,4-phthalazinedione compound and peroxidase are reacted under the existance of a chemiluminescent enhancing agent (d) and an oxidant (c) in chemiluminescence method, surface active agent and an antioxidant additionally coaxist in this chemimluminescence method, A liquid chemiluminescent reagent for determining peroxidase is composed of the surface active agent, antioxidant, chemiluminescent enhancing agent (d), and 2,3- dihydro-1,4-phthalazinedione compound in this method.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a chemiluminescence method and a chemiluminescence reagent applicable to clinical test agents and the like and an enzyme immunoassay method using them.

[0002]

2. Description of the Related Art In a chemiluminescence method in which a 2,3-dihydro-1,4-phthalazinedione compound is reacted with an oxidant in a solution containing a peroxidase in the presence of a chemiluminescence enhancer, the luminescence generated is further improved. A method of coexisting a surfactant and a protein in order to enhance the concentration is known (Japanese Patent Laid-Open Publication Nos. 6-22795 and 6-242).
No. 111). Further, in order to improve the storage stability of the chemiluminescent reagent, there is a method of using a reagent containing a 2,3-dihydro-1,4-phthalazinedione compound as an acidic solution (Japanese Patent Laid-Open No. 62-228936).

[0003]

However, although the method of coexisting a surfactant and a protein has an effect of enhancing luminescence, when it is stored in a solution state, the luminescence ability is remarkably reduced in a short period of time, and the measurement sensitivity is lowered. Will occur.
Therefore, there is a problem that the reagent for luminescence cannot be stored in a solution and the reagent needs to be adjusted at each measurement. Further, in the method of using the reagent as an acidic solution, long-term storage in a solution state is possible, but at the time of luminescence measurement, it is necessary to adjust to a weak alkaline which is an optimum pH of luminescence and cannot be directly used for measurement. There is a problem.

[0004]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that a chemiluminescence enhancer and a surfactant are used together with 2,3-dihydro-1,4-
By using an antioxidant together with the phthalazinedione compound, the present invention has reached the method and reagent of the present invention, which have an effect of enhancing luminescence at the time of measuring luminescence, have an effect of sustaining luminescence, and are stable in solution for a long period of time. That is, a 2,3-dihydro-1,4-phthalazinedione compound (b) and an oxidizing agent (c) are reacted in a solution containing a peroxidase (a) in the presence of a chemiluminescence enhancer (d). In the chemiluminescence method for quantifying peroxidase (a) according to the intensity of chemiluminescence generated, at least one selected from a cationic surfactant, an amphoteric surfactant and a nonionic surfactant together with a chemiluminescence enhancer (d). Of the surfactant (e) of
A chemiluminescence method characterized by using an antioxidant (f) together with a 2,3-dihydro-1,4-phthalazinedione compound (b), and an antioxidant (f) used in the chemiluminescence method Chemiluminescent reagent formed by combining the added solution of 2,3-dihydro-1,4-phthalazinedione compound (b), surfactant (e), chemiluminescence enhancer (d), and oxidant (c) , And the chemiluminescence method or the enzyme immunoassay method using the chemiluminescence reagent.

In the present invention, as the chemiluminescence enhancer (d), conventionally known ones can be used. For example, compounds described in JP-A-59-500252 and JP-A-5-520252
Compounds described in JP-A-9-171839, JP-A-2-
The compounds described in No. 291299 can be used. Of these, the phenol derivative is preferable. P-iodophenol, 4- (cyanomethylthio) phenol, and 4-cyanomethylthio-2-chlorophenol, which have a particularly high enhancing effect, are preferable.

In the present invention, the oxidizing agent (c) is
Examples thereof include hydrogen peroxide, sodium perborate, potassium perborate and the like. Of these, hydrogen peroxide is preferred.

In the present invention, 2,3-dihydro-1,
Examples of the 4-phthalazinedione compound (b) include luminol, isoluminol, N-aminohexyl-N-ethylisoluminol (AHEI) and N-aminobutyl-.
N-ethylisoluminol (ABEI) is mentioned.
Of these, preferred is luminol.

In the present invention, peroxidase (a)
Examples include peroxidase extracted from horseradish, microorganisms, milk, leukocytes and the like. Of these, horseradish peroxidase is preferred. Further, peroxidase (a) is a ligand (eg, antigen, antibody, hapten, protein A, avidin, etc.) used in immunoassay even in a free state.
It may be in the form of a complex bound to biotin).

In the present invention, as the surfactant (e), a cationic surfactant, an amphoteric surfactant and a nonionic surfactant can be used. From the cationic surfactant, the amphoteric surfactant and the nonionic surfactant, It is desirable to use a plurality of selected surfactants in combination.

The cationic surfactant is preferably a quaternary ammonium salt type cationic surfactant, the amphoteric surfactant is a betaine type amphoteric surfactant, and the nonionic surfactant is preferably a sorbitan fatty acid ester ethylene oxide adduct.

Preferred examples of the quaternary ammonium salt type cationic surfactant include distearyl dimethyl ammonium chloride, stearyl dimethyl benzyl ammonium chloride, stearyl trimethyl ammonium chloride, cetyl trimethyl ammonium chloride, benzalkonium chloride,
Examples thereof include didecyldimethylammonium chloride and lanolin fatty acid aminopropylethyldimethylammonium ethyl sulfate.

A preferred example of the betaine-type amphoteric surfactant is coconut oil fatty acid amidopropyl betaine.

A preferred example of the sorbitan fatty acid ester ethylene oxide adduct is a sorbitan oleic acid monoester ethylene oxide adduct.

These surfactants can be used alone, but by using a plurality of surfactants in combination, the effect of enhancing and maintaining the emission is further enhanced. Of these surfactants, the most preferable is a combination of benzalkonium chloride and sorbitan oleic acid monoester ethylene oxide adduct.

The amount of the surfactant (e) used varies depending on the type of the surfactant and the amount of the chemiluminescence enhancer (d), but the weight ratio (e) / (d) = 0.2-200. Is preferred. When the weight ratio (e) / (d) is lower than 0.2, the effect of enhancing and maintaining emission is low or not observed.
On the other hand, when it is higher than 200, adverse effects such as increase in blank value of luminescence and suppression of luminescence reaction are observed. A particularly preferred weight ratio (e) / (d) is in the range of 1-50.

In the present invention, as the antioxidant (f), a conventionally known one having no phenol skeleton can be used. Compounds with a phenol skeleton strongly suppress the luminescence reaction,
It is not preferable because it weakens the intensity of the emitted light. On the other hand, antioxidants having no phenol skeleton show only weak inhibition, and no inhibition is observed especially at low concentrations. Examples of preferable antioxidants (f) include histidine, methionine, sorbitol, terpenes (citral, limonene, etc.), ascorbic acid, thiol compounds (dithiothreitol, methylmercaptan, diallyl sulfide, etc.), chelating agents (ethylenediamine tetra). Sodium acetate, etc.). Most preferred of these is citral.

The amount of the antioxidant (f) used may vary depending on the amounts of the 2,3-dihydro-1,4-phthalazinedione compound (b) and the chemiluminescence enhancer (d) used. The storage ratio is preferably in the range of (f) / (b) = 0.001 to 1. Furthermore, in the reaction solution, the antioxidant (f) and the chemiluminescence enhancer (d) are in a weight ratio (f) /
It is preferable to add it so that (d) = 0.001-1. This range does not suppress the chemiluminescent reaction,
The storage stability of a 2,3-dihydro-1,4-phthalazinedione compound (b) solution is improved.

In the present invention, the 2,3-dihydro-1,4-phthalazinedione compound (b) and the oxidizing agent (c) are combined with the chemiluminescence enhancer (d) in a solution containing the peroxidase (a). React in the presence. This reaction is preferably carried out in an alkaline solution, especially at pH 7
-11 is suitable. The buffer used has the above-mentioned pH.
It is possible to use any type of buffer solution as long as it satisfies the requirements, but phosphate buffer solution, glycine / NaO
H buffer, Tris / hydrochloric acid buffer, Tris / acetate buffer,
Carbonic acid buffer solution, barbital buffer solution, borate buffer solution and the like are preferred.

In the present invention, the 2,3-dihydro-1,4-phthalazinedione compound (b) containing the antioxidant (f) and the chemiluminescence enhancer containing the surfactant (e) are added. A combination of (d) and an oxidizing agent (c) is used as a chemiluminescent reagent used for a luminescence reaction. The 2,3-dihydro-1,4-phthalazinedione compound (b) added with the antioxidant (f) and the chemiluminescence enhancer (d) added with the surfactant (e) are the above buffer solutions. It can be stored and used by dissolving it separately. In order to simplify the measurement operation, a 2,3-dihydro-1,4-phthalazinedione compound to which a surfactant (e) and a chemiluminescence enhancer (d) are added together with an antioxidant (f) It is preferable to store and use the solution (b) (luminescent reagent A). The oxidizing agent (c) is a 2,3-dihydro-1,4-phthalazinedione compound (b) added with an antioxidant (f), and a chemiluminescence enhancer added with a surfactant (e). Apart from (d), the above reaction p
It is stored and used as a solution (luminescent reagent B) dissolved in a solvent that does not change H (usually water). Usually, the luminescent reaction is performed by adding the luminescent reagent A and the luminescent reagent B to a sample containing peroxidase. in advance,
Although it is possible to mix and use the luminescent reagent A and the luminescent reagent B, the storage stability after mixing is reduced.

The concentrations of the chemiluminescence enhancer (d), the 2,3-dihydro-1,4-phthalazinedione compound (b) and the oxidizer (c) used in the present invention are determined by the kind of compound used, The optimum concentration must be set according to the measurement method and conditions. Usually, the chemiluminescence enhancer (d) is 10
^{-4 to} 1.0 (W / V%), 2,3-dihydro-1,4-
The phthalazinedione compound (b) is 100 nmol to 1
mol / liter, oxidizer (c) is 100 nmol-1
It can be set in the range of mol / liter.

The chemiluminescence method and chemiluminescence reagent of the present invention can be used in conventionally known peroxidase-based assay methods and assay kits, particularly immunoassay methods and immunoassay kits.

The enzyme immunoassay method using peroxidase is widely known. The amount of the substance to be measured in the sample is quantified by quantifying the peroxidase activity in accordance with the amount of peroxidase-labeled ligand (eg, antigen, antibody, hapten, protein A, avidin, biotin, etc.). it can. The present invention is applicable to all conventional enzyme immunoassays using peroxidase.

[0023]

The present invention will be further described below with reference to examples, but the present invention is not limited thereto.

Example 1 This example illustrates that the surfactant according to the present invention exhibits luminescence enhancement and sustaining effects.

1) Preparation of luminescent reagent A 0.18 g of luminol (manufactured by Tokyo Kasei) and 0.1 g of 4- (cyanomethylthio) phenol were added to 0.1 M (mol / mol).
l), dissolved in 1 liter of Tris / hydrochloric acid buffer of pH 8.5. 0.1% of the surfactant shown in Table 1 was added to this solution.
It was added to give a luminescent reagent A. Even when a plurality of surfactants were added at the same time, the concentration of the final surfactant was adjusted to 0.1%. These solutions were refrigerated until used. The surfactants used were products of Sanyo Kasei Kogyo Co., Ltd., except for Tween80. Tween80 is
A product of DIFCO was used.

[0026]

[Table 1]

2) Preparation of Luminescent Reagent B Hydrogen peroxide was used as the oxidizing agent (c). 200μ
1 of 35% hydrogen peroxide solution was dissolved in 1 liter of deionized water to obtain a luminescent reagent B. It was stored refrigerated until used.

3) Immunoreaction Reagent and Immune Reaction Operation Using a commercially available enzyme immunoassay reagent "Glazyme AFP-EIA TEST" (Wako Pure Chemical Industries) using peroxidase as a label, immunization was carried out according to the attached instruction. The reaction was carried out. That is, a sample 20 containing AFP in a test tube.
μl with 300 μl buffer for immune reaction and antibody beads 1
The pieces were reacted at 37 ° C. for 1 hour. After washing the beads with a buffer solution, add 300 μl of the peroxidase-labeled antibody solution, and add 37
Reacted at 1 ° C for 1 hour. After washing with a buffer solution, enzyme activity was measured.

4) Luminescence measurement procedure The enzyme activity was measured as follows. The test tube (12 × 75 mm) containing the above beads was set in a sample holder of luminescence reader BLR-201 type manufactured by Aloka Co., Ltd., and 200 μl of luminescence reagent A and luminescence reagent B20 were set.
0 μl was added to start the luminescence reaction. 1 from the start of luminescence reaction
The light emission amount per minute was measured every 3 seconds.

5) Measurement results The amount of luminescence and the change in the amount of luminescence when the standard AFP solution (640 ng / ml) was measured are shown in FIGS. 1 and 2. When a surfactant was added, it showed remarkable enhancement of emission and maintenance effect.

Example 2 This example shows the effect of enhancing and maintaining luminescence when a plurality of surfactants are used at the same time.

A luminescent reagent A was prepared in the same manner as in Example 1. The added surfactant is cation GC-50, Tw.
Using een80, it was added alone (0.5% addition) or simultaneously (0.25% addition each). In the same manner as in Example 1, standard AFP 640 ng / ml was measured and the amount of luminescence was measured.

The results are shown in FIG. The effect of enhancing and maintaining luminescence is higher when the surfactants are added at the same time than when the surfactants are used alone.

Example 3 This example shows that the antioxidant according to the present invention contributes to the storage stability of luminescent reagent A.

1) Preparation of luminescent reagent A 0.18 g of luminol (manufactured by Tokyo Kasei) and 0.1 g of 4- (cyanomethylthio) phenol were added to 0.1 M (mol / mol).
l), dissolved in 1 liter of Tris / hydrochloric acid buffer of pH 8.5. To this solution, 0.1% of surfactants (cation G-50 and Tween 80) and 0.00% of antioxidants were added.
1% was added to obtain luminescent reagent A. These solutions were stored at 4 ° C. and 40 ° C. until use, and the measurement was performed immediately after preparation and 2 weeks later. The antioxidants used are histidine, methionine, sorbitol, citral, ascorbic acid, dithiothreitol, sodium ethylenediaminetetraacetate.

2) Measuring method An immunoreaction was carried out in the same manner as in Example 1. Sample is standard A
FP 0 ng / ml and standard AFP 640 ng / ml were used. The peroxidase activity was measured in the same manner as in Example 1. However, the amount of luminescence was shown as an average value for 10 seconds after 40 seconds from the start of the luminescence reaction.

3) Measurement results The results are shown in Table 2. The storage stability was improved by adding the antioxidant.

[0038]

[Table 2]

Example 4 This example shows the effects of the surfactant and antioxidant according to the present invention when the chemiluminescence enhancer (d) is P-iodophenol.

1) Preparation of Luminescent Reagent A 0.18 g of luminol (manufactured by Tokyo Kasei) and 0.1 g of P-iodophenol at 0.1 M (mol / l), pH of 8.
5 of Tris / hydrochloric acid buffer was dissolved in 1 liter. To this solution, a surfactant (cation G-50 and Tween
80) 0.1%, antioxidant (citral) 0.00
1% was added to obtain luminescent reagent A.

2) Measuring method An immunoreaction was carried out in the same manner as in Example 1. The peroxidase activity was measured in the same manner as in Example 1.

3) Results of measurement FIG. 4 shows the change in the amount of luminescence within 1 minute from the start of luminescence when standard AFP 640 ng / ml was measured. Clearly, the enhancement of luminescence and the maintenance effect were recognized.

Immediately after preparation of luminescent reagent A and at 4 ° C. and 40 ° C.
Table 5 shows the measurement results after storage for 2 weeks. Standard AFP 20 ng / ml was used as a sample. The amount of luminescence was shown as an average value for 40 seconds after the initiation of the luminescence reaction. The storage stability is clearly improved.

[0044]

[Table 3]

[0045]

EFFECTS OF THE INVENTION According to the present invention, it is possible to further enhance the sensitivity of the method for determining peroxidase by chemiluminescence by further increasing and maintaining the amount of luminescence. Therefore,
When applied to an immunoassay, a more sensitive immunoassay becomes possible. In addition, since the luminescent reagent can be stably stored in a solution state for a long period of time, reagent preparation at the time of measurement can be simplified and stable luminescence measurement can be performed. Therefore, by applying the present invention, it is possible to make a more sensitive and stable immunoassay reagent.

[Brief description of drawings]

FIG. 1 shows the case where a surfactant according to the present invention is added,
The change in the amount of light emission is shown.

FIG. 2 shows the case where a surfactant according to the present invention is added,
The change in the amount of light emission is shown.

FIG. 3 shows changes in the amount of luminescence when two kinds of the surfactant according to the present invention were added simultaneously.

FIG. 4 shows changes in the amount of luminescence of the present invention when the chemiluminescence enhancer (d) is P-iodophenol.

Claims (13)

[Claims] 1. A 2,3-dihydro-1,4-phthalazinedione compound (b) and an oxidizing agent (c) in a solution containing a peroxidase (a) in the presence of a chemiluminescence enhancer (d). In the chemiluminescence method for quantifying peroxidase (a) according to the intensity of chemiluminescence produced by the reaction, a chemiluminescence enhancer (d) is further selected from cationic surfactants, amphoteric surfactants and nonionic surfactants. 2,3-dihydro-using one or more surfactants (e)
A chemiluminescence method characterized in that an antioxidant (f) is used together with a 1,4-phthalazinedione compound (b). 2. The surfactant (e) is one or more surfactants selected from quaternary ammonium salt type cationic surfactants, betaine type amphoteric surfactants and sorbitan fatty acid ester ethylene oxide adducts. The chemiluminescence method according to claim 1. 3. The surfactant (e) is distearyl dimethyl ammonium chloride, stearyl dimethyl benzyl ammonium chloride, stearyl trimethyl ammonium chloride, cetyl trimethyl ammonium chloride, benzalkonium chloride, didecyl dimethyl ammonium chloride, lanolin ethyl sulphate. The chemiluminescence method according to claim 2, which is one or more kinds of surfactants selected from fatty acid aminopropylethyldimethylammonium, coconut oil fatty acid amide propyl betaine and sorbitan oleic acid monoester ethylene oxide adduct. 4. The chemiluminescence method according to claim 3, wherein the surfactant (e) is a combination of benzalkonium chloride and sorbitan oleic acid monoester ethylene oxide adduct. 5. The chemiluminescence enhancer (d), to which the surfactant (e) is added in a weight (e) / (d) = 0.2 to 200 is used. Any one of the chemiluminescence methods. 6. The chemiluminescence method according to claim 1, wherein the chemiluminescence enhancer (d) is a phenol derivative. 7. The chemiluminescence enhancer (d) is at least one phenol derivative selected from P-iodophenol, 4- (cyanomethylthio) phenol and 4-cyanomethylthio-2-chlorophenol. The chemiluminescent method described. 8. The chemiluminescence method according to claim 1, wherein the antioxidant (f) is an antioxidant having no phenol skeleton. 9. The antioxidant (f) is histidine, methionine, sorbitol, citral, limonene, ascorbic acid, dithiothreitol, methyl mercaptan,
The chemiluminescence method according to claim 8, which is one or more compounds selected from the group consisting of diallyl sulfide and sodium ethylenediaminetetraacetate. 10. The antioxidant (f) is added to the 2,3-dihydro-1,4-phthalazinedione compound (b) at a weight ratio (f) / (b) = 0.001 to 1. And in the solution, the antioxidant (f) and the chemiluminescence enhancer (d)
Is in the range of weight (f) / (d) = 0.001-1, and the chemiluminescence method according to any one of claims 1-9. 11. A 2,3-added antioxidant (f)
A combination of a dihydro-1,4-phthalazinedione compound (b), a chemiluminescence enhancer (d) to which a surfactant (e) is added, and an oxidizer (c).
A chemiluminescent reagent for use in the chemiluminescent method according to any one of 1. 12. A surfactant containing a surfactant (e) and a chemiluminescence enhancer (d) together with an antioxidant (f).
12. A solution of the dihydro-1,4-phthalazinedione compound (b) in combination with an oxidizing agent (c).
A chemiluminescent reagent as described. 13. An enzyme immunoassay method using peroxidase (a) as a label, wherein the quantification of peroxidase (a) is performed by the chemiluminescence method according to any one of claims 1 to 11, or An enzyme immunoassay method, which comprises using the chemiluminescent reagent according to claim 12 or 13.