JP3711543B2 - Chemiluminescence measuring reagent and measuring method - Google Patents

Description

【００１６】
【発明の効果】
本発明の化学発光測定試薬は、特に保存安定性に優れ、また簡便で精度の良い測定が可能である。
【図面の簡単な説明】
【図１】本発明の実施例における種々のpHの発光液Ａ及び発光液Ｂの両試薬を３７℃に保管した苛酷試験により保存安定性を評価したグラフである。なお、１ルミカウントの発光量は１．２×１０^-16Ｗである。
【図２】本発明の実施例におけるＧＨ定量用の検量線を示すグラフである。なお、１ルミカウントの発光量は１．２×１０^-16Ｗである。[0001]
[Industrial application fields]
The present invention relates to a chemiluminescence measuring reagent and measuring method for measuring the enzyme activity of peroxidase by chemiluminescence in the presence of a sensitizer.
[0002]
[Prior art]
In recent years, a method using chemiluminescence has begun to spread instead of a measurement method using a radioisotope. In the method using chemiluminescence, there is a method in which a chemiluminescent substrate or an enzyme is used as a labeling substance that changes in accordance with increase / decrease of a measurement target substance. However, the method of labeling an enzyme is an excellent method in terms of versatility and measurement accuracy of a measurement apparatus resulting from the persistence of light emission. There are various types of enzymes to be labeled, but peroxidase is widely used, and in the system using peroxidase as a labeling substance, a method of measuring chemiluminescence generated mainly in the presence of a chemiluminescent substrate such as luminol and an oxidizing agent is mainly used. ing. A method of sensitizing this reaction with a sensitizer is known as a sensitive method, and various sensitizers have been developed (Methods in Enzymology, Vol. 133, p.331-353, 1986). JP-A-2-291299, JP-A-59-171839, JP-A-59-500002, JP-A-1-503730, and the like.
[0003]
[Problems to be solved by the invention]
As the composition of the reagents, the above-mentioned JP-A-59-171839, JP-A-59-500262, etc. store peroxidase, luminol, sensitizer and oxidizing agent as separate reagents, and at the time of reaction Mixing to initiate the luminescence reaction is described. More specifically, a solution is prepared by mixing luminol and hydrogen peroxide several hours before use, and this is injected into a reaction system containing a peroxidase-antibody complex to which a sensitizer has been added in advance. Is described. JP-A-2-291299 discloses a mixture of luminol and hydrogen peroxide solution mixed about 2 hours before use, and further added with a sensitizer just before use. It is described to be added.
In general, the analytes to which the measurement methods using these chemiluminescence reactions are applied are often very small amounts, and the above methods are not sufficient, and can be measured with higher sensitivity and stability. Development of a reagent with high storage stability is desired.
The present invention solves these problems and provides a simple and accurate chemiluminescence measuring reagent and measuring method that are particularly excellent in storage stability.
[0004]
[Means for Solving the Problems]
That is, the present invention is a reagent for measuring the enzyme activity of peroxidase by chemiluminescence using a chemiluminescent substrate, a reaction sensitizer and an oxidizing agent, and these are pH 9 to 13 containing a chemiluminescent substrate and a reaction sensitizer. The present invention relates to a chemiluminescence measuring reagent stored as a reagent (1) prepared in (1) and a reagent (2) prepared at pH 4 to 8 containing an oxidizing agent, and a chemiluminescence measuring method using this reagent.
[0005]
Chemiluminescence measurement using the reagent of the present invention is particularly limited as long as it is a method for detecting and quantifying the generated luminescence by reacting a chemiluminescent substrate and an oxidizing agent by the catalytic action of peroxidase in the presence of a sensitizer. is not. Peroxidase is generally used as a labeling enzyme, but it can also be used as an unlabeled form. For example, as a specific binding reaction used as a labeling enzyme, various analyzes such as a single 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, a DNA probe method, etc. Available to law.
[0006]
The peroxidase used in the present invention is not particularly limited, but is preferred from the point that a basic isozyme of horseradish peroxidase can be combined with a sensitizer to obtain a high specific light emission amount. The basic isozymes of horseradish peroxidase are known to be B, C, D, and E. Among them, type C is the RZ value (indicating the ratio of hemin to protein) and enzyme activity. Most preferred. This is commercially available, for example, from Toyobo Co., Ltd.
[0007]
Examples of the chemiluminescent substrate used in the chemiluminescent reaction include luminols, lophine, and lucigenin. Luminols are preferable, and specifically, luminol, isoluminol, N-ethylisoluminol, N- (4-aminobutyl). ) -N-ethylisoluminol hemisuccinimide, N- (6-aminohexyl) -N-ethylisoluminol, and the like. Of these, luminol or isoluminol is preferable in terms of stability and luminous efficiency, and luminol is particularly preferable. Since luminol and hydrazine and sulfide ions, which are raw materials for production, are often mixed in reagent grades that are usually available, it is preferable to use luminol that has been purified by repeated recrystallization.
[0008]
As the oxidizing agent used for chemiluminescence, hydrogen peroxide, perborate, urea peroxide and the like are preferable, and hydrogen peroxide is particularly preferable from the viewpoint of ease of handling.
The sensitizer used for the chemiluminescence reaction is not particularly limited as long as it has a luminescence enhancement effect or a luminescence persistence effect, but p-iodophenol, p-bromophenol, phenolindole, 4- [4 ′ -(2'-methyl) thiazolyl] phenol, 4- (4'-thiazolyl) phenol, 4- [4 '-(2'-(3'-pyridyl)) thiazole] phenol, 4- (2'-thienyl) Phenol derivatives such as phenol, 4- [2 '-(4'-methyl) thiazolyl] phenol, phenothiazine-N-propylsulfonate or phenolindophenol, 6-hydroxybenzothiazole, 4- (4-hydroxyphenyl) thiazole Thiazole derivatives such as 3- (10-phenothiazyl) -propyl sulfonate, p-hydroxyphenylpropionic acid Used as such diethylaniline are preferred. Among these, 4- [4 '-(2'-methyl) thiazolyl] phenol, 4- (4'-thiazolyl) phenol, 4- [4'-(2 '-(3'-pyridyl)) thiazole] is preferable. Phenol, 4- (2'-thienyl) phenol, 4- [2 '-(4'-methyl) thiazolyl] phenol, phenothiazine-N-propyl sulfonate, or phenol indophenol, particularly preferred are S / It is 4- [4 '-(2'-methyl) thiazolyl] phenol in that the N ratio (signal to noise ratio) is high.
[0009]
In the reagent of the present invention, the reagent (1) prepared at pH 9 to 13 containing a chemiluminescent substrate and a reaction sensitizer and the reagent (2) prepared at pH 4 to 8 containing an oxidizing agent are used. Store as a liquid. Reagent (2) containing an oxidizing agent does not become a stable reagent even when the pH is too high or too low. The reagent (1) containing a chemiluminescent substrate and a reaction sensitizer becomes unstable when the pH is too low.
In order to achieve the above pH, as a buffer solution used for each reagent, phosphate buffer solution, acetate buffer solution, Tris buffer solution, Good buffer solution and the like can be preferably used for reagent (2) (pH 4 to 8). As the reagent (1) (pH 9 to 13), borate buffer, glycine buffer, ammonium buffer, Tris buffer, Good buffer and the like are preferably used. The combination of these buffers can be arbitrarily selected, and the optimum reaction conditions can be obtained by adjusting the pH and salt concentration of the final reaction solution when both reagents are mixed depending on the respective concentrations and pH.
The preferred pH of the final reaction solution is 8 to 10. If this pH is too low, the sensitivity tends to decrease, and if it is too high, the blank becomes too high.
[0010]
The component concentration in each reagent varies depending on the type of component used, the mixing ratio of reagent (1) and reagent (2), etc., but the final reaction solution concentration should be within the range shown below. . That is, the concentration of the oxidizing agent (for example, hydrogen peroxide) in the final reaction solution is preferably in the range of 0.1 to 10 mM. If the concentration is lower than this, the sensitivity tends to decrease due to the substrate shortage. Tend to decrease.
The concentration of the chemiluminescent substrate (for example, luminol) in the final reaction solution is preferably in the range of 0.1 to 100 mM, and sensitivity outside this range tends to decrease.
The concentration of the reaction sensitizer is not particularly limited because the optimum concentration varies depending on the substance used, the concentration range of peroxidase to be measured, and the like. For example, in the case of p-iodophenol, the range of 0.01 to 10 mM is preferable, and in the case of 4- [4 ′-(2′-methyl) thiazolyl] phenol, it is preferably used in the range of 0.001 to 10 mM. When the concentration of each reaction sensitizer is out of this range, the sensitizing effect tends to decrease.
[0011]
The reagent (1) prepared at pH 9 to 13 containing a chemiluminescent substrate and reaction sensitizer and the reagent prepared at pH 4 to 8 containing an oxidizing agent used in the present invention contains peroxidase to be measured. The reaction vessel may be mixed by dispensing simultaneously or sequentially at the time of measurement, or both solutions may be mixed before measurement to form one solution and dispensed into the reaction vessel at the time of measurement. In addition, when the mixture is preliminarily mixed into one liquid, the sensitivity of the luminescent liquid gradually decreases, and it can be used usually only for a few days at room temperature. Therefore, it is preferable to use it early after mixing.
In order to improve the measurement accuracy, the reagent prepared in the pH range of 4 to 8 containing the oxidizing agent used in the present invention may further contain a protein component, a surfactant and the like.
[0012]
The measuring object to which the reagent of the present invention can be used is not particularly limited as long as it can be measured by directly measuring peroxidase or by using it as a labeling enzyme. For example, since it can be used for enzyme immunoassay, high-sensitivity measurement up to the order of pg / ml is possible. For example, growth hormone (GH), endothelin, thyroid stimulating hormone (TSH), free thyroxine 4 (FT4), etc. It can be applied as a measuring reagent for trace biological components such as peptide hormones and cytokines.
[0013]
Example 1 Effect of pH on Reagent Stability 5 μl of peroxidase solution (horseradish-derived peroxidase C type dissolved in PBS solution, concentration 100 pg / ml) is placed in a strip-type black microwell (manufactured by NUNC). Next, 100 μl of luminescent solution A containing 1 mM hydrogen peroxide (preparing an aqueous solution of pH 9 to 3 with 10 mM phosphate buffer or borate buffer) and 0.16 mM 4- [4 ′-(2′-methyl) thiazolyl Dispens 100 μl of luminescent solution B containing phenol and 10 mM luminol (pH 9-13, prepared so that the final pH is 9 when mixed with the corresponding luminescent solution A with 100 mM borate buffer). The mixture was stirred and mixed, and the amount of chemiluminescence after 5 minutes was measured using a chemiluminescence measuring apparatus (MLR-100, manufactured by Corona Electric Co., Ltd.).
Here, the result of evaluating the storage stability by a severe test in which both reagents of the luminescent solution A and the luminescent solution B were stored at 37 ° C. is shown in FIG. As can be seen, it was found that when the pH of the luminescent solution A is in the range of 4-8 (at this time, the luminescent solution B has a pH of 9-13).
[0014]
Example 2 Application to enzyme immunoassay As an application of this chemiluminescence measuring reagent to enzyme immunoassay, measurement of growth hormone (GH) in serum was examined.
25 μl of serum and 100 μl of biotinylated anti-GH sheep antibody solution were dispensed into strip type black microwells (manufactured by NUNC) sensitized with anti-GH monoclonal antibody, and incubated for 60 minutes with stirring at room temperature. After washing 4 times with PBS containing 0.05% (v / v) Tween 20, 100 μl of streptavidin-labeled peroxidase solution was dispensed and incubated for 10 minutes at room temperature with stirring. After washing 4 times with 0.05% (v / v) Tween 20-containing PBS, 100 μl of the same luminescent solutions A (pH 7) and B (pH 10) as in Example 1 were dispensed. The mixture was agitated and mixed, and the amount of chemiluminescence after 5 minutes was measured using a chemiluminescence measuring device (MLR-100, manufactured by Corona Electric Co., Ltd.).
When a calibration curve was obtained using standard serum with a known GH concentration instead of serum, a good calibration curve was obtained as shown in FIG. Three types of the same specimen were repeatedly measured 10 times, and the simultaneous reproducibility was determined. As a result, good simultaneous reproducibility was obtained as shown in Table 1.
[0015]
[Table 1]

[0016]
【The invention's effect】
The chemiluminescence measuring reagent of the present invention is particularly excellent in storage stability and enables simple and accurate measurement.
[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a graph showing an evaluation of storage stability by a severe test in which both reagents of luminescent solution A and luminescent solution B having various pH values were stored at 37 ° C. in Examples of the present invention. Note that the light emission amount of 1 lumicount is 1.2 × 10 ⁻¹⁶ W.
FIG. 2 is a graph showing a calibration curve for GH determination in an example of the present invention. Note that the light emission amount of 1 lumicount is 1.2 × 10 ⁻¹⁶ W.

Claims (6)

A reagent for measuring the enzyme activity of peroxidase by chemiluminescence using a chemiluminescent substrate, a reaction sensitizer and an oxidizing agent, which is prepared at a pH of 9 to 13 containing a chemiluminescent substrate and a reaction sensitizer (1) and a reagent (2) prepared at pH 4 to 8 containing an oxidizing agent and stored, the chemiluminescent substrate is a luminol, the reaction sensitizer is a phenol derivative, and A chemiluminescence measuring reagent , wherein the oxidizing agent is hydrogen peroxide, perborate or urea peroxide .   The chemiluminescence measuring reagent according to claim 1, wherein the chemiluminescent substrate is luminol or isoluminol. The reagent for measuring chemiluminescence according to claim 1 or 2 , wherein the oxidizing agent is hydrogen peroxide. Reaction sensitizer is 4- [4 '-(2'-methyl) thiazolyl] phenol, 4- (4'-thiazolyl) phenol, 4- [4'-(2 '-(3'-pyridyl)) thiazole] It is phenol, 4- (2'-thienyl) phenol, 4- [2 '-(4'-methyl) thiazolyl] phenol, phenothiazine-N-propyl sulfonate or phenol indophenol . The chemiluminescence measuring reagent according to 1. The reagent for chemiluminescence measurement according to any one of claims 1 to 4 , wherein the peroxidase is horseradish peroxidase labeled. Chemiluminescence measurement method which comprises using a chemiluminescence reagent according to any one of claims 1-5.

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