JP3005238B2 - Chemiluminescence assay - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for enhancing and stabilizing luminescence in a chemiluminescent reaction using peroxidase derived from a fungus belonging to the genus Arthromyces or Coprinus as a catalytic enzyme.

In recent years, the importance of health maintenance and early detection of abnormal conditions in living organisms has been recognized, and simple and quick quantitative analysis of biological components that serve as biochemical indicators as a means to grasp minute metabolic fluctuations and abnormalities in living organisms. Methods, especially methods using enzymatic techniques with excellent specific selectivity, are being developed and applied. At that time, a biological component serving as a biochemical index is quantified by converting it into a measurable signal compound (for example, hydrogen peroxide or NAD (P) ⁺ ) using a selective enzyme.
Hydrogen peroxide is particularly frequently used as such a final signal compound, and an enzyme-coupled reaction system that provides hydrogen peroxide has been energetically developed. In order to quantify hydrogen peroxide as a final signal compound, a chemiluminescence reaction using peroxidase (hereinafter abbreviated as POD) as a catalytic enzyme is widely used. The present invention relates to a method for enhancing and stabilizing luminescence in the chemiluminescence reaction.

(Prior Art) A POD chemiluminescence reaction is represented by the following reaction formula, for example, when hydrogen peroxide is used as an oxidizing agent. Quantitative luminescence (emission of photons) can be derived by this reaction. Although a method in which A generated in the reaction formula is a dye or a fluorescent substance is also known, a method for measuring the amount of luminescence is the most sensitive, and therefore, a biological sample, for example,
It has various advantages, such as the ability to reduce the amount of blood collected during periodic diagnosis and the like, and the ability to sufficiently dilute a biological sample to perform a reaction, thereby reducing the effects of reaction interfering substances in the sample. In addition, the high measurement sensitivity of the method for measuring the amount of luminescence will allow the measurement of biological components that could not be used as biochemical indicators because they did not reach the measurement range in the past. Expected.

Although the detailed mechanism of chemiluminescence by POD is not completely elucidated, studies using horseradish-derived peroxidase (hereinafter abbreviated as HRP) have been widely performed. However, although HRP has the advantage of emitting light under milder conditions than other inorganic light emitting catalysts,
The luminescence catalytic ability of HRP is insufficient for use in microquantitative reactions of biological components. As a means for solving this problem, various compounds that enhance the chemiluminescence of HRP have been found (JP-A-57-171839 and JP-T-59-500252).
Japanese Patent Application Laid-Open No. H11-157, and the use of such a sensitizer has not yet reached a satisfactory level of the chemiluminescence catalytic activity of HRP.

Furthermore, the available HRP is often a mixture of multiple Izozymes, which affects the accuracy and reproducibility of the quantitative reaction, and furthermore, HRP has a complicated behavior of the luminescence reaction,
For example, there is also a problem that it is difficult to control the reaction when performing quantitative measurement, for example, the luminescence reaction speed fluctuates with time and shows a pulsating luminescence pattern.

Therefore, the present inventors have found that P
An attempt was made to search for OD, and found a peroxidase produced by fungi belonging to the genera Arthromyces and Coprinus (hereinafter, these are abbreviated as ARP and CCP).
No. 87 discloses this. The present inventors have also found that the catalytic ability of ARP and CCP for chemiluminescence is at least 100 to 300 times better than that of HRP (JP-A-63-219398).

(Problems to be Solved by the Invention) The present invention further enhances the excellent luminescence catalytic ability of ARP or CCP by using a sensitizer, and is more suitable for a chemiluminescence determination method for trace amounts of biological components. The purpose is to provide.

The present invention also enhances the catalytic activity of these enzymes under the condition that the ratio of luminescence (signal) generated by catalysis by ARP or CCP to non-specific luminescence (noise), that is, the signal / noise ratio is high, and It is an object to provide a chemiluminescence assay which is particularly suitable for the quantification of components.

In addition, the present invention eliminates these drawbacks, since the sensitized chemiluminescence reaction often ceases in a short period of time, maintaining the enhanced luminescence catalyzed by ARP or CCP for as long as possible, thus simplifying the reaction. It is an object of the present invention to provide a chemiluminescence quantification method capable of quantification with a simplified analyzer.

(Means for Solving the Problems) The present inventors have conducted various studies for the above purpose, and as a result, as a chemiluminescence sensitizer for ARP or CCP, hydrazobenzene, indigo, 3-methyl- 2-benzothiazolinone hydrazone / HCl, α-naphthoquinone, indophenol, phenylhydrazine, 8-hydroxyquinoline, phenolphthalein, L-tryptophan, N, N-dimethylindoaniline, calcein, indigo carmine and resorcinol . Among these sensitizers, hydrazobenzene, indigo and 3
-Methyl-2-benzothiazolinone hydrazine / HCl
Is particularly preferable in that the enhanced chemiluminescence is maintained for a long time and the signal / noise ratio is high.

Accordingly, the present invention provides a method for producing chemiluminescence by reacting a luminescent substrate selected from the group consisting of peroxidase; oxidizing agent; luminol and isoluminol, and quantifying any of the components involved in the reaction. This is a method characterized by generating chemiluminescence in the presence of a sensitizer.

The peroxidase used in the present invention is peroxidase (ARP) from the genus Arturomyces or peroxidase (CCP) from a fungus of the genus Coprinus. Examples thereof are described in JP-A-61-0443987. Peroxidases include those produced by genetic engineering. In addition, horseradish-derived peroxidase (HR
P) is not used in the present invention.

As an example for carrying out the method of the present invention, the following conditions can be set: a) ARP or CCP 0.01 μg to 1000 mg / b) Oxidizing agent 0.01 nmol to 100 mmol / c) Luminol or isoluminol 0.5 μmol to 200 mmol / d) Chemiluminescence reaction sensitizer 1μmol ~ 100mmol / e) pH 8 ~ 11 f) Temperature 10 ~ 60 ° C Prepare a mixed solution excluding at least one of the main reaction components of the above a) ~ d). Into a reaction vessel (for example, a test tube). Next, the missing main reaction component is added to the reaction tank to start the chemiluminescence reaction. The light produced by the reaction is quantified by standard measuring equipment, for example a photomultiplier, and the signal is sent to a recorder, oscilloscope or scalar for display or recording. Alternatively, if desired, it may be observed with the naked eye, or further reacted on a photographic plate and quantified. The present inventors measured the luminous intensity by using a luminometer (Lumak / 3M).
(Bio counter M2010, manufactured by 3M Pharmaceutical Co., Ltd.).

According to the method of the present invention, any of an oxidizing agent, a luminescent substrate, a peroxidase, and a chemiluminescent sensitizer can be detected and quantified. Examples of the oxidizing agent include hydrogen peroxide and a peroxide substance (for example, lipid peroxide and the like). Is an important measurement object. When components such as proteins, hormones, steroids, nucleic acids, and metabolic intermediates in a biological sample are quantified by an enzyme-linked immunosorbent assay, peroxidase is directly bound to an antibody to be measured and hydrogen peroxide is added, or Hydrogen peroxide generated in the reaction system by the action of an enzyme (conjugate enzyme) such as alkaline phosphatase or glucose oxidase bound to the antibody may be used, and based on the result, the amount of the original component in the biological sample You can know. The method of the present invention can be used for various analytical purposes other than trace quantification of biological components, with respect to analysis items that can use hydrogen peroxide generated directly or as a signal as an index.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited only to Examples.

Example 1 Enhancement of chemiluminescence (relative to ARP) ARP used was RZ = 2.0 purified by the method described in JP-A-61-0443987 (RZ is the ratio of A403 to A205). ). Luminol and hydrogen peroxide were purchased from Nacalai Tesque. The chemiluminescence reaction sensitizer was purchased from Nacalai Tesque, Tokyo Kasei Kogyo, Sigma or Alditch. Other reagents were purchased from Nacalai Tesque.

The reaction is performed in advance with a 50 mM pyrophosphate / HCl buffer (pH
In 9.6), a solution prepared by dissolving 20 μg / ARP and 1 mmol / luminol was prepared, and 650 μl of this solution was added to dimethyl sulfoxide (DMSO) in a disposable plastic test tube in 0.1%.
After mixing with 50 μl of a chemiluminescence reaction sensitizer dissolved in 1 mM or DMSO as a control, the mixture was placed at the luminescence measurement site of the luminometer and prepared with the above buffer.
The reaction was started by adding 100 μl of 15 mM hydrogen peroxide. The reaction temperature was 25 ° C. Luminescence was recorded by an average luminescence integrator built into the luminometer (in this example, the average luminescence integrated amount for 30 seconds is shown) and an external output signal from the luminometer was recorded by a recorder. Observed average luminescence and signal / noise ratio (S / N)
Are shown in Table 1.

Example 2 Sustained luminescence reaction (relative to ARP) The reaction was performed in advance using a 50 mM pyrophosphate / HCl buffer (pH
In 9.6), a solution was prepared by dissolving 1 μg and 5 μg / ARP and 1 mmole / luminol, and 650 μl of the solution was dissolved in DMSO in a disposable plastic test tube to a concentration of 0.1 to 1 mM. DMSO as control
After mixing with 50 μl, the same procedure as in Example 1 was performed. The reaction was followed for 3 minutes after the start of the reaction, and the persistence of the luminescence reaction was compared as an integrated light amount every 30 seconds. The results are shown in Table 2.

Example 3 ARP was added under the same measurement conditions as in Example 1 except that the reaction was started by adding 1 mM of hydrogen peroxide.
The effect of the concentration of hydrazobenzene, which is one of the sensitizers of the present invention, on the chemiluminescence reaction was investigated using the catalyst as a catalyst.
As a result of measuring as an average integrated light emission for 60 seconds, 0.5 to 1 mM
(The final concentration was 30 to 60 μM), the maximum sensitizing effect was obtained. The results are shown in FIG.

Example 4 Effect of pH on Sensitized Chemiluminescence Reaction The influence of pH on the sensitized chemiluminescence reaction when 1 mM of hydrazobenzene, which is one of the sensitizers of the present invention, was added.
The measurement was performed under the same conditions as in Example 3 in comparison with the case of DMSO (control) to which no sensitizer was added. To investigate the effect of pH,
A 50 mM Tris / HCl buffer (pH 8.3 to 9.5), a 50 mM pyrophosphate / HCl buffer (pP8.7 to 9.6), and a 100 mM glycine / NaOH buffer (pH 9.0 to 11.0) were used. As a result, it was found that the optimum pH shifted to the neutral side as compared with the case where hydrazobenzene was not added, and the luminescence reaction became maximum around the pyrophosphate / HCl buffer solution pH 9.6. The results are shown in FIG. In FIG. 2, chemiluminescence sensitized with hydrazobenzene is shown by a solid line, and chemiluminescence without hydrazobenzene is shown by a broken line, a white circle represents a Tris / HCl buffer solution, a black circle represents a pyrophosphate / HCl buffer solution, Double circles indicate chemiluminescence in glycine / NaOH buffer.

Example 5 Quantitative Range of ARP by Sensitized Luminescence Using hydrazobenzene, one of the sensitizers of the present invention, the quantifiable range of ARP based on the sensitized luminescence reaction was examined under the following conditions. At the same time, chemiluminescence in HRP (RZ = 3.5: manufactured by Toyobo Co., Ltd.), particularly HRP by 6-hydroxybenzothiazole which is already known as a sensitizer for HRP.
Sensitized luminometer (Thorpe et al., Anal. Biochem., 14 ).
5 , 96, 1985).

The reaction is performed beforehand in the case of ARP with 50 mM pyrophosphateH
Cl buffer (pH 9.6), 0.1M Tris-HCl buffer (pH 8.5) for HRP, 2 μg to 4 mg / POD (corresponding to 0.16 μg to 325 μg / final concentration of reaction solution), 1 mmole The effect of dissolving luminol was prepared, and 650 μl of this was mixed with 50 μl of the sensitizer or control DMSO dissolved in DMSO to 1 mM in a disposable plastic test tube, and 100 μl of 1 mM hydrogen peroxide was added. The reaction was started by the addition. Other reaction conditions were the same as in Example 1. In the sensitized luminescence system using ARP with hydrazobenzene as a sensitizer, quantification in a lower concentration range is possible and the quantification range is wider than in the sensitized luminescence system using HRP. (Quantification was possible at a reaction final ARP concentration of 0.16 μg to 16.3 μg /). The results are shown in FIG. In Fig. 3, white circles indicate AR.
The circles with P (hydrazobenzene added) and X can quantify ARP (no sensitizer added), the black circles can quantify HRP (6-hydroxybenzothiazole added), and the double circles can quantify HRP (no sensitizer added). Each area is shown.

Example 6 Determination of hydrogen peroxide by sensitized luminescence of ARP The present invention was carried out under the same conditions as in Example 5 except that the concentration of ARP was 2.5 mg / hour and the amount of hydrogen peroxide added for starting the reaction was 1 nM / 4 mM. The range in which hydrogen peroxide can be quantified by the method described above was examined. In a sensitized luminescence system using ARP in which hydrazobenzene was used as a sensitizer, the final reaction concentration was 0.25 to 50.
0 nM hydrogen peroxide was quantifiable. The results are shown in FIG.

Example 7 Increase in the amount of chemiluminescence (relative to CCP) In the case of CCP, as in the case of ARP, in order to confirm that the chemiluminescence is increased by the sensitizer of the present invention, Example 1 was repeated using CCP instead of ARP. The same test was performed. The CCP used had an RZ of 2.1. As a result, the sensitizer of the present invention showed the same sensitizing effect in CCP as in ARP. Third result
It is shown in the table.

Example 8 Sustained luminescence reaction (relative to CCP) The reaction was carried out in the same manner as in Example 2 except that 1 μg / CCP was used and the sensitizer was dissolved in DMSO to 0.1 mM. As a result, a compound showing sustained luminescence reaction in ARP showed the same effect when placed in CCP. The results are shown in Table 4.

Example 9 Quantification of NADH NADH was quantified by the method of the present invention. Hydrazobenzene was used as the luminescence sensitizer. 600 μl of ARP 0.1 mg / and Luminol 1 mmol / previously dissolved in 50 mM pyrophosphate / HCl buffer (pH 9.3), dissolved in the above buffer to 0.1 mM to 1 mM (final concentration 6.25 μM to 62.5 μM) 100 μl of 1.A mixture containing 50 μl of the prepared NADH solution and 50 μl of a 1 mM hydrazobenzene / DMSO solution.
The luminescence reaction was started by adding 6 μM 1-methoxy 5-methylphenazinium methyl sulfate (dissolved in the above buffer). The luminescence measurement method is the same as that in Example 1 except that the luminescence was measured using the average luminescence amount for 60 seconds.
The results are shown in FIG.

Example 10 Determination of Cumene Hydroperoxide Cumene hydroperoxide, one of peroxides, was quantified as an oxidizing agent in a chemiluminescence reaction using ARP. Hydrazobenzene was used as a sensitizer. ARP
At a concentration of 0.4 mg to 10 mg / 50 mM pyrophosphate / HCl buffer (p
H9.3), and the luminescence was measured in the same manner as in Example 1 except that the reaction was started by adding 100 μl of a 1 mM cumene hydroperoxide solution instead of hydrogen peroxide. Table 5 shows the results of sensitized luminescence with hydrazobenzene obtained as the average luminescence amount for 60 seconds.

Example 11 Persistence of luminescence reaction by sensitizer Reaction was carried out under the same conditions as in Example 1 except that the concentration of ARP was changed to 2 μg /. The external output signal emitted from the luminometer was followed by a recorder, and the sensitizer was used. The sensitized light emission pattern by the addition of hydrazobenzene, which is one of them, was examined. Sixth
The figure shows the results. As is clear from this figure, the chemiluminescence enhanced by the sensitizer stably continued for 10 minutes or more.

Example 12 Influence of sensitizer on Vmax and Km The chemiluminescence reaction was carried out under the same conditions as in Example 5 except that the concentration of ARP or HRP was changed to 100 μg / and hydrogen peroxide added for starting the reaction was changed from 1 μM to 1 mM. went. Based on the amount of luminescence at each concentration of hydrogen peroxide obtained in this way, Lineweaver-Burk
From the plot (double reciprocal plot), the Michaelis constant (Km) and the maximum velocity (Vmax) were determined. As a result, it was found that the addition of the chemiluminescence sensitizer of the present invention hardly affected the Michaelis constant, but significantly increased the maximum velocity (Vmax). The results are shown in Table 6.

(Effect of the Invention) According to the present invention, the chemiluminescence catalytic activity is higher than that of HRP,
By using a chemiluminescence sensitizer that further enhances the catalytic ability of RP and CCP, a highly sensitive method for measuring hydrogen peroxide, lipid peroxide, and the like has become possible. The method of the present invention enables the measurement to be performed under conditions with a high signal / noise ratio. Further, the method of the present invention has excellent measurement accuracy because the sensitized chemiluminescence is stably maintained for a long time, and is very suitable for analysis of biological components such as clinical tests.

[Brief description of the drawings]

FIG. 1 is a graph showing the relationship between the sensitizer hydrazobenzene and the amount of chemiluminescence. FIG. 2 is a graph showing the relationship between pH and the amount of chemiluminescence in the presence of a sensitizer. FIG. 3 is a graph showing the results of examining the concentration range of POD used when performing chemiluminescence in the presence of a sensitizer. FIG. 4 is a graph showing the result of quantifying the amount of hydrogen peroxide by the method of the present invention. FIG. 5 is a graph showing the results of quantifying NADH by the method of the present invention. FIG. 6 is a graph showing the duration of chemiluminescence in the presence of a sensitizer.

────────────────────────────────────────────────── ─── Continued on the front page (56) References Methods in Enzymology, Vol. 133 (1986) pp. 331-353 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12Q 1/28 MEDLINE (STN)

Claims (3)

(57) [Claims] 1. A component involved in the reaction of a peroxidase derived from a fungus belonging to the genus Arthromyces or Coprinus; an oxidizing agent; a luminescent substrate selected from the group consisting of luminol and isoluminol; Hydrazobenzene, indigo, 3-methyl-2-benzothiazolinone hydrazone.HCl, α-naphthoquinone, phenylhydrazine, 8-hydroxyquinoline, phenolphthaline, L-tryptophan , N, N-dimethylindoaniline, calcein, indigo carmine, and resorcinol, wherein chemiluminescence is produced in the presence of a chemiluminescence sensitizer. 2. A sensitizer comprising hydrazobenzene, indigo,
3-methyl-2-benzothiazolinone hydrazone / HC
2. The method of claim 1, wherein the method is selected from the group consisting of l. 3. The method according to claim 1, wherein the component to be detected or quantified is hydrogen peroxide as an oxidizing agent, and the hydrogen peroxide is produced by the action of an enzyme from another substance. .